sikorski_chemical functional properties of food components 3rd ed_0849396751

Chemical andFunctional

Properties ofFood Components

Third Edition

9675_C000.fm Page i Wednesday, September 20, 2006 5:45 PM

Chemical and Functional Properties of Food Components Series

SERIES EDITOR

Zdzisław E. Sikorski

Chemical and Functional Properties of Food Components, Third EditionEdited by Zdzisław E. Sikorski

Carcinogenic and Anticarcinogenic Food ComponentsEdited by Wanda Baer-Dubowska, Agnieszka Bartoszek and Danuta Malejka-Giganti

Methods of Analysis of Food Components and AdditivesEdited by Semih Ötleş

Toxins in FoodEdited by Waldemar M. Dąbrowski and Zdzisław E. Sikorski

Chemical and Functional Properties of Food SaccharidesEdited by Piotr Tomasik

Chemical and Functional Properties of Food LipidsEdited by Zdzisław E. Sikorski and Anna Kolakowska

Chemical and Functional Properties of Food ProteinsEdited by Zdzisław E. Sikorski

9675_C000.fm Page ii Wednesday, September 20, 2006 5:45 PM

CRC Press is an imprint of theTaylor & Francis Group, an informa business

Boca Raton London New York

Chemical andFunctional

Properties ofFood Components

EDITED BY

Zdzislaw E. SikorskiGdansk University of Technology

Gdansk, Poland

Third Edition

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CRC PressTaylor & Francis Group6000 Broken Sound Parkway NW, Suite 300Boca Raton, FL 33487-2742

© 2007 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business

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Library of Congress Cataloging-in-Publication Data

Chemical and functional properties of food components / edited by Zdzislaw E. Sikorski. -- 3rd ed.

p. cm.Includes bibliographical references and index.ISBN 0-8493-9675-1 (alk. paper)1. Food--Analysis. 2. Food--Composition. I. Title.

TX545.C44 2007664’.07--dc22 2006047537

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Preface

Water, saccharides, proteins, lipids, and mineral compounds constitute the mainbuilding materials of the tissues and are responsible for the nutritional value andmost sensory properties of foodstuffs. A large number of other constituents presentin lower quantities, especially colorants, flavor compounds, vitamins, prebiotics,probiotics, and additives, also contribute to food quality. These compounds undergovarious biochemical and chemical changes during postharvest storage and processingof raw materials. The extent of changes depends on the chemical properties of thesefood components, on the conditions of storage, and on the parameters of freezing,salting, drying, smoking, marinating, frying, cooking, and other methods of preser-vation or processing.

The material presented in this book emphasizes the role of the chemical prop-erties of different food constituents, and shows how the reactions that take place inthe conditions of storage and processing affect the quality of foodstuffs. In Chapters1 and 2, the content of various food components is described, as well as their rolein the structure of raw materials and the formation of different attributes of quality.The components contained in foods in the largest amounts are presented in detailin Chapters 3 through 7. Chapter 8 deals with the flow properties of food materialsand presents numerous examples of the rheological behavior of various products.Chapters 9 and 11 describe factors affecting the color and flavor of foods, respec-tively, and Chapter 13, the most important food additives. Interactions among dif-ferent constituents are key with respect to many features of the quality of industriallyprocessed and home-prepared foods. These are treated in Chapter 12. Chapters 10and 14 through 20 of the book deal with different aspects of the biological valueand safety of foodstuffs, including allergenic activity, the role of prebiotics andprobiotics, as well as the effect of food on the moods and health of people.

The characteristics that make this volume very different from the second editionare the new contributions on lipids and rheology, the integrating chapter on interac-tions, and new chapters dealing with the safety and biological aspects of foods.

The volume contains a well-documented presentation of the current state ofknowledge concerning food in the form of concise monographs. The text is basedon the personal research and teaching experience of the authors, as well as on criticalevaluation of the current literature. According to the editor’s suggestions, most listsof cited references contain only the indispensable sources. The book is addressed tofood science graduate students, professionals in the food industry, nutritionists,personnel responsible for food safety and quality control, and to all persons interestedin the roles and attributes of different food constituents.

In preparing the book I have had the privilege of working with a large group ofcolleagues from several universities and research institutions in Asia, Australia,Europe, and North America, who have agreed to share their knowledge and experi-

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ence with the reader. Their acceptance of my editorial suggestions and the timelypreparation of the high-quality manuscripts are sincerely appreciated.

I dedicate this volume to the scores of researchers, especially Ph.D. students,whose investigations in universities all over the world contribute to a better under-standing of the nature and interactions of food components, and thus lead to improv-ing the quality of foods. A small fraction of the results of these investigations hasbeen used in preparing this new edition of the book.

Zdzis

ł

aw E. Sikorski

Gda

ń

sk University of Technology

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About the Editor

Zdzis

ł

aw E. Sikorski

earned his B.S., M.S, Ph.D., and D.Sc. in food technologyand chemistry from the Gda

ń

sk University of Technology (GUT), and his

Dr

honoriscausa

from the Agricultural University in Szczecin. He gained practical experiencein breweries, in fish, meat, and vegetable processing plants in Poland and Germany,and on a deep-sea fishing trawler. He was an organizer, professor, and head of theDepartment of Food Chemistry and Technology, and served two terms as dean ofthe Faculty of Chemistry at GUT, 7 years as chairman of the Committee of FoodTechnology and Chemistry of the Polish Academy of Sciences, chaired the scientificboard of the Sea Fisheries Institute in Gdynia for 9 years, and was an elected memberof the Main Council of Science and Tertiary Education in Poland for 11 years. Dr.Sikorski was one of the founders and is now an honorary member of the PolishSociety of Food Technologists. He also was a researcher and professor at Ohio StateUniversity in Columbus, Ohio; the Commonwealth Scientific and Industrial ResearchOrganization (CSIRO) in Hobart, Australia; the Department of Scientific and Indus-trial Research (DSIR) in Auckland, New Zealand; and the National Taiwan OceanUniversity, Keelung. His published works include about 210 papers, 15 books, 9chapters on marine food science and food chemistry in edited volumes, and he holdsseven patents in the area of fish and krill processing. Several of his books haveappeared in multiple editions. He is a member of the editorial board of the

Journalof Food Biochemistry

and of two Polish food science journals. His research dealsmainly with food preservation, the functional properties of food proteins, and theinteractions of food components. In 2003 he was elected a Fellow of the InternationalAcademy of Food Science and Technology.

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Contributors

Agnieszka Bartoszek

Gda

ń

sk University of TechnologyGda

ń

sk, Poland

Maria Bielecka

Polish Academy of SciencesOlsztyn, Poland

Maria H. Borawska

Medical Academy of Bia

ł

ystokBia

ł

ystok, Poland

Emilia Barbara Cybulska

The Elbl

ą

g University of Humanities and Economy

Elbl

ą

g, Poland

Peter Edward Doe

University of TasmaniaHobart, Tasmania

Norman F. Haard

University of CaliforniaDavis, California

Julie Miller Jones

College of St. CatherineSt. Paul, Minnesota

Zenon K

ę

dzior

Agricultural University of Pozna

ń

Pozna

ń

, Poland

Jen-Min Kuo

Department of Seafood ScienceNational Kaohsiung Marine UniversityKaohsiung, Taiwan

Wies

ł

awa

Ł

ysiak-Szyd

ł

owska

Medical Academy of Gda

ń

skGda

ń

sk, Poland

Micha

ł

Nabrzyski


ń

skGda

ń

sk, Poland

Krystyna Palka

Agricultural University of CracowCracow, Poland

Barbara Piotrowska

MerckWarsaw, Poland

Anna Pruska-K

ę

dzior

Agricultural University of Pozna

ń

Pozna

ń

, Poland

Bob Rastall

University of ReadingReading, United Kingdom

Adriaan Ruiter

Professor EmeritusUtrecht UniversityUtrecht, The Netherlands

Gra

ż

yna Sikorska-Wi

ś

niewska


ń

skGda

ń

sk, Poland

Zdzis

ł

aw E. Sikorski

Gda

ń

sk University of TechnologyGda

ń

sk, Poland

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Piotr Siondalski


ń

skGda

ń

sk, Poland

Andrzej Sto

ł

yhwo

Warsaw Agricultural UniversityWarsaw, Poland

Bonnie Sun-Pan

Department of Food ScienceNational Taiwan Ocean UniversityKeelung, Taiwan

Ma

ł

gorzata Szumera


ń

skGda

ń

sk, Poland

Piotr Tomasik

Agricultural University of CracowCracow, Poland

Alphons G.J. Voragen

Wageningen UniversityWageningen, The Netherlands

Jadwiga Wilska-Jeszka

Technical University of

Ł

ód

żŁ

ód

ż

, Poland

Barbara Wróblewska

Polish Academy of SciencesOlsztyn, Poland

Chung-May Wu

Hungkuang Institute of TechnologyChai-Nan University of Pharmacy and

ScienceNational Taiwan Ocean UniversityKeelung, Taiwan

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Contents

Chapter 1

Food Components and Quality ............................................................ 1

Zdzis

ł

aw E. Sikorski and Barbara Piotrowska

Chapter 2

Chemical Composition and Structure of Foods ................................ 15

Krystyna Palka

Chapter 3

Water and Food Quality..................................................................... 29

Emilia Barbara Cybulska and Peter Edward Doe

Chapter 4

Mineral Components.......................................................................... 61

Micha

ł

Nabrzyski

Chapter 5

Saccharides......................................................................................... 93

Piotr Tomasik

Chapter 6

The Role of Proteins in Food .......................................................... 129

Zdzis

ł

aw E. Sikorski

Chapter 7

Lipids and Food Quality .................................................................. 177

Andrzej Sto

ł

yhwo

Chapter 8

Rheological Properties of Food Systems ........................................ 209

Anna Pruska-K

ę

dzior and Zenon K

ę

dzior

Chapter 9

Food Colorants................................................................................. 245


Chapter 10

Food Allergens ................................................................................. 275

Barbara Wróblewska

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Chapter 11

Flavor Compounds in Foods............................................................ 295

Bonnie Sun-Pan, Jen-Min Kuo, and Chung-May Wu

Chapter 12

Interactions of Food Components ................................................... 329

Zdzis

ł

aw E. Sikorski and Norman F. Haard

Chapter 13

Main Food Additives........................................................................ 357

Adriaan Ruiter and Alphons G.J. Voragen

Chapter 14

Food Safety ...................................................................................... 375

Julie Miller Jones

Chapter 15

Prebiotics.......................................................................................... 391

Bob Rastall

Chapter 16 Probiotics in Food............................................................................ 413

Maria Bielecka

Chapter 17 Mood Food....................................................................................... 427

Maria H. Borawska

Chapter 18 Food Components in the Protection of theCardiovascular System..................................................................... 439

Piotr Siondalski and Wiesława Łysiak-Szydłowska

Chapter 19 Mutagenic, Carcinogenic, and Chemopreventive Compoundsin Foods............................................................................................ 451

Agnieszka Bartoszek

Chapter 20 The Role of Food Components in Children’s Nutrition ................. 487

Grażyna Sikorska-Wiśniewska and Małgorzata Szumera

Index...................................................................................................................... 517

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1

1

Food Componentsand Quality

Zdzis

ł

aw E. Sikorski and Barbara Piotrowska

CONTENTS

1.1 Food Components ............................................................................................ 11.1.1 Components in Food Raw Materials and Products............................. 11.1.2 Factors Affecting Food Composition................................................... 31.1.3 The Role of Food Components ........................................................... 4

1.2 Functional Properties of Food Components.................................................... 51.3 The Role of Postharvest Changes, Handling, and Processing

in the Quality of Foods.................................................................................... 61.3.1 Introduction .......................................................................................... 61.3.2 Attributes of Quality ............................................................................ 61.3.3 Safety and Nutritional Value................................................................ 71.3.4 Sensory Quality.................................................................................... 8

1.4 Chemical Analysis in Ensuring Food Quality................................................. 91.4.1 Introduction .......................................................................................... 91.4.2 Requirements of the Producer ............................................................. 91.4.3 Requirements of the Consumer ......................................................... 121.4.4 Limits of Determination .................................................................... 12

References................................................................................................................ 13

1.1 FOOD COMPONENTS

1.1.1 C

OMPONENTS

IN

F

OOD

R

AW

M

ATERIALS

AND

P

RODUCTS

Foods are derived from plants, carcasses of animals, and single-cell organisms.Their main components include water, saccharides, proteins, lipids, and minerals(Table 1.1), as well as a host of other compounds present in minor quantities,albeit of significant impact on the quality of many products. The nonproteinnitrogenous compounds, vitamins, colorants, flavor compounds, and functionaladditives belong here.

The content of water in various foods ranges from a few percent in dried commod-ities (e.g., milk powder), about 15% in grains, 16 to 18% in butter, 20% in honey, 35%in bread, 65% in manioc (cassava), and 75% in meat, to about 90% in many fruits and

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2

Chemical and Functional Properties of Food Components

vegetables. Saccharides are present in food raw materials in quantities ranging fromabout 1% in meats and fish, 4.5% in milk, 18% in potatoes, and 15 to 21% in sugarbeets, to about 70% in cereal grains.

The protein content in foods is present mainly as crude protein (i.e., as N

×

6.25).The nitrogen-to-protein conversion factor (N:P) of 6.25 has been recommended formost plant and animal food products under the assumption that the N content in theirproteins is 16%, and they do not contain nonprotein N. The N content in the proteinsin various foods, however, is different because it depends on the amino acid compo-sition. Furthermore, the total N consists of protein N and of N contained in numerousnonprotein compounds, such as free peptides and amino acids, nucleic acids and theirdegradation products, amines, betains, urea, vitamins, and alkaloids. In some foodsthe nonprotein N may constitute up to 30% of total N. In many of these compoundsthe C:N ratio is similar to the average in amino acids. However, the N content in urea,at 47%, is exceptionally high. The average conversion factor for the estimation of trueprotein, based on the ratios of total amino acid residues to amino acid N determinedfor 23 various food products, is 5.68 and for different classes of foods is between 5.14and 6.61

(Table 1.2). The N:P factor of 4.39, based on analysis of 20 different vege-tables, has been proposed by Fujihara et al. (2001) for estimating the true proteincontent in vegetables. A common N:P factor of 5.70 for use with respect to blendedfoods or diets has been recommended by Sosulski and Imafidon (1990).

TABLE 1.1Typical Products as Rich Sources of the Main Food Components

Water Saccharides Proteins Lipids Minerals Vitamins

JuicesFruitsMilkVegetablesJelliesLean fish Lean meat

SaccharoseHoneyCerealsChocolatePotatoCassavaFruits

SoybeanBeansMeatFishWheatCheeseEggs

OilsLardButterChocolateNutsEgg yolkPork

VegetablesFruitsMeatFish productsDairy productsCerealsNuts

VegetablesFruitsFish liverMeatCerealsMilkYeast

TABLE 1.2The Nitrogen to Protein Conversion Factors in Foods

Product Factor Product Factor

Dairy productsEggMeat and fishCereals and legumes

6.02–6.155.73

5.72–5.825.40–5.93

PotatoLeafy vegetablesFruitsMicrobial biomass

5.185.14–5.30

5.185.78–6.61

Source: Data from Sosulski, F.W. and Imafidon, G.I. 1990.

J. Agric. Food Chem.

38,1351, 1990).


Food Components and Quality

3

Crude protein makes up from about 1% of the weight of fruits and 2% of potatoes,3.2% of bovine milk, 12% of eggs, 12 to 22% of wheat grain, about 20% of meat,to 25 to 40% of various beans. During their development, cereal grain and legumeseeds deposit large quantities of storage proteins in granules also known as proteinbodies. In soybeans these proteins constitute 60 to 70% of the total protein content,and the granules are 80% proteins.

The lipid content in foods is given in nutrition information labeling predomi-nantly as

total fat

, which is often called

crude fat

. This is a mixture of various classesof lipids, mainly different triacylglycerols. The lipids of numerous food fishes, suchas orange roughy, mullet, codfish, and shark, as well as some crustaceans andmollusks also include wax esters. Some shark oils are very rich in hydrocarbons,particularly in squalene. Furthermore, the lipid fraction of food raw materials harborsdifferent sterols, vitamins, and pigments that are crucial for metabolism. Thus thecomposition of the extracted crude fat depends on the kind of food and the polarityof the solvent used for extraction. Lipids constitute from less than 1% of the weightof fruits, vegetables, and lean fish muscle, 3.5% of milk, 6% of beef meat, and 32%of egg yolk, to 85% of butter.

1.1.2 F

ACTORS

A

FFECTING

F

OOD

C

OMPOSITION

The content of different components in food raw materials depends on the speciesand variety of the animal or plant crop; on the conditions of cultivation and time ofharvesting of the plants; on the feeding, conditions of life, and age of the farmanimals or the fishing season for fish and marine invertebrates; and on postharvestchanges that take place in the crop during storage. The food industry, by establishingquality requirements for raw materials, can encourage producers to control, withinlimits, the contents of the main components in their crops; for example, starch inpotatoes, fat in various meat cuts, pigments in fruits and vegetables and in the fleshof fish from aquaculture, or protein in wheat and barley, as well as the fatty acidcomposition of lipids in oilseeds and meats. The contents of desirable minor com-ponents can also be effectively controlled; for example, the amount of naturalantioxidants to retard the oxidation of pigments and lipids in beef. Contaminationof the raw material with organic and inorganic pollutants can be controlled byobserving recommended agricultural procedures in using fertilizers, herbicides, andinsecticides, and by seasonally restricting certain fishing areas to avoid marine toxins.The size of predatory fish like swordfish, tuna, or shark, which are fished commer-cially, can be limited to reduce the risk of excessive mercury and arsenic in the flesh.

The composition of processed foods depends on the recipe applied and onchanges taking place due to processing and storage. These changes are mainlybrought about by endogenous and microbial enzymes, active forms of oxygen,heating, chemical treatment, and processing at low or high pH (Haard 2001). Exam-ples of such changes are listed below:

• Leaching of soluble, desirable and undesirable components, such as vita-mins, minerals, and toxins during washing, blanching, and cooking

• Dripping after thawing or due to cooking


4


• Loss of moisture and volatiles due to evaporation and sublimation • Absorption of desirable or harmful compounds during salting, pickling,

seasoning, frying, or smoking • Formation of desirable or harmful compounds due to enzyme activity,

such as the development of typical flavor in cheese or decarboxylation ofamino acids in fish marinades

• Generation of desirable or objectionable products due to interactions ofreactive groups induced by heating or chemical treatment, such as flavorsor carcinogenic compounds in roasted meats, or

trans

-fatty acids in hydro-genated fats

• Formation of different products of oxidation of food components, mainlyof lipids, pigments, and vitamins

• Loss of nutrients and deterioration of dried fish due to attacks by flies,mites, and beetles

1.1.3 T

HE

R

OLE

OF

F

OOD

C

OMPONENTS

The indigenous water that is immobilized in the plant and animal tissues by thestructural elements and various solutes contributes to buttressing the conformationof the polymers, serves as a solvent for different constituents, and interacts inmetabolic processes.

Polysaccharides, proteins, and lipids serve as the building material of differentstructures of the plant and animal tissues used for food. The structures made of thesematerials are responsible for the form and tensile strength of the tissues, and createthe necessary conditions for metabolic processes to occur. Compartmentalizationresulting

from these structures plays a crucial biological role in the organisms. Someof the main components, as well as other constituents, are bound to different cellstructures or are distributed in soluble form in the tissue fluids.

Many saccharides, proteins, and lipids are stored for reserve purposes. Polysac-charides are present in plants as starch in the form of granules and in muscles asglycogen. Other saccharides are dissolved in tissue fluids or perform different bio-logical functions, such as in free nucleotides or as components of nucleic acids, orin being bound to proteins and lipids. Proteins also play crucial metabolic roles inplants and animals as enzymes and enzyme inhibitors, participate in the transportand binding of oxygen and metal ions, and perform immunological functions.

The distribution of lipids in food raw materials depends on their role in the livinganimal and plant organisms. In an animal body, lipids occur primarily as an energy-rich store of neutral fat in the subcutaneous adipose tissue; as kidney, leaf, and crotchfat; as the intramuscular fat known as marbling; and as intermuscular or seam fat.In fatty animals, the largest portion of lipids is stored as depot fat in the form oftriacylglycerols. In lean fish species, most of the fat occurs in the liver. The lipidscontained in the food raw materials in low quantities serve mainly as componentsof protein-phospholipid membranes and have metabolic functions.

The main food components supply the human body with the necessary buildingmaterial and source of energy, as well as elements and compounds indispensablefor metabolism. Some plant polysaccharides are only partly utilized for energy.



5

However, as dietary fiber they affect, in different ways, various processes in thegastrointestinal tract.

Many of the minor components originally present in the raw materials arenutritionally essential, such as vitamins. Some of them, although not indispensable,can be utilized by the body, including most free amino acids, or impart desirablesensory properties to food products. Numerous groups, including tocopherols,ubiquinone, carotenoids, ascorbic acid, thiols, amines, and several other nonproteinnitrogenous compounds serve as endogenous muscle antioxidants, playing an essen-tial role in postmortem changes in meat (Decker et al

.

2000). Other minor compo-nents are useless or even harmful if present in excessive amounts. Most food rawmaterials are infected with different microorganisms, putrefactive and often patho-genic, and some contain parasites and the products of microbial metabolism. Avariety of compounds are added intentionally during processing, to be used aspreservatives, antioxidants, colorants, flavorings, sweeteners, and emulsifyingagents, or to fulfill other technological purposes.

1.2 FUNCTIONAL PROPERTIES OF FOOD COMPONENTS

The term

functional properties

has a broad range of meanings. The term

technolog-ical properties

implies that the given component present in optimum concentration,subjected to processing at optimum parameters, contributes to the expected desirablesensory characteristics of the product, usually by interacting with other food con-stituents. Hydrophobicity, hydrogen bonds, ionic forces, and covalent bonding areinvolved in these interactions. Thus, the functional properties of food componentsare affected by the number of accessible reactive groups and by the exposure ofhydrophobic areas in the given material. Therefore, in a system of given water activityand pH, and in the given range of temperature, the functional properties can be toa large extent predicted from the structure of the respective saccharides, proteins,and lipids. They can also be improved by appropriate, intentional enzymatic orchemical modifications of the molecules, mainly those that affect the size, chargedensity, or the hydrophilic/hydrophobic character of the compounds, or by changesin the environment of both the solvent and other solutes.

The functional properties of food components make it possible to manufactureproducts of desirable quality. Thus, pectins contribute to the characteristic textureof ripe apples and make perfect jellies. Other polysaccharides are efficient thickeningand gelling agents at different ranges of acidity and concentration of various ions.Alginates in the presence of Ca

2+

form protective, unfrozen gels on the surface offrozen products. Some starches are resistant to retrogradation, thereby retardingstaling of bread. Fructose retards moisture loss from biscuits. Mono- and diacylg-lycerols, phospholipids, and proteins are used for emulsifying lipids and stabilizingfood emulsions and foams. Antifreeze proteins inhibit ice formation in variousproducts, and gluten plays a major role in producing the characteristic texture ofwheat bread.

Technologically required functional effects can also be achieved by intentionallyemploying various food additives, such as food colors, sweeteners, and a host of


6


other compounds. These additives are per se not regarded as foodstuffs, but are usedto modify the rheological properties or acidity, increase the color stability or shelflife, and act as humectants or flavor enhancers.

During the two most recent decades, the term

functional

has been predominantlygiven to a large group of products and components, also termed designer foods,pharmafoods, nutraceuticals, or foods for specific health use, which are regarded ashealth enhancing or potentiating the performance of the human organism. Thesefoods, mainly drinks, meals, confectionery, ice cream, and salad dressings containvarious ingredients, including oligosaccharides, sugar alcohols, or choline, which areclaimed to have special physiological functions like neutralizing harmful compoundsin the body and promoting recovery and general good health (Goldberg 1994). Foodscontaining prebiotics (various oligosaccharides) and probiotics (mainly dairy prod-ucts) are treated in detail in Chapters 15 and 16 of this volume, respectively.

1.3 THE ROLE OF POSTHARVEST CHANGES, HANDLING, AND PROCESSING IN THE QUALITY OF FOODS

1.3.1 I

NTRODUCTION

The chemical nature of food components is of crucial importance for all aspects offood quality. It determines the nutritional value of the product, its sensory attrac-tiveness, development of desirable or deteriorative changes due to interactions withother constituents and to processing, and susceptibility or resistance to spoilageduring storage. Food components, which contain reactive groups, many of themessential for the quality of the products, are generally labile and easily undergodifferent enzymatic and chemical changes, especially when treated at elevated tem-perature or in conditions promoting the generation of active species of oxygen.

1.3.2 A

TTRIBUTES

OF

Q

UALITY

The quality of a food product—the characteristic properties that determine its degreeof excellence—is a sum of the attributes contributing to the consumer’s satisfactionwith the product. The composition and the chemical nature of the food componentsaffect all aspects of food quality. The total quality reflects at least the followingattributes:

• Compatibility with the local or international food law regulations andstandards regarding mainly the proportions of main components, presenceof compounds serving as identity indicators, contents of contaminants andadditives, hygienic requirements, packaging, and labeling

• Nutritional aspects, such as the contents and availability of nutritionallydesirable constituents, mainly proteins, essential amino acids, essentialfatty acids, saccharides, vitamins, fiber, and mineral components

• Safety aspects affected by the concentration of compounds that mayconstitute health hazards for consumers and affect the digestibility and



7

nutritional availability of the food, such as heavy metals, toxins of variousorigins, some enzymes and enzyme inhibitors, factors decreasing theavailability of some metal components, pathogenic microorganisms, andparasites

• Sensory attributes, such as the color, size, form, flavor, taste, and rheolog-ical properties, obviously affected by the chemical composition of theproduct, and by changes resulting from processing and culinary preparation

• Shelf life under specific storage conditions• Convenience aspects related to the size and ease of opening and reclosing

the container, the suitability of the product for immediate use or fordifferent types of thermal treatment, ease of portioning or spreading, andtransport and storage requirements

• Ecological aspects regarding suitability for recycling of the packagingmaterial and pollution hazards

For many foods, one of the most important quality criteria is freshness. This isespecially so in the case of numerous species of vegetables, fruits, and seafood. Fishof valuable species in a state of prime freshness, suitable to be eaten raw, may sellat a market price ten times higher than the same fish after several days storage inice, even though the stored fish is still very fit for human consumption.

1.3.3 S

AFETY

AND

N

UTRITIONAL

V

ALUE

Food is regarded as safe if it does not contain harmful organisms or compounds inconcentrations above the accepted limits (see Chapter 14). The nutritional value offoods depends primarily on the levels of nutrients and nutritionally objectionablecomponents in the products.

Processing may increase the safety and biological value of food by inducingchemical changes increasing the digestibility of the components or by inactivatingundesirable compounds, such as toxins or enzymes catalyzing the generation of toxicagents from harmless precursors. Freezing and short-term frozen storage of fishinactivates the parasite

Anisakis

, which could escape detection during visual inspec-tion of herring fillets used as raw material for cold marinades produced under mildconditions. Thermal treatment brings about inactivation of myrosinase, the enzymeinvolved in hydrolysis of glucosinolanes. This arrests the reactions that lead to theformation of goitrogenic products in oilseeds of

Cruciferae

. Heat pasteurization andsterilization reduce to an acceptable level the number of vegetative forms and spores,respectively, of pathogenic microorganisms. Several other examples of such improve-ments in the safety and biological quality of foods are covered in the followingchapters of this book.

There are also, however, nutritionally undesirable side effects of processing, suchas destruction of essential food components as a result of heating, chemical treatment,and oxidation. As is generally known, the partial thermal decomposition of vitamins,especially thiamine, loss of available lysine- and sulfur-containing amino acids, orgeneration of harmful compounds such as carcinogenic heterocyclic aromaticamines, lysinoalanine and lanthionine, or position isomers of fatty acids, not present


8


originally in foods. In recent years new evidence of side effects has been accumulatedwith respect to chemical processing of oils and fats. Commercial hydrogenation ofoils brings about not only the intended saturation of selected double bonds in thefatty acids and thereby the required change in the rheological properties of the oil,but also results in the generation of a large number of

trans-trans

and

cis-trans

isomers that are absent in the unprocessed oils.

1.3.4 S

ENSORY

Q

UALITY

Many of the desirable sensory attributes of foods stem from the properties of theraw material. The natural color of meat, fish muscles and skin, vegetables, and fruitsdepends on the presence of a host of different pigments, which are water or lipidsoluble. Chlorophylls impart the green color to vegetables, but also to olive oil. Somenatural oils are yellow or red due to different carotenoids. Carotenes are also presentin the flesh oil of redfish (

Sebastes marinus

), while different carotenoproteins areresponsible for the vivid colors of fish skin. Many hydroxycarotenoids (xanthophylls)occur in plants in the form of esters of long-chain fatty acids. The red, violet, orblue color of fruits and flowers is caused by anthocyanins. Betalains impart colorto red beets. The flavor, taste, and texture of fresh fruits and vegetables, as well asthe taste of nuts and milk, depend on the presence of natural compounds. Theseproperties are in many cases carried through to the final products.

In other commodities, the characteristic sensory attributes are generated as aresult of processing. The texture of bread develops due to interactions of proteins,lipids, and saccharides among themselves and with various gases, while that ofcooked meats appears as the results of thermal protein denaturation. The bouquetof wine is due to the presence of volatile components in the grape as well as theresult of fermentation of saccharides and a number of other biochemical and chemicalreactions. The delicious color, flavor, texture, and taste of smoked salmon are gen-erated in enzymatic changes in the tissues and the effect of salt and smoke. Theflavor of various processed meats develops due to thermal degradation of predomi-nantly nitrogenous compounds, the generation of volatile products of the Maillardreaction, interactions of lipid oxidation products, and the effect of added spices.Optimum foam performance of beer depends on the interactions of peptides, lipids,the surface-active components of hops, and gases. The flavor, texture, and taste ofcheese result from fermentation and ripening, while the appealing color and flavorof different fried products are due to reactions of saccharides and amino acids.

The sensory attributes of foods are related to the contents of many chemicallylabile components. These components, however, just as most nutritionally essentialcompounds, are prone to deteriorative changes in conditions of severe heat treatment,oxidizing conditions, or application of considerably high doses of chemical agents,such as acetic acid or salt, which are often required to ensure safety and sufficientlylong shelf life of the products. Thus, loss in sensory quality takes place, for example,in oversterilized meat products due to the degradation of sulfur-containing amino acidsand development of an off-flavor; toughening of the texture of overpasteurized hamor shellfish due to excessive shrinkage of the tissues and drip; deterioration of thetexture and arresting of ripening in herring preserved at too high a concentration of salt.



9

Optimum parameters of storage and processing ensure the retention of thedesirable properties of the raw material and lead to the development of intendedattributes of the product. In the selection of these parameters, the chemistry of foodcomponents and of the effect of processing must be studied. The eager foodtechnology student can find all the necessary information in at least two excellenttextbooks on food chemistry by Belitz et al. (2001) and Fennema (1996), in numer-ous books on food lipids, proteins, and saccharides, as well as in current interna-tional journals.

1.4 CHEMICAL ANALYSIS IN ENSURING FOOD QUALITY

1.4.1 I

NTRODUCTION

The aspects of food quality described in Section 1.3.2 can be assured only byapplying appropriate control in the manufacturing process and storage, based onsensory, physical, chemical, biochemical, and microbial techniques. For the purposesof analysis, appropriate techniques and hardware are used, from the most simpleprocedures and gadgets, to the very sophisticated analytical instruments known inanalytical chemistry. A rational system of control is necessary for the producers ofthe raw material, the food processor, the retailer, and even for consumer organiza-tions. The results of chemical and microbiological analyses are indispensable forselecting the most suitable parameters of processing and for their implementation,for designing and operating the hazard analysis and critical control points systemof quality assurance in processing plants, and for securing safety of the food productsavailable in the market.

1.4.2 R

EQUIREMENTS

OF

THE

P

RODUCER

Thanks to the possibility of rapid and reliable determination of food compositionand contaminants by applying contemporary techniques, the raw materials can beoptimally used for manufacturing various products; loss in quality and health hazardscan be avoided.

In the relationship between the primary producer and the food processor, therequirements regarding the contents and characteristics of the most important com-ponents, as well as freshness grades of the raw materials are agreed upon, often inthe form of contracts. Depending on the commodity, it may be saccharose in sugarbeets; fat in milk or in mackerel as raw material for hot smoking; the color ofvegetables and egg yolks, depending on the concentration of carotenoid pigments;the proportion of lean tissue and marbling in pig or beef carcasses; connective tissuein meats used for least-cost formulations of sausages; the contents and character-istics of gluten in wheat grains; starch and protein in barley used for malting; extractin tomatoes; oil in oil-bearing raw materials; free fatty acids and peroxide value infat-containing commodities; trimethylamine, hypoxanthine, or other freshness indi-cators in marine fish; or the elasticity of kamaboko, the Japanese-type fish cake.These components and characteristics are usually determined using standard, simple


10


chemical or physicochemical analyses or enzymatic sensors. For example, the textureof kamaboko is commonly determined by folding a 5-mm-thick slice of the productand observing the formed edge. The highest quality kamaboko can be folded twicewithout any cracking; the lowest-quality product falls apart after the first folding.Although this test is very simple, it may decide the price of a large consignment ofsurimi or kamaboko (Suzuki 1981). Nowadays many companies supply the hardware,reagents, and analytical procedures for numerous applications in the food plant andfor water field analysis (Table 1.3). For example, to assist in the routine analyses indairy production, many tests based on photometric or reflectometric techniques areoffered, such as reflectometric detection of alkaline phosphatase for controlling milkpasteurization, photometric control of lactose fermentation and determination ofurea, or photometric assay of ammonia in milk. Thanks to enormous progress inanalytical methodology and instrumentation, the food chemist can use automatedequipment for assaying water, proteins, lipids, saccharides, fiber, and mineral com-ponents. Online analyses provide for continuous control of processing parameters.

TABLE 1.3Examples of Some Tests Offered for Rapid Food Analysis

Measured parameter Example of food Technique

Ascorbic acid Apple purée, apple sauce, banana purée, candies, fruit and vegetable juices

Reflectometric determination after reaction with molybdophosphoric acid to phosphomolybdenum blue

Calcium Beer Photometric determination with cresolphthalexone

Cheese Reflectometric determination after reaction with phthalein purple

Milk Photometric determination with glyoxal-bis(2-hydroxyanil) or reflectometric determination after reaction with phthalein purple

Wine Photometric determination with cresolphthalexone

Chloride Meat and sausage products,pickled cabbage

Photometric determination as Fe(III)-thiocyanate in hot water extract with the Fe(III)-Hg(II)-thiocyanate method

Chromium Dairy products Photometric determination with diphenylcarbazide after decomposition with H

2

SO

4

and perhydrolCopper Various foods Photometric determination with cuprizon

subsequent to decomposition with H

2

SO

4

and perhydrol

Formaldehyde Fish products Reflectometric determination after reaction with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole

Glucose Jam, juices Reflectometric determination after reaction with glucose oxidase and peroxidase



11

The characteristic freshness attributes of different foods are usually evaluated bysensory methods and by determination of specific indices, predominantly by biochem-ical sensors. A typical example may be the examination of fish freshness by a tastepanel, and by chemical tests or biochemical sensors suitable for assaying the volatileodorous compounds and products of nucleotide catabolism (Figures 1.1 and 1.2).

The results of these kinds of analyses serve as the basis for technological decisionsregarding the suitability of the raw materials for further storage or the given treatment,as well as for adjusting the processing parameters; they also often decide the priceof the commodity.

FIGURE 1.1

Degradation of ATP in fish muscle and the K-value as a freshness indicator.

FIGURE 1.2

Typical sensory and chemical tests of fish freshness: (1) flavor of cooked fish aftercooking, (2) K-value, (3) volatile base nitrogen, (4) trimethylamine nitrogen, (5) flavor of raw fish.

K = 100(Ino + Hx)/ATP + ADP + AMP + IMP + Ino + Hx

Uric acid

Xanthine

Hypoxanthine (Hx)

Inosine (Ino)

Adenosine(Ado) inosine monophosphate (IMP)

Adenosine monophosphate (AMP)

Adenosine diphosphate (ADP)

Adenosine triphosphate (ATP)↓

↓

↓

↓

↓

↓ ↓ ↓↓

↓ ↓

Time of storage in ice [days]

K V

alue

, %

e or

scy

orns

Se

A N

T

M

N,

se

ba ilet

laV

o

5100

80

60

40

20

13

4

2

14121086420 16


12


The producer needs chemical analysis to ascertain that the raw material used inhis plant does not contain any harmful components or contaminants in quantitieshigher than those accepted by national or international regulations; for example,nitrates (V) and (III) in vegetables, pesticide residues in various crops, heavy metalsin many plant and animal tissues including Hg in large predatory fish, histamine infish meat, or mycotoxins in peanuts.

1.4.3 R

EQUIREMENTS

OF

THE

C

ONSUMER

The results of routine analyses performed by the producer and by food inspectionlaboratories must ascertain that most consumer expectations regarding nutritious andwholesome food of high sensory quality be fulfilled. The consumer generally requiresthat foods offered on the market contain the components typical for the type ofproduct and that their proportions are those represented on the label. This concerns,for example, the contents of protein and fat in meat products, milk fat in butter,vitamin C in fruit juices, the unique fatty-acid composition of the product sold asextra virgin olive oil or as n-3 polyenoic fatty acids–rich preparation, absence ofpork in products declared as being made of other meats, or meat of fish species otherthan that specified in comminuted commodities. Food adulteration is an age-old viceand chemical analysis helps to combat it. The nutrition-cautious person looks on thelabel for information regarding essential amino acids, polyunsaturated fatty acids,vitamins, mineral components, fiber, and recently functional additives or geneticallymodified products. Many consumers carefully study the labels on packaged foodsbecause their health or even life may depend on the information regarding thepresence of different ingredients rich in allergens in the produce, such as gluten orpeanuts. However, small amounts of such compounds may originate from residuesin processing machinery or stem from additives used by the processor. The safetyof food products is safeguarded by determining, for example, heavy metals and theirspeciation (see Chapter 4), polycyclic aromatic hydrocarbons (PAHs) in oils, heavilysmoked fish and meat products, acrylamide in French fries, mycotoxins in a varietyof commodities, and various additives. For determination of the very large numberof hazardous components, additives, and impurities, many specialized chromato-graphic, spectroscopic, and physical techniques, as well as enzyme, microbial, andimmunological sensors are used (Tunick 2005).

1.4.4 L

IMITS

OF

D

ETERMINATION

By applying efficient procedures of enrichment and separation of analytes combinedwith the use of highly selective and sensitive detectors, it is now possible to determinedifferent additives and contaminants, as well as the products of various chemicaland biochemical reactions in foods in extremely low concentrations. This is oftennecessary because the national and international bodies responsible for the safety offoods require that the producers conform to regulations allowing very low amountsof various natural toxic compounds and contaminants in their produce. The contentsof nitrates in potatoes and other vegetables for children less than three years of agein Poland should not exceed 250 mg NO

3

/kg. The tolerance for various pesticide



13

residues ranges in different foods from about 0.01 to 20 mg/kg (Lehotay and Mas-tovska 2005). According to German regulations introduced in 1973, the content ofbenzo[a]pyrene (BaP), the recognized representative of the carcinogenic PAHs,should be no higher than 1

µ

g/kg in smoked meat products; for meats treated withsmoke preparations, the upper limit of 0.03

µ

g/kg has been set by the EuropeanUnion. In Europe the countries producing olive residual oil have established amaximum level of 2

µ

g/kg for each of the eight highly carcinogenic PAHs, but notabove 5

µ

g/kg for the total amount of all eight compounds. In smoked meat andfishery products, in baby foods, and in food oils, according to the new regulation ofEC, No. 208/2005, the maximum permissible level of BaP is 5

µ

g/kg, 1

µ

g/kg, and2

µ

g/kg wet weight, respectively.In selecting the most appropriate analytical procedure for detection or determi-

nation of a compound in a food sample, the properties of the matrix must beconsidered. This is especially important in the step of separation of the analyte fromthe food material, be it by digestion, membrane techniques, solvent extraction,supercritical fluid extraction, sorption, headspace technique, or steam distillation.An interesting treatment of suitability of various procedures for extraction of lipidsfrom food samples is presented in Chapter 7. In gas chromatography/mass spec-trometry (GC/MS) determination of acrylamide, the methanol extraction of theanalyte from the food sample during several days in a Soxhlet apparatus yields about7 times higher results than homogenizing with the solvent followed by centrifuging.The detection limit of acrylamide in foods is actually about 10

µ

g/kg wet weight(Food and Agriculture Organization/World Health Organization [FAO/WHO] 2002).

By using procedures comprising extraction of hydrocarbons from the foodmatrix, cleanup, separation by gas chromatography (GC) or high-performance liquidchromatography (HPLC), followed by detection and quantification by mass spec-trometry or in fluorescence detectors, it is possible to determine the individualcarcinogenic PAHs at concentrations on the order of 0.1 or even 0.01

µ

g/kg wetweight (Sto

ł

yhwo and Sikorski 2005). The accuracy of the results depends signifi-cantly on the quality of standards used for calibration. Certified reference materialsare now available containing up to 15 PAHs in food samples. For quantitative analysisinternal GC/MS calibration with stable isotopes added prior to extraction and an MSdetector in selected ion mode may be used (van Rooijen 2005). In studies and routinemonitoring regarding nutritional requirements and food safety aspects, many toxicelements are determined in trace concentrations of 0.01 to 10 mg/kg or even inultratrace amounts of less than 10

µ

g/kg by using mainly spectrometric techniques(Capar and Szefer 2005). The lowest dose-inducing symptoms of allergy in highlysensitive persons is about 0.1 mg of peanut or egg protein. This means that theapplied chemical examination must guarantee the detection of a few micrograms ofpeanut material in 1 gram of food (Williams et al. 2005).

REFERENCES

Belitz, H.D., Grosch, W., and Schieberle, P. 2001. Lehrbuch der Lebensmittelchemie. 4th ed.,Springer Verlag, Berlin.


14 Chemical and Functional Properties of Food Components

Capar, S.G. and Szefer, P. 2005. Determination and speciation of trace elements in foods, inMethods of Analysis of Food Components and Additives, Ötles, S., Ed., CRC Press,Boca Raton, FL, chap. 6.

Decker, E.A., Livisay, S.A., and Zhou S. 2000. Mechanism of endogenous skeletal muscleantioxidants: chemical and physical aspects, in Antioxidants in Muscle Foods. Nutri-tional Strategies to Improve Quality, Decker, E., Faustman, C., and Lopez-Bote, C.J.,Eds., Wiley-Interscience, New York, chap. 25.

FAO/WHO (Food and Agricultural Organization/World Health Organization). 2002. HealthImplications of Acrylamide in Food, Report of a Joint FAO/WHO Consultation,Geneva, 25–27 June.

Fennema, O.R., Ed. 1996. Food Chemistry, 3rd ed., Marcel Dekker, New York.Fujihara, S., Kasuga, A., and Aoyagi, Y. 2001. Nitrogen-to-protein conversion factors for

common vegetables in Japan, J. Food Sci. 66, 412.Goldberg, I. 1994. Functional Foods: Designer Foods, Pharmafoods, Nutraceuticals, Chap-

man and Hall, New York.Haard, N.F. 2001. Enzymic modification in food systems, in Chemical and Functional Prop-

erties of Food Proteins, Sikorski, Z.E., Ed., Technomic Publishing Co., Lancaster,PA, chap. 7.

Lehotay, S.J. and Mastovska, K. 2005. Determination of pesticide residues, in Methods ofAnalysis of Food Components and Additives, Ötles, S., Ed., CRC Press, Boca Raton,FL, chap. 12.

Sosulski, F.W. and Imafidon, G.I. 1990. Amino acid composition and nitrogen-to-proteinconversion factors for animal and plant foods, J. Agric. Food Chem. 38, 1351.

Stołyhwo, A. and Sikorski, Z.E. 2005. Polycyclic aromatic hydrocarbons in smoked fish—acritical review, Food Chem., 91, 3.3.

Suzuki, T., 1981. Fish and Krill Proteins: Processing Technology, Applied Science PublishersLtd., London.

Tunick, M.H. 2005. Selection of techniques used in food analysis, in Methods of Analysis ofFood Components and Additives, Ötles, S., Ed., CRC Press, Boca Raton, FL, chap. 1.

van Rooijen, J.J.M. 2005. The control of polycyclic aromatic hydrocarbons in food ingredi-ents, International Review of Food Science and Technology, Winter 2005/2006, 116.

Williams, K.M. et al. 2005. Determination of food allergens and genetically modified com-ponents, in Methods of Analysis of Food Components and Additives, Ötles, S., Ed.,CRC Press, Boca Raton, FL, chap. 11.


15

2 Chemical Composition and Structure of Foods

Krystyna Palka

CONTENTS

2.1 Introduction .................................................................................................... 152.2 Protein Food Products.................................................................................... 16

2.2.1 Meat.................................................................................................... 162.2.2 Milk and Milk Products..................................................................... 192.2.3 Eggs.................................................................................................... 20

2.3 Saccharide Food Products.............................................................................. 212.3.1 Cereal and Cereal Products ............................................................... 212.3.2 Potatoes .............................................................................................. 232.3.3 Honey ................................................................................................. 252.3.4 Nuts .................................................................................................... 252.3.5 Seeds of Pulses .................................................................................. 25

2.4 Edible Fats...................................................................................................... 252.5 Fruits and Vegetables ..................................................................................... 26References................................................................................................................ 28

2.1 INTRODUCTION

Foods are edible fragments of plant or animal organisms in a natural or processedstate, which after being eaten and digested in the human organism, may be a sourceof different nutrients. Taking as a base the dominant nutritional component, foodproducts may be divided into four groups:

1. Protein food products2. Saccharide food products3. Edible fats4. Fruits and vegetables

The particular groups of chemical constituents participate in building the struc-ture of food products as components of specialized tissues. For this reason thischapter presents, in addition to chemical composition, the morphology of the selectedproducts from each group.

9675_book.fm Page 15 Monday, September 18, 2006 5:58 PM


2.2 PROTEIN FOOD PRODUCTS

2.2.1 MEAT

Meat is the edible part of animal, chicken, or fish carcasses. Its chemical compositionis as follows: 60 to 85% water, 8 to 23% protein, 2 to 15% lipids, 0.5 to 1.5%saccharides, and about 1% inorganic substances (Table 2.1). These quantities changesignificantly depending on type, age, sex, level of fattening, and part of animalcarcass. The largest fluctuations are observed in the contents of water and lipids.

Water is a solvent of organic and inorganic substances and an environment of bio-chemical reactions. It also participates in the maintenance of meat protein conformation.

TABLE 2.1Chemical Composition of Foods Rich in Proteins

ProductWater

%

Crude proteinN×6.25

%Lipids

%Saccharides

%

Mineralcomponents

%

Mus

cle

food

Beef, lean 71.5 21.0 6.5 1.0 1.0Pork, lean 72.0 20.0 7.0 1.0 1.0Veal 75.0 20.0 3.5 1.0 1.0Lamb 71.5 19.5 7.0 1.5 1.0Chicken

Light meat 75.0 23.0 2.0 1.0Dark meat 76.0 20.0 4.5 1.0

Herring 60.0 18.0 15.5 0.5–1.5Oyster 85.0 7.5 1.5 0.5–1.5

Milk

and

milk

pro

duct

s Cow milk 88.0 3.0 3.5 4.5 1.0Sheep milk 82.0 6.0 6.5 4.5 1.0Sour cream 25% 68.0 3.0 25.0 4.0 0.5Yogurt, low fat 85.0 5.0 1.0 7.5 0.7Quarg 64.0–75.0 9.0–14.0 12.0–18.0 2.5 1.5Ripened cheese 35.0–50.0 20.0–35.0 20.0–30.0 2.0 5.0Milk powder 3.0 26.0 26.0 38.0 6.0

Egg

s an

d eg

g pr

oduc

t

Whole eggwithout shell 73.5 13.0 12.0 1.0 1.0

White 88.0 11.0 traces 0.5 0.5Yolk 48.5 16.0 32.0 1.0 1.0Whole egg powder 3.5 47.5 43.0 to 0.5 4.0

Source: Adapted from Hedrick, H.B. et al., Principles in Meat Science, Kendal-Hunt Publ. Comp.,Dubuque, 1994; Kirk, R.S. and Sawyer, R., Pearson’s Composition and Analysis of Foods, LongmanScience, London, 1991; Renner, E., Cheese: Chemistry, Physics and Microbiology, Vol. 1, Fox, P.F., Ed.,Chapman & Hall, London, 1993; Sikorski, Z.E., Seafood Raw Materials, WNT, Warsaw, 1992; Tamime,A.Y. and Robinson, R.K., Yoghurt. Science and Technology, CRC Press. Boca Raton, FL, 1999.


Chemical Composition and Structure of Foods 17

Meat proteins include sarcoplasmic, myofibrillar, and connective tissue proteins.Among the sarcoplasmic proteins are heme pigments and enzymes, which influencethe color, smell, and structure of meat. Myofibrillar proteins and collagen are ableto retain and hold water in meat structure and to emulsify fat. Therefore, theyinfluence the rheological properties of meat products.

Mineral elements are in enzymatic complexes and other structures that play animportant biochemical role. They can affect the technological properties of meat,for example, water-holding capacity, as well as the sensory characteristics. Meat isalso a good source of the B group of vitamins.

The main structural unit of striated muscle tissue is a multinucleus cell calledmuscle fiber. Its length varies from several millimeters to hundreds of millimeters,and the diameter is between 10 and 100 µm (Figure 2.1a). The thickness of musclefibers affects the meat’s tenderness. The muscle fiber contains typical somatic cellcompounds, sarcoplasmic reticulum, and myofibrils. The sarcoplasmic reticulum hasthe capacity of reversible binding of calcium ions. Myofibrils are the main structuralelement of muscle fiber, making up 80% of its volume. They have a diameter of 1to 2 µm and are situated parallel to the long axis of the fiber (Figure 2.1b). Thespaces between myofibrils are filled up with a semiliquid sarcoplasm, which formsthe environment of enzymatic reactions and takes part in conducting nervousimpulses into the muscle.

Each myofibril consists of two different protein structures: myosin thick fila-ments (15 nm × 1.5 µm) and thin (7 nm × 1 µm) filaments composed of actin,tropomyosin, and troponin.

Inside the muscle fiber there is also a cytoskeleton—the protein structures assur-ing the integrity of muscle cells. Cytoskeletal proteins such as titin and nebulin arelocated in myofibrils and anchored in the Z-band. Desmin is made up of costamers,which connect the myofibrils; vinculin connects myofibrils and sarcolemma (Figure2.2). Postmortem degradation of cytoskeletal proteins plays a role in the improvementof meat functional properties, especially its tenderness and water-holding capacity.

The muscle fiber is covered by a thin membrane called the sarcolemma and alayer of connective tissue called the endomysium. Bundles of muscle fibers are

FIGURE 2.1 Scanning electron microscope (SEM) micrographs of bovine semitendinosusmuscle: transverse section (a) and longitudinal section (b). F, muscle fiber; MF, myofibril.(From Palka, K., unpublished. With permission.)

10 µm 5 µm



FIG

UR

E 2.

2Sc

hem

atic

str

uctu

re o

f m

uscl

e cy

tosk

elet

on:

titin

(1)

, ne

bulin

(2)

, vi

ncul

in (

3),

skel

emin

(4)

, an

d de

smin

(5)

. C

, co

stam

eres

; C

M,

cell

mem

bran

e; F

, m

uscl

e fib

er;

M,

M-b

and;

Z

, Z

-ban

d.



surrounded by the perimysium, and the whole muscle by the epimysium. At the endsof the muscle, the epimysium forms tendons, which connect the muscle to the bone(Figure 2.3). Both the quantity and kind of connective tissue affect the technologicaland nutritional properties of meat.

2.2.2 MILK AND MILK PRODUCTS

Milk is a liquid secretion of the mammary glands of female mammals, consistingof 80 to 90% water and 10 to 20% dry mass. It is an oil-in-water (O/W) emulsioncomposed of fat and fat-soluble vitamins; the aqueous phase contains proteins,mineral salts, lactose, and water-soluble vitamins. The chemical composition of milk(Table 2.1) depends on species and breed, lactation period, as well as nutritional andhealth conditions of the animal.

Milk proteins are made up of caseins and whey proteins. Milk proteins, caseins,and several enzymes, mainly hydrolases and oxidoreductases, are very important inthe manufacturing of cheeses and yogurts (Figure 2.4). After drying they are usedin the food industry as milk powder, caseinates, and casein hydrolyzates. Nonproteinnitrogenous compounds constitute about 0.2% of milk.

Milk fat is made up of about 98% triacylglycerols and 1% phospholipids. It alsocontains smaller amounts of di- and monoacylglycerols, sterols, higher fatty acids,carotenoids, and vitamins. In cow milk fat, over 500 various fatty-acid residues havebeen identified. The polyenoic fraction constitutes about 3% of the total fatty acids andis composed mainly of linoleic acid and α-linolenic acids. Milk fat is easily digestiblebecause of a relatively low melting temperature and great dispersion (droplets of 5 to10 µm in diameter). Because of the latter, it is susceptible to hydrolysis and oxidation.

The main saccharide of milk is lactose. During heat treatment of milk, lactoseis involved in Maillard reactions. Lactose is used for the production of baby formulas,low-calorie foods, bread, drugs, and microbiological media.

The milk minerals are composed mainly of calcium and phosphorus in the formof calcium phosphate. Phosphorus is also present in milk in the form of phospho-proteins. These components have important nutritional and technological signifi-cance. The total content of Ca and P in milk is about 0.12 and 0.10%, respectively.

About 6 to 9% of milk volume is made up of gases, mainly CO2, N2, and O2.Oxygen present in milk may cause oxidation of unsaturated fatty acids. For thisreason air is removed from milk during processing.

FIGURE 2.3 Schematic structure of skeletal muscle: tendon (1), epimysium (2), perimysium(3), endomysium (4), sarcolemma (5), myofibril (6), muscle fiber (7), and bundle of musclefibers (8).



Milk contains vitamins essential for the growth and development of youngorganisms, especially vitamins from the B group and vitamin A. The quantity ofvitamin A depends on the season.

2.2.3 EGGS

The hen egg consists of a shell, the egg white, and yolk (Figure 2.5). The 0.2- to0.4-mm thick shell constitutes up to 10 to 12% of the egg mass and consists of about3.5% organic and 95% mineral components. The shell has a multilayer structure.Two of the layers are made of keratin and collagen fibers. The next two layers arecalcinated and on the surface are covered by a thin membrane (cuticula), whichcontains two-thirds of the shell pigments. The shell protects the egg against micro-biological contamination and makes the exchange of gases possible.

FIGURE 2.4 SEM micrograph of protein matrix in yogurt. (From Domagała, J., unpublished.With permission.)

FIGURE 2.5 Schematic structure of a hen egg: shell (1), membranes (2), air chamber (3),rare white (4), dense white (5), yolk (6), and chalazae (7).



The egg white (about 60% of the egg’s mass) is composed mainly of water anda mixture of proteins, and also has a multilayer structure. Starting from the shellthere are four fractions of white: external thin, external thick, internal thin, andinternal thick, which make up 23, 57, 17, and 2.5%, respectively, of the egg whitemass. Mucin structures called chalazae keep the yolk in a central position in theegg. During long storage, the chalazae lose their elasticity, and the egg white losespart of its water due to evaporation.

The egg yolk (about 30% of the egg’s mass) has a spherical shape, a diameterof about 3 to 3.5 cm, and a color ranging from dark to light orange, depending onthe quantity of lipids and carotenoid pigments in the fodder. It is surrounded by athin and elastic vittelin membrane built of keratin and mucin fibers. The egg yolk,being an O/W emulsion stabilized by lecithin, has a very high viscosity. The viscosityof the yolk decreases during storage, as a result of water permeation from the whitethrough the vittelin membrane. Egg yolks are utilized as a stabilizer in the manu-facturing of mayonnaise.

The chemical composition of the egg (Table 2.1) is rather stable. As the onlysource of food for the embryo, it contains all substances essential for life. There isabout 6.6 g of very well-balanced proteins in one egg. About two-thirds of the yolkmass are lipids, mainly unsaturated. Cholesterol makes up about of 2.5% of the drymass of the yolk. The egg is also a source of vitamins A, B, D, E, and K, and thebest dietary source of choline. The minerals S, K, Na, P, Ca, Mg, and Fe are in freeform or bound to proteins and lipids.

Eggs and egg products, thanks to their texture-improving properties, emulsifyingeffect, and foaming ability, are multifunctional additives used in food technology inliquid or dried form.

2.3 SACCHARIDE FOOD PRODUCTS

2.3.1 CEREAL AND CEREAL PRODUCTS

Cereals are fruits of cultivated grasses that may be used as raw materials for pro-duction of food and feed. The major cereals are wheat, rye, barley, oats, millet, rice,sorghum, and maize. The share of cereal products in the human diet is estimated as50 to 60%.

The shape of grains varies from elongated (rye) to spherical (millet), but theanatomical structures of cereal grains are rather similar. The essential anatomicalelements of cereal grains are seed coat (bran), endosperm, and germ. Commerciallythe most important cereal is wheat. A wheat grain is about 1-cm long and has adiameter of 0.5 cm. It is egg-shaped with a deep crease running along one side anda number of small hairs, called the beard, at one end (Figure 2.6).

The grain is surrounded by a five-layer coat called bran, which makes up 15%of the mass of the whole grain. It is rich in B vitamins and contains about 50% ofthe total mass of minerals in the grain. The bran consists of cellulose and is indi-gestible for humans. It is separated during flour production and used as animal fodder.

The germ, about 3% of the mass of the grain, is situated at the base of the grain.It contains the embryo, which is rich in lipids, proteins, B vitamins, vitamin E, and



minerals, mainly iron. A membranous tissue called the scutellum separates the germfrom the endosperm. It is a rich source of thiamine—about 60% of all its contentin the grain.

The starchy endosperm makes up 80 to 90% of the wheat grain and is a reserveof food for the germ. The starch granules are embedded in a protein matrix, whilethe periphery of the endosperm is composed of a single aleurone layer. The aleuronelayer is rich in proteins and contains high amounts of minerals, vitamins, andenzymes, but it is usually removed during milling. Considering the size, most of thestarch granules in the endosperm cells of wheat may be located in two ranges: large(15 to 40 µm in diameter) and small (1 to 10 µm in diameter), whereas those in thesubaleurone endosperm cells are 6 to 15 µm in diameter.

The chemical composition of cereal (Table 2.2) is dependent on the species,means of cultivation, and the time and conditions of growth, harvest, and storage.

Starch constitutes about 80% of the grain dry mass. In bread making the mostimportant properties of starch are its water-holding capacity, gelatinization, andsusceptibility to hydrolysis.

The protein content of cereal grains is in the range of 7 to 18%. From the techno-logical point of view, proteins, mainly gluten proteins, as well as enzymes (amylases,proteases, and lipases) are important during dough making. Cereal grains also contain2 to 4% lipids, mainly triacylglycerols of unsaturated fatty acids and phospholipids.

The mineral elements, mainly P and K, and to a smaller extent Mg and Ca, makeup about 2% of the grain mass. Vitamins of the B group and vitamin E are alsopresent in grains.

The milling technique can be modified to increase or decrease the yield of flourfrom a given amount of grain. The percentage of flour produced is termed the

FIGURE 2.6 Schematic structure of wheat grain: longitudinal section (a) and transversesection (b). Beard (1), bran (2), endosperm (3), crease (4), scutellum (5), germ (6).



extraction rate of flour. Whole flour, containing the bran, germ, scutellum, andendosperm of the grain, has an extraction rate of 100%. The extraction rate of 70%means that the flour is almost entirely composed of crushed endosperm. As thepercentage of flour increases, the amount of dietary fiber in flour increases, too. Thisis an important nutritional aspect of cereal products.

2.3.2 POTATOES

The potato is a swollen underground stem or tuber that contains a store of food forthe plants. In the tuber, a bud end and a stem end can be distinguished. The hollows,called eyes, are spirally arranged around the tuber surface. The tuber section isdivided into pith, parenchyma, vascular system, cortex, and periderm (Figure 2.7).

TABLE 2.2Chemical Compositions of Cereals and Cereal Products

ProductWater

%

Crude proteinN×6.25

%Saccharides

%Lipids

%

Mineralcomponents

%

Gra

ins

Wheat 15.0 11.0 68.5 2.0 1.5Rye 15.0 9.0 70.5 1.5 1.5Maize 15.0 10.0 67.0 4.5 1.5Rice paddy 15.0 7.5 75.5 0.5 1.0Millet 15.0 10.5 65.0 4.0 3.0

Flou

r

Wheat flour 97% 13.5 10.0 70.5 3.0 1.5Wheat flour 50% 13.5 8.5 75.0 1.5 0.5Rye flour 97% 13.5 7.5 73.0 2.0 1.5Rye flour 60% 13.5 5.5 78.5 1.5 0.5

Bre

ad

Wheat bread 37.5 8.0 57.5 1.5 2.0Rye bread 46.0 6.5 45.0 1.0 2.0Rusks 7.0 8.5 75.0 5.5 1.5

Source: Adapted from Fox, B.A. and Cameron, A.G., Food Science: A Chemical Approach, Hodder andStoughton, London, 1986; Kent, N.L., Technology of Cereals with Special Reference to Wheat, PergamonPress, Oxford, 1975.

FIGURE 2.7 Schematic structure of potato, longitudinal section: eye (1), periderm (skin) (2),parenchyma (3), vascular ring (4), and pith (5).



Each potato tuber is a single living organism, and its water is indispensable inall the vital processes. Water transports any substances moving in the interior of thetuber. It also protects the tubers against overheating (by transpiration). Water con-stitutes about 75% of the potato (Table 2.3). The second major constituent of potatois starch (about 20%). With regard to starch content, there are potato cultivates of alow (to 14%), medium (15 to 19%), and high (above 20%) starch content. Potato isalso a valuable source of ascorbic acid—up to 55 mg/100 g. Its ash consists of about60% K and 15% P2O5. The chemical composition of potato tubers changes duringstorage due to evaporation and catabolic processes.

In many parts of the world, potatoes are the main saccharide source in humanfood and animal fodder and are also widely used as raw material for starch manu-facture and in the fermentation industry.

The shape and size of starch granules are specific for different starchy rawmaterials (Figure 2.8).

TABLE 2.3Chemical Composition of Potato and Honey

Potato [%] Honey [%]

Water 76.0 Water 17.0Dry matter: 24.0 Saccharides: 82.5

Starch 17.5 Fructose 38.5Saccharides 1.5 Glucose 31.0Proteins 2.0 Maltose 7.0Cellulose 1.0 Trisaccharides 4.0Lipids 0.5 Saccharose 1.5

Mineral components 1.0 Proteins, vitamins, and mineral components 0.5

Source: Adapted from Lisińska, G. and Leszczyski, W., Potato Science and Technology, ElsevierApplied Science, London, 1989; Ramsay, I., Honey as a food ingredient, Food Ingredients andProcess. Int., 10, 16, 1992.

FIGURE 2.8 SEM micrographs of starch granules in different starchy raw materials: potato(a), wheat (b), and maize (c). (From Juszczak, L., unpublished. With permission.)

a b c

20 µm 20 µm20 µm



2.3.3 HONEY

Honey is produced by honeybees from the flower nectar of plants. Fresh honey is aclear, very aromatic, dark amber-colored liquid. It is very sticky and hygroscopic,with a density of about 1.40 g/cm3. Honey is an oversaturated solution of glucoseand fructose, and easily crystallizes. After crystallization its color is brighter. It is avery stable product. At a temperature of 8 to 10°C and humidity of 65 to 75% itmay be stored for many years. Honey is a high-calorie food easily assimilated bythe human organism.

Honey is used in the manufacturing of alcoholic beverages (i.e., in wine pro-duction). In medicine it is prescribed for heart, liver, stomach, skin, and eye ill-nesses. In the food industry honey is used as a very effective sweetener (25%sweeter than sucrose), a very well-binding, concentrating, and covering additive,and a taste intensifier.

The chemical composition of honey (Table 2.3) is dominated by glucose andfructose. Honey also contains many other valuable components, such as enzymes,organic acids, mineral elements, nonprotein nitrogenous compounds, vitamins,aroma substances, and pigments.

2.3.4 NUTS

Nuts are composed of a wooden-like shell and a seed, covered by a yellow or brownskin. Each part makes up about 50% of the nut mass. Inside of the seed is a germ.The seeds of nuts consist of about 60% lipids rich in unsaturated fatty acids, 16 to20% easily digested proteins, 7% saccharides, vitamin B1 (10 mg/100g) and C(30–50 mg/100 g), and P, Mg, K, and Na. Because of their high quantity of easilyassimilated nutrients, nuts may be used in the diets of convalescents and children.

2.3.5 SEEDS OF PULSES

To this group belong peas, beans, lentils, soybeans, and peanuts. All of them havefruits in the form of pods. Their shape and size depend on the cultivar. Inside thepod there are seeds used as raw material in the food industry.

The dry mass of pulse seeds consists of saccharides (14 to 63%), proteins (28to 44%), and lipids (1 to 50%). The other constituents are mineral elements (mainlyK and P), and vitamins from the B group. Soybean is the most valuable pod plant,due to its high quantity and good quality of protein. Soy products in the form ofmeat extenders and analogues are used all over the world. Soybean is also a rawmaterial in the oil industry.

2.4 EDIBLE FATS

Food products such as butter, lard, margarine, or plant oils are regarded as visiblefats. They make up about 45% of the total fat consumed by humans, while theinvisible fats, which are natural components of foods such as meat, fish, eggs, andbakery products, make up about 55%.



Visible fats are composed mainly of triacylglycerols. They also contain fat-soluble vitamins A, D, and E, and additives added during processing, for example,antioxidants, colorants, or preservatives.

The consistency of fats depends on the content of unsaturated fatty-acid residues.The oils of plant and fish origin are rich in long-chain polyenolic fatty acids.

Butter consists of 16 to 18% water, 80 to 82.5% lipids, 0.5 % proteins, and 0.5%saccharides.

2.5 FRUITS AND VEGETABLES

Fruits and vegetables are rich sources of vitamins and minerals, as well as terpenes,flavonoids, tannins, chinons and phytoncides. They make food more attractivebecause of smell and color.

Fruits and vegetables are living organisms and their chemical compositions arevery changeable. The predominant constituent of fruits and vegetables is water,which may represent up to about 96% of the total weight of the crop. The water infruits and vegetables may be in free or bound form. A relatively high amount of freewater improves the taste of fruits and vegetables consumed in their raw state, aswell as the accessibility of soluble components. Most of the solid matter of fruit andvegetables is made of saccharides and smaller amounts of protein and fat.

The total saccharide content in the fresh weight of fruits and vegetables rangesfrom about 2% in some pumpkin fruits to above 30% in starchy vegetables. Gener-ally, vegetables contain less than 9% saccharides. The polysaccharides (celluloseand hemicelluloses) are largely confined to the cell walls. The di- and monosaccha-rides (sucrose, glucose, and fructose) are accumulated mainly in the cell sap. Theproportions of the different saccharide constituents can fluctuate due to metabolicactivity of the plant, especially during fruit ripening.

The majority of proteins occurring in fruits and vegetables play enzymatic rolesthat are very important in the physiology and postmortem behavior of the crop. Theprotein content in vegetables is lower than 3%, except in sweet maize (above 4%).In fruits it ranges from below 1% to above 1.5%. Proteins are found mainly in thecytoplasmic layers.

The lipids of fruits and vegetables are, like the proteins, largely confined to thecytoplasmic layers, in which they are especially associated with the surface mem-branes. Their content in fruits and vegetables is always lower than 1%. Lipid andlipidlike fractions are particularly prominent in the protective tissues at the surfacesof plant parts—the epidermal and corky layers.

Plant tissues also contain organic acids formed during metabolic processes. Forthis reason fruits and vegetables are normally acidic in reaction. The quantity oforganic acids is different, from very low, about 2 milliequivalents of acid/100 g insweet maize and pod seeds to very high, up to 40 milliequivalents/100 g in spinach.For the majority of fruits and vegetables the dominant acids are citric acid and malicacid, each of which can, in particular examples, constitute over 2% of the freshweight of the material. Lemons contain more than 3% citric acid. Tartaric acidaccumulates in grape and oxalic acid in spinach. The fruits in general show a decreasein overall acidity during the ripening process.



The total amount of mineral components in fruits and vegetables is in the rangeof 0.1% (in sweet potatoes) up to about 4.4% (in kohlrabi). The most abundantmineral constituent in fruits and vegetables is potassium (Table 2.4). Generally,vegetables are a better source of minerals than fruits. The mineral elements influencenot only the growth and crop of fruits and vegetables, but also their texture (Ca),color (Fe), and metabolic processes (microelements).

The diversity of form shown by fruit and vegetable structures is extremely wide.Among the vegetables there are representatives of all the recognizable morphologicaldivisions of the plant body—whole shoots, roots, stems, leaves, and fruits.

Fruits may also be classified into a number of structural types. The individualseed-bearing structures of the flower (called carpels) constitute the gynoecium. Theseed-containing cavity of a carpel is called the ovary, and its wall develops into thepericarp of the fruit. The edible fleshy part of a fruit most commonly develops fromthe ovary wall, but it may also be derived from the enlarged tip of stem from whichfloral organs arise, and sometimes leaflike structures protecting the flowers may alsobecome fleshy, for example, in pineapple.

Most of the metabolic activity of plants is carried out in the tissue calledparenchyma, which generally makes up the bulk of the volume of all soft edibleplant structures. The epidermis, which is sometimes replaced by a layer of corkytissue, is structurally modified to protect the surface of the organ. The highly spe-cialized tissues collenchyma and sclerenchyma provide mechanical support for theplant. Water, minerals, and products of metabolism are transported through the plantvia the vascular tissues, xylem and phloem, which are the most characteristic ana-tomical features of plants in the cross-section.

The structure of fruits is dominated by soft parenchymatous tissue, while con-ducting and supporting structures are rather poorly developed. An exception is thepineapple, in which conducting tissues are very prominently represented. The subtlestructure and proportions of individual tissues influence the texture, properties, andsuitability for processing of fruits and vegetables.

TABLE 2.4Mineral Components of Fruits and Vegetables (mg/100 g of raw mass)

Component mg Rich source

K 350 Parsley (above 1000 mg)Na 65 CeleryCa 150 Spinach (up to 600 mg)Mg 50 Sweet maizeP 120 Seeds and young growing partsCl 90 CeleryS 80 Plants with higher quantity of proteinsFe 2 Parsley (up to 8 mg)

Source: Adapted from Duckworth, R.B., Fruit and Vegetables, PergamonPress, London, 1966.



REFERENCES

Duckworth, R.B., Fruit and Vegetables. Pergamon Press, London, 1966.Fox, B.A. and Cameron, A. G., Food Science: A Chemical Approach. Hodder and Stoughton,

London, 1986.Hedrick, H.B. et al., Principles of Meat Science. Kendal/Hunt Publish Comp., Dubuque, IA,

1994.Kent, N.L., Technology of Cereals with Special Reference to Wheat. Pergamon Press, Oxford,

1975.Kirk, R.S. and Sawyer, R., Pearson’s Composition and Analysis of Foods. Longman Science,

London, 1991.Kristensen, L. and Purslow, P.P., The effect of ageing on the water-holding capacity of pork:

role of cytoskeletal proteins, Meat Science, 58, 17–23, 2001.Lisińska, G. and Leszczyński, W., Potato Science and Technology. Elsevier Applied Science,

London, 1989.Ramsay, I., Honey as a food ingredient, Food Ingredient and Processing International, 10,

16, 1992.Renner, E., Nutritional aspects of cheese, in Cheese: Chemistry, Physics and Microbiology,

Vol. 1, Fox, P.F. (Ed.). Chapman & Hall, London, 1993, p. 557.Sikorski, Z.E., Seafood Raw Materials. WNT, Warsaw, 1992 (in Polish).Tamime, A.Y. and Robinson, R.K., Yoghurt. Science and Technology. CRC Press, Boca Raton,

FL, 1999.


29

3 Water and Food Quality

Emilia Barbara Cybulska and Peter Edward Doe

CONTENTS

3.1 Introduction .................................................................................................... 303.2 Structure and Properties of Water.................................................................. 31

3.2.1 The Water Molecule........................................................................... 313.2.2 Hydrogen Bonds ................................................................................ 313.2.3 Properties of Bulk Water ................................................................... 333.2.4 Thermal Properties of Water.............................................................. 363.2.5 Water as a Solvent ............................................................................. 373.2.6 Water in Biological Materials............................................................ 41

3.2.6.1 Properties ............................................................................ 413.2.6.2 Water Transport .................................................................. 43

3.3 Water in Food................................................................................................. 443.3.1 Introduction ........................................................................................ 443.3.2 Sorption Isotherms and Water Activity ............................................. 46

3.3.2.1 Principle .............................................................................. 463.3.2.2 Measurement of Water Activity ......................................... 483.3.2.3 Water Activity and Shelf Life of Foods............................. 48

3.3.3 Bottled Water ..................................................................................... 503.3.3.1 Classification....................................................................... 503.3.3.2 Natural Mineral Water ........................................................ 50

3.3.4 Bottled Water Other than Natural Mineral Water ............................. 523.3.4.1 Definition ............................................................................ 523.3.4.2 Water Defined by Origin .................................................... 523.3.4.3 Hygiene, Labeling, and Health Benefits ............................ 53

3.3.5 Water Supply, Quality, and Disposal................................................. 533.3.5.1 Water Supply ...................................................................... 533.3.5.2 Water Quality: Standards and Treatment ........................... 54

3.3.6 Water Pollution .................................................................................. 563.3.7 Wastewater Treatment and Disposal.................................................. 57

References................................................................................................................ 58



3.1 INTRODUCTION

Water is the most popular and most important chemical compound on our planet. Itis a major chemical constituent of the Earth’s surface and it is the only substancethat is abundant in solid, liquid, and vapor form. Because it is ubiquitous, it seemsto be a mild and inert substance. In fact, it is a very reactive compound characterizedby unique physical and chemical properties that make it very different from otherpopular liquids. The peculiar water properties determine the nature of physical andbiological world.

Water is the major component of all living organisms. It constitutes 60% or moreof the weight of most living things, and it pervades all portions of every cell. Itexisted on our planet long before the appearance of any form of life. The evolutionof life was doubtlessly shaped by physical and chemical properties of the aqueousenvironment. All aspects of living cells’ structure and function seem to be adaptedto water’s unique properties.

Water is the universal solvent and dispersing agent, as well as a very reactivechemical compound. Biologically active structures of macromolecules are sponta-neously formed only in aqueous media. Intracellular water is not only a medium inwhich structural arrangement and all metabolic processes occur, but an active partnerof molecular interactions, participating directly in many biochemical reactions as asubstrate or a product. Its high heat capacity allows water to act as a heat buffer inall organisms. Regulation of water contents is important in the maintenance ofhomeostasis in all living systems.

Only 0.003% of all freshwater reserve participates in its continuous circulationbetween the atmosphere and the hydrosphere. The remaining part is confined inthe Antarctic ice. The geography of water availability has determined, to a largedegree, the vegetation, food supply, and habitation in the various areas of the world.For example, Bangladesh has one of the world’s highest population densities, madepossible through the regular flooding of the Ganges River and the rich silts itdeposits in its wake. In Bangladesh, the staple food—rice—grows abundantly andis readily distributed. In other societies, the food must be transported long distancesor kept over winter.

Human well-being is closely linked to the availability of water and food. Anexpected increase in the world population by the year 2050 (65% or 3.7 billion) willcreate enormous pressure on freshwater resources and food production. Agricultureis by far the largest consumer of water and the key issue is to look for ways toimprove water use efficiency. The solution lies in producing more food from existingwater and land resources (Wallace and Gregory, 2002).

Stability, wholesomeness, and shelf life are significant features of foods that are,to a large degree, influenced by the water content. Dried foods were originallydeveloped to overcome the constraints of time and distance before consumption.Canned and frozen foods were developed next. The physical properties, quantity,and quality of water within food have a strong impact on food effectiveness, qualityattributes, shelf life, textural properties, and processing.


Water and Food Quality 31

3.2 STRUCTURE AND PROPERTIES OF WATER

3.2.1 THE WATER MOLECULE

Water is a familiar material, but it has been described as the most anomalous ofchemical compounds. Although its chemical composition, HOH or H2O, is univer-sally known, the simplicity of its formula belies the complexity of its behavior. Itsphysical and chemical properties are very different from compounds of similarcomplexity, such as HF and H2S. To understand the reasons for water's unusualproperties, it is necessary to examine its molecular structure in some detail.

Although a water molecule is electrically neutral as a whole, it has a dipolarcharacter. The high polarity of water is caused by the direction of the H-O-H bondangle, which is 104.5°, and by an asymmetrical distribution of electrons within themolecule. In a single water molecule, each hydrogen atom shares an electron pairwith the oxygen atom in a stable covalent bond. However, the sharing of electronsbetween H and O is unequal because the more electronegative oxygen atom tendsto draw electrons away from the hydrogen nuclei. The electrons are more often inthe vicinity of the oxygen atom than in the vicinity of the hydrogen atom. The resultof this unequal electron sharing is the existence of two electric dipoles in themolecule, one along each of the H-O bonds. The oxygen atom bears a partial negativecharge δ–, and each hydrogen a partial positive charge δ+. Because the molecule isnot linear, H-O-H has a dipole moment (Figure 3.1). Because of this, water moleculescan interact through electrostatic attraction between the oxygen atom of one watermolecule and the hydrogen of another.

3.2.2 HYDROGEN BONDS

Such interactions, which arise because the electrons on one molecule can be partiallyshared with the hydrogen on another, are known as hydrogen bonds. The H2Omolecule, which contains two hydrogen atoms and one oxygen atom in a nonlineararrangement, is ideally suited to engage in hydrogen bonding. It can act both as adonor and as an acceptor of hydrogen. The nearly tetrahedral arrangement of the

FIGURE 3.1 Water molecule as an electric dipole.

O

104.5°H H

+

–Dipolemoment

O

H

Η

δ– δ–

δ+δ+

δ–δ–

δ+

δ+



orbital about the oxygen atom allows each water molecule to form hydrogen bondswith four of its neighbors (Figure 3.2).

An individual, isolated hydrogen bond is very labile. It is longer and weakerthan a covalent O-H bond (Figure 3.3). The hydrogen bond’s energy, that is, theenergy required to break the bond, is about 20 kJ/mol. These bonds are intermediatebetween those of weak van der Waals interactions (about 1.2 kJ/mol) and those ofcovalent bonds (460 kJ/mol).

Hydrogen bonds are highly directional; they are stronger when the hydrogenatom and the two atoms that share it are in a straight line (Figure 3.4).

Hydrogen bonds are not unique to water. They are formed between water anddifferent chemical structures, as well as between other molecules (intermolecular)or even within a molecule (intramolecular). They are formed wherever an electrone-gative atom (oxygen or nitrogen) comes in close proximity to a hydrogen atomcovalently bonded to another electronegative atom. Some representative hydrogenbonds of biological importance are shown in Figure 3.5.

FIGURE 3.2 Tetrahedral hydrogen bonding of five water molecules.

FIGURE 3.3 Two water molecules connected by hydrogen bonds.

H

H

H

H

H

H

H

H H

HO

O

O O

O

1

2

34

5



Intra- and intermolecular hydrogen bonding occurs extensively in biologicalmacromolecules. A large number of the hydrogen bonds and their directionalityconfer very precise three-dimensional structures upon proteins and nucleic acids.

3.2.3 PROPERTIES OF BULK WATER

The key to understanding water structure in solid and liquid form lies in the conceptand nature of the hydrogen bonds. In the crystal of ordinary hexagonal ice (Figure 3.6),

FIGURE 3.4 Directionality of the hydrogen bonds.

FIGURE 3.5 Some hydrogen bonds of biological importance.



each molecule forms four hydrogen bonds with its nearest neighbors. Each HOHacts as a hydrogen donor to two of the four water molecules, and as a hydrogenacceptor from the remaining two. These four hydrogen bonds are spatially arrangedaccording to tetrahedral symmetry (Bjerrum, 1952)

The crystal lattice of ice occupies more space than the same number of H2Omolecules in liquid water. The density of solid water is thus less than that of liquidwater, whereas simple logic would have the more tightly bound solid structure moredense than its liquid. One explanation for ice being lighter than water at 0°Cproposes a reforming of intermolecular bonds as ice melts, so that on average, awater molecule is bound to more than four of its neighbors, thus increasing itsdensity. But as the temperature of liquid water increases, the intermolecular dis-tances also increase, giving a lower density. These two opposite effects explain thefact that liquid water has a maximum density at a temperature of 4°C. At any giveninstant in liquid water at room temperature, each water molecule forms hydrogenbonds with an average of 3.4 other water molecules (Lehninger et al., 1993). Theaverage translational and rotational kinetic energies of a water molecule are approx-imately 7 kJ/mol, the same order as that required to break hydrogen bonds; there-fore, hydrogen bonds are in a continuous state of flux, breaking and reforming withhigh frequency on a picosecond time scale. A similar dynamic process occurs inaqueous media with substances that are capable of forming hydrogen bonds.

At 100°C liquid water still contains a significant number of hydrogen bonds,and even in water vapor there is strong attraction between water molecules. The verylarge number of hydrogen bonds between molecules confers great internal cohesionon liquid water. This feature provides a logical explanation for many of its unusualproperties. For example, its large values for heat capacity, melting point, boilingpoint, surface tension, and heat of various phase transitions are all related to theextra energy needed to break intermolecular hydrogen bonds.

FIGURE 3.6 Structure of ice.



That liquid water has structure is an old and well-accepted idea; however, thereis no consensus among physical chemists as to the molecular architecture of thehydrogen bond’s network in the liquid state. It seems that the majority of hydrogenbonds survive the melting process, but obviously rearrangement of molecules occurs.The replacement of crystal rigidity by fluidity gives molecules more freedom todiffuse about and to change their orientation. Any molecular theory for liquid watermust take into account changes in the topology and geometry of the hydrogen bondnetwork induced by the melting process.

Many models have been proposed, but none has adequately explained all prop-erties of liquid water.

Historically, there are two competing theoretical approaches used to describe themolecular structure of liquid water: (1) the continuum (uniform) models, and (2) themixture (cluster) models (Starzak and Mathlouthi, 2003; Dautchez et al., 2003).

According to continuum models, liquid water is depicted as a continuous, three-dimensional network in which water molecules are interconnected by somewhatdistorted hydrogen bonds; hydrogen bonding is almost complete, the structuralparameters (distances and angles) and bond energies have continuous distribution;all water molecules are qualitatively the same and a whole-water sample is consid-ered as a single entity with temperature-dependent local structure. The continuummodels are incompatible with particular water properties such as the compressibilityminimum and the density maximum.

Most mixture models describe liquid water as an equilibrium mixture of a fewclasses of different structural species more or less defined. A paper by Röntgen(1892) in which water was described as a saturated solution of ice in a liquidcomposed of simpler molecules, started the mixture model history. Over the nextcentury a variety of structural arrangements, based on the equilibrium of smallwater aggregates were proposed and used to explain the properties of water andaqueous solutions.

The most popular, the flickering clusters model (Figure 3.7), suggests that liquidwater is highly organized on a local basis: the hydrogen bonds break and reformspontaneously, creating and destroying transient structural domains (Frank andQuist, 1961; Frank and Wen, 1957). However, because the half-life of any hydrogenbond is less than a nanosecond, the existence of these clusters has statistical validityonly; even this has been questioned by some authors who consider water to be acontinuous polymer.

Experimental evidence obtained by x-rays and neutron diffractions strongly sup-ports the persistence of a tetrahedral hydrogen bond order in the liquid water, butwith substantial disorder present. Since the high-resolution Raman technique becameavailable the spectra have been carefully analyzed in favor of the mixture models.

The first computer simulation was performed by Rahman and Stillinger (1971)with a model of 216 water molecules. The view that emerges from these studies isthe following: liquid water consists of a macroscopically connected, random networkof hydrogen bonds. This network has a local preference for tetrahedral geometry,but it contains a large proportion of strained and broken bonds, which are continuallyundergoing topological reformation. The properties of water arise from the compe-tition between relatively bulky ways of connecting molecules into local patterns



characterized by strong bonds and nearly tetrahedral angles and more compactarrangements characterized by more strain and bond breakage (Stillinger, 1980).

With the advent of supercomputers, a flood of quantitative studies on waterstructure based on quantum and statistical mechanics have been carried out. Anumber of models have been proposed in which more and more complicatedstructural units as liquid water components have been suggested (Starzak andMathlouthi, 2003).

According to a model proposed by Wiggins (1990, 2002), two types of structurecan be distinguished: high-density water and low-density water. In the high-densitywater, the bent, relatively weak hydrogen bonds predominate over straight, strongerones. Low-density water has many icelike straight hydrogen bonds. Although hydro-gen bonding is still continuous through the liquid, the weakness of the bonds allowsthe structure to be disrupted by thermal energy extremely rapidly. High-density wateris extremely reactive and more liquid, whereas low-density water is inert and moreviscous. A continuous spectrum of water structures between these two extremes canbe imagined. The strength of water–water hydrogen bonding, which is the sourceof water density and reactivity, has great functional significance; this explains water’ssolvent properties and its role in many biological events.

A common feature of all theories is that a definite structure of liquid water isdue to the hydrogen bonding between molecules, and that the structure is in thedynamic state as the hydrogen bonds break and reform with high frequency.

3.2.4 THERMAL PROPERTIES OF WATER

The unusually high melting point of ice, as well as the heat of water vaporizationand specific heat, is related to the ability of water molecules to form hydrogen bondsand the strength of these bonds.

FIGURE 3.7 Flickering clusters of H2O molecules in bulk water.



A large amount of energy, in the form of heat, is required to disrupt the hydrogen-bonded lattice of ice. In the common form of ice, each water molecule participatesin four hydrogen bonds. When ice melts, most of the hydrogen bonds are retainedby liquid water, but the pattern of hydrogen bonding is irregular, due to the frequentfluctuation. The average energy required to break each hydrogen bond in ice hasbeen estimated to be 23 kJ/mol, while the energy to break each hydrogen bond inwater is less than 20 kJ/mol (Ruan and Chen, 1998).

The heat of water vaporization is much higher than that of many other liquids. Asis the case with melting ice, a large amount of thermal energy is required for breakinghydrogen bonds in liquid water, to permit water molecules to dissociate from oneanother and to enter the gas phase. Perspiration is an effective mechanism of decreasingbody temperature because the evaporation of water absorbs so much heat.

A relatively large amount of heat is required to raise the temperature of 1 g ofwater by 1°C because multiple hydrogen bonds must be broken in order to increasethe kinetic energy of the water molecules. Due to the high quantity of water in thecells of all organisms, temperature fluctuation within cells is minimized. This featureis of critical biological importance because most biochemical reactions and macro-molecular structures are sensitive to temperature. The unusual thermal properties ofwater make it a suitable environment for living organisms, as well as an excellentmedium for the chemical processes of life.

3.2.5 WATER AS A SOLVENT

Many molecular parameters, such as ionization, molecular and electronic structure,size, and stereochemistry, will influence the basic interaction between a solute anda solvent. The addition of any substance to water results in altered properties of thatsubstance and of the water itself. Solutes cause a change in water properties becausethe hydrate envelopes that are formed around dissolved molecules are more organizedand therefore more stable than the flickering clusters of free water. The propertiesof solutions that depend on a solute and its concentration are different from thoseof pure water. The differences can be seen in such phenomena as the freezing pointdepression, boiling point elevation, and increased osmotic pressure of solutions.

The polar nature of the water molecule and the ability to form hydrogen bondsdetermine its properties as a solvent. Water is a good solvent for charged or polarcompounds and a relatively poor solvent for hydrocarbons. Hydrophilic compoundsinteract strongly with water by an ion–dipole or dipole–dipole mechanism, causingchanges in water structure and mobility and in the structure and reactivity of thesolutes. The interaction of water with various solutes is referred to as hydration. Theextent and tenacity of hydration depends on a number of factors, including the natureof the solute, salt composition of the medium, pH, and temperature.

Water dissolves dissociable solutes readily because the polar water moleculesorient themselves around ions and partially neutralize ionic charges. As a result, thepositive and negative ions can exist as separate entities in a dilute aqueous solutionwithout forming ion pairs. Sodium chloride is an example where the electrostaticattraction of Na+ and Cl– is overcome by the attraction of Na+ with the negative



charge on the oxygen and Cl– with the positive charge on the hydrogen ions (Figure3.8). The number of weak charge–charge interactions between water and the Na+

and Cl– ions is sufficient to separate the two charged ions from the crystal lattice.To acquire their stabilizing hydration shell, ions must compete with water mol-

ecules, which need to make as many hydrogen bonds with one another as possible.The normal structure of pure water is disrupted in a solution of dissociable solutes.The ability of a given ion to alter the net structure of water depends on the strengthof its electric field. Among ions of a given charge-type (e.g., Na+ and K+ or Mg2+

and Ca2+), the smaller ions are more strongly hydrated than the larger ions, in whichthe charge is dispersed over a greater surface area. Most cations, except the largestones, have a primary hydration sphere containing four to six molecules of water.Other water molecules, more distant from the ion, are held in a looser secondarysphere. Electrochemical transfer experiments indicate a total of 16 molecules ofwater around Na+ and about 10 around K+. The bound water is less mobile anddenser than HOH molecules in bulk water. At some distance, the bonding arrange-ments melt into a dynamic configuration of pure water.

Water is especially effective in screening the electrostatic interaction betweendissolved ions because, according to Coulomb’s law, the force (F) between twocharges q+ and q– separated by a distance r is given as:

F = q+ ⋅ q–/εr2 (3.1)

where ε is the dielectric constant of the medium. For a vacuum, ε = 1 Debye unit,whereas for bulk water, ε = 80; this implies that the energies associated with electrostaticinteractions in aqueous media are approximately 100 times smaller than the energiesof covalent association, but increase considerably in the interior of a protein molecule.

In thermodynamic terms, the free energy change, ∆G, must have a negative valuefor a process to occur spontaneously.

∆G = ∆H – T∆S (3.2)

where ∆G represents the driving force, ∆H (the enthalpy change) is the energy frommaking and breaking bonds, and ∆S (the entropy change) is the increase in randomness.

Solubilization of a salt occurs with a favorable change in free energy. As saltsuch as NaCl dissolves, the Na+ and Cl– ions leaving the crystal lattice acquire greater

FIGURE 3.8 Hydration shell around Na+ and Cl–.



freedom of motion. The entropy (∆S) of the system increases; where ∆H has a smallpositive value and T∆S is large and positive, ∆G is negative.

Water in the multilayer environment of ions is believed to exist in a structurallydisrupted state because of conflicting structural influences of the innermost vicinalwater and the outermost bulk-phase water. In concentrated salt solutions, the bulk-phase water would be eliminated, and the water structure common in the vicinity ofions would predominate. Small or multivalent ions, such as Li+, Na+, H3O+, Ca2+,Mg2+, F–, SO4

2–, and PO43–, which have strong electric fields, are classified as water

structure formers because solutions containing these ions are less fluid than purewater. Ions that are large and monovalent, most of the negatively charged ions andlarge positive ions, such as K+, Rb+, Cs+, NH4

+, Cl–, Br–, I–, NO3–, ClO4–, and CNS–

disrupt the normal structure of water; they are structure breakers. Solutions contain-ing these ions are more fluid than pure water (Fennema, 1996).

Through their varying abilities to hydrate and to alter water structure and itsdielectric constant, ions influence all kinds of water–solute interactions. The con-formation of macromolecules and the stability of colloids are greatly affected by thekinds and concentrations of ions present in the medium.

Water is a good solvent for most biomolecules, which are generally charged orpolar compounds. Solubilization of compounds with functional groups such asionized carboxylic acids (COO–), protonated amines (NH3

+), phosphate esters, oranhydrides is also a result of hydration and charge screening.

Uncharged but polar compounds possessing hydrogen bonding capabilities arealso readily dissolved in water, due to the formation of hydrogen bonds with watermolecules. Every group that is capable of forming a hydrogen bond to anotherorganic group is also able to form hydrogen bonds of similar strength with water.Hydrogen bonding of water occurs with neutral compounds containing hydroxyl,amino, carbonyl, amide, or imine groups. Saccharides dissolve readily in water, dueto the formation of many hydrogen bonds between the hydroxyl groups or carbonyloxygen of the saccharide and water molecules. Water–solute hydrogen bonds areweaker than ion–water interactions. Hydrogen bonding between water and polarsolutes also causes some ordering of water molecules, but the effect is less significantthan with ionic or nonpolar solutes.

The introduction into water of hydrophobic substances such as hydrocarbons,rare gases, and the apolar groups of fatty acids, amino acids, or proteins is thermo-dynamically unfavorable because of the decrease in entropy. The decrease in entropyarises from the increase in water–water hydrogen bonding adjacent to apolar entities.Water molecules in the immediate vicinity of a nonpolar solute are constrained intheir possible orientations, resulting in a shell of highly ordered water moleculesaround each nonpolar solute molecule (Figure 3.9a). The number of water moleculesin the highly ordered shell is proportional to the surface area of hydrophobic solute.In the case of dissolved hydrocarbons, the enthalpy of formation of the new hydrogenbonds often almost exactly balances the enthalpy of creation in water, a cavity ofthe right size to accommodate the hydrophobic molecule. However, the restrictionof water mobility results in a very large decrease in entropy.

To minimize contact with water, hydrophobic groups tend to aggregate; this processis known as hydrophobic interaction (Figure 3.9b). The existence of hydrophobic



substances barely soluble in water but readily soluble in many nonpolar solvents,and their tendency to segregate in aqueous media, has been known for a long time.However, the origin of this hydrophobic effect is still somewhat controversial. Theplausible explanation is that hydrophobic molecules disturb the hydrogen bondedstate of water, without having any compensatory ordering effects. Apolar moleculesare water structure formers; water molecules cannot use all four possible hydrogenbonds when in contact with hydrophobic, water-hating molecules. This restrictionresults in a loss of entropy, a gain in density, and increased organization of bulk water.

Amphipathic molecules, compounds that contain both polar or charged groupsand apolar regions, disperse in water if the attraction of the polar group for watercan overcome possible hydrophobic interactions of the apolar portions of the mol-ecules. Many biomolecules are amphipathics: proteins, phospholipids, sterols, certainvitamins, and pigments have polar and nonpolar regions. When amphipathic com-pounds are in contact with water, the two regions of the solute molecule experienceconflicting tendencies: the polar or charged hydrophilic regions interact favorablywith water and tend to dissolve, but the nonpolar hydrophobic regions tend to avoidcontact with water. The nonpolar regions of the molecules cluster together to presentthe smallest hydrophobic area to the aqueous medium, and the polar regions arearranged to maximize their interactions with the aqueous solvent. In aqueous media,many amphipathic compounds are able to form stable structures, containing hundredsto thousands of molecules, called micelles. The forces that hold the nonpolar regionsof the molecules together are due to hydrophobic interactions.

The hydrophobic effect is a driving force in the formation of clathrate hydratesand the self-assembly of lipid bilayers. Hydrophobic interactions between lipids andproteins are the most important determinants of biological membrane structure. The

FIGURE 3.9 Cagelike water structure around the hydrophobic alkyl chain (a) and hydropho-bic interactions (b).

Hydrophylic head group”

(a) (b)



three-dimensional folding pattern of proteins is also determined by hydrophobicinteractions between nonpolar side chains of amino acid residues.

3.2.6 WATER IN BIOLOGICAL MATERIALS

3.2.6.1 Properties

Water behaves differently in different environments. Properties of water in hetero-geneous systems, such as living cells or food, remain a field of debate (Mathlouthi,2001; Rückold et al., 2003). Water molecules may interact with macromolecularcomponents and supramolecular structures of biological systems through hydrogenbonds and electrostatic interactions. Solvation of biomolecules such as lipids, pro-teins, nucleic acids, or saccharides resulting from these interactions, determines theirmolecular structure and function.

Various physical techniques, such as nuclear magnetic resonance (NMR), x-raydiffraction, and chemical probes (exchange of H by D), indicate that there is a layerof water bound to protein molecules, phospholipid bilayers, and nucleic acids, aswell as at the surface of the cell membranes and other organelles.

Water associated at the interfaces and with macromolecular components mayhave quite different properties from those in the bulk phase. Water can be expectedto form locally ordered structures at the surface of water-soluble, as well as water-insoluble macromolecules and at the boundaries of the cellular organelles. Biomac-romolecules generally have many ionized and polar groups on their surfaces andtend to align near polar water molecules. This ordering effect exerted by the mac-romolecular surface extends quite far into the surrounding medium.

According to the association–induction theory proposed by Ling (1962), fixedcharges on macromolecules and their associated counterions constrain water mole-cules to form a matrix of polarized multilayers having restricted motion, comparedwith pure water. The monolayer of water molecules absorbed on the polar sorptionsite of the molecule is almost immobilized and thus behaves, in many respects, likepart of the solid or like water in ice. It has different properties from additional waterlayers defined as multilayers. The association–induction theory has been shared bymany researchers for many years. Unfortunately, elucidation of the nature of indi-vidual layers of water molecules has been less successful, due to the complexity ofthe system and lack of appropriate techniques.

Measurements of the diffusion coefficients of globular protein molecules insolution yield values for molecular size that are greater than the corresponding radiidetermined by x-ray crystallography. The apparent hydrodynamic radius can becalculated from the Stokes-Einstein relation:

D = kBT/6πηaH (3.3)

where D is the diffusion coefficient, kB is the Boltzmann constant, T is the temperature,η is the solution viscosity, and aH is the molecule radius (Nossal and Lecar, 1991).

Similarly, studies utilizing NMR techniques show that there is a species ofassociated water that has a different character than water in the bulk phase. By these



and other methods, it was found that for a wide range of protein molecules, approx-imately 0.25 to 0.45 g of H2O are associated with each gram of protein.

The hydration forces can stabilize macromolecular association or prevent mac-romolecular interactions with a strength that depends on the surface characteristicof the molecules and the ionic composition of the medium.

The interaction between a solute and a solid phase is also influenced by water.Hydration shells or icebergs associated with one or the other phase are destroyed orcreated in this interaction and often contribute to conformational changes in mac-romolecular structures—and ultimately to changes in biological and functional prop-erties important in food processing.

Biophysical processes involving membrane transport are also influenced byhydration. The size of the hydration shell surrounding small ions and the presenceof water in the cavities of ionic channels or in the defects between membrane lipidsstrongly affect the rates at which the ions cross a cell membrane.

The idea that intracellular water exhibits properties different from those of bulkwater has been around for a long time. The uniqueness of the cytoplasmic waterwas deduced from:

The observation that cells may be cooled far below the freezing point of asalt solution iso-osmotic with that of the cytoplasm.

Properties of the cytoplasm, which in the same conditions should bind waterlike a gel.

Osmotic experiments in which it has often been observed that part of cellwater is not available as a solvent. This water has been described as osmot-ically inactive water, bound water, or compartmentalized water.

According to a recent view, three different kinds of intracellular water can bedistinguished: a percentage of the total cell water appears in the form of usual liquidwater. A relevant part is made up of water molecules that are bound to different sitesof macromolecules in the form of hydration water, while a sizeable amount, althoughnot fixed to any definite molecular site, is strongly affected by macromolecular fields.This kind of water has been termed vicinal water. Most of the vicinal water surroundsthe elements of the cell cytoskeleton. Vicinal water has been extensively investigated,and it has been found that some of its properties are different from those of normalwater. It does not have a unique freezing temperature, but an interval ranging from–70 to –50°C; it is a very bad solvent for electrolytes, but nonelectrolytes have thesame solubility properties as in usual water; its viscosity is enhanced, and its NMRresponse is anomalous (Giudice et al., 1986).

The distribution of various types of water inside living cells is a question thatcannot be answered yet, especially because in many cells marked changes have beennoted in the state of intracellular water as a result of biological activity. The possibilitythat water in living cells may differ structurally from bulk water has prompted asearch for parameters of cell water that deviate numerically from those of bulk water.

The diffusion coefficient for water in the cytoplasm of various cells has beendetermined with satisfactory precision. It has been found that the movement of watermolecules inside living cells is not much different and is reduced by a factor of



between 2 and 6, compared with the self-diffusion coefficient for pure water. Accord-ing to Mild and Løvtrup (1985), the most likely explanation of the observed valuesis that part of the cytoplasmic water, the vicinal water close to the various surfacestructures in the cytoplasm, is structurally changed to the extent that its rate of motionis significantly reduced compared with the bulk phase.

In heterogeneous biological materials and foods, water exists in different states.It is thought that water molecules in different states function differently.

Water associated with proteins and other macromolecules has traditionally beenreferred to as bound water. However, to designate such water as bound can bemisleading because for the most part, the water molecules are probably only tran-siently associated, and at least a portion of the associated water has to be constantlyrearranged due to the thermal perturbations of weak hydrogen bonds.

Water molecules are constantly in motion, even in ice. In fact, the translationaland rotational mobility of water directly determines its availability. Water mobilitycan be measured by a number of physical methods, including NMR, dielectricrelaxation, electron spin resonance (ESR), and thermal analysis (Chinachoti, 1993).The mobility of water molecules in biological systems may play an important rolein a biochemical reaction’s equilibrium and kinetics, formation and preservation ofchemical gradients and osmotic pressure, and macromolecular conformation. In foodsystems, the mobility of water may influence the engineering processes, such asfreezing, drying, and concentrating, chemical and microbial activities, and texturalattributes (Ruan and Chen, 1998). Water determines quality, stability, shelf life andphysical properties of food. It has influence on rheological, thermal, mass transfer,electrical, optical, and acoustic physical properties (Lewicki, 2004).

3.2.6.2 Water Transport

Water transport is associated with various physiological processes in whole livingorganisms and single cells.

When cells are exposed to hyper- or hypoosmotic solutions, they immediatelylose or gain water, respectively. Even in an isotonic medium, a continuous exchangeof water occurs between living cells and their surroundings. Most cells are so smalland their membranes are so leaky that the exchange of water molecules measuredwith isotopic water reaches equilibrium in a few milliseconds.

The degree of water permeability differs considerably between tissues and celltypes. Mammalian red blood cells and renal proximal tubules are extremely permeableto water molecules. Transmembrane water movements are involved in diverse phys-iological secretion processes.

How water passes through cells has begun to become clear only in the last fewyears. Water permeates living membranes through both the lipid bilayer and specificwater transport proteins. In both cases water flow is passive and directed by osmosis.Water transport in living cells is therefore under the control of ATP (adenosinetriphosphate) and ion pumps.

The most general water transport mechanism is diffusion through lipid bilayers,with a permeability coefficient of 2 to 5 × 104 cm/sec. The diffusion through lipidbilayers depends on lipid structure and the presence of sterol (Subczyński et al.,



1994). It is suggested that the lateral diffusion of the lipid molecules and the waterdiffusion through the membrane is a single process (Haines, 1994).

A small amount of water is transported through certain membrane transport pro-teins, such as a glucose transporter or the anion channel of erythrocytes (aquapores).

The major volume of water passes through water transport proteins. The firstisolated water transporting protein was the channel-forming integral protein fromred blood cells. The identification of this protein has led to the recognition of afamily of related water-selective channels, the aquaporins, which are found in ani-mals, plants, and microbial organisms. In addition to water, they permeate someother small molecules. The pore is formed by six membrane-spanning helices. Inthe membrane they aggregate into tetramers, but each monomer acts as a separatewater channel. Water flow through the protein channel is controlled by the numberof protein copies in the membrane. In red blood cells, there are 200,000 copies/cellthat account for up to 90% of the water permeability of the membrane; in apicalbrush border cells of renal tubules, it constitutes 4% of the total protein (Engel etal., 1994). It is assumed that another important function of aquaporins is the detectionof osmotic and turgor pressure gradients (Hill et al., 2004).

3.3 WATER IN FOOD

3.3.1 INTRODUCTION

Water, with a density of 1000 kg ⋅ m–3, is denser than the oil components of foods;oils and fats typically have densities in the range 850 to 950 kg ⋅ m–3. Glycerols andsugar solutions are denser than water.

Unlike solid phases of most other liquids, ice is less dense than liquid water,and ice has a lower thermal conductivity than water. These properties have an effecton the freezing of foods that are predominantly water based; the formation of an icelayer on the surface of the liquids and the outside of solids has the effect of slowingdown the freezing rate.

Because a molecule of water vapor is lighter (molecular weight = 18) than thatof dry air (molecular weight about 29), moist air is lighter than dry air at the sametemperature. This is somewhat unexpected in that the popular conception is thathumid air (which contains more water) is heavier than dry air.

At room temperature, water has the highest specific heat of any inorganic ororganic compound with the sole exception of ammonia. It is interesting to speculatewhy the most commonly occurring substance on this planet should have one of thehighest specific heats. One of the consequences of this peculiarity in the food industryis that heating and cooling operations for essentially water-based foods are moreenergy demanding. To heat a kilogram of water from 20 to 50°C requires about 125kJ of energy, whereas heating the same mass of vegetable oil requires only 44 kJ.

A sponge holds most of its water as liquid in the interstices of the spongestructure. Most of the water can be wrung out of the sponge, leaving a matrix of air



and damp fibers. Within the sponge fibers the residual water is more strongly held—absorbed within the fiber of the sponge.

If the sponge is left to dry in the sun, this adsorbed water will evaporate leavingonly a small proportion of water bound chemically to the salts and to the celluloseof the sponge fibers. As with the familiar example of water in a sponge, water isheld in food by various physical and chemical mechanisms (Table 3.1).

It is a convenient oversimplification to distinguish between free and bound water.The definition of bound water in such a classification poses problems. Fennema(1996) reports seven different definitions of bound water. Some of these definitionsare based on the freezability of the bound component and others rely on its avail-ability as a solvent. He prefers a definition in which bound water is that which existsin the vicinity of solutes and other nonaqueous constituents and exhibits propertiesthat are significantly altered from those of “bulk water” in the same system.

Moisture content can be measured simply by weighing a sample, then ovendrying it, usually at 105°C overnight—the difference in mass being the moisture inthe original sample. However, much confusion is caused by reporting the moisturecontent simply as a percentage without specifying the basis of the calculation. Itshould be made clear whether the moisture content is calculated on a wet basis(moisture content divided by original mass) or on a dry basis (moisture contentdivided by the bone dry or oven dry mass). Even the term bone dry mass can causeconfusion among non-English speakers; it was once misinterpreted as the mass ofthe dry bones! In foods containing significant quantities of fat or salt, for example,moisture content may be calculated as the mass of water in a sample divided by thedry solids that are not salt or fat, in which case the moisture content should bereported as salt free, fat free, dry basis.

TABLE 3.1Classification of Water States in Foods

Class of water Description

Proportion of typical90% (wet basis)moisture content in food

Constitutional An integral part of nonaqueous constituent <0.03%Vicinal Bound water that strongly acts with specific hydrophilic

sites of nonaqueous constituents to form a monolayer coverage; Water–ion and water–dipole bonds.

0.1 to 0.9%

Multilayer Bound water that forms several additional layers around hydrophilic groups; water–water and water–solute hydrogen bonds

1 to 5%

Free Flow is unimpeded; properties close to dilute salt solutions; water–water bonds predominate.

5% to about 96%

Entrapped Free water held within matrix or gel, which impedes flow 5% to about 96%

Source: After Fennema, O.R., Food Chemistry, 2nd ed., Marcel Dekker, Inc., New York, 1985.



3.3.2 SORPTION ISOTHERMS AND WATER ACTIVITY

3.3.2.1 Principle

Since 1929 it has been recognized that the chemical and microbial stability, andhence the shelf life of foods, is not directly related to its moisture content, but to aproperty called water activity (Tomkins, 1929). Essentially, water activity is themeasure of the degree to which water is bound within the food, and hence isunavailable for further chemical or microbial activity.

Water activity is defined as the ratio of the partial pressure of water vapor inor around the food to that of pure water at the same temperature. Relative humidityof moist air is defined in the same way except that by convention, relative humidityis reported as a percentage whereas water activity is expressed as a fraction. Thusif a sample of meat sausage is sealed within an airtight container, the humidityof the air in the head space will rise and eventually equilibrate to a relativehumidity of, say, 83%, which means that the water activity (aw) of the meatsausage is 0.83.

The relationship between water activity and moisture content for most foods ata particular temperature is sigmoid-shaped in a curve called the sorption isotherm(Figure 3.10). The phrase equilibrium moisture content curve is also used. Sorptionisotherms at different temperatures can be calculated using the Clausius–Clapeyronequation from classical thermodynamics, namely,

where:

is the absolute temperature

is the heat of sorption

is the gas constant

A complication arises from one of the methods of measuring sorption isothermsfor a food. A food that has previously been dried and then is rehydrated will havea different sorption isotherm (adsorption isotherm) from that which is in the processof drying (desorption isotherm). This difference is due to a change in the water-binding capacity in foods that have been previously dried.

Many mathematical descriptors for sorption isotherms have been proposed. Oneof the more famous is that of Brunauer, Emmett, and Teller (1938) (the BET isotherm),which is based on the concept of a measurable amount of monomolecular layer(vicinal) water for a particular food. Wolf, Speiss, and Jung (1985) compiled 2201references to sorption isotherm data for foods. An example of the type, detail, andaccuracy of sorption isotherm data available in the literature is presented in Table 3.2.

d a

d T

H

Rw(ln )

( / )1= ∆

T

∆H

R



Iglesias et al. (1975) propose the following three-parameter equation to fit sorp-tion isotherm data for a range of foods:

aw = exp(a′ θr)

FIGURE 3.10 A typical sorption isotherm for a food.

TABLE 3.2Sorption Isotherm Data for Cod and Corn

Product SpecificationsTemperature

(°C) Xm r a′

Coda Adsorption 30 7.68 1.2398 1.3490Cornb Desorption 4.5 8.30 2.2345 1.9748

Desorption 15.5 7.68 2.4862 2.0949Desorption 30 7.30 2.5663 1.7950Desorption 38 6.35 2.3711 1.8618Desorption 50 6.89 2.1203 1.5936Desorption 60 5.11 2.2185 1.7430

a Adsorption, after Jason, AC., A study of evaporation and diffusion processes in the dryingof fish muscle, in Fundamental Aspects of the Dehydration of Foodstuffs, Soc. Chem. Ind.,London, 1958, p. 103.b Desorption, after Chen, C.S. and Clayton, J.T., Trans. A.S.A.E., 14, 927, 1971.

Source: From Iglesias, H.A, Chirife, J, and Lombardi, J.L., J. Food Technol., 10, 289, 1975.

Desorption

Adsorption

Water activity

Moi

stur

e co

nten

t (dr

y ba

sis)

0.2 0.4 0.6 0.8 1.0

4.0

3.0

2.0

1.0



where: a′ and r = the parameters as listed in Table 3.2θ = X/Xm X = the equilibrium moisture contentXm, in units of g/100 g dry basis = the BET monomolecular moisture content for

the particular food as listed in Table 3.2

However, there are nearly as many equations for sorption isotherms as there areresearchers in this field.

3.3.2.2 Measurement of Water Activity

Many methods of measuring water activity have been developed. These includedirect vapor pressure measurement, equilibration with a stable hygroscopic substancethat has a known sorption behavior, and various types of hygrometers (Doe, 1998).

Water activity is most conveniently measured by the measurement of relative humid-ity in the head space over a food sample in a sealed container. Commercially availableinstruments for water activity determination use various methods for measuring therelative humidity: hair hygrometer (Lluft), electrical hygrometer (Nova Sina), and dew-point temperature (Aqualab). The hygrometer-based instruments are prone to drift, andmust be calibrated regularly against saturated solutions of various inorganic salts.Hygrometer-based instruments are also prone to hysteresis at high humidities.

The Aqualab CX2 water activity meter (Decagon Devices Inc., Pullman, WA)detects water condensation on a chilled mirror (dewpoint temperature). The instru-ment is sensitive to less than 0.001 water activity units. Readings take 5 minutes orless and are accurate to ± 0.003 water activity units.

Care must be taken with any measurement of water activity to ensure that thesample is representative of the food being tested. Dried fish, for example, will havemoisture and salt content, and hence water activity, varying widely from thin,exposed flesh to the relatively moist interior. If the worst-case scenario for thegrowth of potentially toxic or spoilage organisms is of interest, the sample of fleshfor water activity determination should be excised from the thickest, most moistregion of the fish.

3.3.2.3 Water Activity and Shelf Life of Foods

Many of the chemical and biological processes that cause deterioration of foods,and ultimately spoilage, are water dependent. Microbial growth is directly linked towater activity. No microbes can multiply at a water activity below 0.6. Dehydrationis arguably the oldest form of food preservation; the sun drying of meat and fish hasbeen traced to the beginning of recorded history. Drying relies on removing water,thus making it unavailable for microbial growth.

Salting or curing has the same effect. A saturated solution of common salt hasa water activity of close to 0.75. Thus by adding sufficient salt to foods, the water



activity can be lowered to a level where most pathogenic bacteria are inactivated,but the moisture content remains high.

Intermediate moisture content foods (IMF), such as pet food and continentalsausages, rely on fats and water-binding humectants such as glycerol to lower wateractivity. Fat, being essentially hydrophobic, does not bind water, but acts as a fillerfor IMF to increase the volume of the product.

The effect of several humectants is for each to sequester an amount of waterindependently of the other humectants that may be present in the food. Each thuslowers the water activity of the system according to the equation in Ross (1975):

awn = aw0 ⋅ aw1 ⋅ aw2 ⋅ aw3 ⋅ etc

where:awn = the water activity of the complex food systemaw0, etc. = the water activities associated with each component of the system

For example, the water activity of a food with a moisture content of 77% (wetbasis) and a salt content of 3% (wet basis) can be calculated as follows: 100 g ofthe food comprises 77 g of water, 20 g of bone dry matter, and 3 g of salt. Thecontribution to the water activity due to the salt can be calculated (according toRaoult’s law of dilute solutions) and the molecular weights of water (18) and salt(58.5) as

aw1 = (77 × 18) / (77 × 18 + 3 × 58.5) = 0.89

The water activity for the salt-free solid matter of the food is found from itssorption isotherm at that moisture content, aw0 = 0.90, say. Thus the water activityof the salted food is

awn = aw0 ⋅ aw1 = 0.9 × 0.89 = 0.8

None of the dangerous pathogenic bacteria associated with food, such as Clostrid-ium or Vibrio spp. which cause botulism and cholera, can multiply at water activityvalues below about 0.9. Thus, drying or providing sufficient water-binding humectantsis an effective method of preventing the growth of food-poisoning bacteria.

Only osmophilic yeasts and some molds can grow at water activities in the range0.6 to 0.65. Thus, by reducing the water activity below these values, foods aremicrobially stable. That is, unless the packaging is such that the food becomes locallyrewet, in which case local spoilage can occur, for example, when condensation occurswithin a hermetically sealed package subject to rapid cooling.

There are various chemical reactions that proceed, and may be accelerated, atlow values of water activity. Maillard reactions leading to lysine loss and browncolor development peaks at aw values around 0.5 to 0.8. Nonenzymatic lipid oxidation



increases rapidly below aw = 0.4. Enzymic hydrolysis decreases with water activitydown to aw = 0.3 and is then negligible.

3.3.3 BOTTLED WATER

3.3.3.1 Classification

Water in glass or plastic bottles or cartons is becoming increasingly popular, notonly in areas where the tap water supply quality may be substandard, but also wheredrinking bottled water is seen as culturally accepted behavior. Health benefits areclaimed for some products. Marketing of bottled water has been so successful thatconsumers spend from 240 to over 10,000 times more per liter for bottled waterthan for tap water.

Tap water in some municipalities may contain hazardous chemicals and micro-organisms; it may also contain additives such as fluoride to provide perceived healthbenefits to communities. Bottled water is required by law to meet standards ofchemical and biological safety. In some respects, standards for bottled water aremore stringent than those for tap water because of the shelf life requirement forbottled water.

There are basically three types of bottled water:

1. Natural mineral water must come from an underground source. It receivesno treatment other than filtration or carbonation, and is bottled at thesource. Natural mineral water must meet tight microbiological standardsand be regularly tested.

2. Spring water may come from other sources. It must also be bottled at thesource and meet microbiological and chemical standards. Permitted treat-ments are filtration and carbonation; however, spring water does not haveto be stable in composition.

3. Table water is basically any water in a bottle. It could come from an under-ground source, but might be tap water. Table water may or may not be treated.It could have additives to change its flavor or chemical composition.

3.3.3.2 Natural Mineral Water

The Codex Alimentarius Commission (CAC) has published a Standard for NaturalMineral Waters (CAC, 1981). This provides guidance for industry and regulatingbodies that may choose to follow this standard or make parts of it mandatory bylaw. The standard defines natural mineral water as being clearly distinguishable fromordinary drinking water because it is:

• Characterized by its content of certain mineral salts and their relativeproportions and the presence of trace elements or of other constituents

• Obtained directly from natural or drilled sources from underground water-bearing strata for which all possible precautions should be taken withinthe protective perimeters to avoid any pollution of, or external influenceon, the chemical and physical qualities of natural mineral water



• Consistent in composition and stable in its discharge and its temperature,due account being taken of the cycles of minor natural influences

• Collected under conditions that guarantee the original microbiologicalpurity and chemical composition of essential components

• Packaged close to the point of emergence with particular hygiene precautions• Not subjected to any treatment other than those permitted by this standard

The standard also defines naturally carbonated natural mineral water, noncarbon-ated natural mineral water, decarbonated natural mineral water, natural mineral waterfortified with carbon dioxide from the source, and carbonated natural mineral water.

The standard lists permissible levels for a number of chemical substances aslisted in Table 3.3.

The standard requires that natural mineral water shall not pose a microbiologicalrisk to the consumer. Escherichia coli or thermotolerant coliforms must not bepresent in a 250-mL sample. If no more than two total coliform bacteria, fecalstreptococci, Pseudomonas aeruginosa, or sulfide-reducing anaerobes are detectedin a 250-mL sample, a second examination must be carried out; if more than twoare detected the batch is rejected.

TABLE 3.3Permissible Levels for Chemicals in Natural Mineral Water

Chemical Permissible level (mg/dm3 unless otherwise specified)

Antimony 0.05 Arsenic 0.01 calculated as total AsBarium 0.7 Borate 5 calculated as BCadmium 0.003 Chromium 0.05 calculated as CrCopper 1 Cyanide 0.07 Fluoride (See note 1 below)Lead 0.01Manganese 0.5 Mercury 0.001 Nickel 0.02 Nitrate 50 calculated as nitrateNitrite 0.02 as nitrite (see note 2 below)Selenium 0.01

Note 1: If more than 1 mg/dm3 fluoride the product must be labeled: contains fluoride.If more than 2 mg/dm3, product must be labeled: The product is not suitable for infantsand children under the age of seven years.Note 2: Set as a quality limit (except for infants).

Source: From CAC (Codex Alimentarius Commission), Codex Standard for NaturalMineral Water, Codex Standard 108-1981, Food and Agriculture Organization of theUnited Nations (FAO), Rome, Rev. 1, 1997.



Natural mineral water containers must be hermetically sealed and safe frompossible adulteration or contamination.

The label must identify the product as natural mineral water and must declarethe chemical composition. No claims of medicinal effects may be shown, and anyhealth benefit claims must be true and not misleading.

3.3.4 BOTTLED WATER OTHER THAN NATURAL MINERAL WATER

3.3.4.1 Definition

There is a Codex standard for packaged water for drinking purposes other thannatural mineral water (CAC, 2001). Such water may contain minerals and carbondioxide naturally occurring or intentionally added, but may not contain sugars,sweeteners, flavorings, or other foodstuffs.

No packaged water may contain substances that emit radioactivity in quantitiesthat may be injurious to health. All packaged water shall comply with the Guidelinesfor Drinking Water Quality published by the World Health Organization (WHO,2004). Addition of minerals must comply with the relevant Codex standards.

The standard distinguishes between “waters defined by origin,” which originatefrom a specific underground or surface resource and do not pass through a communitywater system, and “prepared waters,” which may originate from any supply. Preparedwaters can be subjected to antimicrobial treatments, which modify the physical andchemical properties of the original water, provided that such treatment satisfies theWHO Guidelines for Drinking Water Quality.

3.3.4.2 Water Defined by Origin

Water defined by origin is limited in treatment prior to packaging to:

• Reduction and/or elimination of dissolved gases (and resulting possiblechanges in pH)

• Addition of carbon dioxide (and resulting change in pH) or reincorporationof the original carbon dioxide present at emergence

• Reduction and or elimination of unstable compounds such as iron, manga-nese, sulfur (as So or S--) compounds and carbonates in excess, under normalconditions of temperature and pressure, of the calcocarbonate equilibrium

• Addition of air, oxygen, or ozone on condition that the concentration ofby-products resulting from ozone treatment is below the tolerance estab-lished under the health limits for chemical and radiological substances

• Decrease and/or increase in temperature• Reduction and/or separation of elements originally present in excess of

maximum concentrations or of maximum levels of radioactivity setaccording to the health limits for chemical and radiological substances

• Antimicrobial treatments may be used to conserve the original microbialfitness for human consumption



3.3.4.3 Hygiene, Labeling, and Health Benefits

The standard recommends that packaged waters comply with the RecommendedInternational Code of Practice—General Principles of Food Hygiene (CAC/RCP1999), also the Code of Hygienic Practice for Bottled/Packaged Drinking Waters(Other than Natural Mineral Waters) (CAC/RCP 2001).

There are requirements in the standard for approval and inspection of the sourcefor waters defined by origin. Labeling must comply with the Codex General Standardfor the Labelling of Prepackaged Foods (CODEX STAN 1-1985, Rev 1-1991)together with any additional provisions in national legislation.

Where appropriate in the case of waters defined by origin, labeling may specify“naturally carbonated” or “naturally sparkling” or “fortified with carbon dioxide.”For all waters, “carbonated” or “sparkling” may be used if the carbon dioxide doesnot come from the same source. If there is no visible release of carbon dioxide, thewords noncarbonated, nonsparkling, or still may be used.

The total dissolved content of packaged waters may be shown, and in the caseof waters defined by origin the chemical composition that confers the characteristicsof the product may be shown.

Where required by local laws, the precise location of the source of a waterdefined by origin must appear on the label.

Water bottled from a tap water distribution system that has not undergone furthertreatment (e.g., the addition of carbon dioxide or fluoride) must bear the words “froma public or private distribution system.”

The standard prohibits any claims for medicinal (preventive, alleviative, or cur-ative) effects. Claims for other health benefits may only appear if true and notmisleading.

Any place name may not form part of the trade name unless it refers to waterdefined by origin collected from that place.

The chemical and microbiological safeguards placed on bottled water make it asafer alternative to tap water in places where community water supplies may besubstandard or compromised by drought or flood.

The mineral content of bottled water may be an important and necessary sup-plement to dietary mineral intake. For a very exhaustive presentation of the contentsof mineral components in different food sources and their role in nutrition, seeChapter 4.

3.3.5 WATER SUPPLY, QUALITY, AND DISPOSAL

3.3.5.1 Water Supply

Just as water is an integral part of any food, the supply, quality, and disposal ofwater is of prime consideration in the establishment and operation of all foodprocessing. Potable (drinkable) water may be required for addition to the product,and will certainly be necessary for cleanup. Nonpotable water may be required forheat exchangers and cooling towers. Boiler feed water must be conditioned within



close limits of pH and hardness. Brennan et al., in their book Food EngineeringOperations (1990), list four types of water used in the food and beverage industries:

1. General-purpose water2. Process water3. Cooling water4. Boiler feed water

The site and consequent viability of a food processing plant may well dependon a guaranteed, regular supply of suitable quality water and an environmentallyacceptable method of disposal. Developed countries now have strict regulations forthe emission of wastewater. Developing countries are becoming increasingly awareof the problems of wastewater disposal. In a recent symposium in Indonesia, a fish-drying processor was asked what his main technical problems were. He cited waterpollution—not for reasons of meeting environmental control regulations, but becausethe fish farmers further down the river were complaining about his wastewater.

3.3.5.2 Water Quality: Standards and Treatment

There are a number of international standards for potable (drinkable) water qualityin existence. The World Health Organization (WHO) has a standard for potable waterquality as part of the Codex Alimentarius. The standard detailed in Table 3.4 is fromthe United States Environmental Protection Agency.

There is also a large EC directive relating to the quality of water intended forhuman consumption (80/778/EEC), which is contained in the Joint Circular fromthe Department of the Environment, Circular 20/82, London, and the Welsh OfficeCircular 33/82, Cardiff, issued on August 19, 1982.

In most cases, water will require some treatment to assure it is of the requiredstandard to meet food hygiene requirements and not constitute a public health hazard.Surface water from rain runoff into rivers or impoundments is likely to containatmospheric solutes, minerals from the ground, organic matter from vegetation,microbial contamination from birds and wild and domestic animals, and humanwaste. Water from underground aquifers will have much of the surface contaminationfiltered out, but is likely to be high in dissolved mineral content.

Water treatment to bring the quality to within the required standard may involvescreening, sedimentation, coagulation and flocculation, filtration, and other physicalor chemical treatments to remove microorganisms, organic matter, or dissolvedminerals.

Metal screens are used to remove particles larger than about 1 mm in size.Settling ponds remove smaller particles. Insoluble, suspended matter is usuallyremoved by sand filters. Coagulating and flocculating agents act to bind smallerparticles into clumps, which then settle or can be screened or filtered.

Microorganisms can be inactivated by heat, chemical disinfection, UV radiation,or ultrasonic treatment. Most town water supplies are chlorinated or have ozoneadded for chemical disinfection.



Treatment to remove dissolved mineral matter is more complex. Dissolvedbicarbonates of calcium, magnesium, sodium, and potassium cause alkalinity; sol-uble calcium and magnesium salts cause hardness. Alkalinity and hardness may needto be adjusted for some food processing operations. For example, the formation ofa head on beer is critically dependent on water hardness. Excessively hard watermay cause discoloration and toughening of certain foods. On the other hand, hardnessmay be required to prevent excessive foaming in cleanup operations.

Iron and manganese salts may be present in water supplies forming organicslimes, which tend to clog pipes. Aeration, filtering, and settling are effective for

TABLE 3.4Primary Maximum Contaminant Levels (mg/dm3 unless specified)

Contaminant Level (mg/dm3 unless specified)

Arsenic 0.05Barium 1Cadmium 0.010Chromium 0.05Lead 0.05Mercury 0.002Nitrate (as N) 10Selenium 0.01Silver 0.05Fluoride 4.0Endrin 0.0002Lindane 0.004Methoxychlor 0.1Toxaphene 0.0052.4 D 0.12,4,5 TP Silvex 0.01Total trihalomethanes 0.10Trichloroethylene 0.005Carbon tetrachloride 0.0051,2 dichloroethane 0.005Vinyl chloride 0.002Benzene 0.005Para-dichlorobenzene 0.0751,1,dichloroethylene 0.0071,1,1,trichloroethane 0.2Radium 226 and 228, combined 5 pC/lGross alpha particle activity 15 pC/lGross beta particle activity 4 millirem/yearTurbidity 1 tu up to 5 tuColiform bacteria 1 per 100 ml, monthly average

Source: From the U.S. Environmental Protection Agency (EPA), The Safe DrinkingWater Act, Program summary, U.S. Environmental Protection Agency, Washington,DC, October 1996.



the removal of iron bicarbonates. Insoluble oxides of manganese are formed throughchlorination.

Excessive amounts of dissolved gases, carbon dioxide, oxygen, nitrogen, andhydrogen sulfide cause problems in boiler feed water, corrosion, and bacterial for-mation. Treatment is by boiling and venting off the noncondensable gases, or bychemical dosing.

Small amounts of hydrocarbons, such as kerosene and diesel, cause tainting infoods. Separating fuels from processing areas, personal hygiene, cleaning stationsaround food processing operations, and good housekeeping can prevent this problem.

3.3.6 WATER POLLUTION

Polluted water is described as polluted if it poses a risk to the health of humans,fish, or other animals.

Microbiologically polluted water contains bacteria and other microorganismsthat may be hazardous or toxic. Human and animal wastes from sewage and farmyardrunoff are the principal sources of microbiological pollution. Polluted water can bean indirect hazard as fish and shellfish may become contaminated and eaten.

Water can be polluted by organic and inorganic chemicals. Domestic and indus-trial pesticides are a major source of chemical pollution, as are detergents. Many ofthese substances are slow to degrade and may be concentrated in the food chainwith disastrous consequences for fish and bird life.

Biodegradable substances tend to be oxygen depleting resulting in a reductionof aerobic bacteria and fish. Food waste is high in biochemical oxygen demand(BOD). Food wastes contain large quantities of organic matter, which breaks downnaturally by oxidation; however, this oxygen demand is at the expense of othernatural biochemical processes in waterways, which become oxygen depleted andlifeless if the BOD is too high. BOD is defined as the quantity of oxygen (in unitsof mg/dm3) required for microorganisms to oxidize the waste at a particular tem-perature (20°C) in 5 days. Food wastes can range in BOD from 500 to 4000 mg/dm3,which is higher than for domestic sewage (200 to 400 mg/dm3).

An excess of nutrients, such as phosphorous and nitrogen in polluted water, willlead to an excessive growth of plant matter in waterways, and algal blooms. Besidesclogging waterways and adding toxins, this extra plant material contributes to theBOD of the water.

Suspended matter, even if chemically and biologically inert, can contribute topollution. These particles will eventually settle out and cause silting, and an anaerobicenvironment at the bottom of waterway.

Water pollution is most effectively prevented by removing the pollutantsbefore they get into the waterways. This can be accomplished through goodhousekeeping practices, such as avoiding the discharge of fatty material anddetergents into the domestic sewerage system, proper design of landfill areas,pretreatment of industrial wastes, separation of storm water from sewage, so asnot to overload treatment plants.



3.3.7 WASTEWATER TREATMENT AND DISPOSAL

The ultimate aim of any food processing operation is to have an environmentallyneutral impact. Reuse, recycle, and sustainability are today’s catchwords. For a foodoperation to be truly environmentally sustainable it should recycle all water notincorporated in the product or vented to the atmosphere. The reality is that it iscurrently considered uneconomic to recycle wastewater from food processing oper-ations. Current practice is to treat wastewater to limit its effect on receiving waters.

Treatment of wastewater mirrors the water treatment methods described above, thatis, a combination of physical, chemical, and biological unit operations and processes.

A physical operation treats suspended rather than dissolved pollutants. Thepollutants may be simply allowed to settle out or float to the top naturally, or thetreatment may be aided mechanically, which will cause smaller particles to sticktogether, forming larger particles that will settle or rise faster—a phenomenon knownas flocculation. Chemical flocculants may also be added to produce larger particles.

Filtration through a medium such as sand as a final treatment stage can result invery clear water. Ultrafiltration, nanofiltration, and reverse osmosis force waterthrough membranes and can remove colloidal material and even some dissolvedmatter. Absorption (adsorption, technically) on activated charcoal is a physical oper-ation that can remove dissolved chemicals. Air or steam stripping can be used toremove pollutants that are gasses or low-boiling liquids from water, and the vaporsthat are removed in this way are also often passed through beds of activated charcoalto prevent air pollution. These last processes are used mostly in industrial treatmentplants, though activated charcoal is common in municipal plants for odor control.

Wastewater from food processing operations usually contains significant solidmatter, which can be removed by physical operations. Fats and oils can be skimmedfrom the surface of settling tanks, and heavier suspended matter can be removed assludge, which can then be dewatered, dried, and used as animal feed, fertilizer orfuel. An alternative method for the removal of oils and fats is by aeration, in whichair bubbles blown from the bottom of a settling tank carry fine solids and grease tothe surface.

Chemical treatments of wastewater are much the same as described above forprocessed water.

BOD can be effectively reduced by biological processes. Both aerobic andanaerobic fermentation of the organic material is used. Depending on the scale ofthe operation, bioreactors range in size from 7.5 m in diameter and 2 to 3 m in depthto lagoons 1 to 2 m deep covering several hectares. However, for long-term sustain-able operation there must be provision for sludge removal. Where the area fortreatment is not a limitation, and there is sufficient isolation for smell not to be adeterrent, wastewater is sprayed directly on the ground where it breaks down underthe action of sunlight and in-ground bacteria.

A typical municipal treatment plant begins with a preliminary stage in whichlarge or hard solids are removed or crushed. The effluent then passes through aprimary settling basin in which organic suspended matter will either settle out or



float to the surface to be skimmed off. The next part of the process is usually referredto as secondary treatment wherein the remaining dissolved or colloidal organic matteris removed by aerobic biodegradation. This promotes the formation of less offensive,oxidized products. The treatment unit should be sufficiently large to remove enoughof the pollutants to prevent significant oxygen demand in the receiving water afterdischarge.

Sewage and wastewater can also be treated anaerobically. Closed reactors facil-itate odor control, although anaerobic lagoons are also used. Such lagoons are deeperthan for aerobic types with grease allowed to accumulate on the surface to controlodor emission. Methane produced as an end product of the biochemical pathwaycan be used for heating the reactors in cold weather. A problem with the anaerobicdigestion process is its sensitivity to pH and temperature variation, and the suscep-tibility of the active microorganisms to chemical disinfectants.

REFERENCES

Bjerrum, N., Structure and properties of ice, Science, 115, 385, 1952.Brennan, J.G. et al., Food Engineering Operations, 3rd ed., Elsevier Applied Science, London,

1990.Brunauer, S., Emmet, P.H., and Teller, E., Adsorption of gases in multilayers, J. Am. Chem.

Soc., 60, 309, 1938.CAC (Codex Alimentarius Commission), Codex Standard for Natural Mineral Water, Codex

Standard 108, 1981, Rev. 1, 1997.CAC (Codex Alimentarius Commission), Code of Hygienic Practise for Bottled/Packed Drink-

ing Water (Other than Natural Mineral Waters) Codex Standard 227, 2001.CAC/RCP (Codex Alimentarius Commission/Recommended International Code of Practice)

General Principles of Food Hygiene. FAO, Rome, 1-1969 Rev. 3 (1997). Amended1999.

CAC/RCP (Codex Alimentarius Commission/Recommended International Code of Practice)Code of Hygienic Practice for Bottled/Packaged Drinking Waters (other than NaturalMineral Waters). FAO, Rome, 48-2001.

Calabrese, E.J., Gilbert, C.E., and Pastides, H., Eds., Safe Drinking Water Act: Amendments,Regulations, and Standards, Lewis Publishers, Chelsea, MI, 1989.

Chen, C.S. and Clayton, J.T., The effect of temperature on sorption isotherms of biologicalmaterials, Trans. A.S.A.E., 14, 927, 1971.

Chinachoti, P., Water mobility and its relation to functionality of sucrose-containing foodsystems, Food Technol., 47, 134, 1993.

CODEX STAN (Codex Standard) Codex General Standard for the Labelling of PackagedFoods. FAO, Rome, 1-1985 (Rev. 1-1991).

Dauchez, M., Peticolas, W., Debelle, L., and Alix, A.J.P., Ab initio calculations of polyhedraliquid water, Food Chem., 82, 23, 2003.

Doe, P.E., Ed., Fish Drying and Smoking Production and Quality, Technomic Publishing Co.Inc., Lancaster, PA, 1998, p. 22.

Engel, A., Waltz, Th., and Agre, P., The aquaporin family of membrane water channels, Curr.Opin. Struct. Biol., 4, 545, 1994.

Fennema, O.R., Water and ice, in Food Chemistry, 3rd ed., Fennema, O.R. (Ed.), MarcelDekker, Inc., New York, 1996.



Frank, H.S. and Quist, A.S., Pauling’s model and the thermodynamical properties of water,J. Chem. Phys., 34, 604, 1961.

Frank, H.S. and Wen, W.Y., Structural aspects of ion-solvent interaction in aqueous solutions:A suggested picture of water structure, Disc. Faraday Soc., 24, 133, 1957.

Giudice, E. et al., Water in biological systems, in Modern Bioelectrochemistry, Gutmann, F.and Keyzer, H., Eds., Plenum Press, New York, 1986.

Haines, Th.H., Water transport across biological membranes, FEBS Lett., 346, 115, 1994.Hill, A.E., Sachar-Hill, B., and Sachar-Hill, Y., What are aquaporins for? J. Membrane Biol.,

197, 1, 2004.Iglesias, H.A., Chirife, J., and Lombardi, J.L., An equation for correlating equilibrium mois-

ture content in foods, J. Food Technol., 10, 289, 1975.Jason, A.C., A study of evaporation and diffusion processes in the drying of fish muscle, in

Fundamental Aspects of the Dehydration of Foodstuffs, Soc. Chem. Ind., London,1958, 103.

Lehninger, A.L., Nelson, D.L, and Cox, M.M., Principles of Biochemistry, 2nd ed., WorthPublishers, Inc., New York, 1993.

Lewicki, P., Water as the determinant of food engineering properties. A review, Journal ofFood Engineering, 61, 483, 2004.

Ling, G.N., A Physical Theory of the Living State, Ginn (Blaisdel), Boston, MA, 1962.Mathlouthi, M., Water content, water activity, water structure and stability of foodstuffs, Food

Control, 12, 409, 2001. Mild, K. and Løvtrup, S., Movement and structure of water in animal cells. Ideas and

experiments, Biochim Biophys. Acta, 822, 155, 1985.Nossal, R. and Lecar, H., Eds. Molecular and Cell Biophysics, Addison-Wesley Publishing

Co., Reading, MA, 1991.Rahman, A. and Stillinger, F.H., Molecular dynamic study of liquid water, J. Chem. Phys.,

55, 3336, 1971.Röntgen, W.K., Über die Konstitution des flüssigen Wassers, Ann. Physik, 45, 91, 1892.Ross, K.D., Estimation of water activity in intermediate moisture foods, Food Technol., 29,

26, 1975.Ruan, R.R. and Chen, P.L., Water in Foods and Biological Materials, Technomic Publishing

Co. Inc., Lancaster, PA, 1998.Rückold, S., Isengard, H.-D., Hanss, J., and Grobecker, K., H., The energy of interaction

between water and surfaces of biological reference materials, Food Chemistry, 82,51, 2003.

Starzak, M. and Mathlouthi, M., Cluster composition of liquid water derived from laser-Raman spectra and molecular simulation data, Food Chemistry, 82, 3, 2003.

Stillinger, F.H., Water revisited, Science, 209, 451, 1980.Subczyński, W.K. et al., Hydrophobic barriers of lipid bilayer membranes formed by reduction

of water penetration by alkyl chain unsaturation and cholesterol, Biochemistry, 33,7670, 1994.

Tomkins, R.G., Studies of the growth of moulds. 1., Proc. R. Soc. B, 105, 375, 1929.U.S. Environmental Protection Agency (EPA), The Safe Drinking Water Act, Program Sum-

mary, U.S. Environmental Protection Agency, Washington, DC, October 1987.Wallace, J.S. and Gregory, P.J., Water resources and their use in food production systems,

Aquat. Sci., 64, 363, 2002.Wiggins, Ph.M., Role of water in some biological processes, Microbiol. Rev., 54, 432, 1990.Wiggins, Ph.M., Water in complex environments such as living systems, Physica A, 314, 485,

2002.



Wolf, W., Spiess, W.E.L., and Jung, G., Sorption Isotherms and Water Activity of FoodMaterials, Science and Technology Publishers, Hornchurch, Essex, U.K., 1985.

World Health Organization (WHO), Guidelines for Drinking Water Quality, 3rd ed. WorldHealth Organization, Rome, 2004.


61

4

Mineral Components

Micha

ł

Nabrzyski

CONTENTS

4.1 The Contents and Role of Minerals Present in Foods .................................. 614.2 Interaction with Dietary Components ........................................................... 63

4.2.1 Introduction ........................................................................................ 634.2.2 Effect on Absorption.......................................................................... 634.2.3 Building Body Tissue and Regulating Body Processes .................... 67

4.3 Role in Food Processes.................................................................................. 674.3.1 Effect on Oxidation............................................................................ 674.3.2 Effect on Rheological Properties....................................................... 724.3.3 Other Effects ...................................................................................... 72

4.4 The Effect of Storage and Processing on the Mineral Componentsin Foods.......................................................................................................... 73

4.5 The Chemical Nature of Toxicity of Some Mineral Food Components ...... 744.5.1 Introduction ........................................................................................ 744.5.2 Aluminum........................................................................................... 764.5.3 Arsenic ............................................................................................... 794.5.4 Mercury .............................................................................................. 844.5.5 Cadmium ............................................................................................ 854.5.6 Lead.................................................................................................... 864.5.7 Interactions of Elements .................................................................... 88

References................................................................................................................ 90

4.1 THE CONTENTS AND ROLE OF MINERALS PRESENT IN FOODS

Minerals represent from 0.2% to 0.3% of the total intake of all nutrients in the diet.They are so potent and so important that without them the organism would not beable to utilize all the other food components. The main mass of these mineralsconstitute the

macroelements

, and the

trace elements

constitute less than one-hun-dredth of one percent of the total mass of daily eaten nutrients. Foods that are goodsources of some minerals are given in Table 4.1. Dietary minerals are necessary formaintenance of normal cellular metabolism and tissue function.

These nutrients participate in a multitude of biochemical and physiologicalprocesses important for health. Because of their broad biochemical activity, many


62


of these compounds are intentionally used as functional agents in a variety of foods.On the other hand, some cations may also induce a diversity of undesirable effectsthat influence the nutritional quality of foods. Minerals play an important role inplant life. They function as catalysts of biochemical reactions, are responsible forchanges in the state of cellular colloids, directly affect cell metabolism, and areinvolved in changes in protoplasm turgor and permeability. They often become thecenters of electrical and radioactive phenomena in living organisms. Minerals areusually grouped in two categories: the macroelements required in human diets inamounts greater than 100 mg, and the microelements required in milligram quantitiesor less. The macroelements include Ca, Mg, P, Na, K, and Cl. The microelementsare Fe, Zn, Cu, Mn, I, Co, Ni, Mo, Cr, F, Se, V, B, Si, and a few others whosebiological functions have not yet been fully recognized.

Actually, mineral deficiency states are more likely to occur than is vitamin insuf-ficiency. At increased risk of mineral deficiencies are people who eat low-calorie diets,the elderly, pregnant women, people using certain drugs such as diuretics, vegetarians,and those living in areas where soils are deficient in certain minerals. There is increas-ing evidence that those humans whose nutritional status is suboptimal in certain traceelements, such as Se, may be at greater risk for some forms of cancer and heart disease.Suboptimal intake can be due to soil depletion, the effects of acid rain, the overrefiningand overprocessing of foods, and other factors.

Minerals occur in foods in many chemical forms. They are absorbed from theintestines as simple cations, as part of an anionic group, or in covalent or noncovalentassociations with organic molecules. The chemical form of minerals in foods stronglyinfluences their intestinal handling and biological availability. Thus, iron in the formof hemoglobin in meats is more bioavailable than inorganic iron. This may also betrue for selenium in selenomethionine, and for the organic chelates of dietary chro-mium and zinc. Factors that affect mineral solubility or their reduction to a suitableform for cellular uptake, or those that influence the transfer through the mucosa ortransport into circulation, govern the rate and efficiency of uptake of the minerals.

TABLE 4.1The Contents of Selected Minerals in Some Foods

Mineral Range in foods (mg/100 g)

Calcium potato, 10; sardines in tomato sauce, 440; Swiss cheese, 960Potassium cheese, >100; wheat meal, 1.30; beef, liver, >300; wheat seeds, >500Magnesium milk, 9; sardines in tomato sauce, 27Zinc bee honey, fish, meat, 0.08–1.2; oysters, >100Iron egg white, 0.2; egg yolk, 7.2; pork, beef, >2; porcine liver, >20 Copper ham, salmon, tuna meat, 0.03–0.5; wheat germ, 0.9–7; oysters, 6–17Chromium kidney, liver, beef, <0.0015–0.004; cheese drowned, whole-meal bread, paprika,

pepper, curry, 0.01–0.05; spices, >0.1–0.5Fluoride fish and its products, 0.03–0.8; black teas, 3–34Iodide marine fish, oysters, shrimps, lobsters, 0.02–0.1; milk powder, 0.06Selenium milk, 0.002; salmon and tuna, canned, 0.08–0.12; potato and tortilla chips, 1.0


Mineral Components

63

For example, iron and zinc are much more bioavailable from human breast milkthan from cow’s milk or comparable infant formula. The intrinsic molecular asso-ciations of these minerals with low-molecular-weight binding compounds in humanmammary secretions are thought to convey this enhanced absorbability (Rosenbergand Solomons, 1984). Some minerals can produce chronic toxicity when absorbedand retained in excess of the body’s demands. Homeostatic mechanisms, oftenhormonally mediated, regulate the absorption of certain minerals and thereby protectagainst excessive accumulation. Recently developed speciation analysis makes itpossible to determine the forms of minerals that are present in food and in theenvironment that may cause specific physiological or pharmacological effects in thehuman organism.

4.2 INTERACTION WITH DIETARY COMPONENTS

4.2.1 I

NTRODUCTION

The interactions between minerals and between minerals and other substances inthe diet number in the hundreds. The term

interaction

is used to describe interrela-tionships between minerals and other nutrients present in the diet, and may be definedas the effect of one element (nutrient) on one or more other elements, and thus maycause some positive or negative biochemical or physiological consequences in theorganism. At the molecular level, such interactions occur at specific sites on proteinslike enzymes, receptors, or ion channels.

4.2.2 E

FFECT

ON

A

BSORPTION

Various nutritional and nonnutritional components of the diet, other nutrients invitamin and mineral supplements, contaminants, and also some medications caninteract with minerals in the gastrointestinal tract and influence their absorption. Forexample, amino acids may perform as intraluminar binders for some trace minerals.Large, complex, and poorly digestible proteins, on the other hand, may bind mineralstightly and diminish their absorption. Triacylglycerols and long-chain fatty acidsmay form soaps with calcium and magnesium and decrease the bioavailability ofthese two nutrients. There are clear indications that iron deficiency promotes cad-mium retention and may thus decrease the tolerance of high environmental or dietarycadmium levels. Evidence from a variety of experiments on animals suggests thatiron deficiency also promotes lead uptake and retention; the evidence for humans iscontroversial, but in some studies substantial increases in lead uptake have occurredwhen dietary iron and iron status were low.

Intestinal parasites, dietary fiber, phytates, and excessive sweating interfere withzinc absorption. Phytates, oxalates, and tannates can interfere with the absorptionof a number of minerals. Certain medications such as tetracycline can also inhibitabsorption of minerals, while others such as diiodoquin or dilantin may actuallypromote uptake of minerals. Apparently, chemically similar minerals share channelsfor absorption, and the simultaneous ingestion of two or more such minerals mightresult in competition for absorption.


64


Minerals like Fe, Mn, Co, Cu, Cr, Ni, and Zn generally function in the organismas cations complexed with organic ligands or chelators, such as proteins, porphyrins,flavines, and pterins. These transition metals have incompletely filled the 3-dimen-sional orbitals (the exception is zinc). One of the characteristics of these metals isthe ability to form complex ions. The electron configuration of metals at the valenceshell, in the elemental as well as in the cationic state, are as listed below:

As can be seen from the above arrangement, copper exists in either the monovalentor divalent ion, and the sum of its biological importance is related to its ability to oscillatebetween the cuprous (I) or cupric (II) state, having the ability to accept or to donateelectrons.

Among them, the divalent zinc (3d

10

) is not strictly a transition metal becauseits orbital d is filled, but its atomic structure is very similar to the metals discussed, andthus it is ready to form complexes analogous to those formed by the transition metalions.

Some other divalent cations like cadmium Cd

2+

(4d

10

), and lead Pb

2+

(6d

10

6s

2

),which are not transition metals, also have a pronounced tendency to form coordinatecovalent bounds with ligands that contain the electron donor atoms like, such as N, O,and S, which are extensively found in proteins. There is a degree of selectivity of metalions for electron donor atoms; copper prefers nitrogen, while zinc prefers sulfur, butseveral different donor atoms complex with each metal ion. The amino acid residues inproteins serve as rich sources of electron donor atoms; for example, the imidazole groupof histidine supplies nitrogen, the carboxyl groups of aspartic and glutamic acids supplyoxygen, and the thiol group of cysteine supplies sulfur for complexation.

The interaction between a metal atom and the ligands can be thought of as a Lewisacid-base reaction. Ligands as a Lewis base are capable of donating one or moreelectron pairs, and a transition metal atom (either in its positively charged state) actsas a Lewis acid accepting or sharing a pair of electrons from the Lewis bases. Thusthe metal-ligand bonds, sharing a pair of electrons, form coordinate covalent bond.

Interesting data of competitive interaction in biological systems exist betweenthe divalent cations such as zinc and cadmium, or zinc, copper, and iron. Zinc,cadmium, and mercury have similar tendencies to form complexes with a coordina-tion number of 4 and a tetrahedral disposition of ligands around the metal. Theyhave, as divalent cations, a very similar electronic structure to the monovalent cationof copper (3d

10

). The consequence of this is the possibility of isomorphous replace-ment among these elements in biological systems.

The essential microelements prefer coordination numbers of 4 or 6, and theycomplex with four or six ligands. Copper and zinc in the cationic form have filledtheir orbitals (they are d

10

ions) and both Cu

1+

and Zn

2+

form tetrahedral complexes(Cu

1+

may lose one of its d orbital electrons and become a d

9

ion (Cu

2+

), and thusform square planar complexes).

On the other hand, the ferrous d

6

ion (Fe

2+

) formsan octahedral configuration. It is possible to predict that Cu

1+

, having the same dorbital configuration as Zn

2+

and Cd

2+

, will interact with both of them, and that Zn

2+

will be antagonistic to Cu

1+

rather than to Cu

2+

.

Element Cr Mn Fe Co Ni Cu ZnElemental state 3d

5

4s

1

3d

5

4s

2

3d

6

4s

2

3d

7

4s

2

3d

8

4s

2

3d

10

4s

1

3d

10

4s

2

Divalent cationic state 3d

4

3d

5

3d

6

3d

7

3d

8

3d

9

3d

10


Mineral Components

65

It has been demonstrated in systematic studies of the interactions between Cd,Zn, Cu, and Fe, in chicks, rats, and mice, that an increased dietary intake of cadmiumcan cause increased mortality, poor growth, and hypochromic microcytic anemia.The growth rate could be restored by zinc supplementation, and the mortality andseverity of anemia signs could be reduced by copper supplementation of the diet.Zinc also restores the activity of heart cytochrome oxidase.

Cu prevents the degeneration of aortic elastin

.

Diets low in calcium promotesignificant increases in the absorption and retention of lead and cadmium in ratorganisms, and have a marked effect on all pathologic changes ascribed to theirtoxicity. High calcium and high phytate in diets appear to restrict lead uptake. Lowintake of phosphorus has a similar effect on Pb retention, like low intake of Ca,and the effects of Ca and P deficiency are additive (Bremner, 1974; WHO, 1996).Other dietary components such as lactose have been implicated in the enhancedabsorption of calcium from milk. Pectins, cellulose, hemicellulose, and polymersproduced by Maillard reactions during cooking, processing, or storage may bindminerals in the lumen and thus reduce their biological availability. Interactionbetween and among minerals, or with anionic species, are important determinantsof mineral absorption. Absorption of iron is hindered by fiber and phosphates andpromoted by ascorbic acid, copper, and meat protein. Ascorbic acid also enhancesthe absorption of selenium but reduces the absorption of copper. High protein intakeappears to increase the excretion of calcium, whereas vitamin D ingestion promotesthe retention of calcium.

When imbalances that are not physiological among competitive nutrients existas a result of leaching from water pipes, storage in unlacquered tin cans, or improperformulation of vitamin and mineral supplements, nutritionally important conse-quences of mineral–mineral interaction can result. To participate in a nutritionallyrelevant process for the organisms as a whole, a mineral must be transported awayfrom the intestines. The concentration of circulating binding proteins, and the degreeof saturation of their metallic binding sites, may influence the rate and magnitudeof transport of recently absorbed minerals (Rosenberg and Solomons, 1984).

Minerals require a suitable mucosal surface across which to enter the body.Resection or diversion of a large portion of the small bowel obviously affects mineralabsorption. Extensive mucosal damage due to mesenteric infarction or inflammatorybowel disease or major diversion by jejunoileal bypass procedures reduces theavailable surface area. Minerals such as copper or iron, whose absorption primarilyoccurs in the proximal intestine, are affected differently from those absorbed moredistally, e.g., zinc. Furthermore, the integrity of the epithelium, the uptake of fluidsand electrolytes, the intracellular protein synthesis, energy-dependent pumps, andhormone receptors must be intact. Intrinsic diseases of the small intestinal mucosamay impair mineral absorption. Conditions such as celiac sprue, dermatitis herpeti-formis, infiltrative lymphomas, and occasionally inflammatory bowel disease pro-duce diffuse mucosal damage. Protein energy malnutrition causes similar damage,and tropical enteropathy affects part of the population of developing countries livingunder adverse nutritional and hygienic conditions. Absorption of most metals fromthe gastrointestinal tract is variable (see Table 4.2), and depends on many externaland internal factors. Thus the quantity of metal ingested rarely reflects that which


66


is bioavailable. In fact, under most circumstances, only a small fraction of ingestedmetals are absorbed, while the great majority passes out of the gut in the feces.

The Recommended Dietary Allowances (RDA) represent standards of nutritionset by the National Research Council, Food and Nutrition Board (1989). It containsthe levels of essential nutrients that are adequate to meet the nutritional needs of thenormal healthy population. Individuals may differ in their precise nutritional require-ments. To take into account these differences among normal persons, the RDAprovides a margin of safety, that is, it sets the allowances high enough to cover the

TABLE 4.2Mean Daily Intake and Recommended Dietary Allowances (RDAs)

1

or Safe and Adequate Intake (SAI)

2

(as well as percentage of absorption of minerals from the gastrointestinal tract according to published data by U.S. National Academy Press, 1989).

Milligrams per adult person Percentageof absorptionMineral Mean daily intake RDA

1

or SAI

2

Macroelements

Calcium 960–1220 800–1200

1

10–50Chloride 1700–5100 7502

2

high

a

Magnesium 145–358 280–350

1

20–60Phosphorus 1670–2130 800–1200

1

high

a

Potassium 3300 2000

2

high

a

Sodium 3000–7000 500

2

high

a

Microelements

Chromium < 0.15 0.05–0.20

2

< 1 or 10–25 in form of GTF

b

Cobalt 0.003–0.012 0.002

c1

30–50Copper 2.4 1.5–3.02 25–60Fluorine < 1.4 1.5–42 high

a

Iodine < 1.0 0.15

1

100Iron 15 10–15

1

10–40Zinc 12; 18 12–15

1

30–70Manganese 5.6; 8 2–3

2

40Molybdenum > 0.15 0.075–0.250

2

70–90Nickel 0.16–0.20 0.05; 0.3 < 10Selenium 0.06–0.22 0.055–0.070 ~ 70Vanadium 0.012–0.030 0.01–0.025 < 1

Microelements recently considered as essential

Boron 1–3 1–2 high

a

Silicon 21–46; 200 21–46 3; 40

a

More than 40%

b

GTF (glucose tolerance factor)

c

0.002 mg of cobalt containing vitamin B

12


Mineral Components

67

needs of most healthy people. For additional nutrients that are necessary to keep thebody in good health (for which the RDA has not yet been established) there areestimated

safe and adequate daily intakes

(SAI).The functions of minerals in the body involve building tissue and regulating

numerous body processes. Their role in the human body is summarized in Table 4.3.

4.2.3 B

UILDING

B

ODY

T

ISSUE

AND

R

EGULATING

B

ODY

P

ROCESSES

Certain minerals, including calcium, phosphorus, magnesium, and fluorine, are com-ponents of bones and teeth. Deficiencies in children cause growth to be stunted andbone tissue to be of poor quality. A continual adequate intake of minerals is essentialfor the maintenance of skeletal tissue in adulthood. Potassium, sulfur, phosphorus,iron, and many other minerals are the structural components of soft tissue (Solomons,1984; Eshleman, 1984).

Minerals are an integral part of many hormones, enzymes, and other compoundsthat regulate biochemical functions in the human organism. For example, iodine isrequired to produce the hormone thyroxine, chromium is involved in the productionof insulin, and iron is present in hemoglobin, myoglobin, and the cytochromes.Hence, the production of these substances in the organism depends on adequateintake of the involved minerals. Minerals can also act as catalysts. Calcium, forexample, is a catalyst in blood clotting. Some minerals are catalysts in the absorptionof nutrients from the gastrointestinal tract in the metabolism of proteins, fat, andcarbohydrates, and in the utilization of nutrients by the cell.

Minerals dissolved in the body fluids are responsible for nerve impulses and thecontraction of muscles, as well as for water and acid-base balance. They playimportant roles in maintaining the respiration, heart rate, and blood pressure withinnormal limits. A deficiency of minerals in the diet may lead to severe, chronic,clinical signs of disease, frequently reversible after supplementation of the diet withthe respective elements or following total parenteral nutrition. Their influence onbiochemical reactions in living systems also makes it possible to use them inten-tionally in many food processes.

4.3 ROLE IN FOOD PROCESSES

4.3.1 E

FFECT

ON

O

XIDATION

Because minerals are an integral part of many enzymes, they also play an importantrole in food processing, for example, in alcoholic and lactic fermentation, meataging, and in dairy food production. Many compounds used as food additives or forrheological modification of some foods, also contain metallic cations. A number ofthese compounds function as antimicrobials, sequestrants, antioxidants, flavorenhancers, and buffering agents, and sometimes even as dietary supplements.

Some heavy metal ions actively catalyze lipid oxidation. Their presence, evenin trace amounts, has long been recognized as potentially detrimental to the shelflife of fats, oils, and fatty foods. They can activate molecular oxygen by producingsuperoxide, which then, through dismutation and other biochemical changes, turns


68


TAB

LE 4

.3Th

e B

iolo

gica

l R

ole

of S

ome

Min

eral

s

Min

eral

Func

tion

Defi

cien

cySo

urce

s

Mac

roel

emen

ts

Cal

cium

Bon

e an

d to

oth

form

atio

n, b

lood

clo

tting

, ce

ll pe

rmea

bilit

y, n

erve

stim

ulat

ion,

mus

cle

cont

ract

ion,

en

zym

e ac

tivat

ion

Stun

ted

grow

th,

rick

ets,

ost

eom

alac

ia,

oste

opor

osis

, te

tany

Milk

, ha

rd c

hees

e, s

alm

on a

nd s

mal

l fi

sh

eate

n w

ith b

ones

, so

me

dark

gre

en

vege

tabl

es,

legu

mes

Mag

nesi

umC

ompo

nent

of

bone

s an

d te

eth,

act

ivat

ion

of m

any

enzy

mes

, ne

rve

stim

ulat

ion,

mus

cle

cont

ract

ion

Seen

in a

lcoh

olis

m o

r re

nal d

isea

se, t

rem

ors

lead

ing

to c

onvu

lsiv

e se

izur

esG

reen

lea

fy v

eget

able

s, n

uts,

who

le g

rain

s,

mea

t, m

ilk,

seaf

ood

Phos

phor

usB

one

and

toot

h fo

rmat

ion,

ene

rgy

met

abol

ism

, co

mpo

nent

of

AT

P an

d A

DP,

pro

tein

syn

thes

is

com

pone

nt o

f D

NA

and

RN

A, f

at tr

ansp

ort,

acid

–bas

e ba

lanc

e, e

nzym

e fo

rmat

ion

Stun

ted

grow

th,

rick

ets

Milk

, mea

ts, p

oultr

y, fi

sh, e

ggs,

che

ese,

nut

s,

legu

mes

, w

hole

gra

ins

Pota

ssiu

mO

smot

ic p

ress

ure,

wat

er b

alan

ce,

acid

–bas

e ba

lanc

e,

nerv

e st

imul

atio

n, m

uscl

e co

ntra

ctio

n, s

ynth

esis

of

prot

ein,

gly

coge

n fo

rmat

ion

Nau

sea,

vom

iting

, mus

cula

r wea

knes

s, r

apid

he

art

beat

, he

art

failu

reM

eats

, fi

sh,

poul

try,

who

le g

rain

s, f

ruits

, ve

geta

bles

, le

gum

es

Sodi

umO

smot

ic p

ress

ure,

wat

er b

alan

ce,

acid

–bas

e ba

lanc

e,

nerv

e st

imul

atio

n, m

uscl

e co

ntra

ctio

n, c

ell

perm

eabi

lity

Rar

e: n

ause

a, v

omiti

ng,

gidd

ines

s,

exha

ustio

n, c

ram

psTa

ble

salt,

sal

ted

food

s, M

SG a

nd o

ther

so

dium

add

itive

s, m

ilk, m

eat,

fish

, pou

ltry,

eg

gs,

brea

d

Mic

roel

emen

ts

Chr

omiu

mT

riva

lent

chr

omiu

m i

ncre

ases

glu

cose

tol

eran

ce a

nd

play

s ro

le in

lipi

d m

etab

olis

m; u

sefu

l in

prev

entio

n an

d tr

eatm

ent

of d

iabe

tes;

hex

aval

ent

chro

miu

m i

s to

xic

Impa

ired

gro

wth

, gl

ucos

e in

tole

ranc

e,

elev

ated

blo

od c

hole

ster

olW

hole

-gra

in c

erea

ls,

cond

imen

ts,

mea

t pr

oduc

ts,

chee

ses,

and

bre

wer

’s y

east

Cob

alt

Cof

acto

r of

vita

min

B

12

pla

ys a

rol

e in

im

mun

ityR

arel

y ob

serv

ed;

if e

xist

s, a

per

nici

ous

anem

ia w

ith h

emat

olog

ical

and

neu

ralg

ic

man

ifes

tatio

ns m

ay b

e ob

serv

ed d

ue t

o vi

tam

in B

12

defi

cien

cy

Org

an m

eats

(liv

er,

kidn

ey),

fish

, da

iry

prod

ucts

, eg

gs


Mineral Components

69

Cop

per

Nec

essa

ry f

or i

ron

utili

zatio

n an

d he

mog

lobi

n fo

rmat

ion,

con

stitu

ent

of c

ytoc

hrom

e ox

idas

e, a

nd

invo

lved

in

bone

and

ela

stic

tis

sue

deve

lopm

ent

Ane

mia

, ne

utro

peni

a, l

euco

peni

a, s

kele

tal

dem

iner

aliz

atio

nL

iver

, kid

ney,

oys

ters

, nut

s, f

ruits

, and

dri

ed

legu

mes

Iron

Hem

oglo

bin

and

myo

glob

in f

orm

atio

n, e

ssen

tial

com

pone

nt o

f m

any

enzy

mes

Ane

mia

, de

crea

se i

n ox

ygen

tra

nspo

rt a

nd

cellu

lar

imm

unity

, m

uscl

e w

eakn

ess

Liv

er, l

ean

mea

ts, l

egum

es, d

ried

frui

ts, g

reen

le

afy

vege

tabl

es, w

hole

-gra

in a

nd f

ortifi

ed

cere

als

Man

gane

seC

ofac

tor

of a

lar

ge n

umbe

r of

enz

ymes

, in

the

agi

ng

proc

ess

has

a ro

le a

s an

ant

ioxi

dant

(M

n-su

pero

xide

di

smut

ase)

, im

port

ant

for

norm

al b

rain

fun

ctio

n, f

or

repr

oduc

tion,

and

for

bon

e st

ruct

ure

In a

nim

als:

cho

ndro

dyst

roph

y, a

bnor

mal

bo

ne d

evel

opm

ent,

repr

oduc

tive

diffi

culti

es;

in h

uman

s: s

hort

age

of e

vide

nce

Tea,

who

le g

rain

s, n

uts.

Fru

its a

nd g

reen

ve

geta

bles

; org

an m

eat a

nd s

hellfi

sh c

onta

in

very

abs

orba

ble

man

gane

se

Mol

ybde

num

Cof

acto

r of

the

enzy

mes

: xan

tine

and

alde

hyde

oxi

dase

. co

pper

ant

agon

ist

Red

uces

con

vers

ion

of h

ypox

anth

ine

and

xant

hine

to u

ric ac

id re

sulti

ng in

the d

evel

opm

ent

of x

anth

ine

rena

l ca

lcul

i; de

fici

ency

sta

te

may

be

pote

ntia

ted

by h

igh

copp

er i

ntak

e

Gra

in,

legu

mes

Zin

cC

onst

ituen

t of

man

y en

zym

e sy

stem

s, c

arbo

n di

oxid

e tr

ansp

ort;

vita

min

A u

tiliz

atio

nD

elay

ed w

ound

hea

ling,

im

pair

ed t

aste

se

nsiti

vity

, re

tard

ed g

row

th a

nd s

exua

l de

velo

pmen

t, dw

arfis

m

Oys

ters

, fish

, mea

t, liv

er, m

ilk, w

hole

gra

ins,

nu

ts,

legu

mes

Fluo

ride

Res

ista

nce

to d

enta

l de

cay

Toot

h de

cay

in y

oung

chi

ldre

nD

rink

ing

wat

er r

ich

in fl

uori

de,

seaf

ood,

tea

Iodi

neSy

nthe

sis

of t

hyro

id h

orm

one

that

reg

ulat

e ba

sal

met

abol

ic r

ate

Goi

ter,

cret

inis

m,

if d

efici

ency

is

seve

re.

Iodi

zed

salt,

sea

food

, foo

d gr

own

near

the

sea

Sele

nium

Prot

ects

aga

inst

a n

umbe

r of

can

cers

C

atar

act,

mus

cula

r dy

stro

phy,

gro

wth

de

pres

sion

, liv

er c

irrh

osis

, inf

ertil

ity, c

ance

r, ag

ing

due

to d

efici

ency

of

sele

nogl

utat

hion

e pe

roxi

dase

; ins

uffic

ienc

y of

cel

lula

r im

mun

ity

Bro

ccol

i, m

ushr

oom

s, r

adis

hes,

cab

bage

, ce

lery

, on

ions

, fi

sh,

orga

n m

eats

Bor

on

a

Prev

ents

ost

eopo

rosi

s in

pos

tmen

opau

sal

wom

en,

bene

ficia

l in

tre

atm

ent

of a

rthr

itis,

bui

lds

mus

cle

Prob

ably

im

pair

s gr

owth

and

dev

elop

men

tFo

ods

of p

lant

ori

gin

and

vege

tabl

es

a

Rec

ently

con

side

red

esse

ntia

l; st

ill h

as t

o be

pro

ven.

Sour

ces

: E

schl

eman

M.M

.,

Intr

oduc

tory

Nut

riti

on a

nd D

iet

The

rapy

,

J.B

. L

ippi

ncot

t C

o.,

Lon

don,

198

4; H

endl

er S

.S.,

The

Doc

tor’

s Vi

tam

in a

nd M

iner

al E

ncyc

lope

dia,

Sim

on a

nd S

chus

ter,

New

Yor

k, 1

990.


70


into hydroxyl free radicals. Three cations are involved in the activity of superoxidedismutase (SOD). This enzyme has been patented as an antioxidant agent for foods.Three types of metalloenzymes of SOD exist in living organisms, namely Cu Zn-SOD, Fe-SOD, and Mn-SOD. All three types of SODs catalyze the dismutation ofsuperoxide anions to produce hydrogen peroxide

in vivo

. There is evidence ofincreased lipid oxidation in apples during senescence. SOD activity may also beinvolved in reactions to damage induced by oxygen, free radicals, and ionizingradiation, and could help protect cells from damage by peroxidation products (Duand Bramlage, 1994).

Besides SOD, catalase, ceruloplasmin, albumin and appotransferrin, and chelatingagents ethylenediaminetetraacetic acid (EDTA), bathocupreine, cysteine, and purineare capable of inhibiting the oxidation of ascorbic acid induced by trace metals. Copper-induced lipid oxidation in ascorbic acid–pretreated cooked ground fish may be inhibitedin the presence of natural polyphenolic compounds, the flavonoids, which are effectiveantioxidants and prevent the production of free radicals (Ramanathan and Das, 1993).In the presence of adenosine diphosphate-chelated (ADP) iron and traces of copper,oxygen radicals are generated in the sarcoplasmic reticulum of muscle food. Musclecontains notable amounts of iron, a known prooxidant, and trace amounts of copperalso catalyzing a peroxidative reaction (Hultin, 1994; Wu and Brewer, 1994). Ironoccurs associated with heme compounds and as nonheme iron complexed to proteinsof low molecular weight. Reactive nonheme iron can be obtained through release ofiron from heme pigments or from the iron storage protein, ferritin. Iron is part of theactive site of lipoxygenase, which may participate in lipid oxidation. Reducing com-ponents of tissue such as superoxide anion, ascorbate, and thiols, can convert inactiveferric iron into active ferrous iron. There are also enzymic systems that use reducingequivalents from NADPH (nicotinamide adenine dinucleotide phosphate), to reduceferric iron. A number of cellular components are capable of reducing ferric to ferrousiron, but under most conditions the two major reductants are superoxide and ascorbate(Hultin, 1994). In some cases the reduction of ferric iron can also be accomplishedenzymically utilizing electrons from NADH (reduced nicotinamide adenine dinucle-otide), and to a lesser extent, NADPH (reduced nicotinamide adenine dinucleotidephosphate) by the enzymic system associated with both the sarcoplasmic reticulumand mitochondria. Ferrous iron can activate molecular oxygen by producing superox-ide. Superoxide may then undergo dismutation spontaneously or through the action ofSOD, and produce hydrogen peroxide, which can interact with another atom of ferrousiron to produce the hydroxyl radical. The hydroxyl radical can initiate lipid oxidation.It is generally accepted that ferrous iron is the reactive form of iron in an oxidationreaction. Because it is likely that most iron ordinarily exists in the cell as ferric iron,the ability to reduce ferric to ferrous iron is thus critical.

Development of rancidity and warmed-over flavor—a specific defect that occursin cooked, reheated meat products following short-term refrigerated storage—hasbeen directly linked to autooxidation of highly unsaturated, membrane-bound phos-pholipids and to the catalytic properties of nonheme iron (Oelinngrath and Slinde,1988; Pearson et al., 1977; Hultin, 1994).

Dietary iron may influence muscle iron stores and thus theoretically may alsoaffect lipid oxidation in muscle food such as pork. There appears to be a threshold


Mineral Components 71

for the dietary iron level (between 130 to 210 ppm), above which the muscle andliver nonheme iron and total iron, and muscle thiobarbituric acid reactive sub-stances begin to increase because of porcine muscle lipid oxidation (Miller et al.,1994a, b).

The secondary oxidation products, mainly aldehydes, are the major contributorsto warmed-over flavor and meat flavor deterioration because of their high reactivityand low flavor thresholds. Ketones and alcohols have high flavor thresholds and arelesser causes of flavor deterioration. Exogenous antioxidants can preserve the qualityof meat products. Radical scavengers appear to be the most effective inhibitors ofmeat flavor deterioration. However, different substrates and systems respond indifferent ways. Active ferrous iron may be eliminated physically by chelation withEDTA or phosphates, or chemically by oxidation, to its inactive ferric form.

Ferroxidases are enzymes that oxidize ferrous iron to ferric iron in the presenceof oxygen:

4Fe2+ + O2 + 4H+ → 4Fe3+ + 2H2O

Ceruloplasmin, a copper protein of blood serum, is a ferroxidase. Oxidation offerrous iron to ferric iron tends to be favored in extracellular fluids, while chelationis more likely intracellular (Hultin, 1994).

Sodium chloride has long been used for food preservation. Salt alters both thearoma and the taste of food. The addition of sodium chloride to blended cod muscleaccelerates the development of rancidity. This salt-induced rancidity is inhibited bychelating agents such as EDTA, sodium oxalate, and sodium citrate, and by nordi-hydroguaiaretic acid and propyl gallate. Although sodium chloride and other metalsalts act as prooxidants, they have a strong inhibiting effect on Cu2+-induced rancidityin the fish muscle. The most effective concentration of NaCl for this antioxidanteffect is between 1% and 8% (Castell et al., 1965, Castell and Spears 1968). Theseauthors also showed that other heavy metal ions were effective in producing ranciditywhen added to various fish muscles. The relative effectiveness was the following,in decreasing order: Fe2+ > V2+ > Cu2+ > Fe3+ > Cd2+ > Co2+ > Zn2+, while Ni2+, Ce2+,Cr3+, and Mn2+ had no effect in the concentrations used. Of those tested, Fe2+, V2+,and Cu2+ were by far the most active catalysts. There were, however, importantexceptions. The comparative effectiveness of the metallic ions was not the same formuscle taken from all the species that were tested.

EDTA is reported to be effective as a metal ion sequestrant and is approved foruse in the food industry as a stabilizer and antioxidant. It acts also as an inhibitorof Staphylococcus aureus by forming stable chelates in the media with mutivalentcations, which are essential for cell growth. The effect is largely bacteriostatic andeasily reversed by releasing the complexed cations with other cations for whichEDTA has a higher affinity (Kraniak and Shelef, 1988).

The addition of phosphate-, pyro-, tripoly-, and hexametaphosphate also protectscooked meat from autoxidation, but ortophosphate gives no protection. The mech-anism by which phosphates prevent autooxidation appears to be related to theirability to sequester metal ions, particularly ferrous iron, which are the major proox-



idants (Pearson et al., 1977). The addition of NaCl increases retention of moisturein meat and meat products.

4.3.2 EFFECT ON RHEOLOGICAL PROPERTIES

The interaction between metal ions and polysaccharides often affects the rheologicaland functional properties in food systems.

In aqueous media, neutral polysaccharides have little affinity for alkali metal andalkali earth metal ions. On the other hand, anionic polysaccharides have a strong affinityfor metal counterions. This association is related to the linear charge density of thepolyanions. The linear charge density is expressed as the distance between the per-pendicular projections of an adjacent charged group on the main axis of the molecule.The higher the linear charge density, the stronger the interaction of counterions withthe anionic groups of the molecule. Such anionic hydrocolloids (0.1% solution) asalginate, karaya, arabic, and ghati have high calcium-binding affinities (Ha et al., 1989).An important functional property of alginates is their capacity to form gels with calciumions. This makes alginates extensively suited to preparing products such as fruit andmeat analogs. They are also widely used in biotechnology as immobilization agentsof cells and enzymes. The method involves diffusion of calcium ions through alginateand a cross-linking reaction with the alginate carboxylic group to form the gel (Ha etal., 1989; World Health Organization [WHO], 1993a, 1993b). Carrageenans arereported to stabilize casein and several plant proteins against precipitation with calcium,and are used to prepare texturized milk products (Samant et al., 1993).

4.3.3 OTHER EFFECTS

Sodium reduction in the diet is recommended as means of preventing hypertensionand subsequent cardiovascular disease, stroke, and renal failure. Reducing or sub-stituting NaCl requires an understanding of the effects caused by the new factorsintroduced. Several ways are proposed to reduce the sodium content in processedmeat without an adverse effect on the quality, flavor, gelation, and shelf life of theproducts. This includes a slight sodium chloride reduction, replacing some of theNaCl with another chloride salt (KCl, MgCl2) or nonchloride salt or altering theprocessing methods (Barbut and Mittal, 1985).

The calcium ion is a known activator of many biochemical processes. Calpainplays an important role in postmortem tenderizing of meat. The function of the metalion in this enzyme is believed to be either neutralizaton of the charges on the surface,by preventing electrostatic repulsion of subunits, or effecting a conformationalchange required for association of the subunits. Thus the metal ions must be presentin a specific state to perform this function.

The metallic cation in solution exists as an aqua-complex ion in equilibriumwith their respective hydroxy-complex:

⇔M(H2O)m+

aqua-complex ionMOH(m–1)+ H+

hydroxy-complex(weak base)



The acid ionization constant (pKa) of the aqua-complex ion determineswhether the ion will form complexes with a protein. This depends greatly on thepH of the medium. Because the ionization constant of low charge is 12.6, theywill form a stable complex only with negatively charged protein in alkaline media.They cannot bind to cationic proteins as they do not share electrons to form acovalent bond. These considerations explain why the activity of calpain is optimumin the alkaline pH range. Thus a decrease in its activity at acidic pH values maypartly be due to a change in the electronic state of Ca2+ (Asghar and Bhatti, 1987;Barbut and Mittal, 1985).

Generally, sodium and potassium react only to a limited extent with proteins,whereas calcium and magnesium are somewhat more reactive. The transition metals,for example, ions of Cu, Fe, Hg, and Ag, react readily with proteins, many formingstable complexes with thiol groups. Ca2+, Fe2+, and Cu2+, as well as Mg2+ cationsmay be an integral part of certain protein molecules or molecular associations. Theirremoval by dialysis or by sequestrants appreciably lowers the stability of the proteinstructure with respect to heat and proteases.

4.4 THE EFFECT OF STORAGE AND PROCESSINGON THE MINERAL COMPONENTS IN FOODS

The effect of normal storage on mineral components is rather low, and may beconnected mainly with changes of humidity or contamination. However, majorchanges of mineral components may occur during canning, cooking, drying, freezing,peeling, and all the other steps involved in preserving and food processing for directconsumption.

The highest losses of minerals are encountered in the milling and polishingprocess of cereals and groats. All milled cereals undergo a significant reduction ofnutrients. The extent of the loss is governed by the efficiency with which theendosperm of the seed is separated from the outer seed coat bran, and the germ. Theloss of certain minerals and vitamins is deemed so relevant to health that in manycountries supplementation was introduced for food products to enrich them with thelost nutrients (i.e., iron to bread). In some countries regulations have been issuedconcerning standards for enriched bread. If the bread is labeled “enriched,” it mustmeet these standards. In white flours, the losses of magnesium and manganese mayreach up to 90%. These minerals remain mainly in the bran—the outer part of thegrain. For this reason it seems reasonable to recommend consumption of bread bakedfrom whole meals instead of from white meals. Although recommended, sometimessteady consumption of bran alone, for dietary purposes, should be done with greatcare because it may also contain many different contaminants, like toxic metals andorganic pesticides.

During preparation for cooking or for canning, vegetables should be thoroughlywashed before cutting to remove dirt and traces of insecticide spray. Root vege-tables should be scrubbed. The dark outer leaves of greens are rich in iron, calcium,and vitamins, so they should be trimmed sparingly. Peeling vegetables and fruitshould be avoided whenever possible because minerals and vitamins are frequently



concentrated just beneath the skin. Potatoes should be baked or cooked in their skins,even for hashed browns or potato salad. However, True et al. (1979) showed thatcooking potatoes by boiling whole or peeled tubers, as well as microwave cookingand oven baking may have a negligible effect on the losses of Al, B, Ca, Na, K, Mg,P, Fe, Zn, Cu, Mn, Mo, J, and Se. Microwaved potatoes retain nutrients well, andcontrary to popular belief, peeling potatoes does not strip away their vitamin C andminerals. Whenever practical, any remaining cooking liquid should be served withthe vegetable or used in a sauce, gravy, or soup. To retain minerals in cannedvegetables, the liquid from the can should be poured into a saucepan and heated ata low temperature to reduce the amount of liquid, then added to the vegetables andheated before serving. Low temperatures reduce shrinkage and loss of many othernutrients. Cooking and blanching leads to the most important nutrient losses.

Cooking of vegetables results in leaching 30 to 65% of potassium, 15 to 70%of magnesium and copper, and 20 to over 40% of zinc. Thus it is reasonable to usethis liquid for soup preparation. A study of changes in the cadmium content of riceduring the polishing process showed only a slight decrease (~3%) of the concen-tration of the metal. The effects of cooking (including washing and soaking inwater) showed a slight decrease (average 5%) of the cadmium content of rice. Theeffect of milling on wheat showed that the cadmium content in flours is about halfthat present in the grain. The concentration of cadmium in bran is about twice thatpresent in whole-wheat grain, while in tofu, miso, and soy sauce it is about 65%,80%, and 50%, respectively, of that in unprocessed soybeans. The losses dependboth on the kind of food cooked and the course of the applied process. Steamblanching of vegetables generally results in smaller losses of nutrients becauseleaching is minimized. Frozen meat and vegetables thawed at ambient temperaturelose many nutrients including minerals in the thaw drip. Frozen fruits should beeaten without delay, just after thawing, and together with the secreted juice. Foodsblanched, cooked, or reheated in a microwave oven generally retain about the sameor even a higher amount of nutrients as those cooked by conventional methods(WHO, 2004).

4.5 THE CHEMICAL NATURE OF TOXICITY OF SOME MINERAL FOOD COMPONENTS

4.5.1 INTRODUCTION

A diet consisting of a variety of foods provides the best protection against potentiallyharmful chemicals in food. This is because the body tolerates very small quantitiesof many toxic substances, but has only a limited ability to cope with large quantitiesof any single one. Almost any chemical can have a harmful effect if taken in a largequantity. This is especially true for trace minerals, and to some degree, also formacroelements, as well as vitamins. For this reason, it is important to understandthe difference between toxicity and hazard. Many foods contain toxic chemicals, butthese chemicals do not present a hazard if consumed in allowable amounts.

A number of minerals can produce chronic toxicity when absorbed and retainedin excess of the body’s demands. The proportion of elements accumulated by the



organism is different from the proportion in the environment, and this results in theirconcentration within the organism. Some of the elements are necessary to theorganism for metabolic processes; others, however, which are accumulated in highproportions (sometimes specifically in some organs), do not have any metabolicsignificance for the organism (e.g., aluminum, arsenic, cadmium, mercury, and lead)and are recognized as toxic.

Compounds of aluminum, arsenic, cadmium, lead, and mercury contaminate theenvironment and enter the food supply. However, among them, aluminum com-pounds are less toxic, and some of them are even used as food additives, (i.e., breadleavening, firming, emulsifying agents), and also are used in drugs (e.g., antacids),and as such all these compounds enter the gastrointestinal tract and add to thealuminum compounds naturally present in foods.

Proteins, in addition to other compounds, are the most common naturally occur-ring chelators playing an important role in the transport, metabolism, detoxification,and retention processes in organisms exposed to higher levels of toxic metals.Particularly metallothioneins (Mts), a family of inducible cysteine-rich proteinsubiquitous in mammals, are capable of binding and storing a number of divalent andtrivalent cations including Zn, Cu, Cd, and other transitional metals. These proteinsare composed of about 60 amino acids of which one-third are cysteine. Theirbiosynthesis in organisms is induced by the above-mentioned metals. The highestconcentrations of Mts are found in the kidneys, liver, and intestines. Each moleculeof Mt is able to bind up to seven atoms of cadmium.

Besides contributing to possible cellular defense mechanisms by sequesteringpotentially toxic metals, Mts are also important in overall homeostasis (WHO, 2004).Zinc metallothionein (Zn-Mt) can detoxify free radicals. Cadmium-induced Mt isable to bind cadmium intracellularly, thus protecting the organism against the toxicityof this metal.

Mts occur in the organism in at least four genetic variants. The two major forms(I and II) are ubiquitous in most organs, particularly in the liver and kidney, andalso in the brain. Mt isolated from adult or fetal human livers contained mainly Znand Cu, whereas that from human kidney contained Zn, Cu, and Cd. The metals arebound to the peptide by mercaptide bonds and arranged in two distinct clusters: afour-metal cluster called the α domain and a three-metal cluster called the β clusterat the C terminal of the protein. The cluster is an obligate Zn cluster, whereas theZn in a β cluster may be replaced by Cu or by Cd. Interaction with Mt is the basisfor metabolic interaction between these metals.

Metallothionein III is found in the human brain and differs from I and II byhaving six glutamic acid residues near the terminal part of the protein. Mt III isthought to be a growth inhibitory factor, and its expression is not regulated by metals;however, it does bind Zn. Another proposed role for Mt III is participating in theutilization of Zn as neuromodulator because it is present in the neurons that storeZn in their vesicles.

Metallothionein IV occurs during differentiation of stratified squamous epithe-lium, but it is known to have a role in the absorption or toxicity of cadmium. Mt inthe gastrointestinal mucosa might play a role in the gastrointestinal transport of Cd.Its presence in cells of the placenta impairs the transport of Cd from maternal to



fetal blood and across the blood–brain barrier, but only when the concentration ofCd is low.

In general, toxicity of metals is a function of their physical and chemical prop-erties, their doses, and the health conditions of the organism exposed to these metals.It is also a function of the ability of the metals to accumulate in body tissues. Forthis reason, it is very important to have adequate information about their chemicalstructure. Currently this may be done by applying speciation analysis, which makesit possible to differentiate the chemical form of the examined element and betterassess the safety level of its residue in foods or drinking water.

4.5.2 ALUMINUM

Aluminum was in the past considered a completely indifferent substance. However,it has been discovered that the risk of Al is greatly increased in persons withimpaired kidney function. Alfrey et al. (1976, 1980) (in WHO, 1989), have shownthat dialysis encephalopathy in a large number of patients with renal failure (under-going chronic dialysis) is attributable to high Al content of some water used forthe preparation of dialysates. The patients on dialysis, who died of a neurologicsyndrome of unknown cause (dialysis encephalopathy syndrome), had brain matterAl concentrations of 25 mg/kg dry weight, while in controls 2.2 mg/kg was mea-sured. But whether the metal has a causative role in the pathogenesis of thesediseases still remains unconfirmed.

The concentration of aluminum in human tissues from different geographicregions was found to be widely varied and probably reflected the geochemicalenvironment of individuals and locally grown food products. In healthy humantissues, the Al concentration was usually below 0.5 µ/g wet weight, but higher levelswere observed in the liver (2.6 µg/g), lung (18.2 µg/g), lymph nodes (32.5 µg/g),and bone (73.4 µg/g of ash) (WHO, 1989).

In plants, the negative effects of Al have been demonstrated especially on celldivision and uptake, as well as on the metabolism of other favorable elements suchas Mn, Mg, and P. However, in animals, encountered in natural amounts, aluminumhas been reported to also have several positive physiological effects. It seems to beinvolved in the reactions between cytochrome c and succinyl dehydrogenase, as wellas a cofactor necessary for activation of guanine nucleotide binding, which is impor-tant in protein metabolism. It has also been shown to play an important role in thedevelopment of potent immune response and in endogenous triacylglycerol metab-olism. On the other hand, excess aluminum in patients with renal failure and dialysisencephalopathy may lead to skeletal defects, such as markedly reduced bone forma-tion, resulting in osteomalacia. A further pathological manifestation of Al toxicityis a microcytic hypochromic anemia not associated with iron deficiency. Such prob-lems have practically disappeared since the use of Al-free deionized water for dialysisbecame routine.

Goats given low Al-semisynthetic ratios (162 µg/kg) for 4 years had significantlyreduced life expectancy as compared with that of control goats receiving 25 mg/kg.In studies with mice fed diets containing bread with Al additives, a decreased numberof offspring and ovarian lesions were seen. In another study with rats maintained



on diets containing a mixture of sodium aluminum sulfate and calcium acid phos-phate at different dietary levels (bred for 7 successive generations) there were nonegative effects on birth weight, average weaning weight, and number of weanedanimals. Histopathological examination of kidneys of surviving rats did not revealany significant changes.

Long-term medication of humans with antacids (aluminum hydroxide) mayprovide several grams of Al per day (WHO, 1996). This may result in inhibition ofintestinal absorption and decreased plasma concentration of phosphorus and as aconsequence, an increase in calcium loss. This effect is probably due to the bindingof dietary phosphorus in the intestine by the aluminum, and was not observed whenphosphorus-containing Al salt was used or when the interfering anion and aluminumcompound were taken separately (WHO, 1989).

Aluminum cations, similar to calcium and magnesium cations, are able to formpoorly soluble compounds with fluoride. Thus higher intake of Al by patients usingantacids may also reduce, to some extent, fluoride absorption. There are some datathat Al and fluoride in animal studies (with ruminants and poultry) are mutuallyantagonistic in competing for absorption in the gut, and thus higher intake of Alcompounds in some circumstances may prevent fluorosis. Conversely, feeding ani-mals with higher levels of fluoride in drinking water may prevent deposition ofaluminum in the brain. But still there is no sufficient evidence to conclude thatconsumption of drinking water containing a high but nontoxic level of fluorides willhave a preventive effect on Alzheimer’s disease (Kraus and Forbes, 1992; Schenkeland Klüber, 1987).

Depending on the type of environment, geographical factors, and industrializa-tion, there can be considerable variations in levels of Al in cultivated and naturallygrowing plants, and consequently in animal fodder and human foods. In addition tothe natural origin, some quantities (sometimes even high) of Al in the diets may befrom food containing Al additives as well as from contact of food with aluminumcontainers, cookware, utensils, and food wrappings.

Daily dietary aluminum intakes vary in different countries, but according to datapublished in a 1989 WHO report, ranged from about 2 to 6 mg/day for children andto about 14 mg/day for teenagers and adults. The same levels of intake are given byPennington (1987) who compiled the data on the basis of the Total Diet Study resultsof teenagers and adults realized in 1984. In the same paper there are results fromother countries, ranging from very low to more than 30 mg of Al per person perday. It seems that intake of Al of approximately 2 to 7 mg/day may be ingested bythose consuming diets low in herbs, spices, and tea, diets that do not contain foodwith Al additives, and foods prepared with little contact with aluminum containers,cookware, utensils, or wrappings.

In the normal daily hospital diet, mean daily intake of Al was about 21.3 ± 12.4mg/person, (Nabrzyski and Gajewska, 1995), while in very special hospital diets (inthe Pennington report, 1987), the intake was markedly lower and ranged from 1.8to 7.33 mg/day.

Certain plants were able to absorb high levels of Al, such as black tea (445.0 to1552.0 mg/kg), while herbal or fruit teas contained about 45 mg/kg and herbal teas,



which were partially supplemented with black teas, had up to 538.0 mg/kg(Nabrzyski and Gajewska, 1995; Fairweather-Tait et al., 1987).

The major food sources of Al in daily diets are grain products, which contributefrom 36.5 to 69.4%; the next are milk, yogurt, and cheese, contributing from 11.0to 36.5%; the Al contribution of all other products was only several percent and inmost cases even less than 1% (Pennington, 1987). In wild mushrooms and cultivatedAgaricus bisporus, Müller et al. (1997) found a low level of Al (14 ± 7 mg/kg ofdry matter). The most popular species, such as Boletus and Xerocomus, were lowin Al (30 to 50 mg/kg dry matter). However, several other species of the genusSuillus, Macrolepiota rhacodes, Hyoholoma capnoides, as well as individual samplesof Russula ochroleuca and Amanita rubescens contained high Al concentrations ofabout 100 mg/kg dry matter. It was concluded that none of the investigated speciesof mushrooms contributes significantly to the daily intake of aluminum by humans.

In the edible parts of different seafoods such as fish, crustaceans, and molluscanshellfish, the Al level was very diversified. In the fillets of lean and fatty fish it wasbelow 0.2 mg/kg wet weight, with the exception of the Al concentration in fillets offish caught in coastal waters near a smelting plant, in which up to 1 mg/kg wetweight was found.

The edible parts of crustacean and molluscan shellfish contained up to 5 mg/kgwet weight of Al. A comparison between fillets and different organs of cod showedhigher Al concentration in organs, especially in gills, that are in continuous contactwith the ambient water (above 0.6 mg/kg wet weight), and in the brain and heart(above 0.4 mg/kg wet weight).

In line with the tolerable daily intake by the body of 1 mg Al/kg, the contributionof aluminum from the edible parts of aquatic food does not play a significant rolein daily diets (Ranau et al., 2001). It is suggested that the additional daily intake ofAl resulting from preparing all foods in uncoated Al pans is approximately 3.5 mg.Nowadays, however, most pans are made of stainless steel or Teflon®-coated alumi-num, which diminishes migration of the metal into foods, and the average contribu-tion of Al to the daily diet from the use of such utensils may be less than 0.1 mg.An additional intake of less than 0.1 mg daily could result from the occasionalconsumption of acid fruits and vegetables, assuming that less than 10% of thesefood items are prepared in aluminum pans (Müller et al., 1993).

The quantities of the metal that migrate into foods depends on the acidity of thefood items and can markedly increase when acidic beverages are stored or heatedin aluminum cans. Generally, the quantities of Al that contribute to the daily dietfrom such adventitious sources are rather inconsistent and insignificant.

Several of the intentional Al-containing additives used as stabilizers, buffers, anti-caking and neutralizing agents, dough strengtheners, leavening agents, acid-reactingingredients in self-rising flour or cornmeal curing agents, components in bleaching,and texturizer agents are given in Table 4.4.

An example may be the acidic form of sodium aluminum phosphate that reactswith sodium bicarbonate to cause leavening action. It is used in biscuit, pancake,waffle, cake, doughnut, and muffin mixes, frozen rolls and yeast doughs, cannedbiscuits, and self-rising flours. Sodium aluminum sulfate is also an acidifying agentfound in many household baking powders. The basic form of sodium aluminum



phosphate is used in processed cheese and cheese foods as an emulsifying agent togive cheese products a soft texture and to allow easy melting. Sodium aluminumphosphate is also used as a meat binding agent.

A slice of cake or bread made with baking powder that contains aluminum maycontain 5 to 15 mg of Al. Recently, most commercial baking powders, and somethat are sold for household use, contain monocalcium phosphate rather than alumi-num salts. Aluminum salts used as firming agents in pickled vegetables and somepickled fruits are now replaced by calcium oxide in both industrial and homepickling, although several brands of commercially packed pickles still may containammonium or potassium sulfate. Aluminum silicates are found in anticaking agents,salt, nondairy creamers, and other dry, powdered products.

A selected committee of the Life Sciences Research Office of the Federation ofAmerican Societies for Experimental Biology reviewed the safety of GRAS (gen-erally recognized as safe) substances including aluminum compounds under contractwith the U.S. Food and Drug Administration (FDA) and estimated that the averagedaily intake of emulsifying agents in processed cheese, firming agents, processingaids, stabilizers, and thickeners, containing Al compounds added to foods was about20 mg, and that about 75% of this was in the form of sodium aluminum phosphate(Pennington, 1987).

A low total body burden of Al, coupled with urinary excretion, suggests thateven at high levels of consumption, thanks to regulation by homeostatic mechanisms,only small amounts—about 1% of the normally consumed dose—is absorbed by ahealthy person from the gastrointestinal tract, and is then excreted by healthy kidneys,so that no accumulation occurs (except in patients with renal failure).

In conclusion, there is no known risk to healthy people from the typical dietaryintake of aluminum. Risk arises only from the habitual consumption of gram quan-tities of antacids containing Al over a long period of time, and they rise substantiallyin persons with impaired kidney function.

4.5.3 ARSENIC

Pentavalent and trivalent arsenicals react with biological ligands in different ways.The trivalent form reacts with the thiol protein groups, resulting in enzyme inacti-vation, structural damage, and a number of functional alterations. The pentavalentarsenicals, however, do not react with –SH groups.

Arsenate can competitively inhibit phosphate insertion into the nucleotide chainsof DNA of cultured human lymphocytes, causing false formation of DNA because ofthe instability of the arsenate esters. Dark repair mechanisms are also inhibited, leadingto persistence of these errors in the DNA molecules. Binding difference of the trivalentand pentavalent forms leads to the differences in accumulation of this element. Trivalentinorganic As is accumulated in higher levels than the pentavalent form. The organicarsenic compounds are considered less toxic or nontoxic in comparison to inorganicarsenic of which the trivalent arsenicals are the most toxic forms.

Dietary As represents the major source of arsenic for most of the general pop-ulation. Consumers eating large quantities of fish usually ingest significant amountsof As, primarily as organic compounds, especially those with a structure similar to



TAB

LE 4

.4Li

st o

f Se

lect

ed M

iner

al C

ompo

unds

Use

d as

Foo

d A

ddit

ives

Che

mic

al n

ame

of c

ompo

und

and

(IN

S)Sy

nony

ms

or o

ther

che

mic

al n

ame

Func

tion

al c

lass

and

com

men

tsA

DI,

TA

DI,

PM

TDI

(mg/

kg b

ody

wei

ght)

Cal

cium

alg

inat

e (4

04)

Cal

cium

alg

inat

eT

hick

enin

g ag

ent,

stab

ilize

rA

DI

not

spec

ified

Cal

cium

asc

orba

te (

302)

Cal

cium

asc

orba

te d

ihyd

rate

Ant

ioxi

dant

AD

I no

t sp

ecifi

edC

alci

um b

enzo

ate

(213

)M

onoc

alci

um b

enzo

ate

Ant

imic

robi

al,

pres

erva

tive

AD

I 0–

5.0

Cal

cium

chl

orid

e (5

09)

Cal

cium

chl

orid

eFi

rmin

g ag

ent

AD

I no

t sp

ecifi

edC

alci

um c

itrat

e (3

33)

Tri

calc

ium

citr

ate,

tri

calc

ium

sal

t of

bet

a hy

drox

ytri

carb

ally

lic a

cid

Aci

dity

, re

gula

tor,

firm

ing

agen

t, se

ques

tran

tA

DI

not

spec

ified

Cal

cium

dih

ydro

gen

phos

phat

e (3

41i)

Cal

cium

dih

ydro

gen

tetr

aoxo

phos

phat

e.

Mon

abas

ic c

alci

um p

hosp

hate

. M

onoc

alci

um p

hosp

hate

Buf

fer,

firm

ing,

rais

ing,

leav

enin

g an

d te

xtur

ing,

ag

ent,

and

in f

erm

enta

tion

proc

esse

sM

TD

I 70

,0

Cal

cium

dis

odiu

m e

thy-

le

nedi

amin

etet

raac

etac

e (3

85)

Cal

cium

dis

odiu

m E

DTA

Ant

ioxi

dant

, pr

eser

vativ

e se

ques

tran

tA

DI

0–2.

5

Cal

cium

glu

tam

ate

(623

)M

onoc

alci

um D

I-L

-glu

tam

ate

Flav

or e

nhan

cer,

salt

subs

titut

eA

DI

not

spec

ified

Cal

cium

hyd

roxi

de (

526)

Slak

ed l

ime

Neu

tral

izin

g ag

ent,

buff

er,

firm

ing

agen

tA

DI

not

limite

dC

alci

um h

ydro

gen

carb

onat

e (1

70ii)

Cal

cium

hyd

roge

n ca

rbon

ate

Surf

ace

colo

rant

, an

ticak

ing

agen

t, st

abili

zer

AD

I no

t sp

ecifi

edC

alci

um l

acta

te (

327)

Cal

cium

dila

ctat

e hy

drat

eB

uffe

r, do

ugh

cond

ition

erC

alci

um s

orba

te (

203)

Cal

cium

sor

bate

Ant

imic

robi

al,

fung

ista

tic,

pres

erva

ti ve

agen

tA

DI

0–25

.0 (

as s

um o

f ca

lciu

m,

pota

ssiu

m a

nd s

odiu

m s

alt)

Mag

nesi

um c

hlor

ide

(511

)M

agne

sium

chl

orid

e he

xahy

drat

eFi

rmin

g, c

olor

-ret

entio

n ag

ent

AD

I no

t sp

ecifi

edM

agne

sium

car

bona

te (

504i

)M

agne

sium

car

bona

teA

ntic

akin

g an

d an

tible

achi

ng a

gent

AD

I 0–

5.0

Mag

nesi

um g

luco

nate

(58

0)

Mag

nesi

um g

luco

nate

dih

ydra

teB

uffe

ring

, fi

rmin

g ag

ent

yeas

t fo

od.

AD

I no

t sp

ecifi

edM

agne

sium

glu

tam

ate

DI-

L-

(625

)M

agne

sium

glu

tam

ate

Flav

or e

nhan

cer,

salt

subs

titut

eA

DI

not

spec

ified

(gr

oup

AD

I fo

r α

glut

amic

aci

d an

d its

m

onos

odiu

m,

pota

ssiu

m,

calc

ium

, m

agne

sium

and

am

mon

ium

sal

ts)


Mineral Components 81M

agne

sium

hyd

roge

nph

osph

ate

(343

ii)

Mag

nesi

um h

ydro

gen

orto

phos

phat

e tr

ihyd

rate

, di

mag

nesi

um p

hosp

hate

Die

tary

sup

plem

ent

MT

DI

70 (

expr

esse

d as

ph

osph

orus

fro

m a

ll so

urce

s)M

agne

sium

hyd

roxi

de (

528)

Mag

nesi

um h

ydro

xide

Alk

ali,

colo

r ad

junc

tA

DI

not

limite

dM

agne

sium

hyd

roxi

de c

arbo

nate

(50

4ii)

Mag

nesi

um c

arbo

nate

hyd

roxi

de h

ydra

ted

Alk

ali,

antic

akin

g, c

olor

ret

entio

n, c

arri

er,

dryi

ng a

gent

AD

I no

t sp

ecifi

ed

Mag

nesi

um l

acta

te D

,L-

also

m

agne

sium

lac

tate

L (

329)

Mag

nesi

um D

I-D

,L-l

acta

teB

uffe

ring

age

nt,

doug

h co

nditi

oner

, di

etar

y su

pple

men

tA

DI

not

limite

d

Mag

nesi

um o

xide

(53

0)

Mag

nesi

um o

xide

Ant

icak

ing,

neu

tral

izin

g ag

ent

AD

I no

t lim

ited

Mag

nesi

um s

ulfa

te (

518)

M

agne

sium

sul

fate

Firm

ing

agen

t A

DI

not

spec

ified

Pota

ssiu

m a

ceta

te (

261)

Pota

ssiu

m a

ceta

teA

ntim

icro

bial

, pr

eser

vativ

e, b

uffe

r A

DI n

ot s

peci

fied

(als

o in

clud

es

the

free

aci

d)Po

tass

ium

alg

inat

e (4

02)

Pota

ssiu

m a

lgin

ate

Thi

cken

ing

agen

t, st

abili

zer

AD

I no

t sp

ecifi

ed (

grou

p A

DI

for

algi

nic

acid

and

its

am

mon

ium

, ca

lciu

m a

nd

sodi

um s

alts

)Po

tass

ium

alu

min

osili

cate

(55

5)Po

tass

ium

alu

min

osili

cate

Ant

icak

ing

agen

tN

o A

DI

allo

cate

dPo

tass

ium

asc

orba

te (

303)

Pota

ssiu

m a

scor

bate

Ant

ioxi

dant

AD

I no

t sp

ecifi

ed (

Gro

up A

DI

for

asco

rbic

aci

d an

d its

so

dium

, po

tass

ium

, an

d ca

lciu

m s

alts

)Po

tass

ium

ben

zoat

e (2

12)

Pota

ssiu

m b

enzo

ate

Ant

imic

robi

al,

pres

erva

tive

AD

I 0–5

.0 (e

xpre

ssed

as b

enzo

ic

acid

)Po

tass

ium

bro

mat

e (9

24a)

Pota

ssiu

m b

rom

ate

Oxi

dizi

ng a

gent

AD

I w

ithdr

awn

Pota

ssiu

m c

arbo

nate

(50

1i)

Pota

ssiu

m c

arbo

nate

Alk

ali,

flav

orA

DI

not

spec

ified

Pota

ssiu

m c

hlor

ide

(508

)Po

tass

ium

chl

orid

e, s

ylvi

ne,

sylv

iteSe

ason

ing

and

gelli

ng a

gent

, sa

lt su

bstit

ute

AD

I no

t sp

ecifi

ed (

grou

p A

DI

for

hydr

ochl

oric

aci

d an

d its

m

agne

sium

, po

tass

ium

and

am

mon

ium

sal

ts)

Pota

ssiu

m o

r so

dium

cop

per

chlo

roph

yllin

(14

1ii)

Pota

ssiu

m o

r so

dium

chl

orop

hylli

nC

olor

of

porp

hyri

nA

DI

0–15

Pota

ssiu

m d

ihyd

roge

n ph

osph

ate

(340

i)M

onop

otas

sium

dih

ydro

gen

orto

phos

phat

e,

mon

obas

ic p

otas

sium

pho

spha

teB

uffe

r, se

ques

tran

t, ne

utra

lizin

g ag

ent

MT

DI

70.0

(con

tinu

ed)



TAB

LE 4

.4 (

cont

inue

d)Li

st o

f Se

lect

ed M

iner

al C

ompo

unds

Use

d as

Foo

d A

ddit

ives

Che

mic

al n

ame

of c

ompo

und

and

(IN

S)Sy

nony

ms

or o

ther

che

mic

al n

ame

Func

tion

al c

lass

and

com

men

tsA

DI,

TA

DI,

PM

TDI

(mg/

kg b

ody

wei

ght)

Pota

ssiu

m h

ydro

gen

carb

onat

e (5

01ii)

Pota

ssiu

m b

icar

bona

teA

lkal

i, le

aven

ing

agen

t, bu

ffer

AD

I no

t sp

ecifi

edPo

tass

ium

hyd

roge

n su

lfite

(22

8)Po

tass

ium

hyd

roge

n su

lfite

Pres

erva

tive,

ant

ioxi

dant

AD

I 0–0

.7 (G

roup

AD

I for

sul

fur

diox

ide

and

sulfi

tes,

exp

ress

ed a

s su

lfur

dio

xide

, cov

erin

g so

dium

an

d po

tass

ium

met

abis

ulfi

te,

pota

ssiu

m a

nd s

odiu

m h

ydro

gen

sulfi

te a

nd s

odiu

m th

iosu

lfat

e)Po

tass

ium

glu

tam

ate

(622

)L

-Mon

opot

assi

um L

-glu

tam

ate

Flav

or e

nhan

cer,

salt

subs

titut

eA

DI

not

spec

ified

So

dium

alg

inat

e (4

01)

Sodi

um a

lgin

ate

Thi

cken

ing

agen

t, st

abili

zer

AD

I no

t sp

ecifi

edSo

dium

alu

min

ium

pho

spha

te a

cidi

c (5

41i)

Salp

, sod

ium

tria

lum

iniu

m te

trad

ecah

ydro

gen

octa

phos

phat

e te

trah

ydra

te (

A),

tri

sodi

um

dial

umin

ium

pen

tade

cahy

drog

en

octa

phos

phat

e (B

)

Rai

sing

age

ntA

DI

0–0.

6

Sodi

um a

lum

inum

pho

spha

te b

asic

(5

41ii)

Kas

al,

auto

geno

us m

ixtu

re o

f an

alk

alin

e so

dium

alu

min

um p

hosp

hate

Em

ulsi

fier

AD

I 0–

0.6

Sodi

um a

scor

bate

(30

1)So

dium

L-a

scor

bate

Ant

ioxi

dant

AD

I no

t sp

ecifi

edSo

dium

ben

zoat

e (2

11)

Sodi

um s

alt

of b

enze

neca

rbox

ylic

aci

dA

ntim

icro

bial

, pr

eser

vati v

eA

DI

0–5.

0So

dium

dih

ydro

gen

phos

phat

e (3

39i)

Mon

osod

ium

dih

ydro

gen

mon

opho

spha

te

(ort

hoph

osph

ate)

Buf

fer,

neut

raliz

ing

agen

t, se

ques

tran

t in

ch

eese

, m

ilk,

fish

, an

d m

eat

prod

ucts

MT

DI

70.0

Dis

odiu

m e

thyl

ened

iam

inet

etra

acet

ate

(386

)D

isod

ium

ED

TA,

diso

dium

ede

tate

Ant

ioxi

dant

, se

ques

tran

t, pr

eser

vativ

e,

syne

rgis

tA

DI 0

–2.5

(as

calc

ium

dis

odiu

m

ED

TA)

Sodi

um g

luta

mat

e (6

21)

Mon

osod

ium

L-g

luta

mat

e (M

SG),

glu

tam

ic

acid

mon

osod

ium

sal

t m

onoh

ydra

teFl

avor

enh

ance

rA

DI

not

spec

ified

Sodi

um i

ron

III-

ethy

lene

diam

ine

tetr

aace

tate

trih

ydra

teFe

rric

sod

ium

ede

teat

e, s

odiu

m i

ron

ED

TA

, so

dium

fer

edet

ate

Nut

rien

t sup

plem

ent (

prov

isio

nally

con

side

red

to b

e sa

fe in

foo

d fo

rtifi

catio

n pr

ogra

ms)

AD

I ac

cept

able



Sodi

um o

r po

tass

ium

met

abis

ulfi

te(2

23,

224)

Dis

odiu

m o

r po

tass

ium

pen

taox

odis

ulfa

teA

ntim

icro

bial

, pre

serv

ativ

e, b

leac

hing

age

nt,

antib

row

ning

age

ntA

DI

0–0.

7 (G

roup

AD

I fo

r su

lfur

dio

xide

and

sul

fite

s ex

pres

sed

as S

O2,

cov

erin

g so

dium

and

pot

assi

um s

alt)

Sodi

um n

itrite

(25

0)So

dium

nitr

iteA

ntim

icro

bial

, co

lor

fixa

tive

AD

I 0–

0.1

Sodi

um n

itrat

e (2

51)

Sodi

um n

itrat

e, c

ubic

or

soda

nitr

e, c

hile

sa

lpet

reA

ntim

icro

bial

, co

lor

fixa

tive

AD

I 0–

5.0

Sodi

um p

hosp

hate

(33

9iii)

Tri

sodi

um p

hosp

hate

, tri

sodi

um m

ono-

phos

phat

e, o

rtop

hosp

hate

, sod

ium

pho

spha

te.

Sequ

estr

ant,

emul

sion

sta

biliz

er,

buff

erM

TD

I 70

.0

Sodi

um o

r po

tass

ium

sor

bate

(20

1, 2

02)

Sodi

um o

r po

tass

ium

sor

bate

Ant

imic

robi

al,

fung

ista

tic a

gent

AD

I 0–

25.0

Not

es: I

NS:

Int

erna

tiona

l num

beri

ng s

yste

m (

pare

nthe

tical

val

ues)

, pre

pare

d by

the

Cod

ex C

omm

ittee

for

Foo

d A

dditi

ves

for

the

purp

ose

of p

rovi

ding

an

agre

ed-u

pon

inte

rnat

iona

lnu

mer

ical

sys

tem

for

ide

ntif

ying

foo

d ad

ditiv

es i

n in

gred

ient

lis

ts,

as a

n al

tern

ativ

e to

the

dec

lara

tion

of t

he s

peci

fic n

ame

( Cod

ex A

lim

enta

rius

, vo

l. 1,

2nd

ed.

, FA

O/W

HO

,G

enev

a, 1

992,

Sec

tion

5.1)

.

AD

I: A

ccep

tabl

e D

aily

Int

ake,

an

estim

ate

of t

he a

mou

nt o

f a

subs

tanc

e in

foo

d or

dri

nkin

g w

ater

tha

t ca

n be

ing

este

d da

ily o

ver

a lif

etim

e w

ithou

t ap

prec

iabl

e ri

sk t

o he

alth

.V

alue

s ar

e ex

pres

sed

on a

bod

y-w

eigh

t ba

sis

for

a st

anda

rd h

uman

wei

ghin

g 60

kg.

AD

I no

t sp

ecifi

ed o

r A

DI

not

limite

d: t

erm

s ap

plic

able

to

a fo

od s

ubst

ance

of

very

low

tox

icity

whi

ch,

on t

he b

asis

of

the

avai

labl

e da

ta (

chem

ical

, bi

oche

mic

al,

toxi

colo

gica

l,an

d ot

her)

, as

wel

l as

the

tot

al d

ieta

ry i

ntak

e of

the

sub

stan

ce a

risi

ng f

rom

its

use

at

leve

ls t

o ac

hiev

e th

e de

sire

d ef

fect

and

fro

m i

ts a

ccep

tabl

e ba

ckgr

ound

in

food

doe

s no

t, in

the

opin

ion

of t

he J

oint

FA

O/W

HO

Exp

ert

Com

mitt

ee o

n Fo

od A

dditi

ves

(JE

CFA

), r

epre

sent

a h

azar

d to

hea

lth. F

or t

hat

reas

on, a

nd f

or r

easo

ns s

tate

d in

ind

ivid

ual

eval

uatio

ns,

the

esta

blis

hmen

t of

AD

I in

num

eric

al f

orm

is

not

deem

ed n

eces

sary

. An

addi

tive

mee

ting

this

cri

teri

on m

ust

be u

sed

with

in t

he b

ound

of

good

man

ufac

turi

ng p

ract

ice,

tha

t is

,it

shou

ld b

e te

chno

logi

cally

effi

caci

ous

and

shou

ld b

e us

ed a

t th

e lo

wes

t le

vel

nece

ssar

y to

ach

ieve

thi

s ef

fect

, it

shou

ld n

ot c

once

al i

nfer

ior

food

qua

lity

or a

dulte

ratio

n, a

nd i

tsh

ould

not

cre

ate

a nu

triti

onal

im

bala

nce.

TAD

I: T

empo

rary

AD

I: t

erm

est

ablis

hed

by t

he J

EC

FA f

or s

ubst

ance

s fo

r w

hich

tox

icol

ogic

al d

ata

are

suffi

cien

t to

con

clud

e th

at u

se o

f th

e su

bsta

nce

is s

afe

over

the

rel

ativ

ely

shor

t pe

riod

of

time

requ

ired

to

eval

uate

fur

ther

saf

ety

data

, b u

t ar

e in

suffi

cien

t to

con

clud

e th

at u

se o

f th

e su

bsta

nce

is s

afe

over

a l

ifet

ime.

A h

ighe

r-th

an-n

orm

al s

afet

y fa

ctor

is u

sed

whe

n es

tabl

ishi

ng a

TA

DI,

and

an

expi

ratio

n da

te i

s es

tabl

ishe

d by

whi

ch t

ime

appr

opri

ate

data

to

reso

lve

the

safe

ty i

ssue

sho

uld

be s

ubm

itted

to

JEC

FA.

MT

DI:

Max

imum

Tol

erab

le D

aily

Int

ake

(or

Prov

isio

nal

Max

imum

Tol

erab

le D

aily

Int

ake

[PM

TD

I]),

is a

ter

m u

sed

to d

escr

ibe

the

endp

oint

of

cont

amin

ants

with

no

cum

ulat

ive

prop

ertie

s. I

ts v

alue

rep

rese

nts

perm

issi

ble

hum

an e

xpos

ure

as a

res

ult

of t

he n

atur

al o

ccur

renc

e of

the

sub

stan

ce i

n fo

od o

r d r

inki

ng w

ater

. In

the

case

of

trac

e el

emen

ts t

hat

are

both

esse

ntia

l nut

rien

ts a

nd u

navo

idab

le c

onst

ituen

ts o

f fo

od, a

ran

ge is

exp

ress

ed; t

he lo

wer

val

ue r

epre

sent

s th

e le

vel o

f es

sent

i alit

y an

d th

e up

per

valu

e th

e PM

TD

I.

Sour

ce:

WH

O (

Wor

ld H

ealth

Org

aniz

atio

n),

Food

Add

itive

s Se

ries

, 35

, Tox

icol

ogic

al e

valu

atio

n of

cer

tain

foo

d ad

ditiv

es a

nd c

onta

min

ants

, pa

per

pres

ente

d at

44t

h M

eetin

g of

the

Join

t FA

O/W

HO

Exp

ert

Com

mitt

ee o

n Fo

od A

dditi

ves,

Gen

eva,

199

4, 1

996.



arsenobetaine and arsenocholine, as well as various other arsenic derivates. Fish ofmany species contain arsenic between 1 to 10 mg/kg. Arsenic levels at or above 100mg/kg have been found in bottom-feeders and shellfish. Both, lipid and water-solubleorganoarsenic compounds have been found, but the water-soluble forms, mainly thequartenary arsonium derivates, constitute the larger portion of the total arsenic inmarine animals (Vaessen and van Ooik, 1989; WHO, 1989).

Studies in mice have demonstrated that over 90% of arsenobetaine and arseno-choline were absorbed and about 98% of the administered dose of arsenobetainewas excreted unchanged in the urine, whereas 66% and 9% of single oral doses ofarsenocholine was excreted in the urine and feces, respectively, within 3 days. Themajority of arsenocholine was oxidized in animal organisms to arsenobetaine, andexcreted in this form in the urine. The retention of arsenocholine in the animal bodywas greater than the retention of arsenobetaine. The fate of organic arsenicals inman still has not been fully clarified.

The minimal available information on the organoarsenicals present in fish andother seafood indicates that these compounds appear to be readily excretedunchanged in the urine, with most of the excretion occurring within two days ofingestion.Volunteers who consumed flounder excreted 75% of the ingested arsenicunchanged in urine within 8 days after eating the fish. Less than 0.35% was excretedin the feces. There are no data on tissue distribution of arsenic in humans followingingestion of arsenic present in fish and seafood. Also there have been no reports ofill effects among ethnic populations consuming large quantities of fish resulting inorganoarsenic intakes of about 0.05 mg/kg body weight per day (WHO, 1989).Inorganic tri- and pentavalent arsenicals are metabolized in man, dog, and cow toless toxic methylated forms such as monomethylarsonic and dimethylarsinic acids(Peoples, 1983).

4.5.4 MERCURY

Organic mercury compounds, especially methylmercury, are recognized as moredangerous for man than the inorganic ones.

Most foods (except fish) contain very low amounts of total mercury (<0.01mg/kg), which is almost entirely in the form of inorganic compounds. Over 90% ofmercury in fish and shellfish appears as methylmercury. This is because fish feedon aquatic organisms that contain this compound, originating from microorganismsthat contain biomethylated inorganic mercury. Marlin is the only pelagic fish knownto have more than 80% of the total muscle mercury present as inorganic mercury(Cappon and Smith, 1982). The amount of methylmercury is especially high in large,old fish of predatory species like shark and swordfish. In fish of freshwater species,the mercury content depends on the concentration of mercury in the water andsediment, and on the pH of the water. The concentration of methylmercury in mostfish is generally less than 0.4 mg/kg, although predators such as swordfish, shark,and pike may contain up to several milligrams of methylmercury per kg in themuscle. The intake of methylmercury depends on fish consumption and the concen-tration of methylmercury in the fish consumed. Many people eat about 20 to 30 g



of fish per day, but certain groups eat 400 to 500 g per day. Thus the daily dietaryintake of methylmercury can range from about 0.2 to 3 to 4 µg/kg body mass.

Studies of the kinetics of methylmercury after ingestion show that its distributionin the tissues is more homogenous than that of other mercury compounds, with theexception of elemental mercury. The most important features of the distributionpattern of methylmercury are high blood concentration, a high ratio of erythrocyte-to-plasma concentration (about 20), and high deposition in the brain. Another impor-tant characteristic is slow demethylation, which is a critical detoxification step.Methylmercury and other mercury compounds have a strong affinity for sulfur andselenium. Although selenium has been suggested as providing protection against thetoxic effects of methylmercury, no such effect has been demonstrated.

Various effects have been observed in animals treated with toxic doses, but someof these such as renal damage and anorexia, have not been observed in humansexposed to high doses. The primary tissues of concern in humans are the nervoussystem and particularly the developing brain, and this has been the main reason forthe wide range of epidemiological studies. Neurotoxicological effects induced bymethylmercury in nonhuman primates resemble those seen in humans and supportthe concept that the developing fetus is more sensitive than an adult. Methylmercuryis readily absorbed (up to 95%) after oral exposure, and passes about 10 times morereadily through the placenta and blood–brain barrier, resulting in higher concentra-tions of this compound in the brain of the fetus than of the mother. It is eliminatedfrom the organism mainly via the bile and feces. Neonatal animals have a lowerexcretory capacity than adults.

The dermal absorption of methylmercury is similar to that of inorganic mercurysalts. The LD50 values after oral administration are 25 mg/kg body weight in oldrats, and 40 mg/kg in young rats. Clearance half-life of methylmercury for the wholehuman body is about 74 days, and for blood is 52 days (WHO, 2000).

4.5.5 CADMIUM

Cadmium shares chemical properties with zinc and mercury, but in contrast tomercury, it is incapable of environmental methylation, due to the instability of themonoalkyl derivate. Similarities and differences also exist in the metabolism of Zn,Cd, and Hg. Cadmium retention in body tissues is related to the formation of Cd-metallothionein, a Cd-protein complex of low molecular mass. Metallothionein (Mt)in the gastrointestinal (GI) mucosa plays a role in the GI transport of Cd. Its presencein cells of the placenta impairs the transport of Cd from maternal to fetal blood andacross blood–brain barriers, but only when the concentration of Cd is low. Newbornsare virtually cadmium free, whereas zinc and copper are readily supplied to the fetus.Rapid renal concentration occurs mainly during the early years of life. In plasma,Cd is transported as a complex with Mt, and may be toxic to the kidneys whenexcreted in the glomerular filtrate. Most Cd in urine is bound to Mt. Cadmium boundto Mt in food does not appear to be absorbed or distributed in the same way as itsinorganic compounds. Low dietary concentrations of calcium promote absorption ofCd from the intestinal tract in experimental animals. A low iron status in laboratoryanimals and humans has also been shown to result in greater absorption of Cd. In



particular, women with low body-iron stores, as reflected by low serum ferritinconcentrations, had an average GI absorption rate twice as high as that of a controlgroup of women having about 5%. High iron levels result in decreasing total andfractional Cd accumulation from diets, whereas low iron levels promote accumula-tion of Cd. Studies in rats with reduced iron levels showed that the inclusion ofwheat bran-containing phytate hindered absorption of iron, calcium, and other min-erals in their diets, but increased the uptake of Cd.

After a single dose of cadmium chloride, the LD50 value for rats and mice treatedorally ranges from about 100 to 3000 mg/kg body mass.

The high affinity of Cd for –SH groups and the ability of imparting moderatecovalency in bonds result in increased lipid solubility, bioaccumulation, and toxicity.In humans with normal levels of exposure, about 50% of the body burden is foundin the kidneys, about 15% in the liver, and about 20% in the muscles. The proportionof Cd in the kidney decreases as liver concentration increases. The lowest concen-trations of Cd are found in brain, bone, and fat. Accumulation in the kidney continuesup to 50 to 60 years of age in humans and falls thereafter, possibly due to age-relatedchanges in the functional integrity of the kidney. In contrast, Cd levels in the musclecontinue to increase over the course of life. The average concentration in the renalcortex of nonoccupationally exposed persons aged 50 varies between 11 and 100mg/kg in different regions.

The diet is the major route of human exposure to cadmium. Contamination offoods with Cd results from its presence in soil and water. The concentration ofcadmium in foods ranges widely; the highest average concentrations were found inmollusks, kidney, liver, cereals, cocoa, and leafy vegetables. A daily intake of about60 µg would be required to reach a concentration of 50 mg/kg in the renal cortexat the age of 50, assuming an absorption rate of 5%. About 10% of the absorbeddaily dose is rapidly excreted (WHO, 1989, 2001).

4.5.6 LEAD

The Joint FAO/WHO Expert Committee on Food Additives (JECFA) and other WHOcommittees have recognized that infants and children are the groups at highest risk toPb exposure from food and drinking water. As an anthropogenic contaminant, Pb findsits way into the air, water, and surface soil. Pb-containing products also contribute tothe Pb body burden. The domestic environment, in which infants and children spendthe greater part of their time, is of particular importance as a source of Pb intake. Inaddition to exposure from general environmental sources, some infants and youngchildren, as a result of normal, typical behavior, can receive high doses of Pb throughmouthing or swallowing of nonfood items. Pica, the habitual ingestion of nonfoodsubstances, which occurs among many young children, has frequently been implicatedin the etiology of Pb toxicity. In the United States, on average, 2-year-old childrenmay receive about 45% of their daily Pb intake from dust, 40% from food, 15% fromwater and beverages, and 1% from inhaled air (WHO, 1986).

Pb absorption is heavily influenced by food intake, much higher rates occurringafter fasting than when Pb is ingested with a meal. This effect may be due mainly



to competition from other ions, particularly iron and calcium, for intestinal transportpathways. Absorption is also affected by age; the typical absorption rates in adultsand infants are 5 to 10% and about 50%, respectively. Children absorb Pb from thediet with greater efficiency than do adults (WHO, 2000).

After absorption and distribution in the blood, where most Pb is found inerythrocytes, it is initially distributed to soft tissues throughout the body. Subse-quently, it is deposited in the bone, where it eventually accumulates. The half-lifeof Pb in blood and other soft tissues is 28 to 36 days. Pb that is deposited inphysiologically inactive cortical bone may persist for decades without substantiallyinfluencing its concentration in blood and other tissues. On the other hand, Pb thatis accumulated early in life may be released later when bone resorption is increasedas result of, for example, calcium deficiency or osteoporosis. Pb that is depositedin physiologically active trabecular bone is in equilibrium with blood. The accu-mulation of high concentrations of Pb in blood when exposure is reduced may bedue to the ability of bone to store and release this element.

Dietary Pb that is not absorbed in the gastrointestinal tract is excreted in thefeces. Pb that is not distributed to other tissues is excreted through the kidney, andto a lesser extent by biliary clearance (WHO, 2000).

The biochemical basis of Pb toxicity is its ability to bind to biologicallyimportant molecules, thereby interfering with their function through a number ofmechanisms. At the subcellular level, the mitochondrion appears to be the maintarget organelle for the toxic effects of Pb in many tissues. Pb has been shown toselectively accumulate in the mitochondria. There is evidence that it causes struc-tural injury to these organelles and impairs basic cellular energetics and othermitochodrial functions. It is a cumulative poison, producing a continuum of effects,primarily on the hematopoietic system, the nervous system, and the kidneys. Atvery low blood levels, it may impair normal metabolic pathways in children. Atleast three enzymes of heme biosynthetic pathways are affected. At about 10 µg/100cm3 in blood, Pb interferes with δ-aminolevulinic acid dehydratase (WHO, 1986).Alteration in the activity of the enzymes of the heme synthetic pathway leads toaccumulation of the intermediates of the pathway. There is some evidence thataccumulation of δ-aminolevulinic acid exerts toxic effects on neural tissues throughinterference with the activity of the neurotransmitter γ-amino-butyric acid. Thereduction in heme production per se has also been reported to adversely affectnervous tissue by reducing the activity of tryptophan pyrollase, a heme-requiringenzyme. This results in increased metabolism of tryptophan via a second pathway,which produces high blood and brain levels of the neurotransmitter serotonin. Pbinterferes with vitamin D metabolism because it inhibits hydroxylation of 25-hydroxy-vitamin D to produce the active form of vitamin D. The effect has beenreported in children at blood levels as low as 10 to 15 µg/ 100 cm3 (WHO, 1986).Measurements of the inhibitory effects of Pb on heme synthesis is widely used asa screening test to determine whether medical treatment for Pb toxicity is neededfor children in high-risk populations who have not yet developed overt symptomsof poisoning.



4.5.7 INTERACTIONS OF ELEMENTS

All minerals are toxic at some dose, and the range of safety versus toxicity is highlyvariable. Data concerning the toxicity of the five discussed toxic minerals are pre-sented in Table 4.5 and Table 4.6. The uptake of elements is not entirely independentof one another. Elements of similar chemical properties tend to be taken up together.Sometimes one element has an inhibiting effect on another, or there can be synergisticeffects, such as enhancement of the absorption of calcium in the presence of adequateamounts of phosphorus; cadmium and lead hindering calcium and iron absorption;zinc and copper antagonism and their influence on the ratio of Zn to Cu on copperdeficiency; aluminum and phosphorus or fluoride antagonism, which may result indecreased plasma concentrations of phosphorus; or reduced fluoride absorption bymutually antagonistic competition for absorption in the gut. Interaction with othersubstances, such as drugs as well as food ingredients, should also be taken intoaccount when healthy nutrition is considered.

TABLE 4.5The Provisional Tolerable Weekly Intake (PTWI) of Toxic Elements According to the Joint FAO/WHO Expert Committee on Food Additives for Man

Element PTWI Comments

Aluminum mg/kg body weight 0–7.0Arsenic µg/kg body weight 15.0 For inorganic arsenicCadmium µg/kg body weight 7.0 —Lead µg/kg body weight 25.0 When blood lead levels in children exceed 25 µg/

100 cm3 ( in whole blood), investigations should be carried out to determine the major sources of exposure, and all possible steps should be taken to ensure that lead levels in food are as low as possible

Mercury µg/kg body weight 3.3 as methyl-mercury and 5.0 as total mercury

With the exception of pregnant and nursing women who are at qreater risk of adverse effects from methylmercury; life in utero is the critical period for the occurrence of neurodevelopmental toxicity; PTWI of 1.6 µg/kg bw is considered sufficient to protect the developing fetuses

Note: PTWI: For definition, see notes in Table 4.4.

Sources: WHO (World Health Organization), Food Additives Series 21, 24, 44, 46, and 52. Toxicologicalevaluation of certain food additives and contaminants; Safety evaluation of certain food additives andcontaminants. Papers presented at meetings of the Joint FAO/WHO Expert Committee on Food Additives,Geneva, 1986, 1989, 2000, 2001, and 2004.



TABLE 4.6Metal Toxicity in Man

Metal

Daily intake(mg per adult person)/

Toxic effects Source of exposureAbsorption(percent)

Aluminum Few to more than 30Neurotoxicity. It is supposed that Al may be implicated in the development of an encephalopathy in patients receiving dialyses, in Alzheimer’s disease, and in Parkinsonism dementia. Signs of neurotoxicity may be manifested when the brain concentration exceeds 10 to 20 times the normal level (>2 mg/kg dry weight of the grey matter). A further toxicological manifestation of Al toxicity is a microcytic hypochromic anemia not associated with Fe deficiency. Oral Al has not been associated with Al-induced encephalopathy. There is no risk of toxicity to healthy people from typical dietary intakes.

Grain, some bakery, dairy products made with additives such as buffers, dough strengtheners and leavening agents, acid reacting ingredients in self-rising flour or cornmeal, emulsifying agents for processed cheeses, stabilizers, texturizers, and anticaking agents, thickeners, and curing agents. High levels in black and herbal teas and spices. Al naturally present in water is generally low (0.001–1 mg/dm3), but when water is treated with aluminum sulfate salts (as coagulating agents) in water supplies, the content may rise up to threefold of the normal accepted level (0.2 mg/dm3).

<1–7 %

Arsenic 0.0–0.29Inorganic compounds cause abnormal skin hyperpigmenta-tion, hyperkeratosis, skin and lung cancer. Organoarsenic compounds present in fish are less toxic or nontoxic

Contaminated water, food containing residue of arsenic pesticides, and veterinary drugs. Fish and shellfish are the richest sources of organic compounds: arsenobetaine and arsenocholine.

Organoarsenic compounds>90%, and inorganic trivalent compounds high

Cadmium <0.024– 0.036Accumulates mainly in liver and renal cortex. Nephrotoxicity, decalcification, osteoporosis, osteomalacia, and Itai-itai disease. In early gestation, embryotoxic. Impairs the immune system, and Ca and Fe absorption. Hypertension and cardio-vascular disease. Kidney is the critical organ.

Oysters, cephalopods, crops growing on land fertilized with high contaminated phosphate and sewage sludge, Cd leaching from enamel and pottery glazes, contaminated water.

3–10% Cd bound to metallothio-nein is well absorbed.

(continued)



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Barbut, S. and Mittal, G.S., Rheological and gelation properties of meat batters prepared withthree chloride salts, J. Food Sci., 53, 1296, 1985.

Bremner, I., Heavy metal toxicities, Quarterly Rev. of Biophysics, 7, 75, 1974.Cappon, C.J. and Smith, J.C., Chemical form and distribution of mercury and selenium in

edible seafood, J. Anal.. Toxicol., 6, 10, 1982.Castell, C.H. and Spears, D.M., Heavy metal ions and the development of rancidity in blended

fish muscle, J. Fish. Res. Bd. Can., 25, 639, 1968.Castell,C.H., MacLeam, J., and Moore, B., Rancidity in lean fish muscle. IV Effect of sodium

chloride and other salts, J. Fish. Res. Bd. Can., 22, 929, 1965.Du, Z. and Bramlage, W.J., Superoxide dismutase activities in senescin apple fruit (Malus

domestica borkh), J. Food Sci., 59, 581, 1994.Eschleman, M., Ed., Introductory Nutriiton and Diet Therapy, J.B. Lippincott, London, 1984.Fairweather-Tait, S.J. et al., Aluminium in the diet, Human Nutrition: Food Sc. and Nutrition,

41 F, 183, 1987.FAO/WHO (Food and Agriculture Organization/World Health Organization), Summary of

Evaluations Performed by the Joint FAO/WHO Expert Committee on Food Additives1956–1993, International Life Science Inst. Press, Geneva, 1994.

Ha, Y.W. et al., Calcium binding of two microalgal polysaccharides and selected industrialhydrocolloids, J. Food Sci., 54, 1336, 1989.

Hendler, S.S., Ed., The Doctor’s Vitamin and Mineral Encyclopedia, Simon and Schuster,New York, 1990.

TABLE 4.6 (continued)Metal Toxicity in Man

Metal

Daily intake(mg per adult person)/

Toxic effects Source of exposureAbsorption(percent)

Lead <0.1–0.2At blood levels greater than 40 µg/100 cm3, exerts a significant effect on hemopoietic system resulting in anemia; affects the central nervous system.

Food contaminated from leaching of glazes of ceramic foodware, as well as from motor-vehicle exhaust, atmospheric deposits, canned food, water supply from plumbing system.

5–10% in adults and 40–50% in children.

Mercury <0.02–0.1Methylmercury compounds easily pass the blood–brain and placental barriers; causes severe neurological damage, greater in young children; in animals, also renal damage and anorexia.

Fish and shellfish, meat from animals fed with mercury-dressed grains.

>90% as methyl-mercury compounds, and 15% as inorganic mercuric compounds.



Hultin, H.O., Oxidation of lipids in seafoods, in Seafoods: Chermistry, Processing Technologyand Quality, Shahidi, F. and Botta, J.R., Eds., Chapman and Hall, London, 1994, p. 49.

Kraniak, J.M. and Shelef, L.A., Effect of ethylenediaminetetraacetic acid (EDTA) and metalions on growth of Staphylococcus aureus 196 E in culture media, J. Food Sci., 53,910, 1988.

Kraus, A.S. and Forbes, W.F., Aluminium, fluoride and the prevention of Alzheimer’s disease,Can. J. Publ. Health, 83, 97, 1992.

Miller, D.K. et al., Dietary iron in swine affects nonheme iron and TBARs in pork skeletalmuscles, J. Food Sci. 59, 747, 1994a.

Miller, D.K. et al., Lipid oxidation and warmed-over aroma in cooked ground pork fromswine fed increasing levels of iron, J. Food Sci., 59, 751, 1994b.

Müller, J.P., Steinegger, A., and Schlater, C., Contribution of aluminium from packagingmaterials and cooking utensils to the daily aluminium intake, Z. Lebensm. Unters.Forsch., 197, 332, 1993.

Müller, M., Anke M., and Illing-Gunter, H., Aluminium in wild mushrooms and cultivatedAgaricus bisporus, Z. Lebensm. Unters. Forsch. 22205, 242, 1997.

Nabrzyski, M. and Gajewska, R., Aluminium and fluoride in hospital daily diets and teas, Z.Lebensm. Unters. Forsch., 201, 307, 1995.

National Research Council, Food and Nutrition Board, Recommended Dietary Allowances,10th ed., National Academy Press, Washington, DC, 1989.

Oelingrath, I.M. and Slinde, E., Sensory evaluation of rancidity and off-flavor in frozen storedmeat loaves fortified with blood, J. Food Sci., 53, 967, 1988.

Pearson, A.M., Love, J.D., and Shorland, F.B., “Warmed over” flavor in meat, poultry, andfish, Adv. Food Chem., 23, 2, 1977.

Pennington, J.A.T., Aluminium content of foods and diets, Food Additives Contam., 5, 161, 1987.Peoples, S.A., The metabolism of arsenic in man and animals, in Arsenic Industrial, Biomed-

ical, Environmental Perspectives (Proceedings of the Arsenic Symposium), Lederer,W.H. and. Fensterheim R.J., Eds.,Van Nostrand Reinhold Co., New York, 1983,p. 125.

Ramanathan, L. and Das, N.P., Effect of natural copper chelating compounds on the prooxidantactivity of ascorbic acid in steam-cooked ground fish, Int. J. Food Sci. Technol., 28,279, 1993.

Ranau, R., Oelhlenschlager, J., and Steinhart, H., Aluminium content in edible parts ofseafood, Eur. Food Res. Techn., 212, 431, 2001.

Rosenberg, J.H. and Solomons, N.W., Physiological and pathophysiological mechanism inmineral absorption, in Absorption and Malabsorption of Minerals, Vol. 12., Solomons,N.W., and Rosenberg, J.H, Eds., Alan R. Liss, Inc., New York, 1984, p. 1.

Samant, S.K. et al., Protein-polysaccharide interactions: a new approach in food formulation,Int. J. Food Sci. Technol., 28, 547, 1993.

Schenkel, H. and Kluber, J., Mogliche Auswirkungen einer erhöhten Aluminiumaufnahmeauf Landwirtschaftliche Nutztiere, Obers. Tierernährung, 15, 273, 1987.

Solomons, N.W., Ed., Absorption and Malabsorption of Mineral Nutrients, Alan R. Liss, NewYork, 1984, p. 269.

True, R.H. et al., Changes in the nutrient composition of potatoes during home preparation,III, Min. Am. Potato J., 56, 339, 1979.

Vaessen, H.A.M.G. and van Ooik, A., Speciation of arsenic in Dutch total diets; methodologyand results, Z. Lebensm. Unters. Forsch., 189, 232, 1989.

WHO (World Health Organization), Environmental health criteria, 18, in Arsenic, WHO,Geneva, 1981.



WHO (World Health Organization), Food Additives Series, 21, Toxicological evaluation ofcertain food additives and contaminants, paper presented at 30th Meeting of the JointFAO/WHO Expert Committee on Food Additives, Cambridge, U.K., 1986.

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93

5

Saccharides

Piotr Tomasik

CONTENTS

5.1 Natural Food Saccharides, Occurrence, Role, and Applications .................. 935.2 Carbohydrate Structure .................................................................................. 945.3 Carbohydrate Chirality................................................................................. 1005.4 Carbohydrate Reactivity............................................................................... 101

5.4.1 Chemical and Physical Transformations of Mono-, Di-,and Oligosaccharides Essential in Food Chemistry........................ 1015.4.1.1 Reactions of Aldehyde and Ketone Function .................. 1015.4.1.2 Reactions of the Hydroxyl Groups .................................. 1025.4.1.3 Reactions of the Glycosidic Bond ................................... 1075.4.1.4 Specific Reactions of Saccharides.................................... 108

5.4.2 Chemical and Physical Transformations of Polysaccharides.......... 1095.4.2.1 Depolymerization of Carbohydrates ................................ 1135.4.2.2 Chemical Modification of Polysaccharides without

Attempted Depolymerization ........................................... 1145.4.2.3 Retrograded, Cross-Linked, and Graft Polysaccharides ..... 116

5.4.3 Enzymatic Conversions of Carbohydrates....................................... 1175.4.4 Cereal and Tuber Starches ............................................................... 118

5.5 Functional Properties of Carbohydrates ...................................................... 1195.5.1 Taste ................................................................................................. 1195.5.2 Colorants .......................................................................................... 1215.5.3 Flavor and Aroma ............................................................................ 1225.5.4 Texture.............................................................................................. 1225.5.5 Encapsulation ................................................................................... 1235.5.6 Polysaccharide-Containing Biodegradable Materials...................... 124

References.............................................................................................................. 125

5.1 NATURAL FOOD SACCHARIDES, OCCURRENCE, ROLE, AND APPLICATIONS

Nature commonly utilizes saccharides as a source of energy, structure-forming mate-rial, water-maintaining hydrocolloids, and even sex attractants. Amino acids synthe-size in the concentrated space and polymerize into proteins on already-availablepolysaccharide matrices. All organism cells, including those of animals, contain

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94


saccharide components in their membranes. Frequently, saccharides exist in naturallyderivatized forms, including aminated forms, as in chitin and chitosan; esterified;alkylated as in glycosides; oxidized; reduced; or linked to proteins, lipids, and otherstructures, such as glycoproteins.

Lower monosaccharides, such as aldotrioses and aldo- and ketotetroses, do notexist naturally in a free state. Glyceroaldehyde in phosphorylated form is the productof alcoholic fermentation and glycolic sequence. Erythrose, an aldotetrose, anderythrulose, a ketotetrose, also appear in phosphorylated forms in the pentose cycleof glucose, while ketopentose-ribulose can be found as its phosphate ester. Severalcommon and uncommon saccharides (erlose, turanose, trehalose, isomaltose,melecitose) have been found in honey (Rybak-Chmielewska, 2004). In nature, var-ious saccharide derivatives are also found. Among them are so-called sugar alcohols(alditols). They are the natural products of the reduction of saccharides. The occur-rence and applications of common saccharides, oligosaccharides, polysaccharides,and sugar alcohols are listed in Table 5.1. Algal gums and mucilages (Ramsden,2004) constitute an abundant group of polysaccharides in plants.

5.2 CARBOHYDRATE STRUCTURE

Carbohydrates are either polyhydroxyaldehydes (aldoses, oses) or polyhydroxyke-tones (ketoses, uloses); there is an electron gap at their carbonyl carbon atom.Typically, aldehydes and ketones accept nucleophiles, such as alcohols, to formhemiacetals and hemiketals, respectively. In pentoses, pentuloses, hexoses, hexulo-ses, and higher carbohydrates, one of the hydroxyl groups can play the role of internalnucleophile. Thus, open-chain structures (Structures 5.2 and 5.5) cyclize into internalhemiacetals (Structures 5.4 and 5.6), and hemiketals (Structures 5.1 and 5.3) witheither five- or six-membered cycles. Formation of such rings is thermodynamicallyfavored. Because of the oxygen atom in the rings, these rings are called furanosesand pyranoses, respectively.

Because their carbon atoms are

sp

3

–hybridized, the rings cannot be planar. Theypreferably take the chair conformation (Siemion, 1985).

In some cases, the pyranose ring formation can be either obstructed or blocked,and a furanose ring dominates for the given sugar. The molecular structure ofdisaccharides and higher saccharides is additionally controlled by a potential energybenefit resulting from the formation of intramolecular hydrogen bonds, as in cel-lobiose (Structure 5.7a), lactose (Structure 5.7b), maltose (Structures 5.8 and 5.9)and sucrose (Structure 5.10).

Both maltose structures correspond to two energy minima. Two hydrogen bondsstabilize the sucrose molecule. In order to achieve such stabilization, the fructosylmoiety must take the furanosyl structure.

In polysaccharides, the structural factors are even more important. A number ofdifferent saccharide units in the chain, the branching of the chain, and the presenceof more polar groups (COOH, PO

3

H

2

, and SO

3

H) or less polar groups (OCH

3

andNHCOCH

3

) than the OH group are crucial for the overall macrostructure of thepolysaccharide. Amylose and cellulose are the most regularly built; they form poly-mer chains of

α

-D- and

β

-D-glucose units, respectively. In very random cases,


Saccharides

95

TABLE 5.1Essential Natural Saccharides in Food, Their Occurrence and Applications

Saccharide Occurrence Applications

Monosaccharides and Their Natural Derivatives

Pentoses

D-Apiose Parsley and celeryL-Arabinose Plant gums, hemicelluloses,

saponins, protopectinAlcoholic fermentation, furan-2-aldehyde production

D-Xylose Accompanies L-arabinose Reduction to xylitol; sucrose substitute; alcoholic fermentation; production of furan-2-aldehyde

Hexoses

L-Fucose Mother’s milk, algae, plant mucus and gums

D-Galactose Oligo- and polysaccharides, plant mucus and gums, saponins, glycosides

Diagnostic in liver tests

D-Glucose Plants and animals, honey, inverted sugar, saponins

Alcoholic fermentation; sweetener; energy pharmacopeial material; nutrient; food preservative

D-Mannose Algae, plant mucus, orangesL-Rhamnose Plant mucus and gums, pectins

saponins, glycosides

Hexuloses

D-Fructose Fruits, honey, inverted sugar Noncavity-causing sweetener; sweetener for diabetics; food humidifier and preservative

D-Glucosylamine Chitin, chitosan Pharmaceutical aid; antiarthritic drugs; ion exchanger

L-Sorbose Rowan berries Synthesis of ascorbic acid

Disaccharides (oses)

Lactose Mammalian milk Dairy product taste improver; fermenting component of milk

Maltose Starch, sugar beet, honeySucrose Sugar beet, sugar cane Alcoholic fermentation; common

sweetener; caramel production; food preservation

Polysaccharides

Agar Red algae Microbiological nutrient; gel-forming agent; emulsifier; bread staling retardant; meat texturizer; meat substitute

Alginates Brown algae Thickener; gel-forming agent; food and beer foam stabilizer

(continued)


96


TABLE 5.1 (continued)Essential Natural Saccharides in Food, Their Occurrence and Applications

Saccharide Occurrence Applications

Carrageenans

ι, κ, λ, µ, ν

Red seaweed Gel-forming agent; stabilizer; protein fiber texturizer; milk fat anticoagulant; milk clarifying agent

Cellulose Plants Saccharification to glucose; dietary fiber; chromatographic sorbent

Dextran Frozen sugar beet Chromatographic sorbent (Sephadex); blood substitute

Furcellaran Red seaweed Gel-forming agent; filler; marmalade stabilizer; protein precipitant

Glycogen Liver, muscles Glucose reservoir for organismsGums:

Arabic Senegal acacia Emulsifier; antistaling stabilizer; flavor fixative

Gatti

Anageissus latifolia

tree Emulsifier; stabilizerGuaran Leguminous plants Food; cosmetics; pharmaceutical

thickener and stabilizerKaraya

Sterculiacea

tree (India) Foam stabilizer; thickenerLocust bean Locust bean Thickener; adhesiveTragacanth

Astragalus

species (Middle East) Thickener; stabilizerHemicelluloses:

Arabinogalactan Larch Emulsifier; stabilizerGalactan, mannans,xylans

Plants Alcoholic fermentation; reduction to alcohol

Heparin Liver, lungs Blood anticoagulantHialuronic acid Connective tissues Water absorbentInulin Endive, Jerusalem artichoke PrebioticPectins Plants, mainly apples, citrus, sugar

beetGel-forming agent; beer stabilizer

Protopectin Plants and nonmatured fruits Decomposes to pectins on plant maturation and cooking

StarchAmylose, amylopectin

Tubers, grains, some fruits, sago palm

Food filler; thickener; gel-forming agent; bakery products; saccharification to syrups

Tamarind flour Tamarind tree (India) Thickener; marmalade; jelly; ice cream; mayonnaise stabilizer

Xanthan gum Semiartificial gum Hydrocolloid stabilizer

Alditols

D-Glucitol (sorbitol) Apples, pears, cherries, aprocits Sweetener; softener; crystallization inhibitor

D-Mannitol Plant exudates, olives, palm trees, marine algae, mushrooms, onion

Energy storage in organisms

D-Ribitol Plants, riboflavinD-Xylitol Corncobs, mushrooms Sweetener


Saccharides

97

STRUCTURES 5.1–5.6


1 22 3

O

OH

HOH2CCH2OH

O HHO

O

OH

HOH2CO H

CH2OHHO

O

OH

HOH2CO

CH2OH

H

HO

OHO

HO

OOH

H

CH2OHOHO

HO

OOH

H

CH2OH

OHO

HOO

OH

H

CH2OH

4 5 6

7b7a7

OH

HOOHO

HOOH

O

H

OHO

O

OHCH2OH

CH2OH

OHO

HOO

HO

OH

O

OH

OH

OH

HOCH2

OH

10

Sucrose

OH

OH

OH

OH

O

HO

O

HO

OH

HO

O

O OHOH

OHOH

O

OH

OH

HO

HO

O

Maltose8 9


98


amylose is branched with short chains (Ball et al., 1998). The amylose chain isoriginally long and randomly coiled (Figure 5.1), but it turns into a more ordered,helical structure.

Helical complexes are formed if hydrocarbons, alcohols, lipids, fatty acids, andbarlike anions, such as I

5–

and OCN

–

are present in the amylose environment (Tomasikand Schilling, 1998a, b). Such compounds and anions, potential guests of the complex,either have hydrophobic fragments or are fully hydrophobic. The possibility of reduc-tion of the energy in the system by interactions of hydrophobic sides of amylose andthe potential guest is the driving force of the formation of a helical complex. Thus,its cavity is hydrophobic and all hydroxyl groups of the D-glucose units are situatedon the external surface of the helix, as well as on the edges of its cavity (the V-typeamylose). The number of glucose units in one helix turn depends on the size of theguest molecule inside the helical complex. With KOH as a guest, one turn involvessix glucose units. Inclusion of KBr reduces the turn to four glucose units, whereasinclusion of

tert-

butanol and

α

-naphtol requires the turns of seven and eight glucoseunits, respectively (Tomasik and Schilling, 1998a). An additional stabilization of theamylose helix comes from the double-helix formation (Imberty et al

.,

1991). Depend-ing on the helix–helix interactions, and in consequence, their mutual agreement, A-and B-type amyloses are formed (Figure 5.2). Distinguished C-type amylose is acombination of both A and B patterns. The conformation of

β

-D-glucose units boundin cellulose in the 1

→

4

′

manner offers a particularly strong hydrogen-bonded, cross-linked macrostructure of this polysaccharide.

FIGURE 5.1

Randomly coiled amylose chain and its helical complex, formed as a result ofits interaction with a nonpolar chain fragment of a molecule.

FIGURE 5.2

The crystallographic A- and B-types of amylose depend on the structure ofdouble amylose helices.

X

X


Saccharides

99

Glycans with 1,2-, 1,3-, and particularly 1,6-linked units have a more irregular,loosely joined structure. The heterogenicity of the polysaccharide structural unitsand their volumes introduce further regularities or irregularities in the macrostruc-ture. A decrease in the group polarity, for example, by methylation, and of the numberof units, results in a more irregular polysaccharide structure.

An opposite effect can be achieved in the presence of more polar groups, oreven less polar but suitably oriented groups. For instance, chitin, a polysaccharidewith acetylamino groups, has a very regular, compact structure that provides insol-ubility of chitin in water. This polysaccharide is a common insect carapace-formingmaterial. Because of this property, chitin has found several technical applications.

Amylopectin (Figure 5.3) presents a special case. It is a highly branched homopoly-mer of

α

-D-glucose units. The 1

→

6-linked terminal branches, which occur at aboutevery 8th glucose unit, contain 15 to 30 glucose units. These branches can alsoparticipate in the formation of helical complexes. Some guest molecules may situatein areas around the branching sites in the amylopectin molecule. In spite of the irregular,bulky structure, amylopectin also forms a double helix (Imberty et al., 1991). Becauseof functional properties, the structure of the polysaccharide matrix, the tertiary struc-ture, is also essential. In solution, polysaccharides such as amylose form separate fibrilsthat, upon coiling, turn into either micelles at a low-temperature gradient or gels at ahigh-temperature gradient. Both amylose and amylopectin participate in the starchgranule organization (Gallant et al., 1997). Their seminative mixture has specificfunctional properties. It strongly depends on the amylose-to-amylopectin ratio, size ofgranules (Table 5.2), content of residual components of native starch, such as lipids,proteins, and mineral salts, and random esterification with phosphoric acid, the latterexclusively in the case of potato amylopectin. Phosphorus present in other starches,resides as a component of phospholipids and glycoproteins.

The amylose-to-amylopectin ratio determines the aqueous solubility of starchand the texture of its gels, resulting from the penetration of water into the starchgranules (swelling) and from pushing the granule interior into a solution where thegel network is formed. Empty domains inside the starch granules can be utilized as

FIGURE 5.3

Scheme of the amylopectin molecule.


100


natural microcapsules for volatile and air-sensitive compounds (Korus et al., 2003).The size of the granules is essential for the smoothness of products prepared fromstarch (e.g., puddings, gels). In practice, not all starch granules swell and participatein gel formation. Larger granules are more susceptible to gelation and chemicalmodification. Also, among several starch varieties, potato starch gels significantlymore readily than others (Lii et al., 2003a). The nutritional value of starches usuallyincreases with the content of residual components. However, in several cases, whenstarch is subjected to chemical modifications or is used for specific nonnutritionalpurposes, such “contaminants” are not beneficial.

Cellulose that is completely insoluble in water forms microfibrils, which arecomposed of crystallites and amorphous regions. Such regions may also be distin-guished inside of starch granules. Roughly, they form concentric crystalline andamorphous layers surrounding the hilum, the origin of the granule growth (Gallantet al., 1997). The amorphous regions contain amylopectin (Szymo

ń

ska et al., 2000).The structure of granules is developed on plant vegetation by enzymatic debranchingof so-called plant glycogen (Erlander, 1998). These enzymes reside inside starchgranules and can be activated on starch processing.

5.3 CARBOHYDRATE CHIRALITY

Chirality is a property resulting from a lack of symmetry of molecules. All carbo-hydrates, including polysaccharides, have centers of asymmetry and, therefore, arechiral. Chirality is expressed as the concentration-independent specific rotation, [

α

]

o

:

[

α

]

o

= (

α

⋅

100)/

lc

(5.1)

where

α

is the angle of twist determined polarimetrically,

l

is the length of thepolarimetric tube, and

c

is the concentration of the saccharide in g/100 cm

3

. Chirality

TABLE 5.2Selected Properties of Starches of Various Botanical Origin

Starch origin

Amylosecontent

(%)Granule size

(

µµµµ

m)

Gelationtemperature

(

°

C)

Proteincontent

(%)

a

Lipidcontent

(%)

a

Barley 19.22 5–40 51–59 up to 12.2 up to 2.0Maize 21–24 10–30 67–100 3.2 6.0Oat 23–30 5–15 87–90 up to 3.9 up to 6.2Potato 18 –23 10–100 59–68 1.5 0.6Rice 8 –37 2–10 68–78 up to 28.2 up to 10.0Rye 24 –30 8 –60 55–70 3.2 2.2Triticale 24–34 2–40 55–62 2.7 3.9Waxy maize 1–2 10–30 62–72 up to 3.1 up to 2.0Wheat 24–29 2–36 59–64 2.4 3.6

a

Average values on a dry basis.


Saccharides

101

of freshly prepared aqueous solutions of saccharides is either variable or constantin time, depending on mutarotation (see below).

5.4 CARBOHYDRATE REACTIVITY

5.4.1 C

HEMICAL

AND

P

HYSICAL

T

RANSFORMATIONS

OF

M

ONO

-, D

I

-,

AND

O

LIGOSACCHARIDES

E

SSENTIAL

IN

F

OOD

C

HEMISTRY

5.4.1.1 Reactions of Aldehyde and Ketone Function

Mutarotation

: This transformation concerns only reducing sugars, such as those witha hydroxyl group at the anomeric carbon atom. The molecules transform reversibly,either spontaneously or on acid or base catalysis, through the pyranose and furanosering opening followed by ring closure (Structures 5.11a and 5.14 into 5.12 and5.11b). An open-chain structure is not typical for saccharides. Aqueous D-glucoseat 25

°

C has approximately 0.003% of the open-chain compound.Mutarotation has limited rather diagnostic significance in food chemistry and

technology. Practical use of this reaction is demonstrated in milk powder manufactur-ing. Evaporation of milk at a rate lower than that of mutarotation of lactose yields aproduct with less

α

-lactose isomer, which crystallizes in prism- or pyramid-like form.Fast milk evaporation gives an amorphous mixture of

α

- and

β

-lactose (Structure 5.7b).

Isomerization

: This process involves mainly D-glucose, which is isomerizedinto D-fructose, which is important for food technology. Some oligosaccharides

STRUCTURES 5.11A–5.14

ReductionReduction

O

CH2OH

OHHO

OH

OH

OH

OHHO

O

CH2OH

OHHO

OH

OH OH

OH

HOCH2OH

OH

OH

OHOHOH2C

HO

D-Mannose

11b

HO

D-Glucose

11a

O

CH2OH

OHOH

HO

11

12 14 13 L-Sorbitol(D-Glucitol)

D-Mannitol


102


can also be isomerized (Boruch and Nebesny, 1979). This process is catalyzed byglucose isomerase.

Reduction to alcohols

: The industrial scale reduction involves either NaBH

4

orelectrochemical and catalytic (Raney nickel) hydrogenation. The resulting open-chain polyols, the sugar alcohols (Structures 5.12 and 5.13), have a new chiral center.In consequence, each ketose (Structure 5.14) yields two alcohols, whereas aldoses(Structures 5.11a and 5.11b) yield only one alcohol.

Addition to the carbonyl group

: The internal, cyclic hemiacetal formation is oneof the illustrations of this type of addition. The H

2

N–X nucleophiles, with X beingNH

2

(hydrazine), NHAr (arylhydrazines), OH (hydroxylamine), NHCONH

2

(semi-carbazide), NHCSNH

2

(thiosemicarbazide), or alkyl (primary amine), producehydrazones, arylhydrazones, oximes, semicarbazones, thiosemicarbazones, and alkylamines (Schiff bases), respectively, according to the following path: Structures5.4 + 5.15

→

5.16

→

…

→

5.21.Hydrazones and arylhydrazones (Structure 5.20) react with the second molecule

of the corresponding reagent into osazones (Structure 5.21). The reaction with mono-,di-, and lower oligosaccharides has some analytical value. Reaction with H

2

N–Xnucleophiles in which X = CH

3

COOH, amino acids, nucleotides, and proteins, aswell as NH

3+

(ammonia) produce aldosylamino acids and aldosylamines, respectively(Structure 5.15 with X = CH

3

COOH or H). Aldoses undergo the Amadori rearrangement and subsequently turn into cara-

mels, the natural brown food colorants, and/or heteroaromatic compounds—deriva-tives of pyrrole, imidazole, and pyrazine. Ketones react similarly into ketosylaminoacids or ketosylamines. This reaction is the first step of either thermal or enzymaticchanges (the Maillard reaction) (see Davidek and Davidek, 2004) resulting in thebrowning of food and the development of the aroma of roasted, baked, or friedfoodstuffs.

Oxidation

: The oxidation of aldoses (Structure 5.24) with bromine or chlorinein alkaline solution (hypobromite and hypochlorite, respectively) leads to aldonicacids that readily self-esterify (lactonize) into

δ

- (Structure 5.25) and

γ

- (Structure5.26) lactones residing with free acid (Structure 5.27) in equilibrium.

β

-Conformersoxidize more readily than

α

-conformers.Glucono-

δ

-lactone (Structure 5.25) has found its application in baking powders,raw fermented sausages, and dairy products, where the slow release of acid is required.

The oxidation with Cu

2+

(the Fehling, Benedict, and Barfoed tests) and Bi

3+

(theNylander test) are the only analytical reactions for reducing sugars.

5.4.1.2 Reactions of the Hydroxyl Groups

Esterification

: Saccharides are commonly esterified with acyl chlorides, as well asorganic and inorganic acid anhydrides. These reactions can be run either exhaustivelywith involvement of all hydroxyl groups of the saccharide molecules or selectively.In the latter case, a protection (blocking) of certain hydroxyl groups is required. Forinstance, all hydroxyl groups of D-glucose, except that at C3, can be protected inreaction with acetone in an acidic medium. The resulting 1,2,5,6-di-

O

-isopropy-lidene-

α

-D-glucofuranose (Structure 5.28), after acylation at a nonprotected


Saccharides

103

hydroxyl group to give monoacylated diketal (Structure 5.29), is then decomposedwith carboxylic acid into a 3-monoacylated saccharide (Structure 5.30). Such reactionscan be applied to polysaccharides, although polysaccharides readily esterify withcarboxylic acids simply on heating of their blends (Tomasik and Schilling, 2004).

The acylation of mono- and oligosaccharides and their derivatives, mainly sor-bitol and sucrose, with higher fatty acids yields surface-active agents and fat replacers(Wang, 2004). Hydrolysis of the acyl group can be achieved by either transesterifi-cation (water or alcohol) or ammonolysis.


OH

CH2OH

N

OHN NH2

H

NH2

HO

+ 2NH2NH2

OH

CH2OH

O

OHN X

HHO

+ 2NH2NH2

Amadorirearrangement

OH

CH2OH

O

OHN X

HHO

OH

CH2OH

O

OHN X

H

HHO

2120

1918

17

16154

OH

CH2OH

OH

OHN X

HO

OH

CH2OH

OH

OH

OHN X

H

HO

+ N X

H

HO

CH2OH

OH

OH

OHHO





2726

25244

OH

OHO

OHO

CH2OH

- HBrBr2/OH-

C

OH

CH2OH

OH

OH O

OH

HO

O

CH2OH

OH

OHO

HO

O

CH2OH

OH

OH

O Br

HO

18 22 23

OH

CH2OH

O

OHN R

HHO

OH

CH2OH

O

OHN R

H

HHO

O

CH2OH

OH

OHOHHO

OH

CH2OH

OH

OHN R

HO

D-Gluconic acid

AcOH

Ac2O

Py

Me2C O

H+

O

CH2OH

OH

OHOH

HO

O

OAc

OO

CH2OMe

Me

O

Me

Me

O

OH

OO

CH2OMe

Me

O

Me

Me

O

CH2OH

OH

OHOHHO

4 28

29 30


Saccharides 105

Etherification: This reaction may involve hydrochloride catalyzed glycosidationwith alcohols, exhaustive methylation with dimethyl sulfate in an alkaline medium,exhaustive methylation with methyl iodide in the presence of Ag2O, and glycosida-tion, which is the substitution of an α-halo atom of α-halopentadecyl sugars withmethanol in the presence of Ag2CO3. Commonly, polysaccharides are etherified withalkyl halides and their derivatives with substituents in the alkyl groups such as thecarboxylic, amino, and quarternized amino groups. Reaction with epoxy compoundsalso results in etherification. Such reactions with small alkyl groups are importantfor saccharide structural analysis. Reactions with long-chain alkyl halides andepoxides provide products useful as biodegradable detergents (Wang, 2004). Reac-tions with halocarboxylic acids and haloalkyl amines lead to anionic and cationicstarches. Anionic starches are required components of polysaccharide—protein com-plexes, while cationic starches are designed as components of cellulose pulp.

Halogenation: The hydroxyl group at C1 is readily substituted with a halideatom (X) when a pentacetylated saccharide is treated with HX in acetic acid. Suchsaccharides are suitable for synthesis of desoxysaccharides. Exhaustive replacementof all hydroxyl groups in lower saccharides and polysaccharides can be achievedwith reagents suitable for such reaction with simple alcohols, such as COCl2, SOCl2,POCl3, PCl3, and PCl5. Chlorination of some sugars results in products of increasedsweetness (Table 5.3).

Dehydration: It is an intramolecular elimination of one water molecule producing1,6- (Structure 5.31), 3,6- (Structure 5.32), or 1,2- (Structure 5.34) anhydrosugars,as well as saccharide enol (Structure 5.33), and ketone (Structure 5.35).

Anhydrosugars can be utilized for the synthesis of some derivatives, such asamino sugars and others. Dehydration is the first step of sugar caramelization. Further

TABLE 5.3Relative Sweetness (RS) of Various Substances in 10% Aqueous Solutions(RS of Sucrose = 1.0)

Substance RS Substance RS

Sucrose 1.00 Stachyose 0.10β-D-Fructopyranose 1.80 1′-Chloro-1′-desoxysucrose 0.20Inverted sugar 1.30 4-Chloro-6-desoxysucrose 0.05D-Glucose α- 0.70 6-Chloro-6-desoxysucrose Bitter

β- 0.80 1,4,6′-Trichloro-1,4,6-tridesoxygalactosucrose 20.00D-Mannopyranose α- 0.30 Mannitol 0.40

β- Bitter Sorbitol 0.60D-Galactopyranose 0.32 Xylitol 0.85–1.2Maltose 0.32 Honey 0.97D-Lactose α- 0.20 Molasses 0.74

β- 0.30 Saccharin 200–700D-Galactosucrose Tasteless Cyclamates 30–140Raffinose 0.01 Aspartame 200 Neohesperidin dihydrochalcone 2000



heating of dehydrated saccharides results in the formation of three subsequent com-pounds called caramelan, caramelen, and caramelin (Tomasik et al., 1989).

6 C12H22O11 – 12 H2O = 6 C12H12O9 (caramelan)6 C12H22O11 – 18 H2O = 2 C36H18O24 (caramelen)

6 C12H22O11 – 27 H2O = 3 C24H26O13 (caramelin)


Product bProduct a

41 40 39

383736

35

3433

32314

H2O H2SO4OHO

HO

CH2OH+

-

OHO

HO

CH2OHOAcO

AcO

Br

CH2OAc

AcO

H2O+ - H2O

OHO

HO

OH

CH2OHOHO

HO

OSO3H

CH2OH

OAcO

AcO

CH2OAc

OHO

HOO

CH2OH

OHO

HOO

CH2OH

OHO

HOO H

CH2OH

+H2OOHO

HO

OHOH

CH2O H a

b

ba

HO

OH

OH

O

OHO

HO

O

OH


Saccharides 107

Reduction: A multistep reaction leads to desoxysaccharides. It involves 1-halo-pentacetylated saccharide, which is dehalogenated with zinc into acetylated glucal(Structure 5.35). Hydrolyzed glucal accepts sulfuric acid, the SO3H residue, whichis readily hydrolyzed into desoxysugar (Structure 5.41).

Oxidation: Apart from CO2 and H2O, there are three series of products that resultfrom the oxidation of saccharides. They are 2,3-dialdehydes (Structures 5.43 and5.49) formed on the oxidative cleavage of saccharides (Structures 5.42 and 5.48)with periodates, the sole oxidants providing this course of oxidation [except Pb(IV)].Such dialdehydes are considered toxic. Further oxidation of dialdehydes leads toglyceric acid (Structure 5.45), glyoxalic acid (Structure 5.47), hydropyruvic acid(Structure 5.46), and erythronic acid (Structure 5.51), as shown below for the oxi-dative cleavage of sucrose (Structure 5.42) and maltose (Structure 5.48).

The oxidation of monosaccharides, such as D-galactose (Structure 5.52) withstrong oxidants proceeds at both C1 and C6 atoms, leaving dicarboxylic, aldaricacids (Structure 5.53). Aldaric can lactonize into lactones (Structure 5.54). Lactonesproduce uronic acids (Structure 5.54) when reduced with sodium amalgam.

The oxidation at C1 can be prevented by protection of the 1–OH group. Theglycosidic bond in oligosaccharides offers sufficient protection. Application of weakoxidants offers a direct route to uronic acids.

Complex formation: The hydroxyl groups offer two types of interactions withmolecules having either a clearly dipole character or charge, that is, ions. Thehydrogen atoms of these groups are capable of interactions with electron-excessivesites of dipoles and anions, whereas lone electron pairs of the oxygen atom areelectron donors for cations and the positive side of dipolar molecules.

The complex formation is a general ability of saccharides. Fruitful results werenoted in the case of Ca2+ salts, preferably chloride, and hydrogen carbonate. Thecation forms fairly stable compounds. This property was widely utilized in sugarmanufacture for the separation of sucrose from its syrup. Saccharide alcohols alsocoordinate metal ions. Depending on the cations, their optical rotation is affected toa different extent, but always in the order of Na+ < Mg2+ < Zn2+ < Ba2+ < Sr2+ < Ca2+.Complexes of saccharides with a wide variety of cations were prepared and charac-terized (Angyal, 1989). Saccharides also complex to other, nonmetalic compounds,including organic food components such as polysaccharides and proteins. The prod-ucts of the interactions are sorption complexes with involvement of hydrogen bonds.The energies of the complex formation are low and do not exceed 4 kJ/mole (Tomasiket al., 1995). Nevertheless, they are essential in food texturization and thermalstability (Ciesielski and Tomasik, 1996; 1998; Ciesielski et al., 1998). The complex-ation itself can seriously affect sugar metabolism in the organism.

5.4.1.3 Reactions of the Glycosidic Bond

As a typical acetal bond, a glycosidic bond readily hydrolyzes in an acid-catalyzedreaction. In this manner, di- and oligosaccharides can be split into monosaccharides.It is a common method for the manufacturing of invert sugar, a mixture of α-D-glucoseand β-D-fructose, from sucrose.



5.4.1.4 Specific Reactions of Saccharides

In strongly acidic media, saccharides produce furan derivatives in a sequence ofreactions that are rearrangements and dehydrations followed by cyclization. Similarproducts are available thermally. Pentoses and hexoses give furan-2-aldehyde and5-hydroxymethylfuran-2-aldehyde, respectively. Both products are responsible forthe specific aroma of caramel and burnt sugar.


O

O

OH2CH

O

HC

O

HC

2CHHO

O O

OH2CH

O

HCCH

HO

OH

OH

OH2CH

OH

O

OH

OH

OH2CH

O

O

O

O

OH2CH

O

CH

CH

O

O

OH2CH

O

HC

O

HC

42

2CHHO

OH

OH

OH2CHO

OH

OH

OH2CH

O

O

HO

OOHC

OHCOOOCH

OH2CH

O

O

OOCH

2CHHO

OH2CH

O

OOHC

OH2CH

O

OOHC

OOHC

OH2CH

O

O

OOCH

OH2CH

O

OOHC

OOHC

OCH

OH2CH

OH

OH

OOHC2CO

OOHC

OCHOH2CH

O

OOHCOH2CH

OH

OOHC

O2H, 2Br+ O2H, 2Br+

48

43 49

44 50

+ +H + +

H

3+ -4IO OOHC-H, 4+ -

4IO OOHC2H- ,

45 46 47 51 46 47

acid D-Glyceric

acid Hydroxypyruvic

acid Glyoxalic

acidErythronic


Saccharides 109

In weakly acidic and neutral media, the reactions proceed at a lower rate. Reductones(Structures 5.55 through 5.57), compounds with the carbonyl groups vicinal to an endiolmoiety, which are formed, are stable at pH < 6, and act as natural antioxidants. Theytransform into desoxysugars, uloses (Structure 5.58). The latter undergo cyclization into5-hydroxymethylfuran-2-aldehyde (Structure 5.62). Corresponding osuloses (Structure5.63) in similar sequences of transformations yield diacetylformosine (Structure 5.69).

In acid-based reactions, 2-acetyl-3-hydroxyfuran (isomaltol) (Structure 5.76), 3-hydroxy-2-methylpyran-4-one (Structure 5.77), and maltol (Structure 5.79) areformed. They are responsible for the aroma of baked bread.

Endiols (Structure 5.80) can isomerize into other saccharides in the Lobry deBruyn-van Ekenstein rearrangement, Thus, D-glucose (Structure 5.4) can isomerizeinto mannose (Structure 5.81) and fructose (Structure 5.1) accompanied by a smallamount of D-psicose (Structure 5.83). An alkaline medium provides isomerizationto disaccharides, which turn from aldoses into ketoses, as shown for lactose (Struc-ture 5.7b) isomerized to lactulose (Structure 5.84).

Because the enolization is not restricted to the 2 and 3 positions, a number ofproducts are formed that undergo subsequent aldol condensations and the Cannizzarooxidation. They are all 2-hydroxy-3-methyl, 3,4-dimethyl-2-hydroxy, 3,5-dimethyl-2-hydroxy, and 3-ethyl-2-hydroxy-2-cyclopenten-1-ones; γ−butyrolactone; and suchfuran derivatives as furyl alcohol, 5-methyl-2-furyl alcohol, and 2,5-dimethyl-4-hydroxy-3(2H)-furanone. These are food flavoring agents.

5.4.2 CHEMICAL AND PHYSICAL TRANSFORMATIONS

OF POLYSACCHARIDES

Although starch is the most widely and massively studied, and has found numerouslarge-scale applications, pectic polysaccharides (MacDougall and Ring, 2004), fructans(Praznik et al., 2004), chitin and chitosan (Einbu and Vaarum, 2004), and hemicelluloses,


O

OH

HOOH

OH

CH2OH

O

OH

HOOH

O

COOHO

OH

HOOH

OH

COOH

HNO3O

C

OH

HOOH

OHO

H

COOH

Na/Hg

H+

52 53

54 55

D-Galactose Mucic acid

D-Galactaric acid monolactone

D-Galacturonic acid



are also modified on an industrial scale. The products of their modifications are widelyutilized in food technology and everyday food preparations. Food processing, such ascooking, baking, frying, and pickling, usually induce food carbohydrate transformations.

The functional group reactivity in polysaccharides is, to a great extent, obstructedby their macrostructure. These potential reaction sites, which do not reside on thesurface, may be unavailable for many reagents, as they are either hidden inside themacrostructure or involved in the formation of intra- and intermolecular hydrogenbonds, crucial for macrostructure properties. Many reactions of polysaccharides are


OHO

HOO

OH

HH

CH2OH

H+OHOH2C OH

CH2OH

OH

OH

HO

C

HOH2C

O

O

H

OHOH2C C

O

H

OHO

HOOH

OH

CH2OH

- H2O

O

C

HOO

O

H

HH

CH2OH

H+

O

C

HOO

O

H

H

CH2OH

- H2O

O

CO

O

H

H

CH2OH

OHOH2C OH

CO

H - H2O

4

1

56

57 58

5960

61 62

3-Desoxy-D-glucosulose


Saccharides 111

governed by the heterogenicity of the reaction system. Only randomly reactingpolysaccharides are solvent soluble. Among polysaccharides utilized either on orafter transformation, hemicelluloses are exclusive. They are water soluble or theyswell. Problems of solubility and compactness are encountered, particularly in cel-lulose and starch, which are fibrillar and granular, respectively. The reactivity interms of the rate and degree of transformation can be controlled by either applicationof suitable reagents or loosening of the compact structure with involvement of aphysical action. The solvent effect (water in the case of starch and higher alcohols[Ruck, 1996] in case of cellulose) is the most commonly used tool. But high pressure;sonication with ultrasounds; ultraviolet, microwave, and polarized light (Fiedorowiczet al., 2001), glow plasma (Lii et al., 2002a), corona discharges (Lii et al., 2003b),ionizing radiation, thermolysis; and deep freezing and thawing (Szymońska et al.,2000) might also be suitable for loosening a compact polysaccharide structure(Tomasik and Zaranyika, 1995). Pasting and gelatinization of starch by heating it inwater or immersing it in aqueous alkali delivers pregelatinized starch. Because suchprocessing breaks several intra- and intermolecular hydrogen bonds, pregelatinizedstarch is easily water soluble and chemically more active. When granular starch ispassed under pressure through narrow nozzles, so-called α-starch (nanostarch) withimproved solubility in water is formed. Physical modifications of macrostructurefrequently result in depolymerization of the polysaccharide.

Starch granules are composed of amylose and amylopectin, together formingcrystalline and amorphous regions of the granule. The polysaccharides in an amor-phous region are more susceptible to enzymatic digestion than in crystalline regions.


HO

OH

OH

O

O

CH3

CH3O

O

OH

O

CH3O

O OH

CH3H3C

HO

HO

O

OH

O

O

CH3H

H

HO

O

OH

OH

O

CH3

HO

O

OH

OH

O

CH3H

O

O

OH

O

CH3

H

63 64

67 68 69

65 66



Starch in the latter region is one of four forms of so-called resistant starch (RS).Resistant starch is used as a prebiotic—a nutrient for probiotic bacteria colonizingthe human intestine. Another type of resistant starch is available by chemical modi-fication of starch (Fiedorowicz et al. 2004; Kapuśniak and Marczak, 2005). Controlled


H+

- H2O

O

O

HO

H3CO

O

HO

HO

H3C

H

O

OH

O

HO

H3C

OC

HO

O

H3C

OHO

C

HO

H3C

O

OH

O

O

O

CH3

H

HO

OH

OH

O

O

CH3

H

HO

OH

OH

O

O

CH3

HO

OH

OH

O

O

CH2

H

HO

OH

OH

O

O

CH2OH

H

H

1

OHOH2C OH

CH2OH

OH

OH

70

71 72

7374

75

77

76

7879

Isomaltol

Maltol

a b


Saccharides 113

swelling of starch granules in water can remove a considerable part of the amorphousinterior of the starch granule, producing empty domains within the granules utilizedin microcapsulation (Korus et al., 2003). Such exudation of the amorphous contentof granules begins in the process of starch isolation (Starzyk et al., 2001). Thefunctional properties of starch depend on the amylose-to-amylopectin ratio, For somepurposes, amylose-rich starch (Hylon starch) is more beneficial and, for the others,application of amylopectin-rich (waxy) starch is advantageous. Such starches areusually genetically engineered. However, recently enrichment of granular starchesin linear, amyloselike polysaccharides with illumination of starch by linearly polar-ized light was described (Fiedorowicz et al., 2001).

5.4.2.1 Depolymerization of Carbohydrates

If not utilized in the pulp industry, hemicelluloses are hydrolyzed in the acid-catalyzed process, mainly to monosaccharides and to furan-2-aldehyde (pentosanes)and 5-hydroxymethylfuran-2-aldehyde (Structure 5.62) (hexosanes). Monosaccha-ride-containing syrups, after purification, are either fermented or utilized as woodmolasses for feeding ruminants. In another approach, xylose, the least soluble


HO

OHHOOH

OHCH2OH

OHO

HOOH

OH

OHOH2C OH

CH2OH

OH

OH

OHOH2C OH

CH2OH

OH

HOH2C

O

OH

OHO

OHHO

OOH

O

OH

OH

CH2OH

O

OH

OH

HOH2C

HO

HOO

OH

OH

CH2OH

OHO

HOCH2OH

OHOH

80 81

1 82 83

7b 84Lactulose

D-Psicose



component of the syrup, is allowed to crystallize. The separated xylose is thenhydrogenated over an Ni/Al catalyst at 120°C under 6 × 106 Pa into xylitol. Hemi-celluloses, together with proteins, participate in the Maillard reaction and maycontribute to the overall secondary food aroma of processed foodstuffs (Tomasikand Zawadzki, 1998).

The acid-catalyzed hydrolysis of cellulose, the saccharification, results in split-ting the terminal D-glucose unit of the fibril. Thus, under thorough control of thereaction conditions, D-glucose is the sole product. Glucose syrup is either a sourceof pharmaceutical-grade D-glucose or is fermented to ethanol.

The β-glycosidic bond of cellulose can be split thermally. Perhaps the mostancient polysaccharide processing—dry wood distillation—delivers charcoal, water,tar, methanol, acetone, acetic acid, and gases. The liquid and gaseous fractions resultfrom the thermolysis of thermally split D-glucose. Thermolysis of cellulose withproteogenic α-amino and α-hydroxy acids produces several aromas potentially inter-esting for the food and cosmetic industries. Starch and pectins also generate aromason heating with these acids (Bączkowicz et al., 1991; Sikora et al., 1998). Depoly-merization of starch yields dextrins—one of the most useful products of the foodindustry. The most frequent dextrinization involves proton catalysis or heat. Hydro-chloric acid is a superior catalyzing proton donor, but organic acids are also capableof starch dextrinization. Such processes extended in time can lead to the sacchari-fication of starch into oligosaccharides and, finally, to D-glucose, maltose, andglucose syrups, which are used directly as sweeteners or are fermented. Thermaldextrinization of starch up to 260°C, produces canary-yellow dextrins called Britishgums. They differ in properties and applications from dextrins from acid hydrolysis.Dextrins are also available by UV irradiation and ionizing radiation, as well as othertypes of physical action on starch (Tomasik and Zaranyika, 1995). Dextrins arecommonly used as food thickeners, plasticizers, and adhesives. Depolymerizationof polysaccharides to formaldehyde seems to be particularly promising to the chem-ical industry as a versatile, renewable source of various chemicals and a key processfor utilization of polysaccharides in the 21st century. Reduction of formaldehydedelivers methanol, one of the most important chemical reagents.

5.4.2.2 Chemical Modification of Polysaccharides without Attempted Depolymerization

Polysaccharides offer practically the same kind of reactivity as monosaccharides,except the reactions on the anomeric carbon atom, because in a waste majority ofpolysaccharides the hydroxyl groups at this atom take part in the polysaccharidechain formation via the glycosidic bonds. In the long chains of polysaccharide, onlythe terminal saccharide units carry free hydroxyl groups at the anomeric carbonatom. However, even such minute modification in this position can be reflected bychanges in the rheological properties of polysaccharide solutions, pastes, and gels.There are some polysaccharides naturally containing additional functional groups,other than the hydroxyl groups. Thus, chitin contains acetamido groups. Alginates,many plant gums (Arabic, gatti, karaya, tragacanth, and xanthan, the latter issemisynthetic), pectins, some galactans, and xylans contain the carboxylic groups.


Saccharides 115

Heparin, furcellaran, and carrageenans carry sulfate functions. These groups can beutilized in chemical modifications of those polysaccharides.

Limitations in the possibility of chemical modifications of starch result fromsteric hindrances of the reaction sites, solubility, viscosity of the reaction medium,and susceptibility to side reactions. Furthermore, depolymerization almost alwaysaccompanies the intended modification. As a rule, polysaccharides are soluble,although frequently only sparingly, in water and dimethyl sulfoxide. Polysaccharidessolubilize on xanthation, that is, on reaction with CS2 in an alkaline medium, toform syrups of xanthates. On acidification, the polysaccharides could be recoveredtogether with CS2. This procedure was utilized for several decades for productionof artificial silk from cellulose.

Polysaccharides undergo hydrolysis; reduction to alcohols, oxidation to alde-hydes, ketones, and carboxylic acids; esterification with inorganic (sulfuric, phos-phoric, nitric, boric, and sillylic) and organic acids; etherification; acetylation withaldehydes; halogenation with the same reagents as mono- and disaccharides; ammi-nation (usually via halogenated polysaccharides); carbamoylation with acylamidesor isocyanates; and metallation. Only some of the large number of potential modi-fications achieved approval under the food laws of particular countries. Thus far,only starch, cellulose, and pectin are chemically modified for nutritional purposesand can be used in foodstuffs. If biodegradable materials are also considered, mod-ification of other polysaccharides can be taken into account.

Practical modification of pectins is limited to changes in the degree of theirmethylation. In this manner, the strength of their jellies can be controlled. Modifi-cation of cellulose for use in the food industry is limited to its esterification. Mainlycellulose acetate is produced. It is used for special membranes for treating waterand fruit juices. Among ethers, methyl- and methylhydroxypropyl celluloses deserveparticular attention. They are available by methylation with common methylatingagents and propylene oxide, respectively. Carboxymethyl cellulose (CMC) is theproduct of etherification of cellulose with chloroacetic acid; it usually has a degreeof substitution from 0.3 to 0.9. All 2-, 3-, and 6-hydroxyl groups do react. Modifiedand derivatized cellulose have found their application in the food industry asnondigestible components of low-calorie meals. CMC is a texturizing agent andedible adhesive.

The chemical modification of starch for nutritional purposes involves oxidation,but only with a limited number of oxidants; esterification with a limited numberof reagents; etherification; and complex formation. Metal derivatives might havesome significance as carriers of bioelements and therapeutic agents, primarily inulinsubstitutes (Tomasik et al., 2001). By forming Werner-type complexes with metalions (Ciesielski et al., 2003; Ciesielski and Tomasik, 2004), the polysaccharidescan influence administration, excretion, and functioning of metal ions in livingorganisms. Oxidation of starch to aldaric and uronic acid-type carboxylic starchesfor practical purposes should leave no more than one carboxylic group per each25th glucose unit. The oxidation should be carried out with a possible high depol-ymerization degree and the formation of the smallest possible number of terminalanomeric carbon atoms. Gels from oxidized starches have low viscosity and goodtransparency. Such oxidation is provided by sodium hypochlorite. This oxidant only



randomly oxidizes the 6-hydroxymethyl groups of starch. The latter groups arereadily oxidized with nitrogen oxides. Metal ion-catalyzed air oxidation of starchresults in simultaneous formation of carboxyl and carbonyl starches. The mostuseful esters of starch are phosphates, commonly used as gelating agents, acetates,and adipates—the film-forming materials. Only these preparations are utilized infoods, which have a low degree (0.2 to 0.0001) of esterification. Higher derivatizedstarches are used as fat substitutes and emulsifiers (Wang, 2004). The most impor-tant and widely used starch ethers are carboxymethyl, hydroxyethyl, and hydrox-ypropyl starches.

Some functional groups introduced into starch by esterification or etherificationcan dissociate. For instance, all starch sulfates, phosphates, and carboxylates disso-ciate in aqueous solution, leaving a negative charge on starch. They are called anionicstarches. Such products are capable of forming complexes with metal ions and withproteins (Schmitt et al., 1998). They may be utilized as biodegradable plastics andmeat substitutes. If the starch was etherified with a reagent introducing a tetralky-lammonium salt function, the dissociation develops a positive charge on the moleculeand such starch becomes cationic. Such starches are used in the manufacture ofpaper and as detergents.

Polysaccharides are important complexing agents for inorganic and organicgases, liquids, and solids. Usually surface sorption is involved, but in case of starch,inclusion complexes inside the amylose helix and eventually short helices of amy-lopectin, and capillary complexes involving capillaries between starch granules areformed. All of them exist in a natural, native form; they can also be formed in severalcommon operations of food processing, such as dough formation, foam beating, andscrambling egg yolk with sugar. Formation of inclusion complexes of starch becomesa more common method of protecting some volatile, as well air-sensitive, foodcomponents (microencapsulation). Such complexes are essential for food texturiza-tion and overall stabilization.

5.4.2.3 Retrograded, Cross-Linked, and Graft Polysaccharides

Retrogradation is a very common reaction of gels of starch polysaccharides. It leadsto enhanced molecular-weight systems. Amylose gels retrograde within hours,whereas retrogradation of amylopectin takes days and even weeks. This process ismanifested by dendrite formation in the gel and in bread by bread staling and waterexpulsion. This phenomenon is due to the orientation of chains of polysaccharideswith respect to one another to aggregate with involvement of intermolecular hydro-gen bonds. The retrogradation affinity depends on the starch variety and decreasesin the following order: potato > corn > wheat > waxy corn starch. Evidently, theretrogradation rate and nature of the formed amylose crystals depend on the starchsource, amylose-to-amylopectin ratio, and storage temperature. Low temperaturesaround the freezing point and polar gel additives favor retrogradation. Retrogradedstarch is utilized as a component of low-calorie foods.

Polysaccharide cross-linking frequently occurs when it is acetylated, esterified, oretherified with corresponding bi- and polyfunctional reagents, such as POCl3, polyphos-phates, anhydrides, aldehydes, carboxylic acids, and carboxyamides. Grafting is most


Saccharides 117

commonly performed with vinyl monomers. Grafting competes with homopolymeriza-tion of vinyl monomers. The latter can be suppressed by adjusting the suitable catalystand reaction mechanism. Usually cross-linked and grafted polysaccharides haveenhanced water-binding capacity, lower aqueous solubility, and shear force stability.

5.4.3 ENZYMATIC CONVERSIONS OF CARBOHYDRATES

With few exceptions, enzymatic processes cause degradation of carbohydrates.Enzymes are used in the form of pure or semipure preparations or together withtheir producers, that is, microorganisms. Currently, semisynthetic enzymes are alsoin use. Alcoholic fermentation is the most common method of utilization of monosac-charides, sucrose, and some polysaccharides, such as starch. Hydrolysis of polysac-charides with alpha-amylase, so-called alpha-amylolysis, is the common way ofhydrolysis of starch to maltodextrins. Recently, it has been shown that such reactioncan be stimulated by illumination of the enzyme with linearly polarized light. Theamylolysis can be accelerated by several orders (Fiedorowicz and Khachatryan,2003). Lactic acid fermentation is another important enzymatic process. Lactic acidbacteria metabolize mono- and disaccharides into lactic acid. This acid has a chiralcenter; thus either D(–), L(+), or racemic products can be formed. In the humanorganism, only the L(+) enantiomer is metabolized, whereas the D(–) enantiomer isconcentrated in the blood and excreted with urine. Among lactic acid bacteria, onlyStreptococcus shows specificity in the formation of particular enantiomers, and onlythe L(+) enantiomer is produced. Enzymatic reduction of glucose-6-phosphate(Structure 5.85) into inositol-1-phosphate with cyclase and reduced NAD coenzyme,followed by hydrolysis with phosphatase, presents another nondegrading enzymaticprocess proceeding on hexoses. Inositol (Structure 5.86) resulting in this mannerfrom its phosphate, plays a role in the growth factor of microbes. Its hexaphosphate,phytin, resides in the aleurone layer of wheat grains.

There are also known bacteria that polymerize mono- and oligosaccharides.Leuconostoc mesenteroides polymerizes sucrose into dextrin—an almost linearpolymer of 400 or more α-D-glucose units. Dextran is also generated in frozensugar beets. This causes difficulties in sugar manufacturing if the beets have to bestored at low temperature. Dextran serves as a blood substitute and chromato-graphic gel (Sephadex). Other polysaccharides synthesized by bacteria are levan,a polymer of β-D-fructose, pullulan, a polymer of α-D-glucose, and xanthan gum, apolymer of β-D-glucose and α- and β-D-mannoses.


OH

OH

CH2

O

O PO3H2

OHHO

85

OH

OH

OH

O

HO

OH

86



The enzymatic oxidation of sucrose with glucose oxidase to D-glucose-δ-lactone consumes oxygen dissolved in beer and juices. In this manner the rate ofundesirable processes caused by oxidation, such as color and taste changes, isdecreased. All essential enzymatic polysaccharide transformations deal with deg-radation (Figure 5.4). In the case of cellulose, this degradation leads to glucose.Endoglucanase and cellobiohydrolase attack the amorphous regions of the compactstructure of cellulose, producing D-glucose and cellobiose, respectively. Starch,amylose, and amylopectin are not necessarily as deeply degraded. There are severalamylolytic enzymes capable of starch degradation. They provide high specificityof their action.

Synthesis of cyclodextrins (cycloglucans, Schardinger dextrins) presents a spe-cial case. Slightly hydrolyzed starch is transformed into cyclic products composedof six, seven, and eight α-D-glucose units, α−, β−, and γ-cyclodextrins. The yield ofcyclodextrins declines with the number of glucose units in cycles.

Although higher-membered cyclodextrins are also formed in the reaction mixtures,their minute yield seriously limits their potential applications. Cyclodextrins are some-times cross-linked into cyclodextrin resins with interesting inclusion properties.

There are several other known enzymatic conversions of saccharides, oligosac-charides, and polysaccharides (Bielecki, 2004).

5.4.4 CEREAL AND TUBER STARCHES

All botanical varieties of starches can be primarily classified into tuber and cerealstarches. Only the true sago starch is isolated from the sago palm trunk (Indonesia).

FIGURE 5.4 Enzymatic transformations of starch.

Amylo-1,6- -gluconase (Dextrinase)

Glucoamylase

Glucose

-Amylase

Dextrin

Pullulanaseand

glucogenase Dextrin

Cyclodextrin

Bacillus macerans

-Amylase

Maltose

Glucose

-Glucosidase

Glucoseisomerase

Fructose

-

--


Saccharides 119

Differences in size and shape of the granules, the amylose-to-amylopectin ratio, andprotein (7 to 13% content), lipid (1.5 to 6% content), other carbohydrate (5 to 23%content), and mineral (1 to 3% content), do not necessarily depend on whether agiven starch belongs to one of the above two classes. Potato starch, the tuber starch,has the largest granules (up to 150 µm) and as the sole starch, it has amylopectinesterified with phosphoric acid (Seideman, 1966; Jane et al., 1994). However, starchgranules of wild yam (Diascorea dumetorum), which has one of the finest granulesever seen, also originates from tubers (Nkala et al., 1994). Generally, cereal starchesare richer in lipids (e.g,. cornstarch), and tuber starches are richer in proteins,although the oat starch, a cereal starch, is one of the richest in lipids and proteins(see Table 5.2). Polysaccharides, lipids, and proteins usually reside in granules innative complexes. These complexes are stronger in cereal starches. Thus, potatostarch can readily be isolated relatively free of proteins, whereas the completedefatting of cornstarch could not be done. It might be due to the structure of suchcomplexes. Residual lipids in cornstarch reside inside of the amylose inclusioncomplex, and protein molecules are too large to be included in the amylose helix.The main difference between tuber and cereal starches comes from their crystallo-graphic pattern: A for tuber and B for cereal starches. These two patterns result fromdifferent mutual orientations of amylose helices inside the granule, for example,single (A-type) and double (B-type) (Figure 5.3). These differences determine severalessential properties of both classes of starches: swelling, gelation, and course ofpasting, and affinity to various physical, physicochemical, and chemical modifica-tions. There are also differences in the taste, digestibility, and nutritive value ofparticular starches.

The most common sources of starch in various regions of the world are potato,maize, cassava (manioc, tapioca, yucca), and rice. Recently, interest in wheat starchhas considerably increased. The popularity of a given starch and starchy plants donot go together. For instance, in several regions of the world rye is commonly used,but isolation of starch from it is difficult due to the mucus present in that grain,which obstructs the isolation. The properties of that starch do not justify the highercosts of its isolation.

5.5 FUNCTIONAL PROPERTIES OF CARBOHYDRATES

5.5.1 TASTE

Saccharides are usually associated with sweet taste, although some among them arebitter and nonsweet saccharides (Table 5.3). Except for sucrose, the sweetnessdecreases with the number of monosaccharide units going toward oligo- and polysac-charides, because only one monosaccharide unit interacts with the mucoprotein ofthe tongue receptor. The quantum-mechanical treatment of sweetness, in terms ofinteraction of sweeteners with receptors, was recently given by Pietrzycki (2004).

Because a number of powerful synthetic and natural nonsaccharide sweetenersare available on the market, apart from reduction to saccharide alcohols, otherderivatizations of saccharides, even if they increase their sweetness (Table 5.3), haveno practical significance.



The following carbohydrate sweeteners are in common use (their relative sweet-ness [RS] with respect to a 10% aqueous solution of sucrose is given in Table 5.3).

D-glucose: because of fast resorption, it is a source of immediately availableenergy. It is used in injections and infusion fluids for children and patientsin their recovery period. Its metabolization requires insulin, and it causestooth cavitation.

D-fructose: the most readily water-soluble sugar. It does not crystallize fromstored juices. Because of its hygroscopicity, it retains moisture in sugar-preserved food and intensifies its flavor and aroma. The metabolism of D-fructose delivers less energy than sucrose. This saccharide neither causesnor accelerates tooth cavitation. It accelerates ethanol metabolism. In theorganism, D-fructose metabolizes into glycogen, so-called animal starch,which is the energy reservoir stored in the liver.

Lactose: a sparingly water-soluble (20% at room temperature) sugar presentin mammalian milk (4.8 to 5.1%). It is utilized as a carrier of other sweet-eners. It improves flavor, produces a good image of food processed inmicrowave ovens, and improves the taste of dairy products.

Sucrose: the most common sweetener used for its pleasant taste. It is widelyused as a preservative of marmalades, syrups, and jams. Osmotic phenom-ena are involved. Due to the competition of microorganisms and preservedfoodstuffs for water molecules, the microorganism tissues undergo plas-molysis. Aqueous solutions containing 30% sucrose do not ferment, and60% solutions are resistant to all bacteria but Zygosaccharomyces.

Maltose: a slightly hygroscopic disaccharide of mild and pure sweet impres-sion. Its solutions have low viscosity. Its color is stable regardless of tem-perature.

Starch syrups: these result from starch saccharification. The saccharificationcan be completed in various stages. The first sweet product, maltotetraosesyrup (RS = 0.25), is viscous. As the saccharification proceeds, the viscosityof syrups declines and their RS increases. Syrups are water-soluble, do notretrograde, and are readily digested. Glucose syrup, the final product ofsaccharification, may be converted by isomerization into fructose syrups(Table 5.4) or hydrogenated into D-sorbitol. Apart from the sweetness andlow energetic value (17.5 kJ/g), the texturizing and filling properties ofsyrups are utilized in practice.

Malt extract from barley malt obtained by aqueous extraction: contains 4 to5% sucrose; spare amounts of D-glucose, D-fructose, and maltose; proteins;and mineral salts.

Maple syrup and maple sugar from juice of Acer saccharium maple trees:contains 98% saccharides, 80 to 98% of which is sucrose.

Sugar alcohols (D-sorbitol, D-xylitol, and D-mannitol): perfectly water-sol-uble, soluble in alcohol, and more stable at low and high pH values thansaccharides. Their sweet taste lasts for a prolonged time and is accompaniedby a cool impression. They metabolize without insulin. The energetic valuesof xylitol, sorbitol, and mannitol are 17, 17, and 8.5 kJ/g, respectively.


Saccharides 121

Therefore, they can be used as sweeteners for diabetics and consumers withobesity. Because of hygroscopicity, they are used as food humectants.

Compounded sweeteners, blends of various sweeteners: the composition ofsuch blends depends on the purpose for which they are designed. Usually,they are blends of various saccharides, but sorbitol, sugar syrups, and evenmalic acid are also compounded.

Honey: this natural product has a composition depending on the harvest time,geographical region, and origin and kind of flowers from which the nectarwas collected. Even the variety of insects is a factor. Fructose, glucose, andmaltose constitute approximately 90% the total sugar content. There is alsoa rich variety of free amino acids and other organic acids, minerals, pig-ments, waxes, enzymes, and pollen. The latter may create allergic reactions.Honey may contain toxic components from poisonous plants, although thereare several poisonous plants that give nonpoisonous honey. In some coun-tries, mainly in Eastern Europe, aqueous solutions of honey are fermentedinto honey-flavored wine (mead). Fermentation involves 1 volume of boiledhoney with 0.5 to 4 volumes of water. The fermentation of concentratedsolutions provides a soft beverage with up to 18% alcohol and dry productresulting from the fermentation of diluted honey solutions.

5.5.2 COLORANTS

Sugars are utilized for generation of caramel, a brown colorant for food (Tomasiket al. 1989). For this purpose, sugar is burned (caramelized). Various additives(caustic soda, caustic sulfite, ammonia, and their combinations) catalyze this process.In laboratory tests some proteogenic amino acids and their sodium and magnesiumsalts proved to be suitable catalysts (Sikora and Tomasik, 1994). Catalysts acceleratethe process and decrease caramelization temperature, usually to the region between130 and 200°C providing, simultaneously, their good tinctorial strength. Productsprepared by noncatalyzed burning sugars at 200 to 240°C have poor tinctorialstrength and serve as food flavoring.

There is a concern about harm from the free radical character of caramels. How-ever, they were proven (Barabasz et al., 1990) to be nonmutagenic. Thermal processingof saccharide- and polysaccharide-containing foodstuffs results in development of

TABLE 5.4Saccharide Content (%) in Various Starch Syrups

Syrups

Glucose conversion

Saccharide Low High Very high Maltose Fructose

Glucose 15 43 92 10 7–52Fructose — — — — 42–90Maltose 11 20 4 40 4Higher saccharides 48 13 2 28 3–6



brown color; it originates from caramelization and, in the case of the polysaccharides,dextrinization. Brown-colored dextrins, even if they contain free radicals, are non-mutagenic because such free radicals are unusually stable (Ciesielski and Tomasik,1996). Depending on the catalyst used, caramels differ in their isoelectric point. Ifcolored matter does not match the isoelectric point of the caramel, micelles of thecaramel irreversibly discharge and the caramel separates. Four types and severalclasses of caramels with widely different properties are manufactured. This varietyprovides a selection of a proper colorant to all types of foodstuffs.

5.5.3 FLAVOR AND AROMA

Burning of sugar in noncatalyzed processes results in the formation of particularlyhigh amounts of furan-2-aldehyde and its derivatives. They constitute the flavor andaroma typical of caramel. Many foodstuffs (meat, fish, bakery products, potato, cocoa,coffee, and tobacco) on thermal treatment (baking, roasting, frying) develop specificaromas. They are volatile derivatives of pyrazine, pyrrole, and pyridine formed onthermal reactions of saccharides and proteins, nucleotides, and amino acids.

Saccharides and polysaccharides—starch and cellulose (Bączkowicz et al., 1991),pectins (Sikora et al., 1998), and hemicelluloses (Tomasik and Zawadzki, 1998)—heated with amino acids develop scents specific to polysaccharides, amino acids, andreaction conditions. Thus, supplementation of saccharides and polysaccharides withamino acids and proteins, as well as supplementation of protein-containing productswith saccharides, can be useful in generation, modification, and enrichment of flavorand aroma of foodstuffs and tobacco. Scents of plants are developed on the thermalreaction of saccharides with α−hydroxy acids (Sikora et al., 1997).

5.5.4 TEXTURE

Concentrated aqueous solutions of carbohydrates form viscous liquids. That propertyis most commonly utilized in practice for texturizing foodstuffs. Intermolecular inter-actions between the same (Mazurkiewicz and Nowotny-Ro. żańska, 1998) and differentsaccharides (Mazurkiewicz et al., 1993; Obanni and BeMiller, 1997; Lii et al., 2002a;Gibiński et al., 2005) and changes in water activity (Mazurkiewicz et al., 2006) areinvolved. Blending of various saccharides and polysaccharides can result in theformation of numerous edible glues and adhesives. Such interactions are commonlyutilized in texturization of puddings, jellies, and foams. Some oligosaccharides andthe majority of polysaccharides form hydrocolloids, which build up their own mac-rostructure. They give an impression of jelly formation, thickening, smoothness,stabilization against temperature and mechanical shock, aging, and resistance onsterilization and pasteurization. Plant gums, pectins, and alginates are particularlywillingly utilized for this purpose (Lai and Lii, 2004; Ramsden, 2004). Such propertiescan be controlled by the addition of salts because various metal ions form Werner-type complexes with saccharides and polysaccharide ligands. Formation of the cal-cium ion–sucrose complex, commonly utilized in sucrose manufacture, illustrates thatphenomenon well. The effect of the metal ions in texturization is particularly visiblein the case of anionic polysaccharides (potato amylopectin, pectin, alginates, carra-geenans, furcellaran, xanthan gum, and carboxylic starches from starch oxidation).


Saccharides 123

The texturizing effect of a given saccharide or polysaccharide and its variousblends is developed as a function of the time necessary for the formation of a gelnetwork (a physical cross-linking). The pH and temperature may also be essentialfactors. If protons (pH <7) or hydroxyl anions (pH >7) and temperature do not evokeany structural changes in the interacting species, the texturizing effect is reversiblein pH and temperature. If retrogradation does not take place, the texturizing effectis also reversible in time.

Saccharides, oligosaccharides, and polysaccharides also form complexes withproteins and lipids. Such complexes contribute to the texture of foodstuffs.

Apart from combinations of natural saccharides, oligosaccharides, and polysac-charides, chemically modified polysaccharides are also utilized for texturization.Frequently, phosphorylated starches are used as gelling agents. Cross-linked starchesare also important in that respect. The degree of cross-linking is essential. It shouldnot exceed 0.2. Among cross-linked starches, those esterified with phosphoric acidare particularly favored. They are available by reacting starch with meta- and ortho-phosphates as well as POCl3 and PCl5. At the same degree of substitution, phospho-rylated potato starch is superior and phosphorylated cornstarch is the poorest.

A starch sulfate ester is used as a thickener and emulsion stabilizer. It is a typicalanionic starch used as a component of anionic starch–protein complexes constitutingmeat substitutes (Tolstoguzov, 1991, 1995). Other anionic starches, as well as pectins,alginic acid, carrageenans, furcellaran, heparin, xanthan gum, and carboxymethylcellulose are also used in food texturization (Clark and Ross-Murphy, 1987; Dejew-ska et al., 1995; Grega et al., 2003; Schmitt et al., 1998; Lii et al., 2002c, 2003c,2003d; Najgebauer et al., 2003, 2004; Zaleska et al., 1999, 2000, 2001a, b, 2002a, b).

Among many available modified polysaccharides, application of only a few ofthem is legal under the food laws of certain countries. Some restrictions are put onthe method of their manufacture and the purity of such products.

The replacement of saccharide sweeteners (first of all, sucrose) in food withvarious natural and synthetic sweeteners of very high RS (currently, mainly saccharin,aspartame, and cyclamates) is a task. It is also demanded by consumers looking forlow-calorie foods. Diabetics are also looking for food free of insulin-requiringsaccharides and polysaccharides. Following such demands, problems are encoun-tered in providing the anticipated texture of sweet products manufactured withoutsaccharides (Mazurkiewicz et al., 2001).

5.5.5 ENCAPSULATION

Various foodstuffs lose their original, desirable flavor, aroma, taste, and color onprocessing. It is a common result of evaporation of volatile compounds or decom-position of certain food components under the influence of oxygen or light. In thismanner the quality of foodstuffs decreases. In order to avoid such effects, volatileand unstable products are either protected in processed sources or, after processing,foodstuffs are supplemented by fragrances, colorants, and other components. Suchgoals are met by encapsulation and supplementation of microcapsule enclosed addi-tives. Saccharides are suitable for making such microcapsules. Compression ofadditives (guest molecules) with a saccharide forming the matrix of the microcapsule



(the host molecule) is a common practice. It is beneficial if there are some other-than-mechanical interactions between the guest and host that decrease the rate ofevaporation or reaction of the guest from the microcapsule. Granular starch canencapsulate guests in capillaries between granules; gelatinized starch, amylose, andamylopectin can trap certain molecules inside helices generated in contact with theguest molecules.

Coacervation or coprecipitation of host and guest, and suspension of the guestmolecule in polysaccharide gels, followed by drying, is another common procedure.Microcapsules can be made of preswelled granular starches. Potato starch is superiorfor this purpose for its granular size and easy swelling (Lii et al., 2001b; Korus etal., 2003). Lipids can be encapsulated in starches through short microwave heatingwith granular starches (Kapuśniak and Tomasik, 2005).

α−, β−, and γ−cyclodextrins are the most effective compounds for microencap-sulation of food components (Szejtli, 1984, 2004). Cyclodextrins take the form oftoruses with cavities of 0.57, 0.78, and 0.95 nm, respectively. Their height is0.78 nm. The upper and bottom edges of the toruses carry secondary and primaryhydroxyl groups, respectively. All hydroxyl groups reside on the external surfaceof the toruses making cyclodextrins hydrophilic. Simultaneously, their cavity inte-rior is hydrophobic.

Cyclodextrins are water-soluble hosts for hydrophobic guests. The formation ofinclusion complexes is controlled by the dimensional compatibility of the guest andhost cavity. Commercially available dextrins are, in fact, inclusion complexes ofcyclodextrins with two water molecules closing the entrance to the cavity. Theformation of cyclodextrin inclusion complexes is reversible and, therefore, is gov-erned by concentration of the guests competing for a place inside the cavity.

5.5.6 POLYSACCHARIDE-CONTAINING BIODEGRADABLE MATERIALS

There is a growing concern about fully biodegradable plastics—packing and wrap-ping foils, containers, equipment of fast-food restaurants, disposable bags, andsuperabsorbents.

Currently, several products made of polyethylene modified into biodegradablematerial are in use throughout the world. Biodegradability of such materials is affordedby admixture of 6 to 15 natural components, such as starch, cellulose, wood, or proteinsinto polyethylene. Polyurethane foams used as thermal insulators and packing materialscontain up to 20% starch. The level of starch in copolymers of ethylene with vinylchloride, styrene, or acrylic acid may reach 50%. Of course, the effect of biodegradationof such materials has more aesthetic significance than ecological. Although degradationof the finely pulverized synthetic portion of such materials is accelerated, it still takesseveral decades for depolymerization to come to its end.

Apparently, the simplest biodegradable plastics could be prepared of starch solelyby its compression up to 106 kPa, provided starch was moisturized up to its naturalwater-binding capacity (~20w-%) (Kudła and Tomasik, 1991).

Following the idea of full biodegradability of materials, attention has been paidto the compositions of plain carbohydrates with either unmodified or modifiedproteins and of modified carbohydrates with unmodified or modified proteins. Such


Saccharides 125

compositions are processed to generate carbohydrate–protein complexes. The ther-modynamic and electrical compatibilities of components should be reached in orderto afford superior functional properties of the materials. Because of the chemicalnature of proteins (cationic character), carbohydrates should be anionic, that is, ondissociation, the negative charge should be left on the polysaccharide moiety. TheCOOH, PO3H2, and SO3H groups provide such properties. Whenever modificationof a carbohydrate is required to make it anionic, the degree of derivatization shouldnot exceed 0.1. It should neither increase the hydrophilicity of the product nor, ifpossible, decrease its molecular weight.

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Gibiński, M. et al. Thickening of sweet and sour sauces with various polysaccharide combi-nations, J. Food Eng., submitted, 2005.

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129

6

The Role of Proteinsin Food

Zdzis

ł

aw E. Sikorski

CONTENTS

6.1 Chemical Structure....................................................................................... 1306.1.1 Introduction ...................................................................................... 1306.1.2 Amino Acid Composition and Sequence ........................................ 1306.1.3 Hydrophobicity................................................................................. 133

6.1.3.1 Average Hydrophobicity................................................... 1336.1.3.2 Surface Hydrophobicity.................................................... 133

6.2 Conformation ............................................................................................... 1346.2.1 The Native State............................................................................... 1346.2.2 Denaturation ..................................................................................... 136

6.3 Functional Properties ................................................................................... 1386.3.1 Introduction ...................................................................................... 1386.3.2 Solubility .......................................................................................... 139

6.3.2.1 Effect of the Protein Structure and Solvent ..................... 1396.3.2.2 Effect of pH and Ions....................................................... 1406.3.2.3 Importance in Food Processing........................................ 141

6.3.3 Water-Holding Capacity................................................................... 1416.3.4 Gelling and Film Formation ............................................................ 141

6.3.4.1 The Gel Structure ............................................................. 1416.3.4.2 Interactions of Components.............................................. 1426.3.4.3 Binding Forces and Process Factors ................................ 1436.3.4.4 Importance in Food Processing........................................ 144

6.3.5 Emulsifying Properties..................................................................... 1456.3.5.1 The Principle..................................................................... 1456.3.5.2 Factors Affecting Emulsifying ......................................... 1466.3.5.3 Determination of Emulsifying Properties ........................ 146

6.3.6 Foaming Properties .......................................................................... 1476.4 Proteins as Important Components in Foods .............................................. 148

6.4.1 Muscle Proteins................................................................................ 1486.4.2 Milk Proteins.................................................................................... 1496.4.3 Egg Proteins ..................................................................................... 1516.4.4 Legume Proteins .............................................................................. 1526.4.5 Cereal Proteins ................................................................................. 1526.4.6 Mycoprotein ..................................................................................... 153


130


6.5 Effects of Heating ........................................................................................ 1536.5.1 Introduction ...................................................................................... 1536.5.2 Rheological Changes ....................................................................... 1546.5.3 Changes in Color ............................................................................. 1566.5.4 Development of Volatile Compounds.............................................. 1566.5.5 Reactions at Alkaline pH................................................................. 156

6.6 Oxidation...................................................................................................... 1586.7 Enzyme-Catalyzed Reactions ...................................................................... 160

6.7.1 Introduction ...................................................................................... 1606.7.2 The Plastein Reaction ...................................................................... 1606.7.3 Transglutaminase Catalyzed Reactions ........................................... 1626.7.4 Proteolytic Changes in Milk Proteins.............................................. 1646.7.5 Role of Enzymes in Muscle Foods ................................................. 1646.7.6 Other Enzymatic Changes in Food Proteins ................................... 167

6.8 Chemical Modifications ............................................................................... 1676.8.1 Introduction ...................................................................................... 1676.8.2 Alkylation......................................................................................... 1686.8.3 Acylation .......................................................................................... 1696.8.4 N-Nitrosation.................................................................................... 1706.8.5 Reactions with Phosphates .............................................................. 171

References.............................................................................................................. 172

6.1 CHEMICAL STRUCTURE

6.1.1 I

NTRODUCTION

Proteins are linear condensation products of various

α

-L-amino acids (a.a.), whichdiffer in molecular weight, charge, and polar character (Table 6.1), bound bytranspeptide linkages. They differ also in the number and distribution of various a.a.residues in the molecule. The chemical properties and size of the side chain, as wellas the sequence of the a.a., affect the conformation of the molecule, that is thesecondary structure containing helical regions,

β

-pleated sheets and

β

-turns, thetertiary structure or the spatial arrangement of the chain, and the quaternary structureor the assembly of several polypeptide chains.

The conformation affects the biological activity, nutritional value, and functionalrole of proteins as food components.

6.1.2 A

MINO

A

CID

C

OMPOSITION

AND

S

EQUENCE

The proportion of each of the various a.a. residues, calculated as a percentage of thetotal number of residues, ranges in most proteins from 0 to about 30%. In extremecases it may even reach 50%. Among the 225 residues of the molecule of phosvitinin egg yolk, there are 122 Ser, most of them phosphorylated, SerP. The typicalsequences of phosvitin are . . .Asp-(SerP)

6

-Arg-Asp. . . and . . .His-Arg--(SerP)

6

-Arg-His-Lys. . . . In collagens, the content of Gly, Pro, and Ala is 328, 118, and 104residues/1000 residues, respectively. Grain prolamines are very rich in Glu (up to


The Role of Proteins in Food

131

55%) and Pro (up to 30%). Paramyosin, abundant in the muscles of marine inver-tebrates, is rich in Glu (20 to 24%), Asp (12%), Arg (12%), and Lys (9%). Mostfood proteins, however, do not differ very much in their a.a. composition. Generally,the content of acidic residues is the highest, and that of His, Try, and sulfur containinga.a. is the lowest. However, the number of residues capable of accepting a positivecharge is often higher, especially in plant proteins because about 50% of the side-chain carboxyl groups are amidated.

The composition affects the value of food proteins as the source of essential a.a.Most proteins of meat and fish muscles have very high biological value, while cerealproteins are generally poor in Lys. Several major grains are deficient also in Thr,Leu, Met, Val, and Trp. In most collagens there are no Cys and Trp residues. Severalmilk proteins, as well as proteins of other origin, contain short a.a. sequencescorresponding to different peptides known for their biological activity.

The sequence of the a.a. residues in the polypeptide chains is critical for thebehavior of the proteins in food systems. The antifreeze fish serum glycoproteins,which contain several a.a. sequences Thr–X

2

–Y–X

7

, where X is predominantly Ala

TABLE 6.1Selected Properties of Proteinogenic Amino Acids

Amino acid Abbreviation pK

a1

pK

a2

pK

R

Isoelectricpoint

pI

Side chainhydrophobicity

(ethanol

→

water)kJ/mol

Glycine Gly 2.34 9.60 6.0 0.0Alanine Ala 2.34 9.69 6.0 3.1Valine Val 2.32 9.62 6.0 7.0Leucine Leu 2.36 9.60 6.0 10.1Isoleucine Ile 3.26 9.68 6.0 12.4Proline Pro 1.99 10.60 6.3 10.8Phenylalanine Phe 1.83 9.13 5.5 11.1Tyrosine Tyr 2.20 9.11 10.07 5.7 12.0Tryptophan Trp 2.38 9.39 5.9 12.5Serine Ser 2.21 9.15 13.60 5.7 0.2Threonine Thr 2.15 9.12 13.60 5.6 1.8Cysteine Cys 1.71 8.35 10.28 5.0 4.2Methionine Met 2.28 9.21 5.7 5.4Asparagine Asn 2.02 8.80 5.4 0.04Glutamine Gln 2.17 9.13 5.7 0.4Aspartic acid Asp 1.88 3.65 3.65 2.8 2.2Glutamic acid Glu 2.19 4.25 4.24 3.2 2.3Lysine Lys 2.20 8.90 10.56 9.6 6.2Arginine Arg 2.18 9.09 12.48 10.8 3.1Histidine His 1.80 5.99 6.00 7.5 2.1

Note

: pK

a1

, pK

a2

, and pK

R

are the negative logarithms of the dissociation constants of the acidic, basic,and R groups of a.a. in aqueous solution


132


and Y a polar residue, have the ability to interact with ice crystals. The molecule of

β

-casein has a polar N-terminal region (residues 1–43) with a charge of –16, andan apolar fragment, containing 34 of the total number of Pro residues. This sequencefavors the temperature-, concentration-, and pH-dependent associations into thread-like polymers, stabilized mainly by hydrophobic adherences. Lysosyme, a basicprotein of egg white and other organisms, containing four –S–S– cross-links in asingle polypeptide, retains its enzymatic activity in acidic solution even after heatingto 100

°

C. The Bowman-Birk trypsin inhibitor consists of 71 a.a. residues in onepolypeptide chain with loops due to seven –S–S– bonds, and is characteristic for itshigh thermal stability. The bovine serum albumin has one SH group and 17 intramo-lecular –S–S– bridges per molecule.

Many a.a. residues undergo posttranslational enzymatic amidation, hydroxyla-tion, oxidation, esterification, glycosylation, methylation, or cross-linking. Somesegments of the original polypeptide chains may be removed (Figure 6.1). Modifiedresidues present in a given protein can be used for analytical purposes, such ashydroxyproline (ProOH), which is characteristic for collagens.

FIGURE 6.1

Posttranslational modifications in collagen (From Sikorski, Z.E.,

Chemical andFunctional Properties of Food Components

, Sikorski, Z.E., Ed., Technomic Publishing Co.,Inc., Lancaster, PA, 1997. With permission.)

Polyrybosome

Hydroxylases

Procollagen GlycosyltransferasesEndopeptidases

Tropocollagen

Collagen fibers



133

Posttranslational modifications may result in covalent attachment of variousgroups to the proteins. They may change the ionic character of the molecule, forexample the phosphoric acid residues or some saccharides. The residues involvedin phosphorylation and binding of saccharide moieties are Ser, Thr, LysOH, ProOH,His, Arg, and Lys. Among the highly phosphorylated proteins is

α

S

-casein. In thecentral region of

α

S1

-casein, SerP occurs in sequences . . .SerP-Ala-Glu. . ., . . .SerP-Val-Glu. . ., . . .SerP-Glu-SerP. . ., and . . .SerP-Ile-SerP-SerP-SerP-Glu. . . . Such dis-tribution favors oligomer formation due to hydrophobic interactions of the apolarfragments of the molecules, while the charged sequences are exposed to the solvent.High saccharide content is characteristic for the allergenic glycoproteins of soybeans(up to about 40%), several egg white proteins (up to 30%), albumins of cereal grains(up to 15%), whey immunoglobulin (up to 12%), and collagens of marine inverte-brates (up to 10%). In

κ

-casein there is a hydrophobic N-terminal part (residues 1to 105) and a hydrophilic macropeptide (106 to 169), or a glycomacropeptide, witha saccharide moiety (0.5%) composed of N-acetylneuraminic acid, D-galactose, N-acetylgalactosamine, and D-mannose residues.

6.1.3 H

YDROPHOBICITY

6.1.3.1 Average Hydrophobicity

The nonpolar character of an a.a. can be expressed by hydrophobicity, that is, changeof the free energy F

ta

accompanying the transfer of the a.a. from a less polar solventto water. Exposure of an a.a. with a large hydrocarbon side chain to the aqueousphase results in a corresponding decrease in entropy due to structuring of wateraround the chain. The hydrophobicity of the side chain of an a.a. is:

F

tr

= F

ta

– F

tGly

where F

tGly

is the hydrophobicity of GlyThe average hydrophobicity F

tav

of a protein can be estimated as

F

tav

=

Σ

F

ta

/n

where n is the number of a.a. residues in the protein molecule.It is not possible to predict the conformation and behavior of a protein in solution

on the basis of F

tav

. However, proteins of high F

tav

yield bitter hydrolysates.

6.1.3.2 Surface Hydrophobicity

The interior of the native molecule of a globular protein contains most of thehydrophobic a.a. residues. However, some of them form hydrophobic clefts or occuron the surface as patches of various sizes.

Phe, Tyr, and Try in food proteins can be monitored by measuring the intrinsicfluorescence. They absorb ultraviolet radiation and emit fluorescence in the follow-ing order:


134


The intensity of fluorescence and the wavelength of maximum intensity dependupon the polarity of the environment. Thus the Try residue located in a nonpolarregion emits fluorescence at 330 to 332 nm, while at complete exposure to water,the emission wavelength is 350 to 353 nm. Furthermore, electron withdrawinggroups, like carboxyl, azo, and nitro groups, as well as different salt ions, have aquenching effect on fluorescence. Measurements of intrinsic fluorescence and offluorescence quenching have not found, however, wide application in hydrophobicitydeterminations, as they are restricted to the effect of aromatic a.a. residues.

The simplest and most commonly used are hydrophobic probes based on thephenomenon that the quantum yield of fluorescence of the compounds containingsome conjugated double-bond systems is in a nonpolar environment about 100 timeshigher than in water. Thus hydrophobic groups can be monitored by aromatic oraliphatic probes and fluorescence measurements. Most often used is 1-anilinonaph-thalene-8-sulfonate (ANS) (Formula 6.1) and

cis

-parinaric acid (CPA) (Formula 6.2).Also the binding of triacylglycerols or sodium dodecylsulphate may be determined.

(6.1)

(6.2)

6.2 CONFORMATION

6.2.1 T

HE

N

ATIVE

S

TATE

Proteins in a natural environment fold spontaneously from an extended form L, tothe native conformation N, which is affected by the primary structure:

L

↔

N

This is accompanied by a decrease in free energy:

–RTlnK =

∆

G =

∆

H – T

∆

S

where R = gas constant, T = temperature, H = enthalpy, S = entropy, and K =equilibrium constant (K = [N]/[L]).

Phe 260 nm 283 nmTyr 275 nm 303 nmTry 283 nm 343 nm

NH

SO3-

CH3 4CH2 (CH = CH) 2(CH )7 COOH



135

The conformation of proteins in solutions is affected by hydrogen bonds andhydrophobic effects. The hydrogen bonds between water and hydrophilic residues leadto enthalpy changes, while the effects of nonpolar groups in the aqueous environmentbring about changes in entropy. This is reflected in the total free energy change

∆

G

t

∆

G

t

=

∆

H

p

+

∆

H

w

– T

∆

S

p

– T

∆

S

w

where the subscripts

p

and

w

refer to protein and water, respectively.Various forces stabilize the native conformation. The dipole–dipole interactions,

depolarization, and dispersion forces are significant only at very close distance r ofthe atoms because the energy of interactions decreases with r

–6

. The hydrogen bonds,abundant in proteins, differ in energy from approximately 2 to about 12 kJ/Mol,depending on the properties and positioning of the groups involved. The strength ofthe H-bonds does not depend significantly on temperature, but increases with pres-sure. The energy of the ionic bonds is affected by the dielectric constant and mayreach in the hydrophobic core of a globular protein about 21 kJ/Mole between theionized residues of Asp and Lys. The energy of hydrophobic interactions increaseswith temperature and decreases with increasing pressure. Covalent bonds other thanthose in the polypeptide chain, although of highest energy, are generally very limitedin number. However, some proteins rich in such bonds may have high thermalstability, for example, mature collagens containing different cross-links generated inreactions of the oxidized

ε

-NH

2

group of Lys and LysOH

REACTION 6.1

Prot NH2 Prot CH

O

lysyl

oxidase

Prot Prot

O H

C

CH

OH

CH Prot

O H

C

ProtCCH

Prot NH2

ProtO

HC C

H

OCH2 Prot+

N

NH

CH2

Prot

CHProt CH Prot

CH

N

Prot

N

N

CH2 Prot

CHProt CH Prot

C

HO

ProtCH2

N

N

H2O


136


and in the Maillard reaction of the saccharide moieties of the molecules, as well asproteins containing many –S–S– bridges, for example, several proteinase inhibitors.

A very significant effect on the properties of proteins is exerted by their quater-nary structures and micellar associations.

Soybean glycinin, composed of 6 basic and 6 acidic subunits has a structure oftwo superimposed rings. In each ring the 3 acidic and 3 basic subunits are arrangedalternatively. Thus ionic interactions are possible both within each ring and betweenthe rings. Because the conformation of the oligomer is buttressed by noncovalentforces, the addition of urea and changes in pH and ionic strength lead to dissociationof the protein into subunits.

In unheated milk the caseins are present as a colloidal dispersion of particlescontaining about 6 to 7% calcium phosphate, known as micelles, and as smallerparticles without calcium phosphate. These forms are in equilibrium, which isaffected by temperature, pH, and the concentration of Ca

2+

. In fresh milk about 80to 90% of the mass of caseins is in the micellar form. The micelles are porous andhydrated, have a diameter ranging from a few to about 600 nm, and a weight averagemolecular mass of 600 MDa. Numerous investigations have resulted in variousmodels of the structure of casein micelles. According to one group of models, themicelles are formed from several hundred subunits called submicelles differing incomposition and size. In these models the subunits are either linked by calciumphosphate or the calcium phosphate is located as discrete packages within thesubmicelles. According to the nanocluster model there are no subunits, but thepolypeptide chains form a matrix in which calcium phosphate nanoclusterlike par-ticles are embedded (Figure 6.2). The outer parts of the micelles are occupied byhydrophilic polypeptide chains, including the macropeptide of

κ

-casein and Ca

2+

-sensitive peptides, and form a hairy layer (Holt and Rogi

ń

ski, 2001)

6.2.2 D

ENATURATION

The native conformation of proteins is generally stabilized by a small amount ofenergy. The net thermodynamic stability of the native structure of many proteins isas low as about 40 to 80 kJmole

–1

. The unfolding enthalpy of metmyoglobin andlysozyme is about 285 and 368 kJ mole

–1

, respectively. Therefore ionizing radiation,shift in pH, change in temperature or concentration of various ions, or addition ofdetergents or solvents, may cause dissociation of the oligomers into subunits, unfold-ing of the tertiary structure, and uncoiling of the secondary structure (Figure 6.3).These changes are known as denaturation.

Exposure of the a.a. residues originally buried in the interior of the moleculechanges the pI, surface hydrophobicity, and the biochemical properties of proteins.Denaturation may be reversible, depending on the degree of deconformation andenvironmental factors. This may affect the results of assays of enzyme activity usedas a measure of, for example, the severity of heat processing in food operations. Formonitoring milk pasteurization, the determination of

γ

-glutamyltransferase can beused because the enzyme undergoes complete inactivation after 16 s at 77

°

C, andno reactivation has been evidenced (Zehetner et al., 1995).



137

Generally food processing causes irreversible denaturation followed by reactionsof the thermally denatured proteins with other components in the system. This secondstep may lead to loss in food quality, but the denaturation may have beneficial ordetrimental effects in foods. The main effects comprise changes in pI, hydration,solubility, viscosity of solutions, biological activity, and reactivity of a.a. residues.

FIGURE 6.2

The calcium phosphate nanocluster model of a casein micelle. Substructure arisesfrom the calcium phosphate nanocluster-like particles in the micelles (dark spheres). There is asmooth transition from the core to the diffuse outer hairy layer that confers steric stability onthe micelle. (Courtesy Holt, C. and Rogi

ń

ski, H.,

Chemical and Functional Properties of FoodProteins,

Sikorski, Z. E., Ed., Technomic Publishing Co., Inc., Lancaster, PA, 2001.)

FIGURE 6.3

Protein denaturation (a) native molecule, (b) molecule in a changed conforma-tion with ruptured disulfide bridges and ionic bonds, (c) denatured molecule with randomlyextended polypeptide chains. (From Sikorski, Z.E.

Chemical and Functional Properties ofFood Components

, Sikorski, Z.E., Ed., Technomic Publishing Co., Inc., Lancaster, PA, 1997.With permission.)

sss

s

s

s

ss

M2+

(a) (b) (c)


138


6.3 FUNCTIONAL PROPERTIES

6.3.1 I

NTRODUCTION

The functional properties important for the food processor are attributes, which at theproper concentration of the respective components or additives and at appropriateconditions, provide for the desirable characteristics of the product. These propertiesof proteins are displayed in interactions with the surrounding solvent, ions, otherproteins, saccharides, lipids, and numerous other components, as well as in surfacephenomena. The most important properties in food processing can be roughly groupedas seen in Table 6.2. They affect the appearance, color, juiciness, mouth feel, andtexture of a large variety of foods, as well as cutting, mincing, mixing, formation ofdough, fibers, foils, and bubbles, shaping, and transporting of food materials.

The term

functional

with respect to food has, since the mid-1980s, had one moremeaning. It has been used to describe foods or food ingredients that provide foradditional health benefits to consumers beyond those of satisfying the basic nutritionalrequirements, that is, they aid specific bodily functions. Such functional componentshave been found predominantly in foods of plant origin, especially oat products as asource of

β

-glucans; soybean, containing various protease inhibitors, phytosterols,saponins, phenolic acids, phytic acid, and isoflavones; flaxseed rich in

α

-linolenic acidand lignan precursors; garlic, providing diallyl sulfide; broccoli and other cruciferousvegetables, which are rich sources of glucosinolates; citrus fruits containing folate,fiber, and limonoids

;

tea, particularly green tea; and red

wine and

grapes

with theirpolyphenolic constituents. Foods of animal origin also contain functional constituents.Fish, especially marine fish, are rich in n-3 fatty acids;

dairy products are one of thebest sources of Ca, and the fermented commodities supply large beneficial microbialpopulations; beef contains conjugated linoleic acid, and in many proteins there aresequences of a.a., which after digestion yield peptides known for their various biolog-ical activities. In a large number of experiments, the following beneficial health effectsof functional foods have been found: cancer chemopreventive, antihypertensive, cho-lesterol-lowering and reducing the risk of coronary heart disease, increasing cellularantioxidant defense, contributing to maintenance of a healthy immune function, pre-venting osteoporosis, antibiotics, and improving intestinal microbial balance.

TABLE 6.2Functional Properties of Proteins Displayed in Interactions with Different Food Constituents

Interactions with

Water Water and proteins Lipids or gases

Wet ability Viscosity inducing Emulsifying abilitySwelling Gelling Emulsion stabilizationRehydration Fiber forming Foaming abilityWater holding Dough forming Foam stabilizationSolubility Membrane forming



139

The functionalities can be modified by using enzymatic and chemical processesthat change the structure of the proteins. They depend also on the pH, ionic strength,and temperature in the food system. By better understanding of the tertiary structureof many food proteins, it should also be possible to modify their functionality usinggenetic engineering. To evaluate the functional properties of some proteins in dif-ferent systems, the quantitative structure–activity relationship approach may beapplied (Nakai and Li-Chan, 1988).

6.3.2 S

OLUBILITY

6.3.2.1 Effect of the Protein Structure and Solvent

The solubility or extractability of proteins is often defined in food chemistry as thepercent of the total quantity of protein contained in the food material that can beextracted by water or a suitable solvent in specified conditions. It depends on theproperties of the protein and of the solvent, pH, concentration and charge of otherions, ratio of sample weight to solvent volume, particle size of the sample, durationof extraction, and on temperature. Generally, proteins rich in ionizable residues, oflow surface hydrophobicity, are soluble in water or dilute salt solutions, for example,the proteins found in egg white. Proteins abundant in hydrophobic groups readilydissolve in organic solvents. The classification of cereal proteins into albumins,globulins, prolamines, and glutelins soluble in water, dilute salt solutions, 60 to 80%aliphatic alcohols, and 0.2% NaOH, respectively, may also be used for character-ization of other proteins. Stabilization by cross-linking is of crucial importance, forexample, the solubility of collagen from different connective tissues depends on thetype and age of the tissue. Young tropocollagen can be solubilized in a neutral orslightly alkaline NaCl solution, tropocollagen containing intramolecular covalentbonds are soluble in citric acid solution at pH 3, while mature collagen with covalentintermolecular cross-links is not soluble in cold, dilute acids and buffers. It can,however, be partially solubilized in a highly comminuted state or after several hoursof treatment in alkaline media. Differences in solubility are essential for variousprocedures of isolation of individual proteins and groups of proteins from foods(Kristinsson, 2001).

Denaturation may decrease solubility, for example, the fish protein concentrateproduced by extraction of minced fish with a boiling, azeotropic solution of isopro-panol, is scarcely soluble in water. In organic solvents, due to their low dielectricconstant, the energy of interactions between charged a.a. residues is higher than inwater. This may favor unfolding of the molecules and exposing of the hydrophobicresidues, which cannot be counterbalanced by entropy forces. Thermal denaturationfollowed by aggregation due to interactions of the surface-exposed reactive groupsleads generally to loss in extractability. On the other hand, if heating brings aboutdeconformation of the quaternary and tertiary structures it may increase the solubil-ity, for example, in collagen.

Adding antioxidants to defatted soy flour prior to alkaline extraction enhancesthe solubility of the protein isolate in proportion to the decrease in oxidation of thiolgroups (Boatright and Hettiarachchy, 1995).


140


6.3.2.2 Effect of pH and Ions

In water solutions, the solubility of proteins has a minimum at the pI (Figure 6.4). Atsuch pH there is no electrostatic repulsion between the molecules, hence the hydrationlayer alone cannot prevent aggregation. Although the attraction of water dipoles byionized groups of opposite charges in a.a. residues largely offsets the electrostaticbinding between the ions; the net balance of the attraction and change in solvententropy favors salt-bridge formation. At pH values below or above pI of the proteinthe solubility increases due to repelling of the positive or negative ions, as well as dueto increased interaction of the charged polypeptide chains with water dipoles. The pIof a protein may shift slightly with a changing concentration of salts in the solution.

The effect of ions on the solubility of a protein depends on their ionic strength µ,and their effect on the surface tension of the solvent, as well as on the dipole momentand the decrease in the molecular surface area of the protein upon aggregation. Variousions, depending on their size and charge, favor or lower the solubility of proteins. In thelow range of concentration, that is, µ = 0.5 to 1.0, the solubility increases with theconcentration of neutral salts. This is known as salting in. The ions have a screeningeffect on the charged protein molecules. Being surrounded by water dipoles they add tothe hydration layer, which favors solubilization of the macromolecules. At higher con-centration the effect depends mainly on the ability of the salts to affect the water struc-tures. Salts containing polyvalent anions at appropriate concentrations precipitate proteinfrom solutions; this is known as salting out. Most widely used for this purpose is(NH4)2SO4 or Na2SO4. Various proteins precipitate from solution at different percentages

FIGURE 6.4 The effect of pH on the solubility of various proteins. (From Sikorski, Z.E.(Ed.), Chemical and Functional Properties of Food Components, Second Edition, CRC Press,Boca Raton, FL, 2002. With permission.)


The Role of Proteins in Food 141

of saturation of the salts. Thus salting out is used for protein fractionation because bychanging the concentration of the salt, various fractions of the mixture of proteins presentin the original solution can be precipitated.

6.3.2.3 Importance in Food Processing

The solubility versus pH curve can be used for selecting parameters for extractionof proteins from different sources. Adding a required amount of salt to meats duringcutting and mixing in a silent cutter is a prerequisite for extracting myofibrillarproteins from the tissue structures, and for forming a sausage batter of adequatequality. CaCl2 is used to precipitate the whey proteins, while CaSO4 coagulates soyproteins in tofu manufacturing in soybean processing.

Solubility also contributes to gelling and emulsifying. It may also be requiredfor efficient use of various protein isolates as functional food additives in productsdiffering in pH and salt content. The loss in solubility due to abusive treatment isoften indicative of protein denaturation and subsequent cross-linking. Therefore,solubility data, if used to characterize commercial protein products, should be deter-mined using standardized procedures (Kołakowski, 2001).

6.3.3 WATER-HOLDING CAPACITY

The ability of many foods to retain water is affected by the involvement of proteinsin different structures. In meat and fish tissues, the state of water depends oninteractions of water structures with proteins and other solutes. Furthermore, becauseof the fibrous nature and compartmentalization of the muscle, water is also held inthe meat by physical entrapment. Alterations in the spatial arrangement of theproteins and in the integrity of tissue structures caused by biochemical and processingfactors are responsible for shrinking or swelling of the material, and thus for retentionor exudation of water. Classical investigations on the effect of pH, divalent cations,postmortem changes, freezing and thawing, heating, salting, polyphosphates, andcitrates on the water-holding capacity (WHC) of meat were made by Hamm (1960).

WHC has a large impact on the texture and juiciness of meat and fish products.A decrease in WHC brings about excessive cooking loss and thawing drip. Changesin WHC may also be used for evaluating the effect of processing on the structureof proteins and on the quality of muscle foods. To be used as a quality index, WHCshould be determined using standardized procedures. They are based either onmeasurements of loss of water from the original sample due to centrifugation,pressing, or capillary force, or on measuring the quantity of liquid separated underthe action of a force from a sample with added water or aqueous solution.

6.3.4 GELLING AND FILM FORMATION

6.3.4.1 The Gel Structure

A gel consists of a three-dimensional lattice of large molecules or aggregates, capableof immobilizing solvent, solutes, and filling material. Food gels may be formed byproteins and polysaccharides, which may participate in gel formation in the form of



solutions, dispersions, micelles, or even in disrupted tissue structures, as in meatand fish products.

Generally gelation is a two-step phenomenon (Damodaran, 1989). The first stepusually involves dissociation of the quaternary structure of the protein, followed byunfolding. In several proteins, heating to about 40°C is sufficient. Some fish proteinsols turn slowly into gels even at 4°C. Preheating at 25 to 40°C, called ashi or setting,is applied prior to cooking in manufacturing gelled, elastic fish–meat products.During setting, the endogenous transglutaminase catalyzes the formation of cross-links between myosin heavy chains. In ovalbumin solutions, gelling starts at 61 to70°C. In the second step, at higher temperature, the unfolded molecules rearrangeand interact, initially usually with their hydrophobic fragments, forming the lattice.Ovalbumin gels increase in firmness when heated up to about 85°C. Subsequentcooling generally stabilizes the gel structure. If the rate of the structuring stage islower than that of denaturation, the unfolded molecules can rearrange and form anordered lattice of a heat-reversible, translucent gel. Too rapid interactions in thedenatured state lead to an irreversible coagulum due to random associations withinsoluble, large aggregates.

In the gel network there are zones where the polymers interact, and largesegments where the macromolecules are randomly extended. The lattice is respon-sible for the elasticity and the textural strength of the product. In multicomponentgels all constituents may form separate or coupled networks, or one component, notinvolved in network formation, may indirectly affect the gelling by steric exclusionof the active molecules. Such exclusion increases the concentration of the activecomponent in the volume of the solution where the gel is formed. In composite gelsmade from minced squid meat at 1.5% NaCl, the added carrageenan and egg whiteform separate networks, which support the structure, made of squid proteins, whileadded starch fills the lattice, swells, and retains water (Gomez-Guillen et al., 1996).K-carageenan added to pork batter formulation increases the hydration and thermalstability of the gels (Pietrasik et al., 2005). Proteins and polysaccharides that haveopposite net charge, when in mixed solutions, may form different soluble andinsoluble complexes held by ionic bonds. Lipid-filled milk protein gels containingsmall fat globules with a narrow particle size distribution have smooth texture andhigh shear modulus.

The formation of a three-dimensional network of partially unfolded moleculesis also crucial for preparing proteinaceous films. These films are usually made froma protein solution at pH values far from the pI. The process comprises controlleddenaturation of the molecules due to heating or shear, addition of plasticizers,degassing, casting or extruding through a nozzle, and drying to evaporate the solvent.

6.3.4.2 Interactions of Components

The structure of gels depends upon the components and the process parameters.Proteins containing over 30% hydrophobic residues form coagulum-type gels, suchas hemoglobin and egg-white albumin. The gelling-type proteins contain lesshydrophobic residues and are represented by some soybean proteins, ovomucoid,and gelatin.



The interactions of different macromolecules may decrease the gel strength, mayhave no influence on the rheological properties of the gel, or may have a synergisticeffect. Casein micelles in a whey protein matrix may enhance or decrease gelling,depending on pH. Heat coagulation of sarcoplasmic proteins impairs the gelation ofactomyosin in gels made from the meat of pelagic fish.

In minced heated fish products the proteinase catalyzed softening known asmodori may be decreased by adding protease inhibitors from potato, bovine plasma,porcine plasma, or egg white. Also, inhibitors from various legume seeds are effectiveagainst fish muscle proteinases (Benjakul et al., 2001; Matsumoto and Noguchi,1992). The impact of other factors may be controlled by applying optimum process-ing parameters.

6.3.4.3 Binding Forces and Process Factors

The hydrophobic interactions prevail at higher temperatures and probably initiatethe gel lattice formation, while hydrogen bonds increase the stability of the cooledsystem. The electrostatic interactions depend upon pH, charge of the molecules,ionic strength, and divalent ions. Intermolecular –S–S– bridges, as well as covalentbonds formed due to the activity of transglutaminases, may also add to the gelformation. In gelled fish products, –S–S– bonding occurs during cooking at about80°C (Hossain et al., 2001). Gels stabilized mainly at low temperature by hydrogenbonding are heat-reversible; that is, they melt due to heating and can be set againby cooling. Gels stabilized by hydrophobic interactions and covalent bonds areheat stable.

Depending on the properties and concentration of the protein, ionic strength,and pH, even a coagulum-type gel such as that of ovalbumin can be melted byrepeated heating and set again when cooled (Shimizu et al., 1991). Heat-inducedgels may melt under increased pressure at room temperature, while cold-set gels ofgelatin are resistant to such conditions (Doi et al., 1991). Optimum ionic strengthand concentration of Ca2+ are required for producing well-hydrated, heat-set gelsfrom whey proteins.

There is generally a pH range at which the gel strength in the given system isthe highest. It depends on the nature of the polymers participating in cross-linking,and increases with protein concentration. At the pI of the proteins, due to lack ofelectrostatic repulsion, the rate of aggregation is usually high, leading to less ordered,less expanded, and less hydrated gels. In heat-induced gels made of minced, water-washed chicken breast muscle at pH 6.4 and low NaCl concentration, the myofibrils,insoluble at such conditions, form local networks of aggregates with large voidsbetween them. Increasing pH to 7.0 results in a gel with an evenly distributed networkof myofibrils and an additional network of fine strands, smaller intramyofibrillarspaces, increased stress and strain values, and higher water-holding capacity (Fengand Hultin, 2001).

The ovalbumin gel has optimum rheological properties at pH 9, while at pH <6it is brittle and has low elasticity. Transparent ovalbumin gel can be made by heatingat pH other than pI at a certain salt concentration. In a two-step procedure, transparentgels can be made from ovalbumin, bovine serum albumin, and lysozyme over a



broad range of salt concentrations by heating the protein solutions first without salt,and after cooling by repeated heating in the presence of added salt (Tani et al.,1993). The pH range for gelation of whey proteins is 2.5 to 9.5, although near thepI, which is about 5, the gels are opaque, coarse, and may turn into curdlikecoagulum. In the neutral to alkaline pH, gels made of fat-free whey protein isolatesor purified β-lactoglobulin are translucent, smooth, and elastic. In acid conditions,if high shear force is applied for a short time at denaturation temperature, wheyproteins aggregate to microparticles. This leads to well-hydrated gels of a smooth,nonelastic texture, similar to that of a fat emulsion. In a slightly alkaline environmentat temperatures above 60°C, insoluble aggregates are formed due to the denaturationof β-lactoglobulin, the major component of whey proteins. The rheological propertiesof whey protein gels, at different pH, depend also on the concentration of Ca2+.

The –S–S– bridges are responsible for the thermal stability of some gels. Suchbonds add to the elasticity of heat-set whey protein gels at neutral to alkaline pH,but not in acidic conditions when the thiol has low reactivity (Jost, 1993). Ascorbicacid improves the formation of heat-set gels of ovalbumin and fish proteins byundergoing rapid oxidation to dehydroascorbic acid, which affects polymerizationby intermolecular –S–S– bridges. In making edible films from wheat gluten, theexposed SH groups of the protein, heat-denatured in an alkaline solution, form –S–S–cross-links due to air oxidation during drying (Roy et al., 1999).

6.3.4.4 Importance in Food Processing

Gelling is important for the quality of comminuted-type, cooked sausages, and forgelled fish products. The gel strength of such commodities is mainly affected by theproperties of myosin and the processing conditions. Comminuting of the meat withsalt results in unfolding of the myosin microfibrils and increases the surface hydro-phobicity. This leads to hydrophobic associations in the lattice structure. Heating to50 to 80°C favors deconformation of the myosin heads (Figure 6.5) and theirinteractions. Although myosin has the highest gel-forming ability of all muscleproteins, the whole myofibrillar protein fraction, the sarcoplasmic, and the connectivetissue proteins are also capable of gelation. The overall gel strength depends on theconcentration and interactions of different proteins.

Edible films may be used for their barrier properties to prevent the migration ofwater, oil, oxygen, and volatile aroma compounds between food and the environment.The coatings prevent oxidative browning of sliced fruits and vegetables due to theirantioxidant properties. The oxygen radical scavenging capacity of films made of

FIGURE 6.5 A schematic representation of the myosin molecule. (From Sikorski, Z.E. (Ed.),Chemical and Functional Properties of Food Components, Technomic Publishing Co., Inc.,Lancaster, PA, 1997. With permission.)



commercial concentrated whey protein powder has been found to be higher than thatof coatings based on calcium caseinate. Addition of carboxymethyl cellulose to theformulation increases the antioxidant capacity of the products (Le Tien et al., 2001).Films can also find application as enzyme supports and carriers of food ingredients.For edible sachets used for delivering premeasured quantities of ingredients in foodprocessing, the heat-seal ability of the material is important. Antimicrobial agentsadded to edible coatings used for food protection may affect the mechanical strengthand barrier properties of the material (Ko et al., 2001). Different films have uniquefunctional properties best suited to fulfill the needs of specific food applications.

The films can be made of proteins, or as a composite material with saccharidesor lipids. For these purposes collagen, gelatin, casein, total milk proteins, wheyproteins, wheat gluten, corn zein, water-soluble fish proteins, and soy proteins areused. The barrier properties, appearance, tensile strength, thermal stability, and theheat-seal ability of the products depend on the characteristics and proportions of thegelling components, interactions and contents of plasticizers, and conditions offabrication. Films made of whey protein are transparent, bland, and flexible, havevery high oxygen, oil, and aroma barrier properties in an environment of lowhumidity, and are poor protection against water vapor migration. Some of theircharacteristics depend on the degree of thermal denaturation of the protein beforecasting. Increasing the degree of denaturation by heating leads to higher tensilestrength and insolubility, and to lower oxygen permeability of the films (Perez-Gagoand Krochta, 2001). Water vapor permeability may be decreased by laminating alipid layer over a protein film or by uniformly dispersing the lipid in the proteinaceouscomponent. The result depends on the properties and amount of added lipid.

6.3.5 EMULSIFYING PROPERTIES

6.3.5.1 The Principle

Proteins help to form and stabilize emulsions, such as dispersions of small liquiddroplets in the continuous phase of an immiscible liquid. The decrease in the diameterof the droplets due to agitation increases exponentially the interfacial area. The work(W) required for the increase in surface area (∆A) can be decreased by lowering thesurface tension (z):

W = z∆A

due to attachment of proteins to the droplets. The protein film around the lipidglobules, with its electrostatic charge and steric hindrance, prevents flocculation,that is, formation of clusters of globules and thus rapid creaming due to the actionof gravitational force:

V = 2r2g∆P / 9µ

where V = velocity of the droplet, g = gravitational force, ∆P = difference in densityof both phases, µ = viscosity of the continuous phase, and r = radius of the dropletor cluster of droplets.



Stable films around the fat globules also prevent the coalescence of the dispersedphase, that is, joining of the fat globules to form a continuous phase. Furthermore,soluble proteins increase the viscosity of the dispersing phase, thus reducing the rateof creaming and coalescence.

The efficiency of proteins as emulsifiers depends upon their surface hydropho-bicity and charge, steric effects, elasticity or rigidity, and viscosity in solution.Globular proteins, which have stable structures and are very hydrophilic, are goodemulsifiers only when unfolded. However, the emulsifying properties do not increaselinearly with the hydrophobicity of the protein, as they depend on the hydro-phile–lipophile balance (HLB), which is defined as

HLB = 20Wh/Wt

where Wh = weight of hydrophilic groups, Wt = total weight of the molecule. The emulsifiers with HLB <9 are regarded as hydrophobic, HLB 11 to 20 as

hydrophilic, and HLB 8 to 11 as intermediate. There is an effect of protein solubility,as the molecules must migrate to the surface of the fat globules. However, incomminuted sausage batters in the presence of salt, the insoluble proteins also mayparticipate in the formation of fat dispersions. After a few minutes of homogeniza-tion, about 90% of initially insoluble meat proteins of the stroma can be found inthe emulsion layer (Nakai and Li-Chan, 1988). The quantity of protein required forstabilization of an emulsion increases with the volume of the dispersed phase andwith the decrease in diameter of the droplets. The concentration of proteins forminga monomolecular layer at the interface is on the order of 0.1 mg/m2, and the effectiveconcentrations are in the range 0.5 to 20 mg/m2. For a high rate of film formation,the required concentration of protein in the emulsion may be as high as 0.5 to 5%.

6.3.5.2 Factors Affecting Emulsifying

The pH of the environment affects the emulsifying properties by changing the solu-bility and surface hydrophobicity of proteins, as well as the charge of the protectivelayer around the lipid globules. Ions alter the electrostatic interactions, conformation,and solubility of the proteins. However, in many foods, mainly comminuted meatbatters, the concentration of NaCl is considerably high for sensory reasons. Thussmall changes in the salt content within the accepted range may have no significanteffect on the properties of proteins. Heating to about 40 to 60°C, causing partialunfolding of the protein structure without loss in solubility, may induce gelation ofthe protective layer, as well as decrease the viscosity of the continuous phase. There-fore moderate heating may improve the emulsifying properties of proteins.

6.3.5.3 Determination of Emulsifying Properties

Several procedures are used to determine the efficiency of proteins in emulsifyinglipids and the stability, which the proteins impart to the emulsions.

The emulsifying capacity is represented by the volume of oil (cm3) that isemulsified in a model system by 1 g of protein when oil is added continuously to a



stirred aliquot of solution or dispersion of the tested protein. It is determined bymeasuring the quantity of oil at the point of phase inversion. The latter can be detectedby a change in color, viscosity, or electrical resistance of the emulsion, or the powertaken by the stirrer engine. The emulsifying capacity decreases with increasing con-centration of protein in the aqueous volume. It is affected by the parameters ofemulsification depending on the equipment, as well as by the properties of the oil.

The emulsion stability is measured as the final volume of the emulsion aftercentrifuging the initial volume or standing for several hours at specified conditions.It may also be determined as the quantity of oil or cream separated from the emulsion,or the time required for the emulsion to release a specified quantity of oil.

6.3.6 FOAMING PROPERTIES

Food foams are dispersions of gas bubbles in a continuous liquid or semisolid phase.Foaming is responsible for the desirable rheological properties of many foods, suchas the texture of bread, cakes, whipped cream, ice cream, and beer froth. Thus foamstability may be an important food quality criterion. However, foams are often anuisance for the food processor, such as in the production of potato starch or sugar,and in the generation of yeast. Residues of antifoaming aids in molasses maydrastically reduce the yield in citric acid fermentation.

The gas bubbles in food foams are separated by sheets of the continuous phase,composed of two films of proteins adsorbed on the interface between a pair of gasbubbles, with a thin layer of liquid in between. The volume of the gas bubbles maymake up 99% of the total foam volume. The contents of protein in foamed productsis 0.1 to 10% and of the order of 1 mg/m2 interface. The system is stabilized bylowering the gas–liquid interfacial tension and formation of rupture-resistant, elasticprotein film surrounding the bubbles, as well as by the viscosity of the liquid phase.The foams, if not fixed by heat setting of the protein network, may be destabilizedby drainage of the liquid from the intersheet space due to gravity, pressure, orevaporation, by diffusion of the gas from the smaller to the larger bubbles, or bycoalescence of the bubbles resulting from rupture of the protein films.

Factors facilitating the migration of the protein to the interface and formationof the film are important for foaming. The foaming capacity or foaming power—the ability to promote foaming of a system, measured by the increase in volume—is affected mainly by the surface hydrophobicity of the protein. The stability andstrength of the foam, measured by the rate of drainage and the resistance to com-pression, respectively, depend on the flexibility and the rheological properties of thefilm. Other components of the system, mainly salts, sugars, and lipids, affect thefoam formation and stability by either changing the properties of the proteins or theviscosity of the continuous phase. The standard whey protein concentrates, whichcontain 4 to 7% of residual milk lipids, have significantly inferior foaming propertiesthan lipid-free isolates.

Excellent foaming ability is characteristic for the egg-white proteins, especiallyovalbumin, ovotransferrin, and ovomucoid. Chilling of the egg white below roomtemperature or the presence of sugar or lipids decrease the foaming, pH less than 6increases the foaming capacity, while heating of the dried proteins at 80°C for a few



days before use increases the foam stability. The foaming of whey protein isolatecan be improved by addition of Ca2+ or Mg2+. The ions are effective only immediatelyafter the salts are added. According to Zhu and Damodaran (1994), the ions mightcause unfolding and polymerization of the proteins at the interface via ionic linkages.On the other hand, prolonged incubation of the isolate solution with the salts slightlyreduces the film-forming ability, possibly by promoting aggregation and micelliza-tion of the protein.

6.4 PROTEINS AS IMPORTANT COMPONENTSIN FOODS

6.4.1 MUSCLE PROTEINS

The meat of slaughter animals, fish, mollusks, and crustaceans is predominantlyused to prepare different dishes, a variety of canned, smoked, marinated, salted, anddried products, as well as a large number of sausage assortments and gels. In theseproducts the functional properties of the muscle proteins are responsible for thedesirable sensory attributes. However, an increasing proportion of the raw material,that is less suitable for producing high-quality products by the meat, poultry, andfish industry, is used for manufacturing protein concentrates, preparations, andhydrolysates. These products can be incorporated into various food commodities fornutritional reasons and as functional ingredients.

Much research effort has been devoted to work out optimum parameters forproducing different protein concentrates from fish and krill. While the products havehigh nutritional value and many are tasteless and odorless, some, manufactured indenaturing conditions, lack the desired functional properties. A good example is thefish protein concentrate obtained by extraction with a hot, azeotropic solution ofisopropanol. On the other hand, a concentrate of myofibrillar proteins known assurimi, produced mainly from fish and to a lesser extent from poultry and meat, ishighly functional.

Surimi is originally a Japanese product obtained by washing minced fish fleshseveral times with fresh water. This treatment removes most of the sarcoplasmicproteins, including enzymes and pigments, nonprotein nitrogenous compounds, var-ious odorous substances, other soluble components, and fat. The remaining myo-fibrillar proteins have higher gel-forming ability than the original protein mixture.Nowadays, fish surimi is produced mainly onboard vessels. The typical commercialsurimi is made from Alaska pollock. In order to prevent deterioration of its functionalproperties during frozen storage, different cryoprotectants, predominantly saccha-rides, are added prior to freezing (Figure 6.6). Surimi is used mainly for manufac-turing traditional Japanese gelled products, obtained by mixing it with salt, grinding,forming, and steaming or broil-cooking (kamaboko) or frying (tempura). Anothergrowing outlet is the production of a variety of fabricated specialties, includingmolded (shrimp-type) and fiberized (crab-leg type) shellfish analogs. The mostimportant topics in this area in the last two decades have been the suitability of fishof different lean and fatty species, including freshwater fish, as raw material forsurimi (Luo et al., 2001); mechanisms of protein changes in frozen stored material



and selection of optimum cryoprotectants; the effect of different ingredients on thetexture and water binding of various gelled products; as well as other factors affectinggelation of surimi (Niwa, 1992; Jiang et al., 1998).

6.4.2 MILK PROTEINS

Milk proteins have found various applications in formulated foods, and as meatextenders. Initially only the caseins were used, but the recovery of whey proteinsand their fractions is economically also feasible.

FIGURE 6.6 A flow sheet of the process used for manufacturing surimi.

Water

Water

Water

Cryoprotectants

3x

Wastewater,Scales

Wastewater,Scales

Heads, Viscera

Skin, Fins,and Bones

Water-SolubleComponents

Scraps ofSkin, Scales

Surplus Water

Meat Separatingand Mincing

Washing

HeadingGutting

Washing

Leaching

Draining

Refining

Dewatering

Mixing

Surimi

White Fish



Least soluble is acid casein. Its solubility and foaming properties can beimproved by glycosylation of the ε-NH2 groups. Sodium and calcium caseinatesprepared by acid precipitation and neutralization are soluble, very heat stable, havehigh WHC, emulsifying, foaming, and gelling ability. Rennet casein has low solu-bility in the presence of Ca2+. Precipitates produced by heat denaturation of the wheyproteins and coprecipitation with casein by the addition of acid or Ca2+ salts aremore soluble and have higher nutritional value than the acid and rennet casein.

Different whey protein concentrates and isolates are manufactured by complex-ing with phosphates or polysaccharides, by gel filtration, ultrafiltration, or heatdenaturation. At pH of about 4.2 and 55 to 65°C α-lactalbumin undergoes isoelectricprecipitation due to dissociation of the Ca2+ and the hydrophobic interactions(Bramaud et al., 1995). Other minor whey proteins precipitate, while the solubleβ-lactoglobulin is separated, concentrated by ultrafiltration, neutralized, andspray-dried. The product has superior WHC and gelling properties in meat prod-ucts. β-lactoglobulin, however, denatures and forms insoluble aggregates at temper-atures above 60°C at pH 8. This is a limitation for the use of whey protein isolatesin pasteurized products. The thermal stability of β-lactoglobulin can be improvedby controlled hydrolysis. Doucet et al. (2001) have shown that hydrolysis of awhey protein isolate by Alcalase to a degree, when only less than 16% and 4% ofβ-lactoglobulin and α-lactalbumin, respectively, remained unchanged, induced gelformation. Gelation was preceded by the formation of aggregates. The structure ofthe gel was stable over a range of temperature from 30 to 65°C. Other, undenaturedforms of whey proteins are also used as foam stabilizers and gelling agents; someare soluble under acid conditions. The purified α-lactalbumin fraction is more suit-able for infant food formulations than the whole whey protein concentrate becausehuman milk does not contain β-lactoglobulin.

Milk proteins have high nutritional value due to the very favorable a.a. compo-sition, and they do not contain significant amounts of antinutritional factors, exceptfor some allergenic activity.

Furthermore, several milk proteins have antimicrobial properties. The wheyprotein lactoferrin has antibacterial activity due to its iron-sequestering ability andthe strongly bactericidal N-terminal domain. Lactoperoxidase in the presence ofH2O2 in milk oxidizes thiocyanate (SCN–), derived from enzymatic hydrolysis ofcyanogenic glycosides from the cow’s feed, to different oxidized forms, which inhibitthe growth of bacteria due to oxidation of SH groups, NADH, and NADPH. In thepolypeptide chains of various milk proteins are some a.a. sequences that can beliberated as biologically active peptides with some positive effects on the functioningof the human organism. Generally these oligopeptides contain predominantly theresidues of hydrophobic a.a., Lys, and Arg. They are very resistant to the digestivepeptidases. Especially rich in biologically active peptides are β-lactoglobulin, α-lactalbumin, lactoferrin, αs1-casein, β-casein, and κ-casein. The peptides Val-Pro-Pro and Ile-Pro-Pro (known as inhibitors of the angiotensin I-converting enzyme),which decrease the systolic and diastolic blood pressure, have been isolated frommilk fermented in the presence of Lactobacillus helveticus. Many other fermentedmilk products have been found to contain other peptides capable of decreasing bloodpressure, including



Tyr-ProIle-Pro-AlaTyr-Leu-Leu-PheTyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-AsnMet-Pro-Phe-Pro-Lys-Tyr-Pro-Val-Gln-Pro-PheArg-Pro-Lys-His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-GlnArg-Pro-Lys-His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln

Various milk proteins contain, in their polypeptide chains, some a.a. sequencesthat correspond to those of peptides having opioid, immunomodulating, Ca2+ binding,antioxidant, antibacterial, or antiviral activity. Glycomacropeptide, the product ofhydrolysis of κ-casein, has the ability to bind some pathogenic bacteria, preventadhesion of bacteria and viruses, promote the growth of bifidobacteria, exert immu-nomodulatory activity, and cause gastric hyposecretion. The antihypersensitive, opi-oid, immunomodulatory, and Ca2+ binding milk peptides, the antiviral properties ofvarious milk components, as well as the antimicrobial activity of lactoperoxidase,lactoferrin, lactoferricins, casein peptides, and peptides from α-lactalbumin havebeen concisely treated by Holt and Rogiński (2001).

A large number of biologically active peptides have also been liberated bydifferent hydrolytic enzymes and acid hydrolysis from other sources, including theproteins of fish muscle, soybeans, corn, wheat, barley, and rice. Angiotensin I-converting enzyme inhibitory activity was also recently found in hydrolysates ofanimal by-products—defatted cracklings of pork and chicken feathers (Karamać etal., 2005).

6.4.3 EGG PROTEINS

Egg albumen is used in various food formulations because of its foaming propertiesand heat-gelling ability, while egg yolk serves as an emulsifying agent. The func-tional properties of egg proteins have been thoroughly treated by Ternes (2001). Thestrength of egg-white gels can be increased by preheating the dry protein at 80°Cbefore use (Kato et al., 1990a). In the gels formed by preheated egg white, themolecular weight of the aggregates is much smaller than in gels made of nonpre-heated proteins. Preheating of dry egg white also confers on the protein lowerenthalpy and temperature of denaturation. This results in increased flexibility of themolecules, leading to more cohesive interfacial films, that is, improved surfacefunctional properties. The more cohesive films composed of overlapping polypep-tides are more capable of expanding under stress than the films formed from non-preheated proteins (Kato et al., 1990b).

Egg-white and egg-yolk gels can also be prepared without heating by applyinghigh pressure (Hayashi et al., 1989). The albumen and the yolk from fresh eggs formstiff gels after exposure for 30 min to a pressure above 6,000 kg/cm2 and 4,000 kg/cm2,respectively. The pressure-induced gels are more adhesive, more elastic, and moredigestible than the boiled egg.

Lysozyme, which makes up 3.5% of total egg-white proteins and can be easilyseparated by ion-exchange techniques, is recognized as a safe, antimicrobial agent



to be used for food preservation. It is stable up to about 100°C, has maximum activityat pH 5.3 to 6.4, and inhibits several pathogenic bacteria, including Listeria mono-cytogenes, Clostridium botulinum, Yersinia enterocolitica, and Campylobacter jejuni(Kijowski and Leśnierowski, 1999).

6.4.4 LEGUME PROTEINS

Soybean proteins are used in a variety of traditional products, such as soymilk orfermented items, defatted flour, grits, concentrates, or isolates. The traditional prod-ucts are prepared according to procedures known in the Orient involving waterextraction, cooking, coagulation of the proteinaceous curd, roasting, and fermenta-tion, often in combination with other components. Different forms of concentratedor isolated protein products are prepared by milling, toasting, extraction of fat andsaccharides, and isolation of protein fractions. The antinutritional factors present insoybeans are usually inactivated or removed in processing. The “beany” or “painty”off-flavor in soymilk due to volatiles generated in lipoxygenase-catalyzed reactionsis prevented by thermal denaturation of the enzyme before or during grinding withwater (Kwok and Niranjan, 1995). The protein isolates may be tailor-made, that is,various fractions are separated in order to have the desirable a.a. composition andfunctional properties.

Grits, flours, and isolates for food applications are also produced from otherlegumes, mainly peanuts, beans, broad beans, and peas (Lampart-Szczapa, 2001).

6.4.5 CEREAL PROTEINS

Cereals are an important source of proteins because the grains contain, dependingon the species, from about 7.5 to 14% of N×6.25. They are used in foods mostly asflour—the main raw material for producing breads, noodles, cakes, and other com-modities of the baking industry. The biological value of cereal proteins is signifi-cantly lower than that of meat proteins because of a deficiency of lysine, and thecontent of methionine residues in the proteins of barley, maize, oats, rye, and wheatis also too low. The prolamins and glutelins present among the storage proteins ofwheat, rye, triticale, barley, and oats may evoke in some persons various symptomsof dietary intolerance. A very serious disease caused by gluten intolerance is celiacdisease, known also as celiac sprue. This enteropathy is caused by the gliadinpeptides altered by the human tissue transglutaminases. These peptides are recog-nized as foreign by local intestinal T cells. Such stimulation of the immune responseleads to accumulation of lymphocytes, plasma cells, and macrophages in the laminapropria and an increased number of lymphocytes in the surface layer of the epithe-lium, which results in shortening and flattening of intestinal villi. In patients whodo not exclude gluten from their diets, the disease may cause lymphoma of thegastrointestinal tract.

The most important role of wheat storage proteins in food processing is thatplayed by gluten in the formation of dough and in the texture of bread crumbs,noodles, and cakes (see Chapter 12). Cereals also contain different enzymes, whichcatalyze various changes in proteins, saccharides, lipids, and other constituents



during storage in the grains and flour, as well as during baking of bread and malting.A brief but comprehensive presentation of cereal grain proteins has been presentedby Wrigley and Bekes (2001).

6.4.6 MYCOPROTEIN

One of the results of research in the second half of the 20th century on single-cellprotein for human food and for animal feed was the development of the process ofmanufacturing mycoprotein. The product has been approved in several Europeancountries for general food use. It has the form of insoluble hyphae, typically 400 to700 µm × 3 to 5 µm with low frequency of branching. Mixing with proteinaceousbinders, flavorings, and colors, followed by forming and heating leads to meatproduct analogs. Mycoprotein can also be used in extruded commodities and as fatreplacement in yogurt and ice cream (Rodger, 2001).

6.5 EFFECTS OF HEATING

6.5.1 INTRODUCTION

Heating affects the conformation of proteins, enzyme activity, solubility, and hydra-tion, and leads to thermal and hydrolytic rupturing of peptide bonds, thermal deg-radation, and derivatization of a.a. residues, cross-linking, oxidation, and formationof sensory active compounds. Most of these reactions are reflected in foods indesirable or detrimental changes in color, flavor, juiciness, rheological properties,enzyme activity, and toxicity. These processes are affected by the temperature andtime of heating, pH, oxidizing compounds, antioxidants, radicals, and other reactiveconstituents, especially reducing saccharides. Some of the undesirable reactions canbe minimized by appropriate treatments. Stabilizers, such as polyphosphates andcitrate, which bind Ca2+, increase the heat stability of whey proteins at neutral pH.Lactose present in whey in sufficiently high concentrations may protect the proteinsfrom denaturation during spray drying (Jost, 1993).

The heat-induced unfolding of a protein usually proceeds in several steps becauseseparate domains of the molecule denature at different temperatures, depending onthe forces stabilizing their structure. The molecular transition temperature, that is,the point at which major change in conformation occurs, can be determined byvarious techniques, mainly by differential scanning calorimetry. The susceptibilityof proteins to thermal denaturation depends on their structure, predominantly on thenumber of cross-links, as well as on simultaneous action of other denaturing agents.Salt bridges, side chain–side chain hydrogen bonds, and a large proportion of resi-dues in α-helical conformation increase the thermal stability of many proteins(Kumar et al., 2000). In some proteins the first changes appear at 35 to 40°C. Thepeak maximum temperature of transition of myosin in the myofibrils ranges from43°C in cod muscle to 60°C in the bovine M. semimembranosus (Howell et al.,1991). Sodium chloride decreases the thermal stability of meat proteins by up toabout 30%. In the experiments of Fernandez-Martin et al. (2000), pressurization ofsausage formulations at 300 MPa at 10°C for 20 min significantly destabilized the



proteins. However, the thermal stability of proteins in formulations heated underhydrostatic pressurization was higher than that in control samples. The stabilizingeffect was related to the temperature of heating.

Heating of soluble collagen brings about uncoiling of the superhelix and unfold-ing and separation of the polypeptide chains from each other, if stable bonds do notbind them. This results in a decrease in viscosity of the solution. The denaturationtemperature Td can be measured in the range of rapid change in viscosity. Insolublecollagen fibers shrink due to heat denaturation by up to 75% of their original length.The shrinkage temperature Ts is usually about 20°C higher than Td. It increases withthe number of cross-links, and in fish collagen with the contents of PrOH. Thedenatured collagen turns into gelatin after prolonged boiling in water, due to partialhydrolysis. This phenomenon affects the rheological properties of cooked meatscontaining much connective tissue.

Thermal changes comprise loss in solubility due to aggregation of the proteins,for example, β-lactoglobulin and the immunoglobulins, or increase in solubilityresulting from breakdown of superstructures, as in collagen. Heating of many pro-teins to 105 to 140°C at low water content in conditions resembling those duringextrusion cooking leads to increased solubility. This concerns proteins that have anopen random coil structure and low number of –S–S– bonds and are not able toform many other covalent cross-links (Mohammed et al., 2000). Further effects ofheating include formation of gels, such as those in most types of sausages, kamaboko,meat gels, and several types of cheeses, development of gas-retaining structures asin dough and bread, hydrolytic changes, and alteration of the rate of proteolysis,modification of nutritive value, and inactivation of some allergens.

6.5.2 RHEOLOGICAL CHANGES

Proteins are primarily responsible for the texture of meat, poultry, fish, meat andfish gels, cheese, bread, cakes, and many other foods. The texture of muscle foodsis affected mainly by the

• Contents and cross-linking of collagen, and the morphological structureof the tissues, e.g., meat, fish, and squid

• Biochemical state of the muscle, that is, interactions of the proteins of themyofibril, such as those in rigor mortis, cold shortening, thaw rigor, aswell as the proteolytic changes during aging of meat

• Mechanical disintegration of the muscle structure

Abusive treatment during storage, processing, and preparation may lead to tough-ening and partial loss of the gel-forming ability of frozen stored fish, shrinking andtoughening of pasteurized ham, toughening and formation of grainy structure incasein curd, and separation of fat layers in sausages.

The thermal changes in meat commence at about 40°C, and some of the meatproteins in solution coagulate at that temperature. Further heating results in shrinkageof collagen (at 50 to 60°C), followed by gelatinization in a moist environment. Atabout 65°C, hardening of the myofibrils occurs. The final texture of the product



depends upon hardening of the myofibrillar structure and gelatinization of collagenin the given meat at the particular state of postmortem changes, heated accordingto a particular time and temperature regime. The rheological changes in meat andfish are reflected in drip loss (20 to 40% of the original weight), and shrinkage.

Wheat flour, when mixed with water, forms a dough that retains gas and setsdue to heating during the baking of bread. The agent responsible for these propertiesof wheat flour is gluten, which develops in mixing of the flour with water. Dry glutencontains 80 to 90% proteins, 5 to 10% saccharides, 5 to 10% lipids, and minerals.The gluten proteins are composed of 40 to 50% gliadin, 35 to 40% glutenin, and 3to 7% soluble proteins. During mixing with water the flour particles, containingstarch granules embedded in the protein matrix, are progressively hydrated and formthe viscoelastic dough. SH/S–S interchange reactions result in formation of inter-molecular –S–S– bridges. Generally the resistance to extension of the doughincreases to a peak value. Overmixing usually results in a decrease in resistance andis caused by excessive breaking of the –S–S– bonds, not accompanied by theformation of new intermolecular bridges. This ends in depolymerization of theprotein and thus increased solubility and decreased viscosity of the dough. Therequired mixing time can be controlled, within limits, by changing the pH, addingoxidants, or mixing in an inert atmosphere. The gas-retaining ability depends on theviscosity of the hydrated protein-carbohydrate-lipid system. Rice and corn flours donot decrease the gas diffusion rate as effectively, because the dough prepared fromthem is not as viscoelastic as dough that contains gluten. The relatively high gasretention of rye-flour dough is due to the viscosity of soluble pentosans. A similareffect can be achieved in gluten-free dough by adding surfactants, particularly naturalflour lipids, monoacylglycerols, or xanthan gum (Hoseney and Rogers, 1990). Duringbaking the expansion of the dough stops due to polymerization or cross-linking, asa result of SH/SS interchange reactions. The setting prevents further expansion ofthe dough and the collapse of the loaf of bread.

Very severe heating of foods, at much higher temperature and time than requiredfor sterilization, may lead to the formation of isopeptide cross-links between thefree NH2 group of Lys and the carboxylic group of Asp or Glu.

REACTION 6.2

- N ( -glutamyl) lysylamidecrosslink

HN CH C

O

CH2)2

COOH

(

NH2

CH2)4

CHHN C

O

(

+heating

HN CH C

O

CH2)2

C

O

CHN CH

CH2)4

NH

O

(

(



These reactions tighten the structure of the products and may decrease thebiological availability of Lys, as well as the digestibility of the proteins.

6.5.3 CHANGES IN COLOR

Pig, sheep, beef, and whale contain about 0.2 to 2.4, 4.5 to 5.5, 1 to 20, and 50 mgof myoglobin in 1g of muscle, respectively. The total content of chromoproteins inthe red muscles of fish ranges from a few to 20 mg/g, and is about 20 times higherthan in the white muscles. The skeletal muscles of whales contain as much as 5 gof MbFe(II)/100 g. Changes in the chromoproteins due to oxidation and reduction,denaturation, curing, and reactions with sulfur-containing compounds lead to desir-able or undesirable alterations in color.

Beef heated at 58 to 60°C internal temperature is rare, at 66 to 68°C mediumrare, at 73 to 75°C medium, and 80 to 82°C well done. Recommended end pointtemperature in pork is 77°C and in poultry 77 to 82°C.

The reactions of myoglobin in cured, heated meat resulting in the formation ofheat-stable nitrosylhemochromogen and a nitrite–protein complex proceed accordingto Killday et al. (1988) as follows:

In proteinaceous foods rich in saccharides or secondary lipid oxidation products,the Maillard reaction prevails. Various products of this reaction can be determinedand used as indicators of thermal changes in proteins (see Chapter 12).

6.5.4 DEVELOPMENT OF VOLATILE COMPOUNDS

Severe heating of proteinaceous foods leads not only to generation of flavor com-pounds due to the Maillard reaction, but also to thermal degradation of Met and Cysresidues in proteins, as well as those of different low-molecular-weight compounds.These reactions are discussed in Chapter 11 of this volume.

6.5.5 REACTIONS AT ALKALINE PH

Alkaline treatment is used for peeling of fruits and vegetables, for producing proteinisolates, for removing nucleic acids from single-cell protein preparations, and for

REACTION 6.3

+

nitrosyl myoglobinradical

NO2 Globin

NO

Fe2+heating

NO2- +

OH

Fe3+

NH

N

Globin

autoreduction

NO

NO

Fe2+

Globin

NH

Nreduction

NO

Fe2+

Globin (or Globin radical)

NH

N

OH-

metmyoglobin nitrosyl myoglobin nitrosylhemochromogen



inactivating mycotoxins and proteinase inhibitors. Severe changes in reactive a.a.residues take place in protein solutions and in foods at high pH even at temperaturesas low as 50°C. They lead to cross-linking and formation of nontypical a.a. residues.

Cross-linking is based on β-elimination, mainly in Cys, Ser, SerP, or Thr, followedby a nucleophilic addition of the ε-NH2 of Lys, δ-NH2 of ornithine, SH of Cys residues,or of NH3 to the double bond of dehydroalanine or 3-methyldehydroalanine:

Reactions of Cys at alkaline pH also liberate free sulfur and a sulfide ion:

REACTION 6.4

REACTION 6.5

HN CH C

CHR

Y

OOH-

H2O+ HN C C

CHR

O

Y+ -

dehydroalanine residue

where: R=H, CH3; Y=OH, OPO3H2, SH, SR+, SSR

HN C C

CHR

Y

O

HN C C

O

CH2

NH2

CH2)4

CHHN C

O

(

+

(

HN CH C

O

CH2

NH

CH2)4

CHHN C

O

HN CH C

O

CH2

S

S

CH2

CHHN C

O

HN C C

O

CH2

OH- -S

S

CH2

CHHN C

O

H2O+-

S

CH2

CHHN C

O

S+

-OH

-S

CH2

CHHN C

O

CH2

CHN C

O

H2O S -2++

HN C C

O

CH2

S

S

CH2

CHHN C

O



At a given temperature, the overall rate of reaction depends upon the rate ofβ-elimination and on the conformation of the protein. This is because the accessibilityof the dehydroalanine residue for the nucleophilic attack depends on the spatialarrangement of the reacting groups. The reaction can be inhibited by acylating thenucleophilic groups in proteins or by adding thiol compounds, which compete witha.a. residues for the dehydroalanine double bond.

Alkaline conditions favoring cross-linking in proteins also lead to racemizationof a.a.:

L-amino acid ↔ D-amino acid

That is, after the first step in the reaction sequence, the carboanions recombine withprotons to the L and D forms. The rate of racemization is affected mainly by the propertiesof the residues (it is the lowest in aliphatic a.a.) and on the structure of the protein.Generally the rate of the process is about 10 times higher in proteins than in free a.a.

Severe heating at alkaline pH may decrease the digestibility and biological valueof proteins that result from cross-linking and racemization. The rate of absorptionin the organism of some D-a.a. is lower than that of the corresponding L forms. Notall D-a.a. can be metabolized. Furthermore, some of the modified residues, mainlylysinoalanine, induce pathological kidney changes in experimental animals.

6.6 OXIDATION

Oxidation of a.a. residues in proteins is initiated in most proteinaceous foods by radicalsand different reactive forms of oxygen—singlet oxygen 1O2, super oxide anion radicalO2

–, and hydroxyl radical ⋅OH. These reactive oxygen species appear in the livingorganism as the result of natural oxidative metabolism. In food they are generated bylight, ionizing radiation, catalytic action of cations, especially heavy metals, pesticideresidues, and the activity of enzymes. Also lipid peroxides and other oxidation productsare involved. Polyphenols, present in many foods, are prone to oxidation to quinones byoxygen at neutral and alkaline pH. They can also act as strong oxidizing agents in differentproducts. Also H2O2, if abused as a bactericidal agent, such as in the treatment of storagetanks, packaging materials, or proteinaceous meals, may cause oxidation of proteins.

The effect of oxidative changes in proteins depends upon the activity of theoxidizing agent, the presence of sensitizers, such as riboflavin, chlorophyll, and eryth-rosine, temperature, and the reactivity of the a.a. residues. Several tissues containvarious prooxidants, including transition metals and hem pigments, lipooxygenases,

REACTION 6.6

R S- H2C C

C

NH

O

H+ R S CH2 C H

C

NH

O

+ +



and peroxidases, as well as endogenous antioxidants, such as glutathione, superoxide dysmutase, and catalase.

Abstraction of a hydrogen atom on the α-carbon or in the a.a. side chain of aprotein leads to the formation of protein radicals. These may polymerize with otherprotein or lipid radicals. Different scission products may also be formed. H2S andfree sulfur may be generated due to oxidation of the –SH group. The sulfur-contain-ing a.a. are converted to many oxidized compounds, including cysteine sulfenic,sulfinic and sulfonic acids, mono- and disulfoxides, and mono- and disulfones. Thea.a. most prone to oxidation in different model systems and in foods are Met, Cys,Trp, Tyr, and His. Oxidation of Trp leads to kynurenine (Friedman and Cuq, 1988):

and of His to Asp:

REACTION 6.7

REACTION 6.8

HN C O

CH2

CH

COOHH2N

CHO

H2N C O

CH2

CH

COOHH2N

OO

H2N COOHCH

CH2

N

R

O2

HNCH2

CHCOOHH2N H2N COOH

CH

CH2

NNCH2

CHCOOHH2N

kynurenine N-formylkynurenine

COOH

CH2

CHCOOHH2N

sensitizer

h , O2

N

NH

H2N COOHCH

CH2

+ C O

NH2

NH2

+ other products



In food systems the oxidation of proteins is often preceded by lipid oxidation,mainly by the primary lipid peroxide products or the radicals produced due to theirbreakdown (O’Grady et al., 2001).

The products of oxidation affect the flavor of foods, either directly or by reactingwith precursors. Their biological value depends generally upon the degree of changedue to scission, polymerization, and oxidation. Furthermore, in heavily oxidizedfoods the essential a.a. may become limiting in the diet. Formation of protein–proteinand protein–lipid cross-links decreases the digestibility of proteins.

6.7 ENZYME-CATALYZED REACTIONS

6.7.1 INTRODUCTION

Reactions in proteins and other nitrogenous compounds catalyzed by endogenousenzymes are responsible for many desirable and undesirable sensory attributes offoods—color, flavor, and texture. They also contribute to the generation of com-pounds that are nutritionally beneficial or may have detrimental effects on humanhealth. The use of added enzymes or enzyme sources is also an essential part ofmany traditional methods of food processing. Because the conditions of enzymaticreactions are much milder than those applied in chemical treatments, various addedenzymes are increasingly used to modify the functional properties of food proteins.

Among many enzymes involved in modification of proteinaceous foods, the mostimportant are probably various proteinases and peptidases. Proteolysis leads toincreased protein solubility and extractability by hydrolyzing large, highly cross-linked insoluble proteins into smaller units, removing some hydrophobic sequencesor splitting off nonprotein fragments of the molecules. Proteinase catalyzed reactionscan also increase the solubility of proteins by binding of hydrophilic a.a. residuesto the polypeptide chains.

Some proteins affect the sensory and functional properties of foods by exhibitingenzymatic activity in their natural environment or when added intentionally duringprocessing in the form of pure enzyme preparations, enzyme-rich materials, or startercultures of microorganisms. The enzymatic reactions involve not only changes inproteins, but also in lipids, saccharides, and a large number of other food constituents.The examples of effects involving protein changes include loss of prime freshnessin fish after catch, rigor mortis and tenderization of meat, ripening of salted fish,manufacturing of caviar, protein isolates and hydrolysates, softening of fish gels,formation of casein curd and ripening of cheese, fermentation in soybean processing,chill-proofing of beer, and proteolytic changes in wheat flour. Enzymatic modifica-tion of proteins in food systems has been treated exhaustively by Haard (2001) andrecently by Kołakowski (2005).

6.7.2 THE PLASTEIN REACTION

Enzyme catalysis can be used for transformation of different proteins into polypeptideswith molecular mass of up to 3 kDa known as plasteins, which may have a tailor-made a.a. composition and functional properties. These products can be synthesized



at a high concentration of an appropriate endopeptidase (E–OH) as a result oftranspeptidation in a mixture of peptides, generally protein hydrolysates, or byformation of peptide bonds with a.a. esters added to the reaction mixture. In the firstcase, two smaller peptides are formed due to hydrolysis of the intermediary acy-loenzyme R–CO–O–E, or in reaction with some other peptide H2N–R2 a plastein oflarger molecular weight is produced:

Addition of a.a. esters leads to incorporation of desirable a.a. residues into thestructure of the generated plasteins. The esters are used instead of free a.a. in orderto increase the nucleophilic character of the reacting amino group. The protectionof the carboxyl group can be removed after synthesis by hydrolysis in alkalineconditions (Figure 6.7).

The rate and yield of the plastein reaction depend on the pH in the reactionmixture, the properties of the endopeptidase, hydrophobicity of the attached a.a.residues, and the concentration of the substrates. At optimal temperature and pH

REACTION 6.9

FIGURE 6.7 Utilization of the plastein reaction.

R-CO-NH-R1 + E-OH R-CO-O-E + H2N-R1

H2O H2N-R2

R-COOH + E-OH R-CO-NH-R2 + E-OH

UndesirableAmino Acids,Impurities

Amino Acids,Small Peptides

Endopeptidases

Exopeptidases,Organic Solvent

Endopeptidases,Amino Acid Esters

Ethanol

Protein

Plasteins

Extraction

Hydrolysis

Plastein Reaction

Modificationof Peptides



conditions, 50% concentration of the substrates, and enzyme-to-substrate ratio of1:50, the reaction time is usually several tens of hours. Incubation of a proteinhydrolysate, concentrated to 30 to 40%, with ethyl esters of, for example, Lys, Met,or Trp, with an appropriate endopeptidase at pH 4 to 7 at about 37°C leads, after afew days, to the accumulation of peptides of 2 to 3 kDa enriched in the respectivea.a. residues. Also plasteins free of Phe residues can be obtained for phenylketonuricpatients. By hydrolysis of a protein in the presence of pepsin, which cleaves thepeptide bonds close to large a.a. residues, followed by further treatment of thehydrolysate by pronase, which liberates terminal, hydrophobic residues, the phenyl-alanine residues can be split from the peptide chain. The free phenylalanine can beremoved by gel chromatography, while the hydrolysate can be used for preparingplasteins suitable for phenylketonuric patients. The a.a. composition of the plasteinscan also be modified by selecting various proteins as the source of hydrolysates.

The rate of incorporation of hydrophobic a.a. residues is higher than that ofhydrophilic substrates. Therefore, the plastein reaction may serve to decrease thebitterness of the products by positioning the hydrophobic a.a. residues inside thelong polypeptide chains of the plasteins. Furthermore, the selective removal ofhydrophobic a.a. from the hydrolyzate decreases the intensity of its bitter taste.

6.7.3 TRANSGLUTAMINASE CATALYZED REACTIONS

Transglutaminase (TGase), or R-glutaminyl-peptide- γ-glutamyltransferase, has beenfound in many isoforms in a large number of organisms. It catalyses the transfer ofthe acyl group of glutamine residues in proteins or peptides on primary amines ora water molecule:

TGase performs, in plant and animal organisms, various catalytic, regulatory,signaling, and immunological functions, due to its protein cross-linking and modi-fying properties. It is also produced as an extracellular enzyme by Streptoverticilliumsp., Physarium sp., and other microorganisms. The red sea bream TGase, cloned inEscherichia coli, is an intracellular enzyme. Generally the activity of TGase of plantand microbial origin is independent on Ca2+, while the enzyme present in animal

REACTION 6.10

+ RN2H

O

2HNC2)2HC(torP

TGase+ 3HN

O

HOC2)2HC(torP

deamidation

crosslinking

TGase+ 3HNtorP4)2HC(

O

HNC2)2HC(torP

TGase+ 3HNR

O

HNC2)2HC(torP

acyl transfer

+ O2H

O

2HNC2)2HC(torP

+ torP4)2HC(N2H

O

2HNC2)2HC(torP



tissues is Ca2+ dependent. However, the Ca2+ requirement depends not only on thesource of the enzyme, but also on the type of substrate.

TGases occur in the form of a monomer, dimer, or tetramer, soluble in the cytosolor bound in mitochondria and lysosomes. Although they have maximum activity at50°C, they can be effectively used for modification of food proteins in the temper-ature range of 5 to 20°C. The optimum pH range for the activity of TGase of differentorigins is 6 to 9.5. The role of TGase in food processing is due to protein cross-linking, incorporation of Lys and amines, as well as deamidation of Gln residues.The effect of the enzyme activity expressed as cross-linking depends on the concen-tration of NaCl as well as on the properties of the protein substrate (Ashie and Lanier,2000). The TGase-mediated incorporation of amines into a polypeptide chain iseffective in TGase-specific sites, for example, when the Gln residues are locatedclose to Pro, Ser, Thr, or Tyr. Therefore the efficiency of the enzyme treatmentdepends not only on the total number of Gln residues in the protein molecule, butalso on their location in the polypeptide chain (Kędzior, 2004). The yield of thereaction products can be significantly increased by acylation of the Lys residues inthe protein. The deamidation activity of TGases of various origins is affected by theenzymes’ substrate specificity (Ohtsuka et al. 2001).

Endogenous TGase activity is responsible for the formation of ε(γ-glutamyl)lysinecross-links in dried fish, in frozen-stored surimi (Haard et al., 1994), and in poly-merization of myosin heavy chains during setting of surimi in the manufacture ofkamaboko (Kumazawa et al., 1995).

Commercially produced microbial TGase has found numerous applications infood processing. It can be used for increasing the gel strength of kamaboko madeof surimi produced from fish of low gel-forming ability, in manufacturing variousmeat commodities, and for improving the rheological properties of dairy products.TGase derived from Streptoverticillium can induce gelation of glycinin and leguminat 37°C, and the gels are more rigid and elastic than thermally gelled products(Chanyongvorakul et al., 1995). Cross-linking of wheat proteins, especially of thehigh-molecular-weight prolamines, may improve the baking properties of wheat flourthat had been deteriorated by proteolysis induced by proteases from some grainpathogens. TGase-mediated enrichment in Lys may increase the biological value ofwheat proteins, while blocking of some Gln residues may decrease the allergenicproperties of the peptides resulting from prolamine digestion. Reactions catalyzedby added TGase can lead to tailor-made protein preparations, such as edible filmsof defined barrier properties from whey proteins, and to covalent binding of saccha-rides to plant proteins rich in Gln residues (Colas et al., 1993). Ca2+-independentenzymes, added at optimum concentration to fish skin gelatin increases the meltingpoint, strength, and viscosity of the gels. The activity of TGase during storage ofthe product might change the rheological properties of the gel. It can, however, bearrested by heating to 90°C just after incubation of the gelatin solution with theenzyme (Gómez-Guillen et al., 2001). Cross-linking of proteins catalyzed by TGasedoes not impair the nutritional value of the products. Kączkowski (2005) recentlyreviewed the biological role and the possibilities of application of TGase as a protein-modifying factor in food processing.



6.7.4 PROTEOLYTIC CHANGES IN MILK PROTEINS

In cheese manufacturing, a crucial role is played by added chymosin, which cleavesthe glycomacropeptide off the κ-casein in milk acidified due to the activity of thestarter lactic acid bacteria. The loss of the protective action of the hydrophilicglycomacropeptide brings about the formation of the casein coagulum. Endogenousand bacterial proteinases catalyze extensive modifications in milk proteins. Plasmin,the alkaline serine proteinase associated with casein micelles and fat milk membrane,attacks β-casein and αs1-casein. Several endogenous plasmin activators and inhibitorscontrol the degradation of milk proteins. Enzyme activity is highest at pH 7.5, andis only slightly reduced during high-temperature, short-time pasteurization of milk.Extracellular proteinases produced by psychrotropic bacteria present in chilled milkhydrolyze different caseins and whey proteins. These enzymes release plasminogenand plasmine from casein micelles. Therefore, in cheese making, plasmine is notretained in the casein curd, but leaks into the whey. The bacterial proteinases aregenerally heat stable—some of them retain 90% of initial activity even after 9minutes of heating at 121ºC. They can cause a bitter note in UHT milk, age gelationof sterilized milk, and flavor defects in fermented products. Also, proteinases ofsomatic cells present in milk, especially from cows in late lactation, may decreasethe yield of cheese, lead to development of bitterness in pasteurized milk, andgelation in UHT milk after prolonged storage (Holt and Rogiński, 2001).

6.7.5 ROLE OF ENZYMES IN MUSCLE FOODS

The sensory quality of meat and seafood is significantly affected by the endogenousproteases. The lysosomes contain, among other enzymes, at least 12 cathepsins,which can exert a concerted action on proteins and peptides. The optimum pH forthe activity of cathepsins is generally in a low acidic range. However, many enzymesalso retain high activity at pH values 1 or 2 units away from the optimum. Cathepsinsare released from the lysosomes in stored beef into the sarcoplasm. Most of the meatcathepsins hydrolyze at least some proteins of the myofibrils. Proteolysis by cathep-sins has been regarded as one of the factors co-responsible for tenderization of meat.However, not all of these enzymes can have a significant effect on intact meat proteinsin the usual temperature conditions of postslaughter handling and chilling of theanimal carcasses. Furthermore, although they are able to hydrolyze myosin and actin,only insignificant degradation of these main proteins of the myofibrils takes placeduring tenderization of meat. They can, however, participate in the concerted actionof various proteinases in aging meat and fish.

The muscles also contain nonlysosomal proteinases capable of hydrolyzingseveral myofibrillar proteins responsible for the structural integrity of the musclefibers, especially the cytoskeletal proteins. Calpains, the calcium-activated neutralmetalloproteinases, require the cysteinyl thiol group for activation. The highestactivity of calpains against isolated myofibrils is in the pH range 7.0 to 7.5, whileat pH 6 and 4.5 the activity is about 80 and 40% of the maximum value, respectively.One form of the enzyme, called m-calpain, requires at least 0.1 to 0.5 mM Ca2+ foractivation, the optimum concentration being 1 mM; the second form, µ-calpain, is



activated at micromolar concentrations of Ca2+. Due to the loss of the Ca2+ retainingability of the reticulum in the muscle postmortem, the concentration of Ca2+ in thesarcoplasm may increase up to about 0.1 mM. There is a cause-and-effect relationshipbetween the activity of µ-calpain and the decrease of muscle strength (Purslow etal., 2001). The enzyme rapidly degrades the cytoskeletal protein desmin, causingdepolymerization of the intermediate filaments that bind neighboring Z-discs in themyofibril. The major fragments resulting from the breakdown are further cleavedby cathepsins. The known variations in the rate of tenderization of various musclesare due to the differences in the activity of calpains and calpastatin—a family ofendogenous inhibitor proteins that are strictly calpain specific and are located in thesame cellular area as the enzyme. The meat tenderization process also involvesdegradation of other cytoskeletal proteins—titin, nebulin, and desmin (Jiang, 2000).

The changes in proteins catalyzed by endogenous enzymes affect the sensoryquality of various salted, cold-smoked, and marinated fish products. In lightly saltedsalmon, sturgeon, herring, anchovy, Baltic sprats, and fish of many other species, thehydrolytic processes lead to development of tender texture. The flavor becomes pre-dominantly fishy and salty, with meaty, cheesy, and slightly rancid notes. The enzymesinvolved in the ripening of salted, uneviscerated fish like herring are mainly those ofthe pyloric appendages. The suitability of fish for salt ripening is affected by the activityof the endogenous proteases, which fluctuates seasonally and depends on the feedingintensity of the fish. Excessive proteolysis, in products stored too long at too high atemperature, leads to unacceptable softening of the flesh. On the surface of overripesalted fish a white “bloom” of crystallized peptides and a.a., mainly Tyr, may appear.In raw herring marinades the ripening occurs due to muscle cathepsins.

High activity of muscle proteases during the spawning migration may bringabout changes that increase the emulsifying ability of the meat of salmon during thespawning period (Kawai et al., 1990).

Different endogenous proteinases are involved in the disintegration of the struc-ture of fish gels known as modori, which often occurs due to slow cooking in thetemperature range of 50 to 70°C. In such gels hydrolysis of myofibrillar proteins,particularly myosin, occurs. Early reports indicated the heat-stable alkaline protein-ases, found in the muscles of fish of several species, were involved. The activity ofthese enzymes is usually not detectable below 50°C. Further investigations revealedthat cathepins B, L, and L-like contribute to the disintegration of the gel structure byhydrolyzing the myosin heavy chain, light chains, actin, and troponins. Using cysteineproteinase inhibitors can limit these undesirable effects. Jiang (2000) presented a verythorough discussion of the role of different proteinases in modori softening.

A high degree of proteolysis is involved in manufacturing edible fish sauces,silage for animal feeding, and hydrolysates for use as functional ingredients in foodsand as bacterial peptones (Sikorski et al., 1995; Lopetcharat et al., 2001). Fish saucesbelong to a group of traditional fermented products typical in Southeast Asia. Theyare manufactured by salting different small fish, mainly Stolephorus spp, Ristelligerspp., Sardinella spp., Engraulis spp., Clupea spp., Scomber colias, and Decapterusspp., according to a variety of recipes. In various recipes, the fish-to-salt proportionsrange from 1 to 6 and the fermentation time, usually at ambient temperature, fromabout 2 to 18 months. Endogenous and bacterial enzymes are involved in developing



the typical flavor of the sauces. The undiluted filtrate of the autolysate is regardedas first-grade fish sauce; the brine extract of the nonhydrolyzed residue is of lowersensory quality and nutritional value. Various proteolytic enzymes from animal,plant, and microbial sources added in the process can increase the rate of proteolysisand even improve the quality of the products.

Fish silage is typically made from by-catch fish and filleting offal by mincing,acidifying with formic acid or a mixture of formic and sulfuric acid, and proteolysiscatalyzed by endogenous enzymes. After about 2 days at 30 to 40°C most of thetissues are solubilized. The silage containing 70 to 80% water can be used as suchafter removal of the solids and the fatty layer, or the liquid can be concentrated tothe required degree. The product, manufactured commercially on a large scale, isused as a feed component for pigs, poultry, and fish.

Fish hydrolysates are produced mainly from lean fish of underutilized speciesor filleting by-products in a process catalyzed by added proteinases of plant, animal,or microbial origin (Figure 6.8) The product, depending on its quality, can be usedas a source of a.a. in growth media for microorganisms, as a milk replacement inanimal feeding, or as a functional ingredient in foods.

FIGURE 6.8 A flow sheet of the process used for manufacturing fish hydrolysates.

Water, 1:2

Filtration

Mincing

HydrolysisOptimum Temperature

Enzyme Inactivation100oC

Evaporation

Drying

Fish Meat

Endopeptidases

NaOH or HCl

Insolubles

Vapors

Vapors

Fish Peptone



Partial autoproteolysis has been successfully utilized for producing in industrialconditions food-grade, thermally coagulated, frozen protein gel from Antarctic krill.The protein recovery is about 80% of the protein content in the whole krill(Kołakowski and Sikorski, 2000).

Other possible applications of proteolytic enzymes in seafood processing includedescaling of fish, pealing and deveining of shrimp, tenderizing of squid, isolatingpigments from shellfish waste, and reducing the viscosity of fishmeal stickwaters.Squid muscles contain very active proteinases (Kołodziejska, 1999). High proteolyticactivity causes extensive degradation of the myofibrillar proteins in the course ofextraction of these proteins for analytical purposes. There are significant differencesin the autoproteolytic activity in the muscles of squid of different species.

Trimethylamine oxide demethylase, present in the muscles of many gadoidfishes, affects the functional properties of fish proteins indirectly by catalyzing theformation of formaldehyde, which may participate in cross-linking. These reactionsare most important in frozen stored gadoid fish.

6.7.6 OTHER ENZYMATIC CHANGES IN FOOD PROTEINS

Phosphatases may dephosphorylate the caseins. Thiol oxidase may participate in theoxidation of SH groups to disulfides. Wheat lipoxygenase and soybean lipoxygenase,catalyzing oxidation of fatty acids, generate oxidized reaction products, whichimprove the dough-forming properties and baking performance of flour. A similarrole is performed by polyphenol oxidase and peroxidase.

Cyclic adenosine monophosphate dependent protein kinase is useful for phos-phorylation of a.a. residues under mild conditions. The modification makes thesoybean proteins soluble in media rich in Ca2+ and improves their emulsifyingproperties (Seguro and Motoki, 1990).

In a papain-catalyzed reaction, at room temperature, proteins can be acylated andenriched in SH groups using N-acetyl-homocysteinethiolactone (Sung et al., 1983):

6.8 CHEMICAL MODIFICATIONS

6.8.1 INTRODUCTION

Chemical additives and reactions are used to change the color and several propertiesof different foods and isolated proteins. Typical examples include the use of sodiumnitrite in the curing of meat, chemical modifications of dough proteins for improvingthe texture of many baked goods, and applications of polyphosphates in the meatindustry. The intended modification in the properties of proteins can be achieved by

REACTION 6.11

C

H3C CO NH CH

CH2

C

O

SH2

H2N Prot H3C CO NH CH CO NH Prot

CH2)2

SH

(

+papain

pH 10



changing the charge, hydrophobicity, and steric parameters of the molecules due tocross-linking or alterations of the a.a. residues. Ascorbic acid and its oxidationproduct dehydroascorbic acid have been known as flour improvers in the baking ofbread. Many experiments on chemical modification of a.a. residues study the struc-ture–function relationship with respect to proteins in different food systems underthe conditions prevailing during processing and storage.

6.8.2 ALKYLATION

The carbonyl compounds formed due to autoxidation of lipids and in catabolicprocesses postmortem in muscles, or introduced with wood smoke can bring aboutundesirable effects by interacting with a.a. residues. On the other hand, some car-bonyl compounds are used intentionally to modify proteins. Formaldehyde waspreviously used for hardening of the collagen dope in manufacturing sausage casings,protect fodder meals against deamination by the rumen microflora, and bind immo-bilized enzymes on supports.

In reducing conditions, alkylation of amino, indole, thiol, and thioether groups,and binding of saccharides to a.a. residues is possible.

For alkylation of amino, phenol, imidazole, thiol, and thioether groups in a.a.residues of proteins reactions with haloacetates and haloamides can also be used.

The digestibility of such derivatives is generally somewhat lower than that ofthe unmodified proteins.

Malondialdehyde, a typical lipid oxidation product, can form cross-links inproteins by reacting with two amino groups.

There is also a possibility that aliphatic monoaldehydes generated in lipid autox-idation can participate in cross-linking of proteins. Formaldehyde can react in food

REACTION 6.12

REACTION 6.13

REACTION 6.14

REACTION 6.15

pH 9

0°C2Prot NH2 4HCHO NaCNBH3 2Prot N(CH3)2 NaHCNBO3 H2O++++ ++++

+ +Prot SH ICH2COOH Prot S CH2COOH HI

+Prot — NH2 ICH2CONH2 Prot NH CH2 CONH2 HI+

Prot N CH CH2 CH N Prot+O

HC CH2 C

O

HNH22 Prot + H2O



systems with amino, amide, hydroxyl, and thiol groups in a.a. residues of proteins,even at room temperature. Some of these reactions result in cross-linking of theproteins. In cheese, the biogenic formaldehyde reacts with the N-terminal histidineof γ2-casein to form spinacine.

Other carbonyl compounds produced in cheese by the lactic acid bacteria formother derivatives in reactions with histidine (Pellegrino and Resmini, 1996).

6.8.3 ACYLATION

Acylation of nucleophilic groups is intended to increase the hydrophobicity of theprotein, introduce additional ionizable groups, or to contribute to cross-linking.

The acylating agents may react with amino, imidazole, hydroxyl, phenol, andthiol groups in a.a. residues. The rate of reaction depends upon the properties of thenucleophiles, pH, the steric factors resulting from the protein conformation, and thepresence of inhibitors. The amino and tyrosyl groups can be acylated easily, whileHis and Cys derivatives readily hydrolyze. Acylation of hydroxyl groups in a.a.residues of proteins proceeds in the presence of an excess of acetic anhydride onlyafter the amino groups have been acylated. N-carboxyanhydrides of a.a. are suitablefor the formation of isopeptide linkages. The degree of acylation depends on thenature of the protein, the acylating agent, and the process conditions, and may reachup to 95% of Lys residues.

Some food proteins are rich in phosphoric acid residues. The acid may either formester bonds with Ser residues, as in caseins and in egg proteins, or may stabilize thenative conformation of protein micelles by electrostatic interactions with negatively

REACTION 6.16

REACTION 6.17

NH

N

CH2

CHCOOHH2N

NH

N

HN

COOH

spinacine

HCHO

-OOC (CH2)2 C NH

O

ProtO

O

O

H2N Prot+



charged groups and Ca2+, as in the caseins. In soy proteins, Ser and Thr residuescan be esterified and Lys amidated with cyclic sodium trimetaphosphate at pH 11.5and 35°C (Sung et al. 1983):

The Nε-triphospholysine residues protect the NH2 group during heating in analkaline environment and hydrolyze at pH lower than 5. For chemical phosphoryla-tion other reagents are also effective, such as phosphorus oxychloride and phosphoruspentoxide. The reactivity of these compounds may, however, lead to undesirablecross-linking of the proteins:

Acylation of a.a. residues generally improves WHC, as well as the emulsifying andfoaming capacity of the proteins. The pI shifts toward lower values, the solubilityincreases over that of the unmodified protein above the pI and decreases in the acidicrange. This is important, for example, for wheat gluten, which has low solubility in theneutral pH range. The positive change in the functional properties is not in all casesachieved at the highest degree of modification because it depends on the change in surfacehydrophobicity, which is affected by the nature of the protein and the acylating agent.

The biological availability of acylated proteins depends on the kind of the acylgroup and the extent of acylation. Isopeptides are generally well utilized, while theavailability of other derivatives usually decreases with the increasing size of theacylating moiety and the degree of acylation.

6.8.4 N-NITROSATION

The nitrous acid generated in foods at low pH from endogenous or added nitritesdecomposes easily to yield the nitrosating agents nitrous anhydrate and the nitroso-nium ion:

REACTION 6.18

REACTION 6.19

P

O O-

O

O

P

P

O O-

O

O-

OProt Ser OH

Prot Lys NH2

+

pH 11 - 12- H2O

pH 11 - 12

pH<5

Prot Ser O P

O-

O

O-

Prot Lys NO

O-

O

O-

O

P

P

O-O

O-O

P

H

P2O7-4

H+

+ +

+

H+

+-

Prot NH2 POCl3 Prot NH POCl2 H Cl++ +

HOOC Prot

++Prot NH CO Prot Cl2PO2-

H+



Reactions of these compounds with the secondary and tertiary amines containedin many foods lead to carcinogenic N-nitrosoamines:

The rate of N-nitrosation increases with the pKa of the amine and depends on thepH; it is highest at a pH between 2 and 4. Compounds capable of binding thenitrosating agents can inhibit the reaction; in meat curing, sodium ascorbate is veryeffective. Foods low in amines and nitrites contain generally about 1 to 10 ppb, whilecured and heavy smoked meat and fish have up to several hundred ppb of N-nitrosocompounds.

6.8.5 REACTIONS WITH PHOSPHATES

Proteins may form protein–phosphate complexes of low solubility in an acid envi-ronment. The metaphosphates, which are less hydrated than the ortophosphates, formcomplexes that are less soluble than those with ortophosphates. This has been utilizedfor the modification of functional properties of protein concentrates and preparations,for separation of proteins in different food processing operations, and in the treatmentof food plant effluents containing proteins.

Polyphosphates improve the sensory quality of many food products. They preventthe separation of butter fat and aqueous phases in evaporated milk, or of the formationof gel in concentrated milk sterilized by HTST. They also stabilize the fat emulsion

REACTION 6.20

REACTION 6.21

REACTION 6.22

O N O N O H2O+2HO N O

O N O N O

H

N O HO N O+

H+

or

or

RNH

R

O N O N O

+ +

+O N

+-H

RN

RN O

HNO2

NO2-

O N OH

N ON

R

R N

H

O

RN

RR +

RN

RN O + R O N ON O R

RN

RN O N O-O



in processed cheese by disrupting the casein micelles, and thus enhance the hydro-phobic interactions between lipids and casein. Polyphosphates are also used in meatprocessing for increasing the WHC and improving the texture of many cooked prod-ucts. The mechanisms involved in different applications depend upon the propertiesof the phosphates and of the commodities, as well as on the parameters of processing.

In meat products, the increase in WHC, texture improvement, and decrease indrip may be caused by increasing the pH, complexing of Ca2+ and Mg2+, binding ofphosphates to proteins, rupturing the cross-links between myosin and actin, anddissolving some proteins. Phosphates facilitate the manufacture of meat products oflow sodium content. To produce an emulsion-type sausage of acceptable quality, atleast 2.5% salt is required. The salt content can be decreased to 1.5 to 2.0% withoutloss in product quality by adding phosphates in amounts of 0.35 to 0.5%. Generally,proprietary brands of several polyphosphates are used in order to provide the bestquality and prevent precipitation of orto- and pyrophosphates in brines rich in Ca2+.

Polyphosphates are added as cryoprotectants to minced frozen fish. They arealso applied in the form of dips to fish fillets prior to freezing to prevent thaw driplosses, and to improve the texture of canned fish. Most common are 10% solutionsof Me5P3O10 and Me4P2O7 used for 1 to 2 min. Different proprietary mixtures arealso applied, such as Na4P2O7 + Na2H2P2O7 or Na3PO4 + Na4P2O7 + Na2H2P2O7.

REFERENCES

Ashie, I.N.A. and Lanier, T.C., 2000, Transglutaminases in seafood processing, in SeafoodEnzymes. Utilization and Influence on Postharvest Seafood Quality, Haard, N.F. andSimpson, B.K., Eds., Marcel Dekker, New York and Basel, chap. 6.

Benjakul, S., Visessanguan, W., and Srivilai, C., 2001, Porcine plasma protein as proteinaseinhibitor in bigeye snapper (Priacanthus tayenus) muscle and surimi, J. Sci. FoodAgric., 81, 1039.

Boatright, W.L. and Hettiarachchy, N.S., 1995, Soy protein isolate solubility and surfacehydrophobicity as affected by antioxidants, J. Food Sci., 60, 798.

Bramaud, C., Mimar, P., and Daufin, G., 1995, Thermal isoelectric precipitation of α-lactal-bumin from a whey protein concentrate: influence of whey-calcium complexation,Biotechnology and Bioengineering, 47, 121.

Chanyongvorakul, Y., Matsumura, Y., Monaka, M., Motoki, M., and Mori, T., 1995, Physicalproperties of soy bean and broad bean 11S globulin gels formed by transglutaminasereaction, J. Food Sci., 60, 883.

Colas, B., Caer, D., and Fournier, E., 1993, Transglutaminase catalyzed glycosylation invegetable protein. Effect on solubility of pea legumin and wheat gliadins, J. Agric.Food Chem., 41, 1811.

Damodaran, S., 1989, Interrelationship of molecular and functional properties of food pro-teins, in Food Proteins, Kinsella, J. E. and Soucie, W. G., Eds., The American OilChemists’ Society, Champaign, IL, chap. 3.

Doi, E., Shimizu, A., Oe, H., and Kitabatake N., 1991, Melting of heat-induced ovalbumingel by pressure, Food Hydrocolloids, 5, 409.

Doucet, D., Gauthier, S.E., and Foegeding, E.A., 2001, Rheological characterization of a gelformed during extensive enzymatic hydrolysis, J. Food Sci., 66, 711.



Feng, Y. and Hultin, H.O., 2001, Effect of pH on the rheological and structural properties ofgels of water-washed chicken-breast muscle at physiological ionic strength, J. Agric.Food Chem., 49, 3927.

Fernández-Martin, F., Fernández José Carballo, P., and Jiménez-Colmenero, F., 2000, DSCstudy on the influence of meat source, salt and fat levels, and processing parameterson batters pressurization, Eur. Food Res. Technol., 211, 387.

Friedman, M. and Cuq, J.L., 1988, Chemistry, analysis, nutritional value, and toxicology oftryptophan in food. A review, J. Agric. Food Chem., 36, 1079.

Gómez-Guillen, M.C., Sarabia, Q.A.I., Solas, M.T., and Montero, P., 2001, Effect of microbialtransglutaminase on the functional properties of megrim (Lepidorhombus boscii) skingelatin, J. Sci. Food Agric., 81, 665.

Haard, N.F., 2001, Enzymic modification of proteins in food systems, in Chemical andFunctional Properties of Food Proteins, Sikorski, Z.E., Ed., Technomic PublishingCo., Inc., Lancaster, PA, chap. 7.

Haard, N.F., Simpson, B.K., and Pan, B.S., 1994, Sarcoplasmic proteins and other nitrogenouscompounds, in Seafood Proteins, Sikorski, Z.E., Pan, B.S., and Shahidi, F., Eds.,Chapman & Hall, New York, chap. 3.

Hamm, R., 1960, Biochemistry of meat hydration, in Advances in Food Research, Vol. 10,Chichester, C.O., Mrak, E.M., and Stewart, G.F., Eds., Academic Press, New York,chap. 8.

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Hayashi, R., Kawamura, Y., Nakasa, T., and Ohinaka, O., 1989, Application of high pressureto food processing: pressurization of egg white and yolk, and properties of gelsformed, Agric. Biol. Chem., 53, 2935.

Holt, C. and Rogiński, H., 2001, Milk proteins: biological and food aspects of structure andfunction, in Chemical and Functional Properties of Food Proteins, Sikorski, Z.E.,Ed., Technomic Publishing Co., Inc., Lancaster, PA, chap. 11.

Hoseney, R.C. and Rogers, D.E., 1990, The formation and properties of wheat flour doughs,CRC Crit. Rev. Food Sci. Nutr., 29, 73.

Hossain, M.I., Itoh, Y., Morioka, K., and Obatake, A., 2001, Contribution of the polymerizationof protein by disulfide bonding to increased gel strength of walleye pollack surimigel with preheating time, Fisheries Science, 67, 710.

Howell, B.K., Matthews, A.D., and Donnelly, A.P., 1991, Thermal stability of fish myofibrils:a differential scanning calorimetric study, Inter. J. Food Sci. Technol., 26, 283.

Jiang, S.T., 2000, Enzymes and their effects on seafood texture, in Seafood Enzymes. Utili-zation and Influence on Postharvest Seafood Quality, Haard, N.F. and Simpson, B.K.,Eds., Marcel Dekker, New York, Basel, chap. 15.

Jiang, S.T., Ho, M.L., and Chen, H.C., 1998, Purified NADPH-sulfite reductase from Saccha-romyces cerevisiae effects on quality of ozonated mackerel surimi, J. Food Sci., 63, 777.

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Kato, A., Ibrahim, H.R., Takagi, T., and Kobayashi, K., 1990a, Excellent gelation of egg whitepreheated in the dry state is due to a decreasing degree of aggregation, J. Agric. FoodChem., 38, 1868.



Kato, A., Ibrahim, H.R., Watanabe, H., Houma, K., and Koboyashi, K., 1990b, Enthalpy ofdenaturation and surface functional properties of heated egg white proteins in the drystate, J. Food Sci., 55, 1280.

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177

7

Lipids and Food Quality

Andrzej Sto

ł

yhwo

CONTENTS

7.1 General Description ..................................................................................... 1787.1.1 Definition.......................................................................................... 1787.1.2 Nonpolar or Simple Lipids .............................................................. 1787.1.3 Polar or Complex Lipids ................................................................. 179

7.2 Isolation and Analysis of Lipids.................................................................. 1797.2.1 Isolation from Natural Sources........................................................ 1797.2.2 Separation into Classes .................................................................... 1807.2.3 Determination of Fatty Acid Composition of Individual

Lipid Classes by Gas Chromatography........................................... 1817.3 Structures and Composition of Common Lipids ........................................ 182

7.3.1 Fatty Acids and Their Physical Properties ...................................... 1827.3.2

Trans

Fatty Acids in Natural and Partially Hydrogenated

Trans

Fats ......................................................................................... 1847.3.3 Conjugated Linoleic Acid ................................................................ 188

7.4 Determination of the

Trans

Fatty Acid Content in Food Lipids ................ 1897.4.1 Methods of Determination............................................................... 1897.4.2 Biological Significance .................................................................... 192

7.5 Fatty Acid Composition of Common Fats .................................................. 1937.6 Long-Chain Polyenoic Fatty Acids and Their Importance

in Human Nutrition...................................................................................... 1937.7 Triacylgylcerols............................................................................................ 197

7.7.1 Chemical and Functional Properties................................................ 1977.7.2 Modifications.................................................................................... 1997.7.3 Fractionation..................................................................................... 1997.7.4 Lipolysis ........................................................................................... 2007.7.5 Analysis of the Composition of Triacylglycerols ........................... 202

7.7.5.1 HPLC of Triacylglycerols ................................................ 2027.7.5.2 GLC of Triacylglycerols................................................... 203

References.............................................................................................................. 206

9675_C007.fm Page 177 Thursday, September 21, 2006 11:54 AM

178


7.1 GENERAL DESCRIPTION

7.1.1 D

EFINITION

There are several definitions of the term

lipids

. Usually lipids are defined as aheterogeneous group of biological compounds almost insoluble in water but solublein fats, hydrocarbon type and other fat solvents. Fats and oils are example of lipids.

In older manuals lipids are defined as all substances which can be extracted fromthe investigated material with diethyl ether. In fact, the diethyl ether is a mediumpolar solvent, partially soluble in water, which is able to extract from slightly wetnatural samples both so-called

nonpolar

and

polar

lipids.

7.1.2 N

ONPOLAR

OR

S

IMPLE

L

IPIDS

Among naturally occurring nonpolar lipids one may find:

• Alkanes or alkenes: hydrocarbons usually containing a straight saturatedor unsaturated chain of up to 36 carbon atoms, which may be alsobranched, with one or more methyl groups in the side chain. In contrastwith the diversity of methyl-branched alkanes found in insects, n-alkanespredominate in plants. Hydrocarbons with an odd number of carbon atoms(C15 up to C33) are predominant among the least polar components ofplant surface lipids.The commonly known naturally occurring hydrocarbon is squaleneC

30

H

50

, present, for example, in concentrations up to 12% in human serum,in lipids of human milk (0.12%), and in olive oil (0.6%). Considerableamounts of squalene are present in the livers of deep-sea sharks. Otherhydrocarbons commonly occurring are carotenoids, such as beta-carotene,better known as provitamin A.

• Fatty alcohols: the aliphatic alcohols with saturated or unsaturated hydro-carbon chains, usually between C6 and C26, and one to three double bonds.

• Waxes: esters of fatty acids (FA) and long-chain alcohols. Esters of cho-lesterol and fatty acids in human serum are frequently investigated. Amongnaturally occurring waxes are: bees wax, carnauba wax, candelila wax,and many other well-known waxes.

• Sterols: a group of compounds known as phytosterols, which are presentin plants. Cholesterol (CL), lanosterol, and other similar compounds arethe main representatives of animal sterols. Both phytosterols or animalsterols may occur in a free state or esterified with fatty acids known assterol esters; or esterified by sulfuric acid—sterol sulfates or linked to aglucide—known as steryl glycosides.

• Tocopherols: present in practically all naturally occurring plant oils. Thelargest concentration of tocopherols is found in germs. Tocotrienols arespecific for some oils, like palm or amaranth oil. Both tocopherols andtocotrienols are generally known as vitamin E. Triacylglycerols (TAGs),in medicine called

triglycerides

, are triesters of glycerol and FAs. Natural



179

plant oils or animal fats are 96 to 98% composed of TAGs, in which aredissolved other accompanying nonpolar and polar lipids.

• Mono- (MAGs) and diacylglycerols (DAGs): these are dissolved in naturaloils or fats, and are the products of partial hydrolysis of TAGs. Thehydrolysis may be catalyzed by enzymes—lipases present practically inall biological systems, for example, pancreatic lipase or lipases present infungi such as

Rhizopus javanicus.

MAGs contain nonpolar fragments(hydrocarbon chain) soluble in fats, and polar (glycerol) fragments solublein water. Thus they act as emulsifiers. MAGs are an important group ofcompounds manufactured commercially and used as emulsifiers for pro-duction of margarine, spreading fats, ice creams, cosmetics, and manyother commercial products.

7.1.3 P

OLAR

OR

C

OMPLEX

L

IPIDS

These important lipids are widely distributed in plants, bacteria, and animals. Theyare known as the major constituents of cell membranes. Because of the large varietyof complex lipids, their classification is a very difficult task. Usually the polar lipidsare divided into three groups:

• Phospholipids (PLs): lipids with a phosphate residue, one glycerol, or anamino alcohol or a fatty alcohol, with or without one or two FA chains(exceptions are one inositol group, two phosphates, or four FA chains).

• Glycolipids:

lipids containing a glycosidic moiety with a glycerol or anamino alcohol and with a FA chain or chains. Sometimes glycolipids alsocontain phosphate residue.

• Proteolipids:

lipids containing one amino acid residue linked to long-chainalcohol or acids.

7.2 ISOLATION AND ANALYSIS OF LIPIDS

7.2.1 I

SOLATION

FROM

N

ATURAL

S

OURCES

The methods for isolating lipids depend on the type of the sample. The nonpolarlipids are usually isolated by extraction with petroleum ether according to AOACMethod No. 920.39C. This applies mainly to the oil seeds.

Lipids from milk and other dairy products are isolated using mainly the RoseGottlieb method in which the lipids bound to proteins are first liberated with asolution of ammonia in ethanol, and then the extraction is carried out with diethylether. The exact analytical procedure is described in AOAC Method No. 932.06.

The methods of isolating of fats from bakery, chocolate, or chocolate-like prod-ucts or cosmetics are very drastic because some lipids are chemically bound to thematrix of the sample. The isolation of fats includes preliminary preparation by boilingof the sample in an 8M HCl solution, followed by filtration of the resulting mixturethrough a wetted filter paper. The fatty material retained on the filter paper is washedseveral times with warm water and loaded into a cellulose thimble, dried at 100 to


180


101

°

C, and extracted with diethyl ether in Soxhlet glassware. The exact analyticalprocedure is described in AOAC Method No 922.06. The isolation of lipids frombiological sources should not only be quantitative but also nondestructive withrespect to the isolated sample. All isolated lipids must be protected against oxidationby solvents, oxygen, or enzymes in combination with temperature and light. Thehigh sensitivity of the analytical methods needed for low amounts of extracted lipidsrequires the use of very pure solvents and clean glassware.

For the isolation of lipids from organs or tissues, the Folch (1957) or Bligh andDyer (1959) methods are used most frequently. In both methods a mixture ofchloroform and methanol is used, which helps to tear apart weak physical bondsbetween polar lipids and the matrix of the sample. Because the above methods arenot included in the AOAC analytical standards, for the convenience of the readerthe Folch procedure is described in detail below.

In the Folch procedure, one gram of a tissue is homogenized for 1 min in a high-speed blender with 20 ml of a 2:1 (v/v) chloroform and methanol mixture. Afterdispersion the mixture is allowed to stand at room temperature for 15 to 20 min andis occasionally agitated. The homogenate is filtered using soft filter paper into a 50 mlgraduated cylinder equipped with a glass stopcock. The solid residue on the filterpaper is carefully rinsed twice with 4 ml of a mixture of chloroform and methanol2:1(v/v), which is added to the extract. To the final volume (V) of the extract in thegraduated cylinder, 0.2 V of a 0.9% NaCl solution in water is added (the content ofwater in the sample before extraction should be taken into account. The cylinder isshaken for 2 to 3 minutes and allowed to stand in darkness to obtain exact separationof phases. If emulsions are formed, a small amount of powdered NaCl is carefullyadded to the cylinder to facilitate separation of the phases. Alternatively, the mixtureis centrifuged at low speed (2000 rpm) to separate the two phases. The upper phasecontaining dissolved proteins is removed by siphoning. The lower chloroform phasecontaining lipids is dried over anhydrous sodium sulfate and evaporated under vacuumin a rotary evaporator or under a stream of nitrogen. The extracts should be storedunder nitrogen in a freezer below –50

°

C or preferably in dry ice. In order to protectthe extracted lipids against autooxidation, addition to the chloroform phase of smallamounts (50

μ

g) of antioxidants (for example, nordihydroguaiaretic acid [NDGA] orbutylated hydroxytoluene [BHT] is advisable).

7.2.2 S

EPARATION

INTO

C

LASSES

There are several methods of separation of extracted lipids in individual classes. Avery efficient procedure of semipreparative separation by extraction to the solid phase(SPE) has been published by Kaluzny et al. (1985). The method for unskilled personsis slightly inconvenient, but its main feature is very good protection against theoxidation of sensitive components in the extracted lipids.

Slightly less protective and selective, but very frequently used, is the method ofsemipreparative planar chromatography for the separation of extracted lipids intocholesteryl esters (CLE, TAG), CL, and PL.

The procedure is as follows. Commercially available 20

×

20-cm plates coveredwith a 0.25-mm silica gel layer are activated before use at 120

°

C for 1.5 hrs. After



181

cooling down in a dry desiccator a solution of about 30 to 50 mg of lipids in 0.5 mlof a chloroform and methanol solution (1:1) is applied on the plate in a form ofcontinuous strips using an automatic mechanical applicator. After evaporation of thesolvent using a fan, the chromatogram is developed in an unsaturated chamber withthe mobile phase: hexane/diethyl ether/glacial acetic acid 80:20:1 (v/v/v). The devel-opment of the chromatogram is carried out up to the moment when the solvent frontreaches about 2 cm below the upper edge of the plate. Then the plate is removedfrom the developing chamber and carefully covered with a glass plate leaving about4 cm from one side of the plate uncovered. After drying the uncovered part of theplate with a cold air stream, the surface is sprayed with a solution of 2

′

,7

′

dichlo-rofluoresceine in ethanol. The separated lipid classes are visualized under UV. Theposition of the bands of individual lipid classes is marked on a plate with a spatula.After the covering glass is removed, the corresponding (invisible) individual bandsare scraped from the plate. The scrapped silica gel containing separated lipid classesare loaded into preferably glass syringes equipped in the bottom with a filtering discor defatted cotton or glass wool. The lipids are eluted from the scrapped materialwith a mixture of chloroform and methanol (2:1, v/v).The unscraped part of the plateis sprayed with a 25% solution of sulfuric acid, heated at 110

°

C in order to char allorganic material present on the plate. The charred plates may be scanned with anappropriate scanning system and transferred to the computer as a document. Thistype of separation is shown in Figure 7.1.

7.2.3 D

ETERMINATION

OF

F

ATTY

A

CID

C

OMPOSITION

OF

I

NDIVIDUAL

L

IPID

C

LASSES

BY

G

AS

C

HROMATOGRAPHY

After separation into classes, usually the composition of the FAs in each class isdetermined. For that purpose the gas chromatographic (GC) method is used. Before

FIGURE 7.1

Example of semipreparative separation into classes of lipids extracted from ratliver by thin layer chromatography. (Abbr.: CLE = cholesterol esters; TAG = triacylglycerols;CL = cholesterol; PL = phospholipids.) Silica gel plates 0.25-mm layer thickness, mobilephase: hexane/diethyl ether/glacial acetic acid 80:20:1 (v/v/v). (Dzik and Sto

ł

yhwo 2004,unpublished data.)

Margin of the plate sprayed with 2′ ,7′dichlorofluoresceine

CLE

TAG

CL

PL


182


analysis by GC, the FAs present in each lipid class should be converted into theirfatty acid methyl esters (FAME), which are more volatile than the correspondingfree FAs. There are several methods of preparing FAMEs, and each method hassame disadvantages.

The FAMEs in milk fats and other dairy products, which contain butyric acids,are usually prepared by cold esterification in an alkaline medium. The most usefultransesterifying agents are 1 to 2 M sodium or potassium methoxides in anhydrousmethanol. These solutions are stable for several months at 4

°

C until a white precip-itate of bicarbonate salt is formed. Glycerolipids are rapidly transesterified (2 to 5min) at room temperature. This procedure protects against losses of very volatilemethyl butyrate during preparation of FAMEs. However, great care must be takento work with really dry samples, otherwise multiple artifacts are developed, whichlead to unreliable results.

The FAMEs of lipids extracted from human or animal tissues or from marineoils are usually prepared following the AOCS Method No. Ce 1b-89, according towhich the lipids are saponified (under nitrogen) in 0.5N NaOH in methanol, andthen the soaps are converted directly into FAMEs. In the case of samples containingmarine oils or extracts from animal samples in which long-chain (C20 to C24)polyunsaturated FAs (LC-PUFA) are present, the addition of internal standard C23:0is necessary because the response factors of FAMEs containing 3 and more doublebonds differ substantially from those of saturated or monounsaturated FAMEs.

For the convenience of the reader, the parameters of separation by GC andidentification of chromatograms of FAMEs of rat liver lipids and of the mixture ofSupelco 37 Component FAME Mix Cat No: 47885-U (Sigma-Aldrich) are presentedin Figure 7.2.

7.3 STRUCTURES AND COMPOSITION OF COMMON LIPIDS

7.3.1 F

ATTY

A

CIDS

AND

T

HEIR

P

HYSICAL

P

ROPERTIES

Carboxylic acids occur in many molecular forms; however, the majority of FAsfound in lipids are those containing one carboxylic (–COOH) group. Several hundredforms of FAs have been identified, but the number occurring frequently in commonlipids is much smaller—usually between 10 and 30 in plant oils and from 40 to over100 in animal fats. FAs differ from each other by the length of the carbon chain,structure of the carbon chain (normal or branched), number of double bonds, theirposition in the hydrocarbon chain with respect to the carboxylic or terminal-methylgroup, and the configuration

cis

or

trans

or conjugation of double bonds. In thesystematic nomenclature, the position of the double bonds is related to the carboxylicgroup, for example, oleic acid C

18:1

9c (octadecenoic acid-delta-9-

cis

). However inbiochemistry, the more practical designation is that which relates to the end of thehydrocarbon chain—the methyl group. In such case the position of the double bondwith respect to the terminal methyl group is designated as omega (in the case ofoleic acid

Ω

-9) or as n-9.



183

FIG

UR

E 7.

2

Chr

omat

ogra

ms

of F

AM

Es

of r

at l

iver

lip

ids

com

pare

d to

the

mix

ture

of

Supe

lco

37 C

ompo

nent

FA

ME

Mix

Cat

No:

478

85-U

(Si

gma-

Ald

rich

). P

aram

eter

s of

sep

arat

ion:

Col

umn

Rtx

233

0 (R

este

k, C

hrom

atog

raph

ic S

peci

altie

s, C

anad

a), 1

00 m

; ID

0.2

5 m

m, d

f

= 0

.2

μ

m; H

2

flow

32

cm/s

,sp

lit-s

plitl

ess

inje

ctio

n at

235

°

C

; de

t. te

mp.

250

°

C

; co

lum

n te

mp:

ini

tial

155

°

C

; ho

ld 5

5 m

in;

next

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e 1.

5

°

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/min

; fin

al t

emp.

210

°

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; ne

xt h

old.

Iden

tifica

tion

of p

eaks

is

give

n on

chr

omat

ogra

ms

(aut

hor’

s da

ta).

FID

1 A

, (58

90K

UB

/Z11

W00

00.D

)

A

C14:0

C14:1C15:0

C16:0

C18:0

C16:1 n-7

C17:1 n-7

C17:0

C18:1 n-9 trans

C18:2 n-6 trans

C18:1 n-7C18:1 n-9

C18:2 n-6

C18:3 n-6

C18:3 n-3

C18:4 n-3

C20:0

C20:2

C24:0

C20:1 n-9

C20:3 n-9

C20:3 n-6

C20:4 n-6

C20:5 n-3

C22:4 n-6

C22:5 n-6

C22:5 n-3

C22:6 n-3

FID

1 A

, (58

90K

UB

/S37

0000

0.D

)

B

C6:0C6:0

C10:0

C16:1 n-7

C17:1 n-7

C18:1 n-9 trans

C18:2 n-6 trans

C18:2 n-6

C18:3 n-6

C18:3 n-3

C20:1 n-9

C18:1 n-9

C11:0C12:0

C13:0

C14:0

C14:1

C15:1

C15:0

C17:0

C20:0

C20:2

C20:3 n-6

C20:3 n-3C20:4 n-6

C20:5 n-3

C22:6 n-3

C21:0

C22:0

C22:1

C22:2

C18:0

C18:0

C23:0

C24:0

C24:1


184


Monocarboxylic FAs are classified according to the number of double bonds in thehydrocarbon chain as saturated; monounsaturated, di-unsaturated, and polyunsaturated.

Systematic and common names of frequently occurring saturated and unsaturatedFAs are given in Table 7.1, Table, 7.2, and Table 7.3.

7.3.2

T

RANS

F

ATTY

A

CIDS

IN

N

ATURAL

AND

P

ARTIALLY

H

YDROGENATED

T

RANS

F

ATS

On January 1, 2006, all packaged foods that enter interstate commerce in the UnitedStates must list

trans fat

content on their nutrition facts labels. This means that fromthat date,

trans

FAs are considered as saturated fatty acids from a nutritional pointof view, but their content in the composition of saturated FA must be listed separately.

Much earlier in Europe the Danish Veterinary and Food Administration publishedExecutive Order No, 160 on March 11, 2003, according to which, beginning in June2003, the content of

trans

fatty acids in the oils and fats (including emulsions),“should not exceed 2 grams per 100 grams of oil or fat.” According to the same

TABLE 7.1Saturated Fatty Acids

Systematic nameShorthanddesignation

Melting point(

°

C)Boiling point

a

(

°

C)

Butanoic Butyric 4:0 7.9; 5.0a 163.4760

Pentanoic Valeric 5:0 3.2a 185.4760

Hexanoic Caproic 6:0 3.4; 4.0a 204.9760

Octanoic Caprylic 8:0 16.7; 16.0a 237.9760

Nonanoic Pelargonic 9:0 12.5a 255.6760

Decanoic Capric 10:0 31.6; 31.4a 270.0760

Dodecanoic Lauric 12:0 44.2; 44.1a 298.1760

Tetradecanoic Myristic 14:0 53.9; 57.4a 326.2760

Hexadecanoic Palmitic 16:0 63.1; 62.8a 351.5760

Heptadecanoic Margaric 17:0 61.3; 60.85a 363.8760

Octadecanoic Stearic 18:0 69.6; 68.9a 361.1760

Eicosanoic Arachidic 20:0 75.3; 73.35a Destr. 318Docosanoic Behenic 22:0 79.9; 79.95a 30660; 26515

Tetracosanoic Lignoceric 24:0 84.2; 84.15a 272.010

Hexacosanoic Cerotic 26:0 87.7a —Octacosanoic Montanic 28:0 90.9a —Triacontanoic Melissic 30:0 93.6a —Dotriacontanoic Lacceric 32:0 96a —

Note: Pressure indicated by superscript.

Source: Cyberlipid Center, resource site for lipid studies: http://www.cyberlipid.org/index.htm(2006). Source for items designated a; Doss, M.P., 1952, Properties of the Principal Fats:Fatty Oils, Waxes, Fatty Acids, and Their Salts, Technical Research Division of The TexasCompany, New York.


Lipids and Food Quality 185

executive order, products claiming to be free of trans fatty acids shall have less than1 gram per 100 grams of the individual oil or the individual fat in the finished product.

The industrial hydrogenation of plant or fish oils is carried out under slightoverpressure of hydrogen at temperatures on the order of 180°C in the presence ofa catalyst, usually nickel formate, which under conditions of hydrogenation param-eters is decomposed into fine particles of metallic nickel. The hydrogenation ofunsaturated FAs takes place on the active surface of the catalyst. The mechanism ofthe reaction is very complex, thus it will not be discussed in detail here. However,in spite of the saturation of the double bonds with hydrogen, isomerization ofunsaturated FAs occurs during hydrogenation, as shown in Figure 7.3.

Besides geometrical isomerization shown in Figure 7.3, during partial hydrogena-tion of unsaturated FAs, migration of the double bonds along the hydrocarbon chainalso takes place resulting in formation of multiple positional FA isomers. Examplesof structures of positional C16:114t and C16:1 3t isomers are shown in Figure 7.4.

TABLE 7.2Monounsaturated Fatty Acids


Melting point(°C)

Boiling pointa

(°C)

Cis-2-octenoic 10:1 (n-6) 12715

Cis-4-decenoic 10:1(n-6) —Cis-8- decenoic Obtusilic 10:1(n-1) 6a to 0a 148–15023

Cis-9-decenoic Ioleocapric 10:1(n-1) 155–716

Cis-5-lauroleic Lauroleic 12:1(n-7) 15a 164–512

Cis-4-dodecenoic Linderic 12:1(n-8) 1a–1.3a 170–213

Cis-9-tetradecenoic Oleomyristic 14:1(n-5) 4.5a 1446

Cis-5-tetradecenoic Physeteric 14:1(n-9) < 0a 190–20015

Cis-4-tetradecenoic Tzuzuic 14:1(n-10) 18.0a–18.5a 185–813

Cis-9-hexadecenoic Oleopalmitic 16:1(n-7) 0.5; 1a 218–22015

Cis-6-octadecenoic Petroselinic 18:1(n-12) 30; 28.6a 237–23815

Cis-9-octadecenoic Oleic 18:1(n-9) 16.2; 13.2a 234–515; 225–610

Trans-9-octadecenoic Elaidic 18:1(n-9)t 43.6 a 23415

Trans-11-octadecenoic Vaccenic 18:1(n-7)t 39; 43.5a–44.5a 1961–2

Cis-9-eicosenoic Gadoleic 20:1(n-11) 25; 25a 17801

Cis-11-eicosenoic Gondoic 20:1(n-9) 22a —Cis-11-docosenoic Cetoleic 22:1(n-11) 32.5a–33a —Cis-13-docosenoic Erucic 22:1(n-9) 33.4; 33.5a 26415

Trans-13- docosenoic Brassidic 22:1(n-13)t 60 28230; 25610

Cis-15-tetracosenoic Nervonic 24:1(n-9) 39; 39a–39.5a —


Sources: Cyberlipid Center, resource site for lipid studies: http://www.cyberlipid.org/index.htm(2006). Source for items designated a; Doss, M.P., 1952, Properties of the Principal Fats: Fatty Oils,Waxes, Fatty Acids, and Their Salts, Technical Research Division of The Texas Company, New York.



TABLE 7.3Polyunsaturated Fatty Acids


MP(°C)

MeEstersboiling point*

(°C)

9,12-octadecadienoic Linoleic(LA) 18:2(n-6) 5; 5a5.2* 21520; 19810;149.51

6,9,12-octadecatrienoic γ-linolenic (GLA) 18:3(n-6) — —8,11,14-eicosatrienoic Dihomo-γ-linolenic

(DGLA)20:3(n-6) — —

5,8,11,14-eicosatetraenoic Arachidonic (AA) 20:4(n-6) 50 —7,10,13,16-docosatetraenoic — 22:4(n-6) — —4,7,10,13,16-docosapentaenoic — 22:5(n-6) — —9,12,15-octadecatrienoic α-Linolenic (ALA) 18:3(n-3) 11; 11.3*

to 11* 20714; 1551

6,9,12,15-octadecatetraenoic Stearidonic (SDA) 18:4(n-3) 57 —8,11,14,17-eicosatetrenoic — 20:4(n-3) — —5,8,11,14,17-eicosapentaenoic EPA 20:5(n-3) — —7,10,13,16,19-docosapentaenoic DPA 22:5(n-3) — —4,7,10,13,16,19-docosahexaenoic DHA 22:6(n-3) 44 —5,8,11-eicosatrienoic Mead acid 20:3(n-9) — —


Sources: Cyberlipid Center, resource site for lipid studies: http://www.cyberlipid.org/index.htm (2006).Source for items designated a, Doss, M.P., 1952, Properties of the Principal Fats: Fatty Oils, Waxes, FattyAcids, and Their Salts, Technical Research Division of The Texas Company, New York.

FIGURE 7.3 Principal chemical reactions during partial hydrogenation of unsaturated FAs.

Oil(liquid)

180oC Catalyst (Ni) Partially hydrogenatedfat (solid)

H2

COOHH3C

+2H

Isomerization Hydrogenation(geometrical cis-trans ) (saturated fatty acid)

(Natural) Cis double bond Trans double bond



As a result of industrial partial hydrogenation of plant or fish oils, most FAs, inparticular C14:1, C16:1, C18:1, C20:1, C22:1, and C24:1, are converted into multiple positionalisomers in the trans or cis configuration of the double bonds. The typical profilesof the composition of the C18:1 positional FA isomers in trans and cis configurationsin partially hydrogenated soybean oil are covered in the next paragraph (Figure 7.8).

The catalysts used for industrial hydrogenation of oils are usually very selective,which means that during hydrogenation, the rate of introduction of hydrogen intodouble bonds decreases with the number of double bonds in the FA molecule. Thuslinolenic acid C18:3 is hydrogenated faster than LA C18:2, which in turn is hydroge-nated faster than oleic acid C18:1. Consequently, in spite of different rates of hydro-genation of unsaturated FA in the end product, there are present small quantities ofdifferent geometric and positional isomers of PUFA, mainly trans-trans and cis-trans positional isomers of LA.

From the nutritional point of view, this fact creates serious problems because inthe human body, the processes of desaturation and elongation of artificial c,t or t,tisomers of LA result in the creation of artificial LC-PUFA, which after incorporationinto the structure of membranes, may potentially change their physical properties,including fluidity.

The hydrogenation of unsaturated FAs also occurs in nature. The process iscaused by the group of bacteria known as Butyrivibrio fibrisolvens that are presentin the rumen of cows or other ruminants. The mechanism of biohydrogenation ofunsaturated FA is totally different than that which is carried out in industry. Figure7.5 illustrates the example of biohydrogenation of LA C18:2 9,12cc.

FIGURE 7.4 Examples of structures of positional C16:114t and C16:1 3t isomers.

FIGURE 7.5 Course of conversion of linoleic acid C18:2 9c12c to stearic acid C18:0 in therumen caused by the Butyrivibrio fibrisolvens strain of bacteria.

H3CCOOH

H3CCOOH

C === C C C === C 9 10 11 12 13 Linoleic acid C18:2 9cis- 12cis

C === C C === C C 9 10 11 12 13 Conjugated linoleic acid C18:2 9cis-11trans

+2H

C C C === C C 9 10 11 12 13 Vaccenic acid C18:1 11trans

+2H

C C C C C 9 10 11 12 13 Stearic acid C18:0



As a result of the simplified schematic of the reaction presented in Figure 7.5,the intermediate products of biohydrogenation of linoleic acid are conjugated linoleicacid (CLA) C18:2 9c11t and vaccenic acid (VA) C18:1 11t. In nature, however, theprocess of biohydrogenation is more complicated. This is confirmed by the presencein milk fat of not only VA C18:111t, but also of other C18:1 trans-positional isomers,the presence of which indicates that side reactions also take place during biohydro-genation of unsaturated FA.

The different mechanisms of industrial partial hydrogenation of oils and bio-hydrogenation of unsaturated FA in cow rumen results in an entirely differentcomposition of FA positional trans and cis isomers. This is documented in Figure7.8 in which the comparison of the profiles of the composition of C18:1 cis andtrans isomers in industrially partially hydrogenated fats and in cow’s milk fat areshown.

7.3.3 CONJUGATED LINOLEIC ACID (CLA)

CLA has attracted a lot of attention over the past few years. Many claims for thebenefits of CLA have been made, from weight loss, to antioxidant, anticancerand, more recently, to activity in diabetes and cardiovascular disease as well.CLA is a mixture of positional and geometric isomers of LA, which is foundpredominantly in dairy products and meat of ruminants (Pariza et al., 2001). Thetwo double bonds in CLA are primarily in position 9 and 11, or 10, and 12 alongthe carbon chain.

There also can be other geometric or positional isomers (cis or trans configura-tion). Thus, at least 8 different CLA isomers of LA have been identified. Of theseisomers, the C18:2 9c,11t form is believed to be the most common natural form withbiological activity. Preliminary studies indicate that CLA is a powerful anticarcino-gen in the rat mammary tumor model with an effective range of 0.1 to 1% in thediet. This protective effect of CLA is noted even when exposure to carcinogens islimited to the time of weaning (Ip et al., 1994). In turn, the isomer C18:2 10c,12tdecreases the rate of biosynthesis of TAGs in preadipocytes; thus, according to someauthors, the supplementation of diet with C18:2 10c,12t isomer strongly reduces bodyfat and body weight. There are also other opinions regarding the biological activityof CLA. According to Noone et al. (2002) the supplementation of the human dietwith a blend of CLA had no effect on lipid metabolism in healthy human subjects.

FIGURE 7.6 The structure of the C18:2 9c 11t CLA isomer.

HOOC-

9c

11t



As the majority of studies have been carried out using mixtures of isomers in animalmodels, the observations regarding biological activity of CLA with respect to humansneeds to be substantiated before they can be regarded as fact (Wahle et al., 2004).

7.4 DETERMINATION OF THE TRANS FATTY ACID CONTENT IN FOOD LIPIDS

7.4.1 METHODS OF DETERMINATION

As the content of FA trans isomers must be shown on labels in the United Statessince January 1, 2006, and the content of so-called trans fats present in food productsin some European countries became strictly limited in July 2003, the need for anefficient method of determination of trans FA becomes obvious. Such determinationsare difficult because the physical properties of FA positional isomers are very similar.In GC evaluations, some trans FA isomers with double bonds closer to the endmethyl group overlap cis positional isomers with the double bond closer to thecarboxylic group. Because of that fact, the existing method of determinations oftrans isomer content according to AOCS Method 985.21 are strongly criticized.Using present state-of-the-art methods, it is rather impossible to separate in onechromatographic run all positional trans and cis isomers present in a sample of lipidsisolated from any food product. However, the task, which is very laborious, becomesfeasible with the application of some hyphenated techniques.

The general procedure used in the author’s laboratory for the determination ofFA trans isomer profiles is carried out in three stages. In the first stage the lipidsare isolated from the investigated sample, usually with the Folch method, or extractedfrom a sample with diethyl ether. Next the extracts (if necessary) are fractionatedinto lipid classes or directly converted after saponification into FAME using 14%boron trifluoride (BF3) solution in methanol.

In the second stage, the FAMEs are fractionated by silver ion HPLC using theChromSphere Lipids 25-cm, 4-mm ID dp5-μm packing (Chrompack, Raritan, NJ)column; the mobile phase: the logarithmic gradient of methylene chloride (solventB, more polar) in hexane (solvent A, as a less polar diluent). For the detection ofseparated components the laser light scattering detector (LLSD) is used (Stołyhwoet al., 1983, 1984).

The silver ion HPLC is based on the ability of the silver ion to complex withunsaturated compounds. The stability of the complex increases with the increasingnumber of double bonds. The cis isomers are more stable than the trans isomers,and conjugated polyenes form less stable complexes than do those containingmethylene-interrupted double bonds. FAs are resolved practically on the basis ofthe number and the configuration of their double bonds. Silver ion HPLC is veryefficient in separating geometrical isomers. The trans isomers migrate ahead ofthe corresponding cis molecules. The migration order is tt > ct > cc for dienes,and ttt > ctt > cct > ccc for the trienes. The example of silver ion HPLC separationof FAMEs of partially hydrogenated soybean oil (Frede et al., 1997) is shown inFigure 7.7.



Each fraction separated by silver ion HPLC (ChromSphere Lipids) is collectedin microcollectors.

In the next stage, all fractions are rechromatographed by GC using, connectedin series, two 105-meter; 0.25-mm df = 0.2-μm Rt×2330 columns (Restek, Chro-matographic Specialties, Canada). The chromatograms of separated cis and transC18:1 positional isomers isolated from partially hydrogenated soybean oil are shownin Fig. 7.8. For comparison, the fraction of positional C18:1 trans isomers isolatedfrom cow milk fat (Frede 1997) are included in the figure.

As seen in Figure 7.8, the trans C18:1 positional isomers in conditions of analysisare eluted from the column before corresponding C18:1 cis positional isomers. Theisomers with double bonds, which are closer to the carboxylic group, are elutedbefore positional isomers that are closer to the terminal methyl group. The physicalproperties of the positional 6t, 7t, 8t. 13t, and 14t are so similar that they cannot be

FIGURE 7.7 Separation of FAME of refined (a) and partially hydrogenated soybean oil (b)into classes by HPLC/Ag+ (Frede, E., Buchheim,W., and Stołyhwo, A., 1997, New develop-ments in milk fats, in Modern Developments in Food Lipids, Shukla, V.K.S. and Kochhar,S.P., Eds., CentreA/S, Lystrup, Denmark, pp. 171–191. With permission.)



separated to the base line even in conditions of analysis (as above) on the 200-meterGC column having almost one-half million theoretical plates.

As seen in a comparison of Figures 7.8A and 7.8B, the retention times of someof the trans positional isomers overlap positional cis isomers. This fact indicates thatexisting methods of determining the content of trans isomers in food products (AOCSMethod 985.21) are not sufficiently accurate to fulfill actual requirements regardinglabeling of products containing trans isomers or limiting their content in food products

FIGURE 7.8 Separation of individual positional FAME C18:1 isomers of hydrogenated fat,preliminarily separated (and collected) into classes (as in Figure 7.7). Parameters of separation:two 100-m ID 025-mm columns Rt× 2330 in series; column temp. 155°C, flow rate 28cm/s.A) C18:1 trans isomers of partially hydrogenated fat; B) C18:1 cis isomers of partially hydro-genated fat; C) C18:1 trans isomers of low milk fat.

A

C

B

t4t5

t6, 7, 8

t9t10

t11

t12

t13, 14

t15 t 16

c4 c5

c6, 7, 8

c9

c10

c11

c12

c13

c15c14

t5

t6, 7, 8

t9

t10

t11

t12 t13, 14

t15 t16



to less than 2 g of trans isomers per 100 g of fat. Such methods seem to be inappli-cable in the case when a given product is prepared using a mixture of partiallyhydrogenated fats and milk fat.

Figures 7.8A, B, and C are also good examples of the differences in the mech-anisms of industrial partial hydrogenation of oils carried out in autoclaves, andalternatively of biohydrogenation of unsaturated FA, which takes place in the cow’srumen. Indeed, the profiles of trans isomers produced in both processes are totallydifferent. In the case of biohydrogenation as seen in Figure 7.8C, the principal transisomer produced in rumen is VA C18:1 11t, whose content in the composition of transisomers accounts for 68% to 86% (author’s own data) in a season and a method offeeding of cows. In the fall, when the content of oil in the grass seed in the pastureis greatest, cows are consuming more unsaturated FAs, which results in a greaterconcentration of VA (and also of CLA) in the milk fat. The presence of positionaltrans C18:1 isomers, other than VA as referred to Figure 7.8C indicates that in thebiohydrogenation in rumen, besides formation of VA, other side reactions take place.However, the rate of side reactions is much smaller because the content of otherthan C18:1 11t isomers is much smaller than of VA.

Biohydrogenation of unsaturated FA in cow’s rumen forms small amounts ofC18:113 cis isomers, and does not produce substantial amounts of any other C18:1 cispositional isomers.

The industrial hydrogenation of plant oils results in production of multipletrans and cis positional isomers as shown in Figure 7.8A and B. In that case theprincipal C18:1 trans (as in Figure 7.8A) isomers are: 6t + 7t + 8t (24%); 9t: elaidicacid (17.1%); 10t (16.1%) and 11t: VA about 15% in the composition of all C18:1

trans isomers. In a parallel manner they produce multiple C18:1 cis isomers, thequantitative profile of which is as follows (author’s own data): 6c + 7c + 8c(16.8%); 9c (24.7%); 10c (16%); 11c (12.9%); 12c (8.6%); 13c (7.2%) 14c (4.5%);15c (3.3%); and 116c (1.2%).

7.4.2 BIOLOGICAL SIGNIFICANCE

The above data suggest a question: What is the nutritive value of all FA isomersproduced by biohydrogenation or alternatively by industrial partial hydrogenation?At present there is official scientific information indicating that trans fats producedindustrially are harmful with respect to human health. Trans fat is known to increaseblood levels of low-density lipoprotein (LDL), or “bad” cholesterol, while loweringlevels of high-density lipoprotein (HDL), known as “good” cholesterol. It can alsocause major clogging of arteries, type 2 diabetes, and other serious health problems,and was found to increase the risk of heart disease. The trans FA may block thetransport of LC PUFA across the human placenta resulting in a decrease of AA C20:4

(n-6) and DHA C22:6 (n-3) in the fetal brain (Stołyhwo 2000, unpublished) reducingthe baby’s psychomotor abilities when delivered. Many food companies use transfat instead of oil because it reduces cost, extends storage life of products, and canimprove flavor and texture.

VA produced by biohydrogenation plays a very important role in human organisms.It is believed that C18:1 11t undergoes delta 9–desaturation in women’s mammary



glands, resulting in the formation of C18:2 9c, 11t, that is, CLA, which protects thebreast against development of breast cancer. The industrially produced trans C18:1

trans isomers are mainly 6t + 7t + 8t; 9t; and 10t. Their delta 9–desaturation, as itresults from their structure, is rather impossible. If delta 9–desaturation of VA in thehuman mammary gland can finally be proven, the difference between the nutritivevalue of trans isomers created by biohydrogenation or industrial partial hydrogena-tion will become evident.

7.5 FATTY ACID COMPOSITION OF COMMON FATS

For the convenience of the reader, Table 7.4 and Table 7.5 cover the FA compositionof common oils and fats used in the food industry. Because of seasonal variations,the above data should be considered as indicative only.

7.6 LONG-CHAIN POLYENOIC FATTY ACIDS AND THEIR IMPORTANCE IN HUMAN NUTRITION

These FAs, also called PUFAs, have two or more cis double bonds, which are themost frequently separated from each other by a single methylene group. Typicalmembers of this group are LA C18:2 9c 12c or ALA C18:2 9, 12, 15, all cis. The mostcommon PUFAs are listed in Table 7.3.

PUFAs, like saturated or monoenoic FAs, are the source of energy that is liberatedduring their oxidation in the human organism. The oxidation occurs on the side ofthe carboxylic group in such a manner that during each step of oxidation, two carbons(one carboxylic group and one methylene group) are cut out from the FA molecule.Thus, after each step of oxidation, the positions of the double bonds in the moleculeof an FA, with respect to carboxylic acid, change. Moreover, independent of oxida-tion of FAs, in the human organism other important chemical reactions controlledby enzymes are taking place, that is, desaturation or elongation of hydrocarbonchains as shown in Figure 7.10 (see below). Again, after each step of elongation ordesaturation, the position of the double bonds with respect to the carboxylic groupin the FA molecule is changing. Thus in the biochemistry of lipids, the shorthandpresentation of FA molecules becomes impractical.

In the late 1950s, Ralph Holman from the Hormel Institute (University ofMinneapolis, Austin, Minnesota) stated that in spite of changes in the FA moleculestructure caused by oxidation or desaturation and elongations processes, the positionof the double bonds in the skeleton of an FA, if counted from the terminal methylgroup, does not alter. Consequently, Holman suggested counting the position of thefirst double bond in the FA molecule from the terminal omega (as this is the lastletter in the Greek alphabet) methyl group, as shown in Figure 7.9.

It was a revolutionary suggestion that resulted in the discovery of importantfamilies of omega-6 and omega-3 polyunsaturated FAs (as in Figure 7.10), whichmakes it easier to understand the metabolism of PUFAs in the human organism.Later the Committees on Chemical Nomenclature suggested the following shorthandnotations for FAs from the omega-6 and omega-3 families: (n-6) and (n-3).



TAB

LE 7

.4Fa

tty

Aci

d C

ompo

siti

on o

f Veg

etab

le O

ils a

s D

eter

min

ed b

y G

as L

iqui

d C

hrom

atog

raph

y fr

om A

uthe

ntic

Sam

ples

(ex

pres

sed

as p

erce

ntag

e of

tot

al f

atty

aci

ds)

Fatt

y ac

idPa

lm o

lein

Palm

ste

arin

Rap

esee

d oi

l

Rap

esee

d oi

l(l

ow e

ruci

c ac

id)

Saffl

ower

see

doi

lSe

sam

e se

edoi

lSo

ya b

ean

oil

Sunfl

ower

seed

oil

C6:

0N

DN

DN

DN

DN

DN

DN

DN

DC

8:0

ND

ND

ND

ND

ND

ND

ND

ND

C10

:0N

DN

DN

DN

DN

DN

DN

DN

DC

12:0

0.1–

0.5

0.1–

0.5

ND

ND

ND

ND

ND

–0.1

ND

–0.1

C14

:00.

5–1.

51.

0–2.

0N

D–0

.2N

D–0

.2N

D–0

.2N

D–0

.1N

D–0

.2N

D–0

.2C

1638

.0–4

3.5

48.0

–74.

01.

5–6.

02.

5–7.

05.

3–8.

07.

9–10

.28.

0–13

.55.

0–7.

6C

16:1

ND

–0.6

ND

–0.2

ND

–3.0

ND

–0.6

ND

–0.2

0.1–

0.2

ND

–0.2

ND

–0.3

C17

:0N

D–0

.2N

D–0

.2N

D–0

.1N

D–0

.3N

D–0

.1N

D–0

.2N

D–0

.1N

D–0

.2C

17:1

ND

–0.1

ND

–0.1

ND

–0.1

ND

–0.3

ND

–0.1

ND

–0.1

ND

–0.1

ND

–0.1

C18

:03.

5–5.

03.

9–6.

00.

5–3.

10.

8–3.

01.

9–2.

94.

8–6.

12.

0–5.

42.

7–6.

5C

18:1

39.8

–46.

015

.5–3

6.0

8.0–

60.0

51.0

–70.

08.

4–21

.335

.9–4

2.3

17.7

–28.

014

.0–3

9.4

C18

:210

.0–1

3.5

3.0–

10.0

11.0

–23.

015

.0–3

0.0

67.8

–83.

241

.5–4

7.9

49.8

–59.

048

.3–7

4.0

C18

:3N

D–0

.6N

D–0

.55.

0–13

.05.

0–14

.0N

D–0

.10.

3–0.

45.

0–11

.0N

D–0

.3C

20:0

ND

–0.6

ND

–1.0

ND

–3.0

0.2–

1.2

0.2–

0.4

0.3–

0.6

0.1–

0.6

0.1–

0.5

C20

:1N

D–0

.4N

D–0

.43.

0–15

.00.

1–4.

30.

1–0.

3N

D–0

.3N

D–0

.5N

D–0

.3C

20:2

ND

ND

ND

–1.0

ND

–0.1

ND

ND

ND

–0.1

ND

C22

:0N

D–0

.2N

D–0

.2N

D–2

.0N

D–0

.6N

D–1

.0N

D–0

.3N

D–0

.70.

3–1.

5C

22:1

ND

ND

>2.

0–60

.0N

D–2

.0N

D–1

.8N

DN

D–0

.3N

D–0

.3C

22:2

ND

ND

ND

–2.0

ND

–0.1

ND

ND

ND

ND

–0.3

C24

:0N

DN

DN

D–2

.0N

D–0

.3N

D–0

.2N

D–0

.3N

D–0

.5N

D–0

.5C

24:1

ND

ND

ND

–3.0

ND

–0.4

ND

–0.2

ND

ND

ND

Sour

ce:

Cod

ex A

limen

tari

us C

omm

issi

on,

Cod

ex S

tand

ard

for

Nam

ed V

eget

able

Oil

s, C

odex

Sta

ndar

d 21

0.



The discovery that unsaturated FAs are essential (Burr and Burr, 1929) was stronglyenhanced by Hollman’s suggestion because it was possible to determine that for thehuman organism, there are only two essential unsaturated FAs—LA C18:2 (omega-6 or

TABLE 7.5Fatty Acid Composition of Animal Fats

LardRendered pork fat

Premier justallow

C10:0 < 0.5 in total < 0.5C14:0 1.0–2.5 2–6C14:ISO < 0.1 < 0.3C14:1 < 0.2 0.5–1.5C15:O < 0.2 0.2–1.0C15:ISO < 0.1 < 1.5C15:ANTI ISO < 0.1C16:0 20–30 20–30C16:1 2.0–4.0 1–5C16:ISO < 0.1 < 0.5C16:2 < 0.1 < 1.0C17:0 < 1 0.5–2.0C17:1 < 1 < 1.0C17:ISO < 0.1 < 1.5C17:ANTI ISO < 0.1C18:0 8–22 15–30C18:1 35–55 30–45C18:2 4–12 1–6C18:3 < 1.5 < 1.5C20:0 < 1.0 < 0.5C20:1 < 1.5 < 0.5C20:2 < 1.0 < 0.1C20:4 < 1.0 < 0.5C22:0 < 0.1 < 0.1C22:1 < 0.5 not detected

Source: Codex Alimentarius Commission, Codex Standard for Named Animal Fats, Codex Standard 211-1999.

FIGURE 7.9 The structure of EPA C20:5 5,8,11,14,17 all cis (C20:5 omega-3).

t

TerminalOmega –CH3 group

35791113151719 1

COOHH3C



n-6) and ALA C18:3 (omega-3 or n-3). Neither LA nor ALA are synthesized in the humanorganism, thus they must be supplied with the diet. Other metabolites are not essentialbecause they are created in the human organism as a result of the action of enzymes,that is, of adequate desaturases and elongases.

The schematic of the metabolism of PUFAs in the human organism is shown inFigure 7.10.

Both LA and ALA, as the “parents” of the n-6 or n-3 FA families, are convertedin the human organism to their metabolites by the successive reactions of desaturationor elongation. Thus, both families compete for the appropriate enzymes. The mostsensitive is the first step, delta 6–desaturation, because this step of FA metabolismis rate limiting. The increase in the concentration of FAs of one family, for example,the n-6 family caused by increased consumption of n-6 FAs (sunflower or maize,known as linoleic oils, do not contain n-3 FAs), causes a decrease in conversion ofALA into their metabolites and vice versa.

Thus from the nutritional point of view, the proportions of n-6 and n-3 FAmetabolites should be balanced as it is schematically shown in Figure 7.11.

FIGURE 7.10 Metabolic pathways of n-3 and n-6 LC-PUFA in the human organism.

Diet

C18:1(n-9) C18:2(n-6) C18:3(n-3)Present in human diet Oleic acid LA ALA

Metabolic processes

Desaturase 6 C18:2(n-9) C18:3(n-6) C18:4(n-3)

-Linolenic GLA SDA

Elongase C20:2(n-9) C20:3(n-6) C20:4(n-3)Dihomo- -linolenic

DGLA

Desaturase 5 20:3(n-9C ) C20:4(n-6) C20:5(n-3)Mead MA TimnodicAA

EPA

Elongase C22:3(n-9) C22:4(n-6) C22:5(n-3) DPAAdrenic

Desaturase 4 C22:5(n-6) C22:6(n-3) DHA Osmond Cervonic



The conversion of LA and ALA may be monitored in a human organism. Thelipids isolated from human tissues by extraction according to the Folch or Bligh-Dyer methods after separation into lipid classes may be converted to FAME andanalyzed by GC. Individual LC PUFA may be easily identified on chromatogramsas shown in Figure 7.12.

7.7 TRIACYLGYLCEROLS

7.7.1 CHEMICAL AND FUNCTIONAL PROPERTIES

The formal presentation of synthesis of TAGs is shown in Figure 7.13.The hydrocarbon chains R may be the same (though this is rare), two may be

the same, or more usually they are different. Because there is a great variety in thechain length and structure of FAs, there is great scope for variation in the makeupof fats and oils. It is also possible that not all hydroxyl groups are esterified, thusother subgroups of acylglycerols may also be present in natural fats and oils.

In MAGs, one FA is esterified to glycerol. Due to the orientation of thatmolecule, two isomeric forms exist as shown in Figure 7.14. R is a saturated or anunsaturated hydrocarbon chain. The external carbons of glycerol are frequentlynamed as “α” and the central one as “β.” One part of a molecule of MAG containshydrophilic –OH groups and another part lipophilic (hydrophobic) acyls of FA.Thus, the MAGs exhibit surface active properties and are commonly used in thefood industry as emulsifiers. The main fields of application are margarines, may-onnaises, and ice creams.

DAGs are FA diesters and occur in two isomeric forms (Figure 7.15). Theirproperties as surface active agents are much weaker than those of MAGs. In foods,DAGs are used as emulsifying agents in the production of fine baked goods, chewinggum, and in replacement of fats. Their main field of application is the cosmeticindustry as one of the components of creams and shampoos.

FIGURE 7.11 Lipid balance: the optimal proportions of n-6 and n-3 fatty acids in the humandiet should be between 5:1 and 3:1.

Flax seed oil (56% ALA); rapeseed oil (11% ALA);soybean oil (6–9% ALA) or fish oils

n-6 FA containing oils: sunflower (67% LA); maize(70% LA); safflower oil (up to 80% LA)



FIG

UR

E 7.

12M

onito

ring

of

LC

PU

FA t

rans

form

atio

ns c

atal

yzed

by

corr

espo

ndin

g de

satu

rase

s an

d el

onga

ses

by G

C (

colu

mn:

Rtx

2330

100

-m I

D0.

25-m

m;

d f 0

.2-μ

m t

emp

in 1

55 t

ime

50;

rate

1.5

°C/m

in;

fin 2

10°C

, the

n ho

ld.

Inj.

Split

/spl

itles

s; h

ydro

gen

32 c

m/s

) (a

utho

r’s

own

data

).

C18

:4(n

-3)

EP

A C

20:5

(n-3

)D

PA

C22

:5

(n-3

)

C18

:2

C18

:1t

C18

:0

C16

:2

C16

:17

C16

:0

C14

:0

C12

:0

GL

A

DG

LA

AA

n-6

ALA

C18

:4

C22

:4

rans

C18

:111

C18

:3

(n-6

)

DG

LA

C20

:3(n

-6)

n-3

C22

:5

C20

:4EPA

DPA

DH

A

C22

:4 C22

:5

LA

C18

:19

AA

C20

:4(n

-6)



7.7.2 MODIFICATIONS

TAGs can be modified by transesterification. This is a term covering a group ofchemical reactions involving a fat and either an FA, an alcohol, or another ester. Asa result of chemical interchange, a new ester is formed.

In acidolysis, an FA in the TAG is replaced by another one according to thereaction in Figure 7.16. Alcoholysis is a reaction of TAGs with alcohol. A goodexample is the synthesis of MAGs in which one portion of TAGs containing onlysaturated FAs and 2 portions of glycerol are subjected to reaction at elevated tem-perature in the presence of a catalyst (NaOH, or ZnO) (Figure 7.17 ). In transester-ification at an elevated temperature in the presence of catalysts, the acyl groups areexchanged between TAGs according to reaction shown in Figure 7.18. These reac-tions give rise to a great variety of possibilities in the industrial modification of thephysical and physiological properties of fats and oils.

7.7.3 FRACTIONATION

That some fats melt before others is obvious to anyone who has looked at butter inhot weather. Clear oil can be seen to be separating from the soft but not molten

FIGURE 7.13 Formal presentation of synthesis of TAGs.

FIGURE 7.14 Isomeric forms of MAGs.

FIGURE 7.15 Isomeric forms of DAGs.

O

H2C OH

HC OH

H2C OH

HO C R

HO C R

HO C R

O

O

O

H2C O C RO

O

H2C O C R

HC O C R+ + 3 H2O

Glycerol Fatty Acids Triacylglycerol

CH2

CH2

O CO R

HO C H

OH

CH2OH

CH2

CH

OH

O CO R

α 1

β 2

α′ 3sn-1- or α-isomer 2- or β-isomer

CH2O

CH

CO R

HO C H

O CO R´

CH2OH3

2

1 CH2

CH

O CO R´

O CO R

sn-1,3- or α, α′-isomer sn-1,2- or α, β-isomer



material. This is the basis of fractionation of fats. The melting points of differentTAGs are shown in Table 7.6.

The melting point (MP) of TAGs increases according to the chain length of FAand decreases according to the number of the double bonds in the TAG molecule.The MP of TAGs containing cis double bonds are much lower than correspondingTAGs with trans double bonds. In order to obtain good separation of TAGs withdifferent MPs it is better to begin with molten fat and to cool it under strictlycontrolled conditions to solidify some parts (fractions). The solid fractions may beremoved as they are formed by decantation, filtration, or centrifugation. The mostpopular fractionated fats on the market are palm oleine and palm stearine. Hydro-genated palm or rapeseed oils are also frequently subjected to fractionation in orderto remove fractions of the TAGs with high melting points.

7.7.4 LIPOLYSIS

In the human organism a TAG molecule cannot be directly absorbed across theintestinal mucosa. It must first be digested into a sn-2 (or β)–MAG isomer and twofree FAs. The enzyme that performs this hydrolysis is pancreatic lipase, which is

FIGURE 7.16 Acidolysis: reaction of TAGs with free FA.

FIGURE 7.17 Alcoholysis: reaction of TAGs with alcohol.

FIGURE 7.18 Transesterification: exchange of acyls between TAGs.

CH2. OOCR1 CH2. OOCR

CH. OOCR2 + R. COOH CH. OOCR2 + R1 COOH

CH2. OOCR3 CH2. OOCR3

CH2. OOCR1 CH2. OH CH2. OOCR1 (or R2 or R3)

CH. OOCR2 + 2 CH. OH 3CH. OH

CH2. OOCR3 CH2. OH CH2. OH

or

CH. OOCR1 CH2. OH

CH. OOCR2 + 3ROH CH. OH + R1. COOR + R2. COOR + R3.COOR

CH2. OOCR3 CH2. OH

CH2. OOCR1 CH2. OOCR2 CH2. OOCR2 CH2. OOCR1

CH. OOCR2 + CH. OOCR3 CH.O OCR2 + CH. OOCR3

CH2. OOCR3 CH2. OOCR4 CH2. OOCR3 CH2. OOCR4



delivered into the lumen of the gut as a constituent of pancreatic juice. Pancreaticlipase catalyses the hydrolysis of FA from positions sn-1 and sn-3 (or α,α′) of TAGsto yield sn-2 (or β)- MAGs. Very little hydrolysis occurs at position sn-2 and thereis limited isomerization to sn-1-MAGs, which may become substrates for furtherlipase action. The enzyme reacts with the TAG at the oil–water interface of largeoil in water (O/W)) emulsion particles, but hydrolysis can occur only after both theenzyme and surface have been modified to allow interaction. Bile salt moleculesaccumulate on the surface of the lipid particles displacing other surface activeconstituents enabling pancreatic lipase to attack the TAG molecule. Schematicallythe enzymatic hydrolysis of TAG is shown in Figure 7.19.

As far as human nutrition is concerned, very important conclusions can be drawnfrom Figure 7.19. Palmitic acid C16:0 is present in human milk or in human fat at aconcentration of 30 to 32% in the FA composition. Almost 96% of palmitic acid inhuman fat is situated in position sn-2 of a TAG molecule. Positions sn-1 and sn-3are usually occupied by short-chain (sn-1) FAs and unsaturated (sn-3) FAs. Thehydrolysis of TAG molecules catalyzed by pancreatic lipase liberates short-chain orunsaturated FAs from sn-1 and sn-3 positions, which are absorbed directly into thebloodstream from the stomach.

In the case of plant oils, the sn-2 position is usually occupied by LA, and thesaturated FAs, including palmitic acid, occupy the external positions sn-1 or sn-3.Such plant oils are commonly added to infant formulas in order to increase thecontent of LA to the level of 9 to 11% in FA composition in order to mimic the FAcomposition of human milk. As a result of enzymatic hydrolysis saturated FAs areliberated in the infant’s gastric system. A part of the liberated saturated FA mayreact with the Ca2+ present in the gastric system. Especially in the case of newbornbabies fed with infant formulas, difficult soluble calcium soaps are removed fromthe organism with the stool causing deficiency of Ca2+ in the organism. The stoolof infants becomes very hard when the mother replaces natural feeding with formulascontaining plant oils. Thus, because of differences of stereospecific structures ofhuman and plant TAGs, addition of plant oils to infant formulas should be considereda poor solution for feeding of newborn babies.

TABLE 7.6Melting Points of Some Saturated and Unsaturated Triacylglycerols with Different Chain Lengths

Triacylglycerol Melting point (°C)

C6 –15C12 15C14 33C16 45C18 55C18:1 (cis) –32C18:1 (trans) 15



7.7.5 ANALYSIS OF THE COMPOSITION OF TRIACYLGLYCEROLS

7.7.5.1 HPLC of Triacylglycerols

A great number of possible isomers and relatively large molecules are the reasonswhy analysis of the composition of the TAGs mixtures is a rather difficult task. Inthe case where di-, tri-, or more unsaturated FAs are present in a TAG molecule, theuse of very efficient GC becomes problematic because high temperatures of columnsmuch over 300°C must be used, which may be destructive with respect to theanalyzed sample. In such cases, less efficient HPLC produces more analytical infor-mation and seems to be very useful.

TAGs are classically named using three letters indicating the diversity of theacylated FA, for example, the homogeneous LLL, OOO, PPP, SSS or the heteroge-neous OOL, SOO, or PSO: L for LA, O for oleic acid, P for palmitic acid, and Sfor stearic acid.

Under isocratic conditions, the logarithm of the elution volume (or retentiontime) of a molecular species is directly proportional to the number of carbon atoms(CN), and inversely proportional to the total number of double bonds (DB) in thethree fatty acyl chains (Stołyhwo et al. 1985). Furthermore, a linear relationship

FIGURE 7.19 Enzymatic hydrolysis of TAG molecule of human milk.

CH2

O

C

CH2

O

CR1

CR3

O

H

CR2

O

O

O

sn-1

sn-2

sn-3Palmiticsaturated

Unsaturated

Saturatedshort chain

Pancreatic lipase

CH2

OH

C

CH2

OHO

H

CR2

O

sn-1

sn-2

sn-3

Palmiticsaturated



exists between the equivalent carbon number (ECN) and the carbon number of theTAG that showed the same unsaturation characteristics.

The principal obstacle in the application of HPLC is the problem of detection.The refractive index (RI) detectors cannot be applied when gradient elution is to beused for the separation of natural mixtures of fats usually containing simultaneouslyfree FA, MAGs, DAGs, and TAGs.

The LLSD is the detector of choice for the analysis of lipid classes including TAGs(Stołyhwo et al. 1983, 1984). The advantage of LLSD lies in the fact that it is sensitivewith respect to all relatively nonvolatile UV-absorbing or nonabsorbing substances.The mobile phase is evaporated before detection of analytes, thus any solvent (exclud-ing buffer solutions), may be used in isocratic or gradient elution systems.

Natural, unaltered TAGs do not absorb UV radiation at wavelengths over 220 nm,thus classical UV 254-nm detectors cannot be used for such purposes. The carbonylgroup of the ester bond in TAGs absorbs UV at a wavelength below 218 nm, but thisis the range at which there is a very limited number of solvents not absorbing UV.Thus, in practice for the analysis of TAGs, the mobile phases may be composed onlyof methanol, propanol-2, acetonitrile, and HPLC gradient-grade hexane.

In spite of the drawbacks of UV, the use of a diode array detector (DAD) inseveral detection channels is very practical in identifying the substances dissolvedin the analyzed sample, for example, carotenoids (detection at 450 to 465 nm);tocopherols (detection at 295 nm), FAs with conjugated double bonds (detection at233 nm for CLA), and 268 nm for triunsaturated FAs with conjugated double bonds.Products of deterioration of lipids by autooxidation can also be detected, for example,peroxides, hydroperoxides, aldehydes, or epoxy acids (detection by UV at 235 to250 nm). An example of the application of multichannel LLSD/DAD detection ofTAGs of evening primrose oil (EPO) and of products of its autooxidation is shownin Figure 7.20 (a, b). With the use of LLSD it is possible to detect all relativelynonvolatile components of EPO. The signal S of the LLSD is dependent on the massm of an analyte approximately according to the equation,

S = km1.37,

where k is the response factor (Stołyhwo et al., 1983, 1984).In channel DAD 215 nm, all analytes absorbing at 215 nm are detected but their

quantities depend on the corresponding peak areas and on molecular coefficients ofabsorbance. Thus tocopherols are easily detected as well as products of autooxidationof TAGs.

In channel DAD 268, the conjugated trienes as intermediate products of auto-oxidation of GLA C18:3(n-6) are detected with good sensitivity. The degree of deteri-oration of EPO caused by peroxidation may be estimated as the ratio of the areas 268nm to 215 nm of the peaks corresponding to the LnLnLn and LnLnL.

7.7.5.2 GLC of Triacylglycerols

The TAGs of fats containing more saturated FA, as, for example, of coconut oil,palm kernel oil, palm oil, cocoa butter, or milk fats may be analyzed by GC. In such



FIGURE 7.20 (a) HPLC of TAGs of natural evening primrose oil (EPO). Detection parallelin channels: LLSD, DAD1A at 218-nm bandwidth (BW) 5 nm; DAD1B at 268-nm BW 10nm. Column: Hypersil ODS 5 μm; mobile phase: acetonitrile/propanol-2/hexane 66/21/13v/v/v. Flow rate 0.8 mL/min, column temp. 18°C.

min0 10 20 30 40 50

nA

30

32

34

36

38

ADC1 A, ADC CHANNEL A of KURS\VITAMX50.D

8.8

07 9

.912

10.

961

11.

208

19.

141

22.

958

27.

639

28.

837

30.

753

35.

101

37.

163

45.

082

46.

175

47.

743

min0 10 20 30 40 50

mAU

5

10

15

20

25

30

35

DAD1 A, Sig=218,5 Ref=550,100 of KURS\VITAMX50.D

6.4

38

10.

145

11.

161

13.

464

19.

348

23.

174

27.

847

29.

120

30.

916

35.

314

37.

378

min0 10 20 30 40 50

mAU

5

10

15

20

25

30

35

DAD1 B, Sig=268,10 Ref=550,100 of KURS\VITAMX50.D

9.0

47

11.

158

19.

098

22.

262

22.

835

28.

792

LLSD

L LL L P

LnLnLn

L L LLnLnL

Tocopherols

DAD 215 nmProducts ofautooxidation

DAD 268 nm

Conjug.trienes



cases, instead of split/splitless injection, it is better to use a cool on column injectionsystem. The TAGs dissolved in isooctane (conc. 0.04%) are injected (0.5 μl) directlyinto the capillary column at an inlet temperature of 77°C, and the temperatureprogram is started. In the author’s laboratory for analysis of TAGs by GC, the Restek65 TAG column (Restek, Chromatographic Specialties, Canada), 30-m 0.25-mm IDdf 0.2-μm column is used.

An example of the separation of TAGs of butter and of butter adulterated with10% sunflower oil is shown in Figure 7.21. The TAGs of sunflower oil contain over80% FAs with a chain length of 18 carbon atoms. Thus the carbon number (CN) ofthose TAGs is CN 54. Adulteration of butter with sunflower oil as shown in Figure7.21 B is evident because natural butter contains only minute amounts (below 0.2%)of TAGs with CN 54.

FIGURE 7.20 (b) HPLC of TAGs of natural evening primrose oil (EPO). Detection parallelin channels: LLSD, DAD1A at 218-nm BW 5 nm; DAD1B at 268-nm BW 10 nm. Column:Hypersil ODS 5 μm; mobile phase: acetonitrile/propanol-2/hexane 66/21/13 v/v/v. Flow rate0.8 mL/min, column temp. 18°C.

nm230 240 250 260 270 280 290

mAU

1

2

3

4

5

6

7

8

9

*DAD1, 19.211 (180 mAU, -

nm220 240 260 280 300

mAU

0

20

40

60

80

100

120

*DAD1, 11.171 (434 mAU, -

nm220 240 260 280 300 320

mAU

0

2.5

5

7.5

10

12.5

15

17.5

20

*DAD1, 13.778 (22.5 mAU,

nm240 250 260 270 280 290

mAU

0

2

4

6

8

10

12

14

*DAD1, 23.291 (1131 mAU

268 nm TocopherolsConjug.trienes

295 nm

Products ofperoxidation 235–250 nm

Conj. trienes 268 nm



REFERENCES

AOAC International, 1990, Method No. 920.39C, Crude fat or ether extract, Gaithersburg,MD.

AOAC International, 1990, Method No. 932.06, Fat in dried milk (Roese Gottlieb MethodIDF-ISO–AOAC), Gaithersburg, MD.

AOCS (American Oil Chemist’s Society), 1993, Method No. 922.06, Acid hydrolysis method,Champaign, IL.

AOCS (American Oil Chemist’s Society), 1993, Method 985.21, Total fatty acid isomers inmargarines: Gas chromatographic method, Champaign, IL.

AOCS (American Oil Chemist’s Society), 1993, Method No. Ce 1b-89, Fatty acid compositionby GLC marine oils (modified), Champaign, IL.

FIGURE 7.21 Separation of TAGs of butter (a) and of butter adulterated with 10% sunfloweroil (b). Column Restek 65 TG cool on column injection at 77°C. Temperature program in80°C time 3 min, next rate 20°C/min, fin 240°C, next rate 3°C/min, fin temp 340°C, nexthold 25 min. Flow rate: hydrogen 22 cm/s.

(a)

CN28

CN 30

34

38 42CN32

CN46 CN 48

CN 50

CN 52

CL

(b)

CN 54



Bligh, E.G. and Dyer, W.J., 1959. A rapid method for total lipid extraction and purification.Can. J. Biochem. Physiol. 37, 911–917.

Burr, G.O. and Burr, M.M., 1929, A new deficiency disease produced by the rigid exclusionof fat from the diet, J. Biol. Chem. 82, 345–367.

Codex Alimentarius Commission, Codex Standard for Named Animal Fate, Codex Standard211-1999, http://www.codexalimentarius.net/download/standards/337/cxs-211e.pdf

Codex Alimentarius Commission, Codex Standard for Named Vegetable Oils, Codex Standard210, http://www.codexalimentarius.net/downloads/standards/336/cxs-210e.pdf

Folch, J., Lees, M., and Sloan-Stanley, G.H., 1957. A simple method for the isolation andpurification of total lipids from animal tissues, J. Biol. Chem., 226, 497–509.

Frede, E., Buchheim,W., and Stołyhwo, A., 1997, New developments in milk fats, in ModernDevelopments in Food Lipids, pp. 171–191, Shukla, V.K.S. and Kochhar, S.P., Eds.,Centre A/S, Lystrup, Denmark.

Ip, C., Scimeca, J.A., and Thompson, H.J., 1994 (August 1). Conjugated linoleic acid. Apowerful anticarcinogen from animal fat sources, Cancer, 74 (3 Suppl), 1050–1054.

Kaluzny, M.A., Duncan, L.A., Merritt, M.V., and Epps, D.E., 1985. Rapid separation of lipidclasses in high yield and purity using bonded phase columns, J. Lipid Res., 26,135–140.

Noone, E.J., Roche, H.M., Nugent, A.P., and Gibney, M.J., 2002. The effect of dietarysupplementation using isomeric blends of conjugated linoleic acid on lipid metabo-lism in healthy human subjects, Br. J. Nutr. 88, 243–251.

Pariza, M.W., Park, Y., and Cook, M.E., 2001. The biologically active isomers of conjugatedlinoleic acid. Prog. Lipid Res., 40, 283–298.

Stołyhwo, A., Colin, H., and Guiochon G., 1983. Use of light scattering as a detector principlein liquid chromatography, J. Chromatog., 265, 1–18.

Stołyhwo, A., Colin H., and Guiochon, G., 1985. Analysis of triglycerides in fats and oilswith the use of light scattering detector, Anal. Chem., 57, 1324– 354.

Stołyhwo, A., Martin, M., Colin H., and Guiochon G., 1984. Study of the qualitative andquantitative properties of the light-scattering detector, J. Chromatog., 288, 253.

Wahle, K.W., Heys, S.D., and Rotondo D., 2004. Conjugated linoleic acids: are they beneficialor detrimental to health? Prog. Lipid Res., 43(6), 553–587.


209

8 Rheological Propertiesof Food Systems

Anna Pruska-Kedzior and Zenon Kedzior

CONTENTS

8.1 Introduction .................................................................................................. 2108.2 Scaling Time of Rheological Behavior of Materials .................................. 2108.3 Types of Rheological Behavior in Simple Deformation............................. 2118.4 Viscous Liquid ............................................................................................. 212

8.4.1 Introduction ...................................................................................... 2128.4.2 The Shear-Dependent, Time-Independent Viscosity

of Non-Newtonian Liquids .............................................................. 2138.4.3 Materials with a Yield Value—The Plastic Behavior Model .......... 2158.4.4 Non-Newtonian Time-Dependent Liquids....................................... 2168.4.5 Temperature and Pressure Dependence of Viscosity ...................... 217

8.5 Viscoelasticity of Food Materials ................................................................ 2178.5.1 Linear Viscoelasticity....................................................................... 2178.5.2 Small Strain Experiments in Simple Shear ..................................... 219

8.5.2.1 Stress Relaxation after Sudden Strain.............................. 2198.5.2.2 Creep and Creep Recovery Test....................................... 2208.5.2.3 Dynamic Assay ................................................................. 221

8.6 Nonlinear Viscoelasticity—Normal Stress Differences .............................. 2238.7 Rheological Properties of Food Macromolecular Systems......................... 224

8.7.1 Flow Behavior of Macromolecular Solution................................... 2248.7.1.1 The Concentration Regimes Effect .................................. 2248.7.1.2 Viscosity of Dilute Solution ............................................. 2258.7.1.3 Viscosity of Semidilute and Concentrated Solutions ...... 2278.7.1.4 Shear Dependence of the Viscosity.................................. 227

8.7.2 Linear Viscoelastic Properties of Food Polymer Systems .............. 2298.7.3 Mechanical Spectra of Food Polymer Systems............................... 231

8.8 Structure–Rheology Relationship in Multiphase Food Materials............... 2358.9 Elastic Solid ................................................................................................. 238

8.9.1 Introduction ...................................................................................... 2388.9.2 Nonlinear Behavior of a Solid......................................................... 238

8.10 Summary ...................................................................................................... 240References.............................................................................................................. 241



8.1 INTRODUCTION

Rheological investigations consist of mechanical excitation of a material understrictly defined conditions. Two basic types of deformation exist: shear strain andextension (elongation) strain. Depending on the stress or strain magnitude, themethod of its application and the length of time the stress or strain acts on a material,shear flow or extensional flow of the material can occur. Deformation caused by anexternal force acting on the material can be large or small. If the deformation issmall enough, the structure of the investigated material does not change. Largedeformation of complex materials usually causes some changes in their macroscopic-scale structure, which can be partially reversible or not.

Rheological methods, like small and large-strain uni- and biaxial deformationin compression or elongation mode, as well as multifrequency small deformationmethods in shear have been applied in food rheology.

8.2 SCALING TIME OF RHEOLOGICAL BEHAVIOROF MATERIALS

Common experience shows the complexity of the rheological properties of foodmaterials when observed over a large time scale. When a stress or a strain isimpressed upon a body, structural rearrangement on the supramolecular level, thatis, 103 to 107 nm occurs inside the material as it responds to the imposed excitation.In any real material, these rearrangements (relaxation) necessarily require a finitetime. Any natural material flows on some time-length scale. The Deborah number(De), a dimensionless quantity describing the “flowness” of a material, allows timescaling in rheology:

(8.1)

Accordingly, a material appears as a liquid if the Deborah number De is small,and as a solid if it is large, with respect to the observation time.

When the changes (relaxation) take place so rapidly that time is negligiblecompared with the time scale of the observation (λ ≈ 0), De approaches 0 and thematerial behaves as a purely Newtonian viscous liquid. For example, characteristicrelaxation time for water in a liquid state is 10–12 s. In the purely viscous material,all the energy required to produce the deformation is dissipated as heat. The time λand De is infinite for the Hookean elastic solid. In a purely elastic material the energyof deformation is stored and may be recovered completely upon release of the forcesacting on it. A material can appear solid-like either because it has a very longcharacteristic time λ or because the deformation process applied to its examinationis very fast. Thus even mobile liquids with low characteristic times can behave likeelastic solids in a very fast deformation process. Water behaves as a solid at veryshort times or equivalently at very high frequencies, that is, at time less than 10–8 s.By contrast, solid-like materials flow as liquids under a sustained stress at sufficiently

Delaxation time

Observation time t= =Re λ


Rheological Properties of Food Systems 211

long times. For instance, crystallized honey flows at room temperature under gravityforces at times of an order 103 to 104 s.

In principle, foods like mayonnaise, sauces, dough, meat, bread crumbs, andfruits, are viscoelastic bodies. Some energy may always be stored during the defor-mation of a material under appropriate conditions, and energy storage is alwaysaccompanied by dissipation of some energy. In a typically viscoelastic material, thetime necessary for the material rearrangements to take place is comparable with thetime scale of the experiment. For typically viscoelastic material, a Deborah numberapproaches unity. For many foods there are situations in which, relating to the timeof experiments, liquids depart from purely viscous behavior and show viscoelasticand elastic properties (and vice versa) (Rao and Steffe, 1992).

Figure 8.1 shows the types of rheological behavior occurring along the Deborahnumber scale: from Newtonian liquids to solid-state and linear viscoelasticity limits.Depending on the type of rheological behavior, the characteristic time t of theDeborah number is expressed as the inverse of deformation rate –1 in simple shearflow tests, or as the product of the amplitude of the oscillatory strain γ0 times itsfrequency ω, (γ0ω)–1 in dynamic tests. As demonstrated in Figure 8.1, strain γ orstrain amplitude γ0 and the Deborah number allow us to characterize different mainclasses of rheological behavior of the continuous fluid phase.

8.3 TYPES OF RHEOLOGICAL BEHAVIOR IN SIMPLE DEFORMATION

The response of linear polymers to simple shear and simple extensional or elonga-tional forces are illustrated in Figure 8.2. The shear force causes both deformationand rotation, whereas the normal extension force results in only deformation without

FIGURE 8.1 Rheological behavior of fluid systems over the time scale. (Adapted fromMacosco, C.V., Rheology: Principles, Measurements, and Applications, VCH Publishers, NewYork, 1994. With permission.)

Deborah number = λ/t or λγ or γ0 λω

stra

in γ

, str

ain

ampl

itude

γ0

γyieldN

ewto

nian

flui

d (η

)

2nd

orde

r flu

id (η

, ψ1, ψ

2)

Ela

stic

sol

id

Linear viscoelasticity (η, λ)

Nonlinear viscoelasticity

.

�γ



rotation. In shear flow, the overall shape of the viscosity curve is similar for linearand branched polymers, although the absolute magnitude of shear viscosity and itsdependence on shear rate is somewhat different. The same polymer systems inextensional flow obey ideal extensional Trouton behavior, but only in the low-shearNewtonian viscosity range (cf. Figure 8.7). Extensional Trouton behavior is definedas ηE ≡ 3η, (η0), where η is Newtonian viscosity, η0 is zero-shear viscosity, and ηE

is uniaxial extensional viscosity. When in a concentrated solution or polymer melt,the molecules are highly ordered by extensional flow, and the branching points ofbranched polymers can act as hooks, resulting in increasing the resistance to flow.Therefore, while linear polymer solutions or melts thin down under extensionalforces, branched polymers tend to stiffen or experience strain hardening. For exam-ple, the strain-hardening phenomenon is commonly observed in wheat dough exten-sional behavior (Dobraszczyk and Morgenstern, 2003).

The use of only steady-state shear rate viscosity appears to be sufficient in mostcases of food processing (pipeline flow, extrusion, mixing, and molding), whereasthe elongation properties become relevant for processing such as converging flow,fiber spinning, or film blowing. Although in most food processing the deformationand flow are very complex and involve uni- and multiaxial phenomena, generallythe uniaxial measurements, in simple shear or simple extension, can satisfactorilyestimate or predict many real process conditions (Bourne, 2002).

8.4 VISCOUS LIQUID

8.4.1 INTRODUCTION

For Newtonian liquids, fundamental relations called constitutive relations betweenstress and strain rate are exhibited by Newton’s law of viscosity:

FIGURE 8.2 The response of linear polymer to simple shear (top) and simple extensionalforces (bottom).



(8.2)

where η, the Newtonian viscosity, is the coefficient independent of strain rate andtime at a given temperature and density, σ is the shear stress, and is theshear rate or strain rate (≡ velocity gradient in simple shear).

The ideal Newtonian fluid is the basis for classical fluid mechanics. In accordancewith Newton’s law, the stress is always directly proportional to shear strain butindependent of the strain itself and time. Many real materials obey this ideal law.Most small molecule liquids like water, dilute true aqueous solutions (e.g., brine bath,vinegar), beverages (coffee, distilled liquors), and oils are generally Newtonian. SomeNewtonian materials also have very high viscosity, such as liquid honey. The New-tonian fluid model may be adequate, although not exact, for dilute suspensions (e.g.,clear fruit juice), emulsions (e.g., milk), and solutions of moderately long-chainmolecules. Table 8.1 shows the viscosity range for some familiar fluid food materials.

8.4.2 THE SHEAR-DEPENDENT, TIME-INDEPENDENT VISCOSITY

OF NON-NEWTONIAN LIQUIDS

Many colloidal suspensions and polymer solutions do not obey a linear Newton’srelation. In this case, the coefficient of apparent viscosity (non-Newtonian viscosity)is dependent on strain rate . Nearly all these materials give aviscosity that decreases with increasing velocity gradient in shear. Some concentratedsuspensions show shear thickening behavior as illustrated in Figure 8.3. Liquidsshowing shear-thinning and shear-thickening non-Newtonian behavior are most oftendescribed using a power law liquid model or Cross model.

The power law liquid model is used to represent the behavior of many liquidfood polymer solutions and some colloidal suspensions and emulsions (Table 8.2).The equation for the power law liquid model may be written in the following form:

. (8.3)

TABLE 8.1Newtonian Viscosity of Some Food Materials

MaterialTemperature

(°°°°C)Viscosity η

(Pa ⋅ s) Reference

Water 20 0.001Cream, 10% fat 40 0.00148 Steffe, 1983Raw milk 20 0.00199Homogenized milk 20 0.0020Whey protein isolate,10% water suspension

50 0.0031 Walkenström et al., 1999

Soybean oil 30 0.04 Steffe, 1983Apple juice, 20° Brix 27 0.0021

σ ηγ= �

�γ γ= d dt�γ = dv dy

η γ σ γ� �( ) = ≠ const

σ γ= < <( )k nn� 0 1



The viscosity of a shear-thinning liquid decreases when the shear rate increases.When the power law model, involving only two disposable constants, explains thebehavior of a liquid adequately at a reasonably broad stress-shear rate range, it

FIGURE 8.3 Flow curves for model viscous fluid material. (a) Newtonian; (b) power lawmodel n > 1; (c) power law model n < 1; (d) Bingham model; (e) Herschel-Bulkley model.

TABLE 8.2Parameters of Rheological Models of Some Non-Newtonian Food Liquids

Power law model Casson model

MaterialTemp.(°C)

Flow behavior

indexn

Consistancycoefficient

kPa ⋅⋅⋅⋅ sn

Casson plastic

viscosityηCk

Pa ⋅⋅⋅⋅ s

Cassonyieldstress

σyPa Reference

Soybean milk 20 0.815 0.01898 Shin and Keum, 2003

Sugar beet molassesBrix (%) 81.46

45 0.756 19.51 Togrul and Arslan, 200460 0.793 9.23

Mustard 25 0.28 17.30 0.0042 15.05 Juszczak et al., 200425 0.29 35.91 0.0106 31.07

Egg yolk(fresh, liquid)

4 0.843 1.683 Telis-Romero et al., 200625 0.854 0.421

46 0.872 0.148Whole liquid egg 20 0.911 0.0294 0.0168 0.01 Ahmed et al., 2003Cheese (Gouda) 55 0.746 3.653 Dimitreli and

Thomareis, 200495 0.759 0.364

a

b

c

d

eσ

σ0

γ•



appears relatively easy to use in analytical or numerical calculation of liquid behaviorin a complex flow situation (e.g., pipeline flow or mixing).

The shear power law model (with n > 1) has often been used for shear-thickeningmaterials, that is, materials that increase in viscosity as the shear rate increases (line con Figure 8.3). Most shear-thickening materials, such as concentrated starch pastesand other swollen particulate systems (some caseinate suspensions), are shear thinningat low shear rates and suddenly become almost rigid as the shear rate is increased.Barnes et al. (1989) have reviewed work on shear thickening of suspensions.

The Cross equation (8.4) has been proposed to describe the viscosity of complexliquids dependent on shear rate:

. (8.4)

The equation relates the viscosity η to the shear rate , introducing two limits, and with a characteristic shear rate . A slope of log η versus log in the

shear-thinning region is related to an exponent n. A physical meaning of the constantvalues and will be explained further (see Section 8.7.1.4 and Figure 8.7).The Cross equation is a universal model suitable when considering colloidal dis-persed and flocculated systems, polymer solutions and melts, and the systems con-taining particles interacting with each other in various ways.

8.4.3 MATERIALS WITH A YIELD VALUE—THE PLASTIC BEHAVIOR MODEL

Materials with a yield value do not flow before a certain stress is reached. Plasticmaterials are often dispersions where the dispersed particles form a network at rest.The stress level required to initiate flow is usually referred to as yield stress σy, andis related to the level of internal structure of the material, which must be destroyedbefore flow can occur. When plastic materials start to flow they can show a straightline or shear-thinning behavior.

Three models of plastic material are most frequently used: the linear Binghamplastic model, and the Herschel-Bulkley and Casson nonlinear models.

Line (d) on Figure 8.3 represents the ideal Bingham plastic model. The flowcurve is linear with an intercept σy on the stress axis:

σ – σy = ηpγ⋅ for σ ≥ σy (8.5)

The constant parameter ηp is called the plastic viscosity. The Bingham modelmay represent, with reasonable accuracy, the behavior of some concentrated suspen-sions and emulsions, such as toothpaste, and food spreads such as margarine, may-onnaise, salad dressing, cheese spread, and ketchup. Nevertheless, behavior of almostall real materials departs significantly from the Bingham model because their flowcurves are not linear, except within a very limited range of strain rates.

η ηη η γ γ

−−

=+ ( )

∞

∞00

1

1 � �n

�γη0 η∞ �γ 0 �γ

η η0 , ∞ �γ 0



The Hershel-Bulkley and the Casson models represent the behavior of a largenumber of nonlinear viscoplastic materials, and are widely used to develop pipelinedesign procedures.

The Hershel-Bulkley model (Figure 8.3e) may be written as

(8.6)

and the Casson model is expressed as

(8.7)

The Casson model appears to give an excellent fit to data of chocolate melt andconfectionery products. Examples of non-Newtonian model parameters calculatedfor some food materials are shown in Table 8.2.

Yield stress is a very important rheological parameter for predicting a product’sprocessing conditions, especially while it is being transported in a pipeline or beinghandled. It is also a significant factor of the product’s sensory perception charac-teristic, mainly its mouth feeling. Quantifying yield stress must be done carefullybecause the value obtained depends on the rheological technique used. Most foodplastic products show relatively low-yield stress values and even sample collectingcan destroy them. For example, yogurt is a highly structured material and extremelysensitive to external forces. Its characterization through the measurement of fun-damental rheological properties demands using specially designed nondestructivedevices.

8.4.4 NON-NEWTONIAN TIME-DEPENDENT LIQUIDS

The viscosity of these kinds of liquids depends on shear rate magnitude and on thelength of time the shear rate is applied. Time-dependent materials change the struc-ture and therefore the viscosity with time, at a constant shear rate or constant shearstress. Depending on the way the structure is affected by shearing, the materials aredivided into two groups: thixotropic and antithixotropic.

Shear-thinning liquids are said to be thixotropic if, after a long rest, when (orσ) is applied suddenly and then held constant, the apparent viscosity is a diminishingfunction of the time of flow. The liquid completely recovers its initial state followinga long enough interval after the cessation of flow. The reversible transition gel ⇔ solis a classic example of thixotropy. Generally speaking, all multiphase and multicom-ponent foods show thixotropic behavior. Food suspensions (mustard, tomato con-centrate), emulsions (soft butter, salad dressings, and mayonnaise) and foams (meatfoams, fish foams, pancake batter, aerated desserts, such as crèmes and mousses,whipped cream) containing artificial dispersants or stabilizers, are time-dependentliquids and show thixotropic behavior. Water and fat content, fillings, thickeners,and emulsifiers are factors influencing apparent viscosity and thixotropic behaviorof this media.

σ σ γ= +ynk �

σ σ η γ1 2 1 2 1 2= + ( )y CA �

�γ



Antithixotropy occurs when, under similar conditions, the apparent viscosity isan increasing function of the duration of flow, and the body recovers its initial stateafter a long enough interval at rest. When the body does not entirely recover itsinitial state, then partial thixotropy or partial antithixotropy takes place. Many foodmaterials, like stirred yogurt, exhibit these intermediate properties, that is, partialthixotropy or partial antithixotropy when pumping, dosing, and storage.

The existence of thixotropy or antithixotropy means that the flow history is animportant factor in the proper design of mechanical processes, where the product’sviscosity continues to change for a long time at given shear conditions.

The term rheopexy has been used with two different meanings: (1) solidificationof a thixotropic system under the influence of a gentle and regular motion, or (2)progressive shear thickening. The first meaning is that for which the term was coined,and its use in the second sense is not recommended. Consequently, use of the termrheopectic fluid (or liquid) to describe a fluid with progressive thickening is notrecommended.

8.4.5 TEMPERATURE AND PRESSURE DEPENDENCE OF VISCOSITY

The temperature dependence of viscosity can often be as important as its shear ratedependence for nonisothermal processing problems. For all liquids, viscosity decreaseswith increasing temperature and decreasing pressure. The viscosity of Newtonianliquids decreases with an increase in temperature following the Arrhenius relationship:

(8.8)

where R = gas constant, T = absolute temperature, Eη = activation energy of viscousflow, and A = experimentally determined constant. It is often found that over quitea wide range, a graph of ln η against 1/T is linear. As far as temperature is concerned,for most industrial food applications involving aqueous systems, interest is confinedfrom 0 to 100°C.

Generally the viscosity of liquids increases exponentially with isotropic pressure.The changes are quite small for pressures differing from atmospheric pressure byabout one bar (Macosco, 1994).

8.5 VISCOELASTICITY OF FOOD MATERIALS

8.5.1 LINEAR VISCOELASTICITY

As a consequence of the structural rearrangements of a material taking place at atime scale comparable to that of the experiment in which they are observed (De ≈1),the relation between stress and strain or rate of strain cannot be expressed only bymaterial constants. The rheological behavior of viscoelastic materials is characterizedby time-dependent material functions.

However, in the limit of infinitesimal deformation, viscoelastic behavior can alsobe described by linear differential equations with constant coefficients. Such behavior

η η= −( )A E RTexp



is termed linear time-dependent or linear viscoelastic behavior. In the most generalcase, the stress becomes a function of the strain, that is, it depends on the strainhistory. Supposing deformation of the material in simple shear, the linear constitutiveequation is based on the principle that the effects of sequential changes in strain areadditive (the Boltzmann superposition principle) (Ferry, 1980; Morrison, 2001). Thisadditivity is an essential feature of linear behavior:

(8.9)

where is the shear rate, G(t) is called the relaxation modulus. The inte-gration is carried out over all past times t ′ up to the current time t.

The strain can be expressed alternatively in terms of the history of the timederivatives of the stress using the following constitutive equation:

(8.10)

where , J(t) is called the creep compliance. Similar to Equation (8.9), theintegration is carried out over all past times t ′ up to the current time t. However, fora viscoelastic material J(t) ≠ 1/G(t) because of the difference between the twomechanical excitation time patterns.

In practice it is found that most materials show linear time-dependent behavioreven in finite deformation as long as the strain remains below a certain limit — thelinear viscoelastic limit. This limit, that is, the magnitude of the strain above whichlinear viscoelastic behavior is no longer observed, varies and is a material property.For instance, the domain of linear viscoelastic behavior of wheat gluten extendsusually up to about 5% strain amplitude in dynamic mode in the 10–3 to 100 rad/sfrequency range, whereas wheat bread dough exhibits nonlinearity above strainvalues ∼0.1%. Linear viscoelastic response of nonstarch polysaccharide solutionsextends up to 50% and higher in the same frequency range.

The experiments designed to determine the linear viscoelastic response of amaterial to an instantaneous stress or strain base on the assumption that up to thetime t0, i.e., the time of stress or strain application, the material remains in rest longenough to relax any prior excitations. A number of small strain experiments are usedin rheology. Some of the more common experiments are transient experiments (stressrelaxation, creep, and creep recovery), and the dynamic experiments in the harmonicregime (sinusoidal oscillation). Different small strain experimental methods are usedbecause they may be more convenient or better suited for a particular material(viscoelastic solid or viscoelastic liquid) or because they provide data over a partic-ular time range. A dynamic experiment at given frequency ω is qualitatively equiv-alent to the transient experiment at time t = 1/ω. Furthermore, it is often not easy totransform results from one type of linear viscoelastic measurement to another.Applications of each of these small strain methods and typical data for severalrheologically different viscoelastic food materials will be considered below.

σ γt G t t t dtt

( ) = − ′( ) ′ ′−∞∫ �( )

�γ γ= ∂ ∂t

γ σt J t t t dtt

( ) = − ′( ) ′ ′−∞∫ � ( )

�σ σ= d dt



8.5.2 SMALL STRAIN EXPERIMENTS IN SIMPLE SHEAR

8.5.2.1 Stress Relaxation after Sudden Strain

In the stress relaxation test, an instantaneous deformation γ0 is applied to a body. Thiscan be done in shear, compression, or extension mode. Deformation or strain is main-tained constant throughout the test, while the stress is monitored as a function of time:

. (8.11)

Relaxation modulus G(t) is a time-dependent analog of equilibrium shear mod-ulus of perfectly elastic solid G.

For viscoelastic solid materials, the relaxation modulus decays to an asymptoticequilibrium modulus Ge. For viscoelastic fluid materials, the relaxation modulusdecays to zero, usually after an extremely long time (Figure 8.4).

The equation for the relaxation modulus as a function of time is usually expressedas the simplest modified Maxwell model describing a situation where the macromo-lecular system shows a very narrow profile of molecular weight distribution:

(8.12)

where G0 is the initial modulus, Ge the equilibrium modulus, and λ is the relaxationtime. In a real situation given the molecular polydispersity of a polymer tested, agroup of n Maxwell elements in parallel represents a discrete spectrum of relaxationtimes, each time λi being associated with a spectral strength Gi (Ferry, 1980):

. (8.13)

FIGURE 8.4 Stress relaxation after sudden strain. Solid line = viscoelastic solid; dotted line= viscoelastic fluid.

σ γt G t( ) = ( )0

G t G G G te e( ) = + −( ) −( )0 exp λ

G t G ti i

i

n

( ) = −=

∑ exp( )λ1

Time, t

Rel

axat

ion

mod

ulus

, G

Ge



Food gels, such as pectins, starch gel, thermally denatured globular proteins,and aggregated proteins from enzymatic or chemical action exemplify this kind ofpolydispersed viscoelastic solid system. The longest relaxation time of these systemstends to infinity, then the corresponding modulus contribution is the equilibriummodulus Ge. The stress relaxation experiment can be performed on both viscoelasticfluids and viscoelastic solids.

8.5.2.2 Creep and Creep Recovery Test

Figure 8.5 gives a schematic representation of a typical creep and creep recoveryexperiment for a viscoelastic fluid. At time t = 0, a constant shear stress σ0 is appliedto the sample and shear deformation γ is recorded as a function of creep time t longenough to reach a steady-state flow of material. At creep time t0, the stress isinstantaneously set to zero and the recoverable part of deformation γr (t, t0) is mea-sured as a function of creep recovery time. The combination of both experiments iscalled the retardation test.

The time-dependent creep compliance in the linear viscoelastic regime can becalculated as

. (8.14)

According to the linear viscoelasticity theory (Boltzmann superposition princi-ple), the creep compliance J(t) can be written for polydispersed materials as a sumof instantaneous elastic compliance (glassy compliance Jg), discrete retardationspectrum 2N positive constants {τk, Jk} of a multiparameter Kelvin-Voigt model, andcontribution of steady-state permanent viscous flow :

(8.15)

where each retardation time τk is associated with a spectral compliance magnitudeJk; viscosity η0 is the steady-state viscosity. For food materials, Jg has a value of~10–9 Pa–1 and can therefore be omitted.

Assume that the real time of the experiment is long enough to reach a steady-state flow of material. According to Figure 8.5, for creep compliance at time t = t0

the following equation is valid:

. (8.16)

The Je0 is the steady-state compliance. As indicated in Figure 8.5, Je

0 and η0 canbe found graphically from creep test data analysis. The inverse of Je

0 is a measureof total viscoelasticity of the material.

J tt( ) =

( )γσ0

t η0

J t J J tt

g k

k

N

k( ) = + − −( )( ) +=

∑1 0

1 exp τη

J t Jt

e00 0

0( ) = +

η



The recoverable compliance Jr(t, t0) (also called elastic compliance) followsfrom the creep or from the recoverable part of creep recovery experiments (seeFigure 8.5) by subtracting the contribution of flow from total compliance

. (8.17)

The retardation experiment is recommended for viscoelastic fluid materials suchas bread dough, biscuit dough, vital wheat gluten, meat dough, meat stuffing, andpastry.

8.5.2.3 Dynamic Assay

In dynamic assay a viscoelastic material is subjected to controlled sinusoidallyvariable excitation (stress or strain). Within the linear response of the material,variations of the noncontrolled variable (stress or strain) tend to become sinusoidalwith a period identical with that of the controlled excitation, but with a differentphase. This can be shown from the constitutive equation as follows:

(8.18)

FIGURE 8.5 Creep and creep recovery test of weak wheat gluten. (Adapted from Pruska-Kędzior, A., Application of Phenomenological Rheology Methods to Quantification of WheatGluten Viscoelastic Properties, The Agricultural University of Poznań Press, Poznań, 2006[in Polish]. With permission.)

J t J tr k k

k

N

( ) = − −( )=

∑ 11

exp( )τ

γ γ ω= 0 sin t



(8.19)

where γ indicates strain, σ the stress associated with it, ω is the angular frequency,and γ0 and σ0 are the amplitudes of the oscillations. The loss angle is the phasedifference δ (ω) between stress and strain.

The complex modulus is the complex number:

(8.20)

where .The real part of the complex modulus is called the storage modulus G′, the

imaginary part is called the loss modulus G″:

(8.21)

and

. (8.22)

It is demonstrated that each dynamic measurement at a given frequency ωsimultaneously provides two independent quantities G′ and G″ as well as tan δ,which is defined as

. (8.23)

The storage modulus G′(ω) is a measure of the mechanical energy stored andrecovered per cycle of sinusoidal deformation, and the loss modulus G″(ω) is ameasure of the energy dissipated per cycle as heat. G′(ω) is related to the elasticcontribution to the reaction of a material on a deformation or stress, and G″(ω) isrelated to the viscous contribution. The data from sinusoidal experiments can alsobe expressed in terms of complex compliance:

(8.24)

where J′ is the storage, and J″ the loss compliance. The relationships among G′,G″, J′, and J″ are as follows:

(8.25)

σ σ ω δ= +( )0 sin t

G expi* = σγ

δ0

0

i = −1

G G iG* = ′ + ′′

G G G* = ′ + ′′2 2

tanδ = ′′ ′G G

J G J iJ* *= = ′ − ′′1

′ = ′′ + ′′

JG

G G2 2



. (8.26)

Similarly, the complex viscosity η* of a viscoelastic material can be presentedas η* = η′ – iη″. Functional relationships G*, G′, G″, η* = f(ω) represented graph-ically are referred to as mechanical spectra.

Examples of viscoelastic food materials’ mechanical spectra as a functionalrelationship G′, G″ = f(ω) are shown in Figures 8.10 and 8.11 and are discussed inSection 8.7.3.

From an industrial point of view, dynamic measurements provide knowledgeabout process-induced structure formation, which has to be considered for productformulation and process design. Dynamic measurements are required to follow thebuildup and breakdown of structures as a function of the parameters of such processesas flow, deformation, heating, and cooling as well as combinations thereof.

8.6 NONLINEAR VISCOELASTICITY—NORMAL STRESS DIFFERENCES

Much labor has been expended in the determination of the linear response ofmaterials. There are many reasons for this. First, there is the possibility of eluci-dating the molecular structure of materials from their viscoelastic response. Second,the material parameters and functions measured in the relevant experiments oftenprove to be useful in industrial quality control. Third, a background in linearviscoelasticity is a helpful introduction to the much more difficult subject of non-linear viscoelasticity.

However, for some technological situations only a nonlinear viscoelasticityapproach can bring satisfactory elucidation of processed material properties. Non-linear viscoelasticity is a phenomenon in which shear flow normal stress appearssignificant. When a viscoelastic material is sheared between two parallel surfaces atan appreciable rate of shear, in addition to viscous stress σ12, there are normal stressdifferences N1 ≡ σ11 – σ22 and N2 ≡ σ22 – σ33. Here “1” is the flow direction, “2” isthe direction perpendicular to the surfaces between which the fluid is sheared, and“3” is the neutral direction. The larger of the two normal stress differences is N1,and this difference is responsible for the processed material’s rod-climbing effect,termed the Weisenberg effect. For example, when cake batter is mixed with an eggbatter, the material climbs up the rod rotating within it. Normal stress differencealso appears important when food sensory properties are considered. It has beenhypothesized that normal stresses generated by oral movements of semisolid foodare sensed by the mouth and contribute to the perception of thickness. This impliesthat the actual force applied in the mouth to the semisolid food is equal to the forceapplied by the tongue muscle, say F0, minus the force representing the normal stressdifference N1, exerted by the food (Terpstra et al., 2005).

For isotropic materials, N1 has always been found to be positive in sign (unlessit is zero). In a cone and plate rheometer, this means that the cone and plate

′′ = ′′′ + ′′

JG

G G2 2



surfaces tend to be pushed apart. N2 is usually found to be negative and smallerin magnitude than N1; typically the ratio –N2 /N1 lies between 0.05 and 0.3(Macosco, 1994). At sufficiently low shear σ12 usually becomes linear in the shearrate, ; that is, the shear viscosity becomes independent of . Similarly,N1 and N2 approach the limits, , at small , and thus the normalstress coefficients:

(8.27)

(8.28)

approach constant values at a small .When the Deborah number is vanishingly small, the fluid can be described as

Newtonian. When the Deborah number is small but not negligible and the flow issteady or near steady, nonlinear effects are weak and can be described by the second-order fluid (Figure 8.1). The second-order fluid equation predicts the existence ofnormal stress differences in shearing (Ferguson and Kemblowski, 1991).

8.7 RHEOLOGICAL PROPERTIES OF FOOD MACROMOLECULAR SYSTEMS

8.7.1 FLOW BEHAVIOR OF MACROMOLECULAR SOLUTION

8.7.1.1 The Concentration Regimes Effect

Important factors determining the rheological behavior of macromolecules are theirsize, shape, and flexibility. An appropriate macromolecular chain stiffness parameteris the ratio of the contour length L of the chain to the length b of the statisticalsegment unit comprising n monomers (Lefebvre and Doublier, 2004). A ratioL/b > 10 would be required for the polymer conformation to be regarded as a coil.Three concentration domains can be distinguished in solutions of polymers: diluteregime (c < c*), semidilute regime (c* < c < c**), and concentrated regime (c > c**)(Figure 8.6).

In a very dilute solution, the volume available to each polymer molecule ismuch higher than that of the individual coil. The coils remain statistically far fromeach other, and encounters bringing them into contact are infrequent. The coilsmaintain the dimensions of an isolated chain. In this low-concentration region, thehydrodynamic forces can be neglected. Only the Brownian motion is acting againststructural forces.

This situation prevails up to the critical overlap concentration c*, semidiluteregime, at which the coils fill the volume of the solution. When the polymer con-centration is increased above c*, there is a progressive interpenetration of the coils,

�γ η σ γ≡ 12 � �γN1

2∝ �γ N22∝ �γ �γ

ψ σ σγ1

11 222

= −�

ψ σ σγ2

22 332

= −�

�γ



concomitant with a contraction of their individual volume. The solution becomes atransient network of entangled chains. In a semidilute regime the coils still retainsome degree of individuality.

At certain concentrations (c** > c*) the polymer solution becomes an entangle-ment network where the chains have completely lost their individual character. Oncethe entanglement network forms, the only characteristic length in the system is nowthe mesh size of the network, which decreases as concentration increases, tendingtoward its limit value b in the polymer melt state. When low-energy interactionsdevelop between chains in the regions of entanglements, the possibility of formingsome junction zones appears. Junction zones exhibit lifetimes much longer thanthose of entanglements. The system has then shifted from the state of an entangledsolution to that of a physical gel.

Food polysaccharide solutions, for instance pectins, guar gum, locust bean gum,carob gum or carrageenans, and food proteins like whey proteins, bovine serumalbumin, ovalbumin, collagen, actin, or myosin as well as polysaccharide-proteinmixtures exhibit this dependence of the molecule’s state on solution concentration.

8.7.1.2 Viscosity of Dilute Solution

The intrinsic viscosity of the macromolecule can be extracted from the viscositymeasurement of the dilute macromolecular solution. The term intrinsic viscosity canbe intuitively misleading. In fact, it is not a viscosity at all, but actually a measure

FIGURE 8.6 Specific viscosity: the reduced concentration master curve for guar and hydrox-yethyl cellulose solutions at 25°C. Guar = empty circles, five samples differing in molecularweight (450 ≤ [η] ≤ 1250 mL/g). Hydroxyethyl cellulose = filled triangles ([η] = 807 mL/g).The lines show the slopes 1.2 and ~5, relative to the dilute and to concentrated regimes,respectively. (Adapted from Lefebvre, J. and Doublier, J.-L., Rheological behavior of polysac-charides in aqueous systems, in Dumitriu. S. (Ed.), Polysaccharides: Structural Diversity andFunctional Versatility, 2nd ed., Marcel Dekker, New York, 2004. With permission.)

c [η]

10-2 10-1 100 101 102

sp

10-2

10-1

100

101

102

103

104

105

106

c*

c**

η



of the hydrodynamic volume of the coil in the case of noncharged polymer chains,or of the asymmetry of the particle in the case of rigid macromolecules. Most polymersolutions show non-Newtonian behavior as a result of the deformation and of theorientation of the polymer coil in flow. Therefore, the intrinsic viscosity is shear ratedependent. For practical reasons, in this section, viscosity and intrinsic viscosity willbe referred to as measurements performed within the Newtonian domain, that is, atshear rates low enough for the dilute solution to display shear rate-independentviscosity. Therefore, the intrinsic viscosity is defined as

(8.29)

and is expressed in mL/g. The quantity ηsp = (η – η0)/η0 is called the specific viscosityof a solution. The specific viscosity is the ratio of the difference between the viscosityof the solution and that of the pure solvent under identical physical conditions. Table8.3 shows examples of the intrinsic viscosity of some macromolecular food solutions.

The relationship between intrinsic viscosity and the molecular weight of poly-merlike macromolecules is usually expressed in the form of the empirical Mark-Houwink equation:

(8.30)

TABLE 8.3Intrinsic Viscosity of Some Polysaccharide Solutions

MaterialTemperature

(°C)

Intrinsic viscosity [η]

(mL/g) Reference

Starches of varying amylose content(solubilized in 0.2 M KOH)

Lourdin et al., 1995

Amylopectin 25 121Waxy maize 25 121Potato 25 270Wheat 25 210Pea 25 250Amylose 25 161

Galactomannans—tara gum(Caesalpinia spinosa)

25 1646 Sittikijyothin et al., 2005

Galactomannans—locust bean gum(Ceretonia siliqua L.)

crude 25 1103purified 25 1496

Cassia javanica galactomannans 25 1160 Goncalves et al., 2004 Wheat water-soluble arabinoxylans(pentosans)

25 220–560 Dervilly-Pinel et al., 2004

Carboxymethylcellulose 25 675–5852 Kulicke et al., 1996

ηη

=

→limc

sp

c0

η = KMvisa



where K and a are empirical parameters depending on the polymer and solventproperties and on the temperature; both are related to chain stiffness. is theviscosity average molecular weight. The value of the exponent a gives informationabout the general conformation of the polymer. For flexible linear chains, a typicallyassumes values between 0.5 and 0.8. For instance, food galctomannans and arabi-noxylans show a = 0.72 and 0.74, respectively, being characteristic of a random coil.Stiff chains display larger values of a, for example, values as high as 1.8 are reportedfor rodlike chain conformations. On the other end of the scale, a < 0.5 indicates somedegree of coil collapse in the case of a linear chain (Lefebvre and Doublier, 2004).

8.7.1.3 Viscosity of Semidilute and Concentrated Solutions

Flow behavior of semidilute and concentrated solutions is illustrated in Figure 8.6,where the data relative to two polysaccharides, guar gum and hydroxyethyl cellulose,differing in structure and in molecular weight, are plotted as specific viscosity ηsp

versus reduced concentration c[η] (Lefebvre and Doublier, 2004). Specific viscosityreflects changes not only in conformation and size, but also in intermolecular inter-actions (association, aggregation), and in the physical state of the system. For c > c*,polymer solutions become non-Newtonian at moderate and even low shear rates; theviscosity values to be considered as those corresponding to low-shear Newtonianviscosity (η0), which are related to the equilibrium state of the solution at rest.

The master curve (Figure 8.6) can be divided into three regions, limited by thecritical concentrations c* and c**. Below c[η] ~ 1, that is, c < c*, the curve is linear,with the slope nd ~ 1.2. This is the dilute regime, where the increase in ηsp isattributable to the hydrodynamic interaction between polymer coils behaving asindependent impermeable spheres.

Above c = c*, the semidilute regime is reached. Progressive coil contraction andincreasing entanglement density govern the rheological behavior of the solution,provided their molecular weight M > Mc. At the upper limit of the semidilute regime,the solution is about 100 times more viscous than the solvent because of the increaseof entanglement density. This part of the curve is often approximated by a linesegment with a slope nsd ~ 2.5. Finally, in the concentrated regime (c > c**), theexperimental data can be fitted with a line segment with a slope nc ~ 4 to 5.

Some differentiation in the values of c*[η] and c**[η] and shifts on the reducedviscosity scale as well as differences in the values of the slope and broadness of thesemidilute regime c**/c*, because of differences in polymers’ structures (flexibility)and in interactions with the solvent were observed (Launay et al., 1997).

8.7.1.4 Shear Dependence of the Viscosity

The non-Newtonian shear behavior is typical for polymer melts and polymer solu-tions at concentration c > c*. This kind of behavior is schematically illustrated bythe flow curve in Figure 8.7, where the steady-state viscosity η is plotted versus theshear rate on bilogarithmic scales. Below a critical shear rate value, , the flowcurve shows the low-shear Newtonian plateau, where the viscosity holds a constantvalue η0. Above critical shear rate value , a shear-thinning region (where the

Mvis

�γ �γ crit

�γ crit



viscosity decreases as shear rate increases) follows. In this region a power law relation accurately fits the data with usually n ~ 0.6 to 0.8. At high shear rates, the

viscosity tends to a second plateau, η∞. Due to instrumental limitations or flow insta-bility, this high-shear Newtonian plateau is rarely observed experimentally, and thenin most cases, can be neglected. The flow curve of a concentrated polymer solutionreflects the effect of shear rate on entanglement density. If is low enough, the systemremains in its equilibrium fully entangled state ( ). As increases, the entan-glement density and the viscosity decrease. Each shear rate value corresponds to agiven entanglement state resulting from the balance between the flow-induced disen-tanglement and reentanglement processes governed by Brownian motion. Concentratedsolutions are viscoelastic by nature; therefore, they do not respond instantaneously tochanges in shear rate or stress value. The constant equilibrium viscosity is reached ata given shear rate only after a long enough time, sometimes even after a few days.

Many equations describing the shear rate dependence on viscosity of polymersolutions and melts have been proposed. The most applied expression is the Crossequation (Section 8.4.2). In the Cross model, the characteristic value of shear rate

0 obtained as the abscissa of the intersection of the line representing the low-shearNewtonian viscosity plateau η0 with the line fitted to the data of the power lawregion of the solution flow curve (see Figure 8.7.). The inverse of 0 gives thecharacteristic relaxation time of the solution . The value is takeninstead as , because is difficult to determine in practice (Launay et al., 1997).Characteristic parameters η0 and depend on the nature of the polymer, itsmolecular weight, its concentration, and on temperature. They may change by severalorders of magnitude from one system to another.

FIGURE 8.7 Flow curve illustrating three regions of the typical macromolecular solutionshear thinning behavior.

(s–1)

η (

Pa·

s)

γ0·

γ·10-3 10-2 10-1 100 101 102 103

10-2

10-1

100

101

102

Power law region

∞

High-shearNewtonianviscosity

0

Low-shearNewtonian viscosity

Criticalshear rate

η

η

η γ∝ −� n

�γη η= 0 �γ

�γ

�γλ γsol = 1 0

� �γ 0

�γ crit �γ crit

λsol



The flow curves of the polymer solution extend most often along several ordersof magnitude of shear rate and of viscosity. Examples of flow curves described withthe Cross model, representing a large range of variability of observed flow propertiesof a polysaccharide are presented in Figure 8.8. Despite large differences in viscosity,presented macromolecular solutions show the same type of behavior, which is provedby the flow master curve.

The master curves are very useful in combining the data for different molecularweights, concentrations, temperatures or solvents. A master curve simplifies treat-ment of data for different systems and conditions. Discrepancies from the mastercurve allow determination of differences in polymer chain structures. Master curvesare built either by graphically superimposing the individual flow curves , byshifting them along the viscosity and the shear rate axes, or are calculated by usingthe reduced variables η/η0 versus . For instance, a “universal” flow curve forpolysaccharides has been thus published. This curve combines the data for a largenumber of different polysaccharides fitted with the Cross model (η∞ omitted) withn = 0.76 (Morris, 1990).

8.7.2 LINEAR VISCOELASTIC PROPERTIES OF FOOD POLYMER SYSTEMS

In the linear domain, viscoelastic behavior is a kind of “fingerprint” of the intactmaterial microstructure, which responds in different way over a certain time scalewithout being changed. Mechanical spectra of viscoelastic materials differ qualita-tively and quantitatively according to the nature of the sample tested. Almost all thedata based on rotational shear rheometry have been obtained through small straindynamic measurements carried out over a narrow, physically feasible frequencyrange, which practically extends from 0.001 to 200 rad/s. Thus, the experimentalwindow frames only a section of the mechanical spectrum limited by the oscillation

FIGURE 8.8 Flow curves of wheat water-soluble arabinoxylans (2% water solutions) (left)and the master curve (right). (Adapted from Pruska-Kędzior, A., Kędzior, Z., Michniewicz, J.,Lefebvre, J., and Kołodziejczyk, P., in Fischer, P., Marti, I., and Windhab, E.J. (Eds): Proceed-ings of 3rd International Symposium on Food Rheology and Structure ISFRS—Eurorheo 2003-01, Laboratory of Food Processing Engineering, ETH, Zurich, 2003, p. 177. With permission.)

η η/η

101 102 103

(Pas

)

10–2

10–1

100

γ (1/s)⋅10-3 10-2 10-1 100 101

0

10-1

100

/γ0

γ ⋅⋅

η γ�( )� �γ γ 0



frequency. Therefore, within the same frequency window, only a part of viscoelasticresponse can be observed and rheological behaviors of the media often remain poorlycharacterized.

Enlargement of the frequency window (time scale) can be easily achieved inthe case of a polymer solution or melt displaying a thermorheologically simplebehavior, that is, a case in which there is similar relaxation behavior with changesin temperature. The method involves application of the time-temperature superpo-sition principle (TTS) to dynamic data obtained at different temperatures over arestricted frequency range. According to the principle, mechanical spectra arecarried out at various constant temperatures, and then superimposed using a hori-zontal shift factor (aT) to form a single master curve. This principle was originallyintroduced by workers in synthetic polymers (William-Landau-Ferry [WLF] equa-tion; Ferry, 1980).

The TTS principle is only valid if its underlying assumptions are met. The firstassumption is that a direct relationship between time (oscillation frequency) andtemperature exists. Second, the polymer structure at the physical and molecular levelis assumed to remain constant over the experimental temperature range. This assump-tion is fulfilled in entanglement polymer systems. An example of a typical mechanicalspectrum based on TTS principle data of an entangled polymer melt is shown inFigure 8.9. Storage G′ and loss G″ moduli as well tan δ are plotted as a function offrequency ω (or time t = 1/ω) on bilogarithmic scales. In this case the spectrumextends over 16 frequency decades covering the entire range of rheological responsesshown by viscoelastic liquids.

The mechanical spectrum (Figure 8.9) comprises four distinct parts based onthe intersection (crossover) of G′ and G″ moduli curves, which correspond to valuesof tan δ equal to one. First, in the low-frequency region viscous flow is predominant.This is a terminal region. In this region G′ and G″ are proportional to ω2 and ω,respectively, with G″ > G′. Slopes 2 and 1 in the terminal region are those expectedfor any linear viscoelastic liquid (Ferry, 1980). In the terminal region, a longest-range molecular motion (slow dissipative process) occurs.

In the second zone, a viscoelastic (rubbery) plateau appears where G′ is nearlyflat and G″ becomes smaller than G′, so that the plateau is delimited by modulicrossover in two points. In this region transient viscoelastic networked structureexists due to physical effects (topological entanglement or particle gel). In a plateauzone, G′ changes relatively slowly while G″ and the loss tangent (tan δ) pass throughtheir minima. The energy losses are small here because the period of oscillation islong compared with the longest relaxation time of an entanglement network strand.However, it is short compared with any relaxation times for motion involving entan-glement or junction zone slippages or disruptions. This characteristic plateau devel-ops up to a high frequency region where G′ and G″ follow dependence on ω1/2.

The third region is called the transition zone, at which the viscous responseagain becomes dominant (G″ > G′). In the transition zone, the viscoelastic behavioris a result of short scale time motions of segments of the chain, which are shorterthan the distance between entanglements (so-called fast dissipative processes).

Finally, at very high frequencies, the moduli cross over for a third time, enteringthe glassy zone. Glassy behavior corresponds to very high frequencies, so no



configuration rearrangements of the polymer backbone chain have time to take place.Energy dissipation occurs only through limited local motions. The polymer behavesas an amorphous or semicrystalline solid.

8.7.3 MECHANICAL SPECTRA OF FOOD POLYMER SYSTEMS

Rheological examination of the viscoelastic behavior of food polymers is limitedexperimentally to the finite range of the viscoelastic spectrum, but rheologicalproperties of food polymers may span many orders of magnitude. No single exper-iment can cover the entire range. The application of the TTS principle to foodbiopolymers and real food systems is usually strongly restricted to a very narrowtemperature range due to irreversible conformational changes of biological macro-molecules at elevated temperatures, as protein denaturation, starch gelation or evenstarch (nonstarch) polysaccharides melt (Figure 8.13). Therefore, the extendedmechanical spectra of these materials obtained using the TTS principle are alsolimited. Combining dynamic measurements with transient tests allows us to partiallyencompass this difficulty (Ferry, 1980; Tschoegl, 1989). For example, data of aretardation test can be converted from time domain to frequency domain andcombined with dynamic test data. This permits us to significantly extend themechanical spectrum of a material down to lower frequencies than those accessible

FIGURE 8.9 Example of mechanical spectrum of entangled polymer system over a widefrequency range. (Adapted from Lefebvre, J. and Doublier, J.-L., Rheological behavior ofpolysaccharides in aqueous systems, in Dumitriu. S. (Ed.), Polysaccharides: Structural Diver-sity and Functional Versatility, 2nd ed., Marcel Dekker, New York, 2004. With permission.)

ω (rad/s)

10–16 10–12 10–8 10–4 100

G' G

" (P

a)

10–6

10–2

102

106

1010

tan δ

10-2

100

102

104

106

1

2

Ter

min

al r

egio

n

Vis

coel

astic

pla

teau

Tra

nsiti

on r

egio

n

Glassy zone



for direct measurement in dynamic mode. Then, often lower crossover of moduliG′ and G″ and the beginning of the spectrum’s terminal region can be observed(Figure 8.10) (Lefebvre et al., 2003).

Many polysaccharides, such as agar, κ-carrageenan, guar, gellan, locust beangum, xanthan, and proteins such as bovine serum albumin or gelatin, polysaccha-ride–polysaccharide mixtures or polysaccharide–protein mixtures are commonlybeing used as structuring agents in the aqueous phase of emulsion food productsand form gel matrices in many semisolid food products.

FIGURE 8.10 Example of the composite mechanical spectrum obtained by combiningdynamic data and retardation test data converted according to the Kashta method. Material= wheat gluten obtained from a milling stream flour. Solid circles = G′, empty circles = G″,data converted from retardation test: solid line = G′, dashed line = G″. (From Pruska-Kędzior,A., Application of Phenomenological Rheology Methods to Quantification of Wheat GlutenViscoelastic Properties, The Agricultural University of Poznań Press, Poznań, 2006 [in Polish].With permission.)

FIGURE 8.11 Mechanical spectra of chemically cross-linked gel (left, wheat water-solublepentosans, 2% concentration) and physical gel (right; native wheat gluten).

ω (rad/s)

10–6 10–5 10–4 10–3 10–2 10–1 100 101 102 103

G’,G

" (P

a)

100

101

102

103

104

ω (rad/s)

10–3 10–2 10–1 100 101 102

G',

G"

(Pa)

10–2

10–1

100

101

ω (rad/s)

10–3 10–2 10–1 100 101 102102

103

104

0NG

'GGe

0

"↓G



Generally, mechanical spectra of protein and polysaccharide solutions obtainedin the frequency range of 10–3 to 102 rad/s are restricted to the terminal region ofthe mechanical spectrum (see Figure 8.9). The study of the viscoelastic behavior ofa dilute polysaccharide or protein solution is difficult and requires special instru-mentation. Such studies have seldom been attempted. An exception is xanthan gum.Very high molecular weight and intrinsic viscoelasticity of this polysaccharide makethis type of investigation available even at concentrations as low as 50 ppm (Lefebvreand Doublier, 2004). A true polysaccharide solution, in general, cannot be directlyprepared at concentrations greater than about 2.5%. The master curve G′(ω) andG″(ω) can be built by a graphical shift procedure using different concentrations, asit can be carried out for the master flow curve. The experimental frequency window,extending over three logarithmic decades, encompasses the transition from the ter-minal region to the very beginning of the plateau region, stopping just beyond thefirst G′, G″ intersection (Figure 8.9).

From certain threshold polysaccharide or protein solution concentrations, thesemacromolecules are able to form networked gel structures. Chemical cross-linkingreactions or physical interactions can drive the gelation processes.

In the case of chemical gelation of polymers, the process is directly controlledby the stoichiometry of the cross-linking. A classical example of chemical covalentgelation of macromolecules important in foods is transglutaminase-mediated cross-linking of proteins via isopeptide bonds and peroxidase-mediated cross-linking ofwater-soluble pentosans (arabinoxylans) via diferulic bonds. Chemical covalent gela-tion is irreversible in terms of sol-gel transition under physical action.

All other food gels are considered as physical gels and can be subdivided intoentanglement gels and particle gels.

The entanglement network is formed by the simple topological interaction ofpolymers. They occur either in the melt or in solution when the concentration andmolecular weight (Mr) becomes greater than some critical entanglement molecularweight Me (Ferry, 1980; Doi and Edwards, 1986). Their mechanical spectra in ausually accessible frequency range encompass a transient viscoelastic plateau (Fig-ure 8.9). The entanglement-like structure of some polysaccharide gels, such as gellanin the presence of co-solutes (sucrose and corn syrup), follows the time-temperaturesuperposition (TTS) principle. A single mechanical spectrum of 0.5% gellan gelconstructed using TTS in a temperature range of 15 to 85°C covers a wide oscillatoryfrequency window in a plateau regime. Gelling polysaccharides modeled previouslyby TTS include κ-carrageenan and glucose, locust bean gum and sucrose, and gellanand glucose systems (Nickerson et al., 2004).

Physical particle gels are structured due to the formation of junction zones witha finite energy and lifetime. Formation of junction zones is always associated withmacromolecular ordered secondary structure. Relatively weak forces, like hydrogenbonds driving the formation of individual junction zones cause the structure of aphysical gel to be strongly dependent on physical factors such as temperature,mechanical excitation, and time. Beta-lactoglobulin, bovine serum albumin, andcollagen proteins represent models of simple protein systems gelling due to thismechanism, and wheat gluten represents the most complex networked system involv-ing this mechanism.



Mechanical spectra of chemical and physical gels differ significantly qualitatively.From a rheological point of view, chemical gel is a viscoelastic solid, and physical gelis a viscoelastic liquid. These differences are exemplified in Figure 8.11 showing themechanical spectra of a chemical gel (2% wheat water-soluble pentosans gel) and wheatgluten as an example of a physical gel-like structure. The mechanical spectra of thesetwo types of gels, recorded in dynamic measurements at the same range of oscillationfrequency, encompass a part of a viscoelastic plateau. For a chemical gel, the G′ curveis almost flat (true rubbery plateau) tending toward an equilibrium modulus Ge at certainlow oscillation frequency values, the largest relaxation time becomes infinite. ModulusG″ theoretically tends toward zero at some infinite time (infinitely low oscillation fre-quency). For a physical gel, G′ decreases with a decrease in oscillation frequency tendingto some equilibrium state, while G″ passes through a minimum and tends, at loweroscillation frequencies, to cross over with G′; in the case shown in Figure 8.11, the cross-over point is beyond the measurement frequency window. At frequencies below the cross-over (in the terminal region) the material enters the steady-state permanent flow.

Rheological properties of viscoelastic liquids and viscoelastic solids can bequantified with characteristic parameters. From mechanical spectra viscoelastic mod-ulus, GN

0 = 1/JN0, a quantitative parameter of the viscoelastic plateau for viscoelastic

liquid and equilibrium elastic modulus, Ge, for the viscoelastic solid can be calcu-lated. Some examples of moduli and characteristic parameters of food viscoelasticmaterials are shown in Table 8.4 and Table 8.5, respectively.

TABLE 8.4Rheological Parameters of Some Viscoelastic Food Materials Measured at a Given Frequency

MaterialTemp.

°°°°C

Yieldstress

σy

Pa

Complexviscosity

η*Pa ⋅⋅⋅⋅ s

Storagemodulus

G’Pa

Lossmodulus

G”Pa Reference

Cream cheesea 18 7300 1588 155739 30603 Kealy, 2006Cream cheese lighta 18 1300 292 28438 6787Caramelb 10 19324 12968 Ahmed et al.,

200650 434 28180 108 42

Egg whiteb

(15% w/wprotein conc.)

20 < 0.01 Ngarize et al., 200490 12.2

Cooled from 90°C 20 73.4Xanthan cryogel(0.5%)c

5 16.1 99.8 15.5 Giannouli andMorris, 2003

a At frequency ω = 100 rad/s.b At frequency ω = 1 rad/s.c Cryogelation at –20°C, values of moduli at frequency ω = 6.28 rad/s.



The steady-state parameters of retardation test: compliance Je0, viscosity η0, and

the longest retardation time τmax = Je0η0, are quantitative parameters of the lower end

of the viscoelastic plateau for viscoelastic liquid (see Figure 8.5).

8.8 STRUCTURE–RHEOLOGY RELATIONSHIPIN MULTIPHASE FOOD MATERIALS

The complex multiphase food systems are composed of dispersed and continuous phases.Liquid droplets compose the dispersed phase of emulsions, and solid particles form thedispersed phase of fine suspensions. Gas bubbles are usually present in dispersed phasesand they represent an important volume fraction in aerated food products. The continuousphase also shows its inner structure due to the presence of macromolecules in colloidaldispersion. Physical and chemical interactions and bonding govern stabilization of themultiphase systems at rest. When these systems start to flow, their structure changes andreaches different equilibrium states depending on the shear rate (Figure 8.12).

TABLE 8.5Characteristic Rheological Parameters of Some Networked Food Systems

MaterialTemp.

°CGN

0

Paω0

rad/s n Reference

WPI a,c 80 820 — — Goncalves et al., 2004WPI + 0.68% GMb 80 320 150 0.5WPI + 1.19% GM 80 230 14 0.5WPI cooled after heatingat 80°C

WPIa,c 20 1500WPI + 0.68% GMb,d 20 1300 100 0.37WPI + 1.19% GMd 20 770 4 0.37Wheat gluten 20 3330 0.20 0.32 Lefebvre et al., 2000Wheat gluten 20 1721 2.37 0.45 Pruska-Kędzior et al., 2005

20 244.3 0.68 0.58Wheat water-solublepentosans gel (1)d

20 11.9 62 0.67 Pruska-Kędzior et al., 2003

Wheat water-solublepentosans gel (2)d

20 13.5 25 0.61

Wheat water-solublepentosans gel (1)e

20 12.8 100

Wheat water-solublepentosans gel (2)e

20 14.9 50

a β-lactoglobulin gels (11% w/w).b GM—Cassia javanica galactomannans.c G′ at G″ minimum.d GN

0 = viscoelastic plateau modulus; ω0 = characteristic frequency of loss peak; n = spread parameterrelated to the loss peak broadness—Cole-Cole fit.e Equilibrium modulus Ge, Winter and Charbon correlation.



In the domain of the lowest shear rates (γ⋅ < γ⋅0) only the Brownian motion isacting against the structural forces. If the concentration of the dispersed phase, orconcentration of the macromolecules in the continuous phase, is high, their stronginteractions generate a yield value σy and the system shows plastic behavior. Forlow enough concentrations of structuring components, the viscosity η0 is independentof the shear rate (lower Newtonian regime).

In the region of moderate and higher shear rate values (γ⋅ > γ⋅0) , hydrodynamicforces become of the same order of magnitude as the structural forces. This inducesnew structures, and if the time these shearing forces act is long enough, a newequilibrium structure is attained. Dispersed phase droplets or bubbles submitted toshear-induced deformations change their initial shape. Dispersed phase solid parti-cles undergo ordering and aggregation–disaggregation processes. The continuousphase also changes its inner structure due to changes, rearrangements, and orderingof inter-macromolecular entanglement. The shear-induced state of arrangement andorientation of the system is unstable. Therefore, if this new structure or viscositystate is required for the product quality or for further processing, it must be fixedby physical or chemical means (for example, fixation of fine grain emulsions).

The significance of determining the viscoelastic properties of the macromo-lecular components of food, such as starch and nonstarch polysaccharides andproteins, appears obvious when their state diagram is considered. Depending onthe degree of macromolecular system hydration and its temperature, the systemproperties span from the glassy state to the flow state, passing through the vis-coelastic (rubbery) state.

FIGURE 8.12 Shear rate dependency of multiphase food materials’ structure and rheologicalproperties. (From Windhab, E.J., in Beckett, S.T. (Ed.), Physico-Chemical Aspects of FoodProcessing, Chapman and Hall, London, 1996. With permission.)

Yieldstress ? 0

Viscosity function for multiphase liquidsystems

Dispersed Phase Cont. Ph.



Dispersed Cont.


Yieldstress ? 0





Dispersed Cont.


Yieldstress ? 0





Dispersed Cont.


Yieldstress ? 0


Yieldstress ? 0


η

η0

η∞

γ0⋅ γ∞

⋅ ⋅γ

Yieldstress σ0

Structural forces Hydrodynamic/viscous forces


Dispersed Phase Cont. Ph.Dispersed Phase Cont. Ph.


Dispersed Phase Cont. Ph.Dispersed Phase Cont. Ph.Dispersed Phase Cont. Ph.

Dispersed Cont.Dispersed Cont.DispersedDispersed Cont.Cont.

Dispersed Phase Cont. Ph.Dispersed Phase Cont. Ph.Dispersed Phase

Dispersed Phase

Dispersed Phase

Dispersed Phase

Cont. Ph.

Cont. Ph.

Cont. Ph.

Cont. Ph.



Figure 8.13 shows an example of a real technological process situation involvingthe degree of hydration and temperature evolution exemplified by the state diagramof wheat starch and protein in the baking process. Three states are distinguished onthe state diagram: glassy, rubbery, and flow. Depending on the temperature and typeof macromolecules, their properties in the rubbery state change from viscoelasticliquid to viscoelastic solid. At room temperature, structural changes of starches andproteins range from the glassy state (amorphous solid) for flour (approximately 14%water) to the viscoelastic rubbery state for dough (80% water). During baking, watercontent decreases from approximately 80% in dough to approximately 40% in thecrumb. Meanwhile, temperature rises from approximately 30°C for dough to crumbfinal temperature in the oven of approximately 96°C. Next, the crumb is cooleddown to room temperature. Proteins and starch behave differently following thiswater content and temperature evolution. Protein systems pass from viscoelasticliquid properties shown by the native gluten network, below protein thermal settingtemperature, through viscoelastic behavior of the denatured gluten protein networkat approximately 96°C (stabilized significantly by hydrophobic bonds that havereplaced destroyed hydrogen bonds) to viscoelastic solid after cooling to roomtemperature. Starch granules behave as rubbery neo-Hookean bodies below gelationtemperature, and act as a neutral filler of a viscoelastic gluten network. Abovegelation temperature, swollen starch granules lose their integrity, and the liquidmixture of amylose and amylopectin enter the flow state above the starch meltingpoint. Once the water content in the crumb diminishes, gelated starch reachesviscoelastic liquid properties at approximately 96°C, and then becomes a viscoelasticgel after the crumb cools to room temperature.

FIGURE 8.13 State diagram of wheat starch and gluten proteins in the baking process. (FromCuq, B., Abecassis, J., and Guilbert, S., State diagrams to help describe wheat bread process-ing, Int. J. Food Sci. Technol., 38, 739–766, 2003. With permission.)



8.9 ELASTIC SOLID

8.9.1 INTRODUCTION

For solids, the fundamental relationship between force and deformation is Hooke’s law:

(8.31)

where σ is a force per unit area (the stress), and γ is the relative length change orstrain in shear. G is a constant of proportionality called the elastic modulus. This isan intrinsic property of solids. Hooke’s law is the basic constitutive equation ofclassical solid mechanics. Frequently, if a solid sample is subject to uniaxial tensileforce (in extension or compression), deformation is described in terms of strain εand the ratio of change in length to undeformed length l0:

(8.32)

where l0 is the original length of the test material sample. Then Hooke’s law iswritten as

(8.33)

where σ11 is tensile (extensional) stress, perpendicular to the surface it acts upon,and E is called tensile or Young’s modulus.

In the limit of the small strain region, the tensile modulus is three times thatmeasured in shear for incompressible, isotropic materials, E = 3G. For compressible,isotropic materials, a parameter µ (Poisson’s ratio) is required to relate the tensileto shear modulus [E = 2G (µ + 1)], where µ ranges from 0.5 for the incompressiblecase to 0.

8.9.2 NONLINEAR BEHAVIOR OF A SOLID

Figure 8.14 explains the principle of rubbery material behavior in tension and incompression. It shows that for a small deformation near zero, the stress is linear withdeformation, but at larger deformations, the stress becomes nonlinear and differentfrom that predicted by Hooke’s law. These materials are described as neo-Hookean.In simple shear, the neo-Hookean model predicts the first normal stress differencethat increases in square with strain (compare a second-order liquid, Equation 8.13):

. (8.34)

Only one normal stress difference N1, for the neo-Hookean solid in shear exists(N2 = 0).

σ γ= G

ε = −l l

l0

0

σ ε11 = E

σ σ γ11 222− = G



FIGURE 8.14 Elastic response of a rubberlike body. (Adapted from Macosco C.V., Rheol-ogy: Principles, Measurements and Applications, VCH Publishers, New York, 1994. Withpermission.)

TABLE 8.6Young’s Modulus of Some Food Solids

MaterialTemperature

(°C)Young’s modulus E

(MPa) Reference

High amylose maize starch foama Ambient 24.7 Lourdin et al., 1995Waxy maize starch foama Ambient 16.5Cassava starch film without plasticizers 25 1200–2800 Mali et al., 2005Cassava starch films with plasticizers 25 <1200Tomato powderc 21.5–79.2d 100–1000 Palzer, 2005Bread crumba Ambient 0.132–0.851 Zghal et al., 2002Bread crumbb Ambient 0.280–0.440Soy protein isolate gele 20 0.0039–0.0042 Renkema, 2004Cheddar cheese 20 0.4–1.79 Hort et al., 1997Avocado pears—0 days postharvest 25 480 Baryeh, 2000Avocado pears—10 days postharvest 25 48

a Foam or crumb cell wall material, Young’s modulus.b Foam or crumb Young’s modulus related to material density.c Young’s modulus at the glass transition temperature.d Depending on the powder dry matter content (89–96% dm).e Depending on the pH and ionic strength.

Strain

0 2 4 6 8

(MPa)

–10

–5

0

5

10

15

20

25

Ten

sile

stre

ssC

ompr

essi

ve s

tres

s

Hooke’s law

neo-

Hooke

an



The neo-Hookean model has been applied to large strain deformation problems.In a number of polymer-processing operations, such as blow molding, film blowing,and thermoforming, deformations are rapid and the polymer melt behaves more likea cross-linked rubber than a viscous liquid. In many real situations, as the time scaleof experiment is shortened, the viscoelastic liquid looks more and more like a neo-Hookean solid; thus in many cases of rapid deformations the simplest and oftenmost realistic model for the stress response of these polymeric materials is the elasticsolid. Meat protein elastin is considered as a neo-Hookean elastic protein. Many realfood materials, from fruits and vegetables, to cheese, meat, and sausages, can beconsidered as neo-Hookean. Some examples of Young’s moduli E values of foodmaterials are shown in Table 8.6.

8.10 SUMMARY

The main aspects of practical applications of rheological methods based on shearare summarized in Figure 8.15. Large deformation tests are dedicated to designingindustrial processes involved in food pipeline transport, mixing, stirring, or centrif-ugation. These tests are also applied to food production online quality control

FIGURE 8.15 Rheological methods applied to investigation of food materials.

FlowPumpingMixingStirring

Food quality controlFood stabilityFood structure

Application

Viscosity η(γ)Yield stress σy

Thixotropy η(γ ,t)Normal stress N1, N2

Steady-stateflow behavior test

Large deformation tests

Storage modulus G'(ω)Loss modulus G"(ω)

Dynamic viscosity η'(ω)Phase angle δ (ω)

Dynamic steady sheartest

MicrostructureParticle size and shape

Morphology

Food quality controlFood stability

Food structure

Application

Relaxation modulus G(t)Relaxation time λ(t)

Relaxation test

Compliance J(t)Retardation time τ(t)

Creep and creeprecovery test

(retardation test)

Transient test

Small deformation tests

Rheology of food macromolecules andcolloid dispersed particles

in shear

.

.



considering its structure formation or preservation. Small deformation tests are usefulparticularly for raw material characterization and designing rheological propertiesof formulated final products in the research and development stage when theirmicrostructure, including macromolecular, has to be described. These tests can alsobe applied in industrial quality control from the point of view of food productstructure and stability control, including texture and sensory appreciation.

However, existing rheological methods still do not answer all demands, whileproperties of food macromolecular systems are considered and new techniques aredeveloped. Optorheological methods involving direct circular dichroism and bire-fringence measurements during material mechanical excitation are introduced fortransparent systems. Rheoacoustic methods extracting rheological parameters fromanalysis of ultrasound propagation within opaque material are examined. Integratedrheo-NMR methods are applied to studying the intramolecular response of materialto mechanical excitation. Image analysis of complex materials and correlation ofstructural fractal dimensions of the components with the rheological parameters ofthe material is being tested. Surface rheology is applied to explain rheologicalproperties of food components at interfaces.

REFERENCES

Ahmed, J., Ramaswamy, H.S., and Pandey, P.K. (2006) Dynamic rheological and thermalcharacteristics of caramels, Lebensm. Wiss. u.-Technol., 39, 216–224.

Ahmed, J. et al. (2003) Effect of high pressure on rheological characteristics of liquid egg,Lebensm.-Wiss. u.-Technol., 36, 517–524.

Barnes, H.A, Hutton, J.F., and Walters K. (1989) An Introduction to Rheology, Elsevier SciencePublishers, Amsterdam.

Baryeh, E.A. (2000) Strength properties of avocado pear, J. Agric. Eng. Res., 76, 389–397.Bourne, M. (2002) Food Texture and Viscosity: Concept and Measurement, 2nd ed., Academic

Press, New YorkDervilly-Pinel, G., Tran, V., and Saulnier, L. (2004) Investigation of the distribution of

arabinose residues on the xylan backbone of water-soluble arabinoxylans from wheatflour, Carbohydr. Polym., 55, 171–177.

Dimitreli, G. and Thomareis, A.S. (2004) Effect of temperature and chemical compositionon processed cheese apparent viscosity, J. Food Eng., 64, 265–271.

Dobraszczyk, B.J. and Morgenstern, M.P. (2003) Rheology and the breadmaking process, J.Cereal Sci., 38, 229–245.

Doi, M. and Edwards, S.F. (1986) The Theory of Polymer Dynamics. Oxford University Press,Oxford.

Ferguson, J. and Kemblowski, Z. (1991) Applied Fluid Rheology, Elsevier Applied Science,London.

Ferry, J. (1980) Viscoelastic Properties of Polymers, 3rd ed., John Wiley and Sons, Inc., NewYork.

Giannouli, P. and Morris, E.R. (2003) Cryogelation of xanthan, Food Hydrocolloids, 17, 495–501.Goncalves, M.P. et al. (2004) Rheological study of the effect of Cassia javanica galactoman-

nans on the heat-set gelation of a whey protein isolate at pH 7, Food Hydrocolloids18, 181–189.



Hort, J., Grys, G., and Woodman, J. (1997) The relationship between the chemical, rheologicaland textural properties of cheddar cheese, Lait, 77, 587–600.

Juszczak, L. et al. (2004) Rheological properties of commercial mustards, J. Food Eng., 63,209–217.

Kealy, T. (2006) Application of liquid and solid rheological technologies to the texturalcharacterisation of semi-solid foods, Food Res. Int., 39, 265–276.

Kulicke, W.-M. et al. (1996) Characterization of aqueous carboxymethylcellulose solutionsin terms of their molecular structure and its influence on rheological behaviour,Polymer, 37, 2723–2731.

Launay, B., Cuvelier, G., and Martinez-Reyes, S. (1997) Viscosity of locust bean, guarand xanthan gum solutions in the Newtonian domain: critical examination of the log(ηsp)0 –log C[η]0 master curves, Carbohydr. Polym., 34, 385–395.

Lefebvre, J. and Doublier, J.-L. (2004) Rheological behaviour of polysaccharides aqueoussystems, in Polysaccharides, Structural Diversity and Functional Versatility, 2nd ed.,Dumitriu, S., Ed., Marcel Dekker, New York, chap. 13.

Lefebvre, J. et al. (2000) Temperature-induced changes in the dynamic rheological behaviorand size distribution of polymeric proteins for glutens from wheat near-isogenic linesdiffering in HMW glutenin subunit composition, Cereal Chem., 77, 193–201.

Lefebvre, J. et al. (2003) A phenomenological analysis of wheat gluten viscoelastic response inretardation and in dynamic experiments over a large time scale, J. Cereal Sci., 38, 257–267.

Lourdin, D., Della Valle, G., and Colonna, P. (1995) Influence of amylose content on starchfilms and foams, Carbohydr. Polym., 27, 261–270.

Macosco, C.V. (1994) Rheology: Principles, Measurements and Applications, VCH PublishersInc., New York.

Mali, S. et al. (2005) Water sorption and mechanical properties of cassava starch films andtheir relation to plasticizing effect, Carbohydr. Polym., 60, 283–289.

Morris, E.R. (1990) Shear thinning of “random coil” polysaccharides: characterization bytwo parameters from a simple shear plot, Carbohydr. Res., 13, 85.

Morrison, A. (2001) Understanding Rheology, Oxford University Press., New York.Ngarize, S., Adams, A., and Howell, N.K. (2004) Studies on egg albumen and whey protein

interactions by FT-Raman spectroscopy and rheology, Food Hydrocolloids, 18,49–59.

Nickerson, M.T., Paulson, A.T., and Speers, R.A. (2004) Time-temperature studies of gellanpolysaccharide gelation in the presence of low, intermediate and high levels of co-solutes, Food Hydrocolloids, 18, 783–794.

Palzer, S. (2005) The effect of glass transition on the desired and undesired agglomerationof amorphous food powders, Chem. Eng. Sci., 60, 3959–3968.

Pruska-Kędzior, A. et al. (2003) Rheological properties of water-soluble wheat pentosanssolutions and gels obtained from various cultivars of common wheat, in Proceedingsof 3rd International Symposium on Food Rheology and Structure ISFRS—Eurorheo2003-01, February 9–13. 2003, Zurich, Switzerland, Fischer, P., Marti, I., andWindhab, E.J., Eds., Laboratory of Food Processing Engineering, ETH, Zurich,175–179.

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Renkema, J.M.S (2004) Relations between rheological properties and network structure ofsoy protein gels, Food Hydrocolloids, 18, 39–47.

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Sittikijyothin, W., Torres, D., and Goncalves, M.P. (2005) Modelling the rheological behaviourof galactomannan aqueous solutions, Carbohydr. Polym., 59, 339–350.

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Togrul, H. and Arslan, N. (2004) Mathematical model for prediction of apparent viscosity ofmolasses, J. Food Eng., 62, 281–289.

Tschoegl, N.W. (1989) The Phenomenological Theory of Linear Viscoelastic Behavior. AnIntroduction, Springer Verlag, Berlin, pp. 433–435.

Walkenström, P. et al. (1999) Effects of flow behaviour on the aggregation of whey proteinsuspensions, pure or mixed with xanthan, J. Food Engineering, 42, 15–26.

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245

9 Food Colorants


CONTENTS

9.1 Introduction .................................................................................................. 2459.2 Carotenoids................................................................................................... 246

9.2.1 Structure ........................................................................................... 2469.2.2 Occurrence ....................................................................................... 2499.2.3 Carotenoids Used as Food Colorants .............................................. 2519.2.4 Physical and Chemical Properties ................................................... 2529.2.5 Biological Activity ........................................................................... 253

9.3 Chlorophyll................................................................................................... 2559.4 Heme Pigments ............................................................................................ 2599.5 Anthocyanins................................................................................................ 260

9.5.1 Occurrence and Structure ................................................................ 2609.5.2 Chemical Properties ......................................................................... 2629.5.3 Biological Activity ........................................................................... 2649.5.4 Stability of Anthocyanins in Food................................................... 264

9.6 Betalains ....................................................................................................... 2659.7 Quinone Pigments ........................................................................................ 2689.8 Turmeric and Curcumin (E 100) ................................................................. 2689.9 Riboflavin (E 101) ....................................................................................... 2699.10 Caramel (E 150)........................................................................................... 2709.11 Melanoidins.................................................................................................. 2709.12 Melanins ....................................................................................................... 2719.13 Synthetic Organic Colors............................................................................. 272References.............................................................................................................. 274

9.1 INTRODUCTION

Color is an important indicator of food quality. The consumer associates food colorwith good processing and safety. However, color cannot be studied without con-sidering the human sensory system. Perception of color is related to three factors:spectral composition of the light source, physical object characteristics, and eyesensitivity. Fortunately the human eye views color in a fairly uniform manner andit is not difficult to replace the eye, in food analysis, with some sensor instrumentor photocell.



The color of food is the result of the presence of natural pigments or of addedsynthetic organic dyes. The definition of a natural colorant is variable from onecountry to another, but generally, natural colorants include the pigments occurring inunprocessed food, and those that can be formed upon heating, processing, or storage.The natural pigments may be divided into four, not necessarily homogenous, groups:

• Isoprenoid derivatives—carotenoids • Porphyrins—chlorophylls and hemes • Phenolics—anthocyanins, betalains, quinones, curcuminoids • Miscellaneous naturally occurring colorants—riboflavins, caramels, mel-

anoidins, and melanins

All natural pigments are unstable and participate in different reactions, so thefood color is strongly dependent on conditions of storage and processing.

The use of synthetic organic colors has been recognized for many years as themost reliable and economical method of restoring some of the food’s original shadeto the processed product. Synthetic colors are superior to natural pigments in tinc-torial power, stability, ease of application, and cost effectiveness. However, from thehealth safety viewpoint they are not accepted by consumers, so over the past yearsincreasing interest in natural food colorants has been observed.

9.2 CAROTENOIDS

9.2.1 STRUCTURE

Carotenoids are the most widespread and important group of pigments in nature.They play a crucial role in plants as accessory pigments for light harvesting and inprevention of photooxidative damage, as well as acting as attractants for pollinators.

Carotenoids comprise a group of structurally related colorants that are mainlyfound in plants, algae, and several lower organisms. Nature produces around 108

tons per year of carotenoids. Most of them are found in marine algae and greenleaves. At present, about 600 carotenoid compounds have been identified. Their basicstructure is a symmetrical tetraterpene skeleton, formed by head-to-tail condensationof two C20 units, which is modified by cyclization, addition, elimination, rearrange-ment, and substitution, as well as oxidation.

Based on their composition, carotenoids are divided into two classes:

• Carotenes, which are pure polyene hydrocarbons; they contain only carbonand hydrogen atoms, including acyclic lycopene and bicyclic β-, α- andλ-carotene (Formulas 9.1 to 9.4)

• Xanthophylls containing oxygen in the form of hydroxy (lutein), epoxy(violaxanthin), and oxo (canthaxanthin) groups (Formulas 9.5, 9.6, and 9.9)

Carotenoids with fewer than 40 carbon atoms are also known. The compoundsderived by the loss of one or both end fragments are called apocarotenoids, for example,β-apo-8′-carotenal or diapocarotenoids such as bixin (Formulas 9.10 and 9.11).


Food Colorants 247

All carotenoids contain a system of conjugated double bonds that influence theirphysical, biochemical, and chemical properties. In principle, each of the polyenechain double bonds could exist in a cis or trans conformation, thus creating a numberof isomers. In practice, however, only a few isomers exist. The reason for this is

FORMULA 9.1

FORMULA 9.2

FORMULA 9.3

FORMULA 9.4

FORMULA 9.5

1

3 58 10 12

7

Lycopene

14

15 14' 12'

15'

10' 8'

7'

6'

4'

2'

HO

OH

Lutein



steric hindrance, making cis isomers less stable than the trans form. Thus, the vastmajority of natural carotenoids are in the all-trans configuration.

The carotenoids owe their characteristic yellow, orange, or red colors to theabsorption of light in the 400- to 500-nm range as a result of the presence of thechromophore of conjugated double bonds. A minimum of seven conjugated doublebonds are required for the yellow color to appear. The increase in the number ofdouble bonds results in a shift of the major adsorption band to the longer wave-lengths. In lycopene and β-carotene there are 11 conjugated double bonds, but insome molecules, such as canthaxanthin, two carbon–oxygen double bonds extendthe chromophore chain. Food processing and storage can cause isomerization ofcarotenoids and affect the color because increasing the number of cis bonds resultsin gradual lightening of the color The cis isomers not only absorb less strongly

FORMULA 9.6

FORMULA 9.7

FORMULA 9.8

FORMULA 9.9

HO

OH

O

O

Violaxanthin

HO

OH

O

OH

Capsanthin

O Canthaxanthin

O


Food Colorants 249

than the all-trans isomers, but they also show a so-called cis peak at 330 to 340nm (Barua et al., 2000).

9.2.2 OCCURRENCE

It is generally considered that de novo biosynthesis of carotenoids is observed onlyin photosynthetic higher plants, mosses, and algae, and nonphotosynthetic bacteriaand fungi. All photosynthetic tissues contain carotenoids: higher plants in the chlo-roplasts, although the color is masked by the chlorophylls, and some bacteria inphotosynthetic membranes. The chloroplasts, present in green unripe fruits, in mostcases gradually change into chromoplasts on ripening, and carotenoid synthesis,often of novel pigments, is greatly stimulated. Typical examples are the tomato andred pepper. Oxygen but not light is required for carotenoid synthesis.

In all photosynthetic organisms, carotenoids have two major functions: as acces-sory pigments for light harvesting and in the prevention of photooxidative damage.In plants they are essential components of the light-harvesting antennae, where theyabsorb photons and transfer the energy to chlorophyll.

The other function of carotenoids is to protect against photooxidation processesthat are caused by the excited triplet state of chlorophyll. Carotenoid molecules,with nine or more conjugated carbon–carbon double bonds, can absorb triplet-stateenergy from chlorophyll, and thus prevent the formation of harmful singlet oxygen.

The main carotenoids of green leaves are lutein, violaxanthin, cryptoxanthin,and β-carotene; the others are produced in smaller quantities. Common carotenoidsin fruits are β-carotene, lycopene, and different xanthophylls. The last are usuallypresent in esterified form. Some of the carotenoids, such as β-carotene, lycopene,and zeaxanthin, are widely distributed and so become important as food components.However, the content of carotenoids usually does not exceed 0.1% of dry weight(Table 9.1). In plant products, carotenoids may occur as simple or as very complexmixtures; some of the most complex are found in citrus fruits. The simplest caro-tenoids usually exist in animal products because the animal organism is limited in

FORMULA 9.10

FORMULA 9.11

CHO

CH3O

O

COHO

Bixin



TAB

LE 9

.1Th

e Q

uant

ites

of

Diff

eren

t C

arot

enoi

ds i

n So

me

Vege

tabl

es a

nd F

ruit

s (m

g/10

0 g

edib

le p

orti

on)

Veg

etab

le o

r fr

uits

ββββ -C

arot

ene

αααα -C

arot

ene

Lyco

pene

Lute

inV

iola

xant

hin

X-C

rypt

oxan

thin

Tota

l

Kal

e8.

680.

15—

18.6

5.81

0.12

34.7

6Sp

inac

h3.

680.

09—

9.54

3.04

—17

.31

Let

tuce

1.68

0.04

—2.

922.

360.

038.

48W

hite

cab

bage

0.03

4—

—0.

080.

070.

002

0.25

Red

pap

rika

3.

78—

3.45

0.25

0.13

1.01

30.3

7a

Gre

en p

apri

ka

0.11

0.01

—0.

410.

120.

002

0.70

Tom

ato

0.89

0.15

11.4

40.

21—

—12

.7B

rocc

oli

0.32

——

0.8

0.18

0.01

11.

56C

arro

t9.

544.

89—

0.36

——

15.9

9B

lack

berr

y0.

130.

02—

0.65

0.06

0.00

80.

90St

raw

berr

y0.

006

0.00

02—

0.04

0.00

30.

0005

0.05

Nec

tari

ne0.

400.

14—

0.98

0.51

0.08

2.40

Apr

icot

0.90

0.02

—0.

040.

020.

061.

13G

rape

frui

t0.

59—

2.77

0.02

0.00

50.

012

3.50

Ora

nge

0.01

30.

006

—0.

020.

220.

050.

40

aIn

clud

ing

13.9

4 m

g/10

0 g

caps

anth

in/c

apso

rubi

n an

d 5.

0 m

g/10

0g o

f ca

psol

utei

n. (

Ada

pted

fro

m H

. Mul

ler ,

Z. L

eben

sm U

nter

s Fo

rsch

A 2

04, 8

8–94

, 199

7.)


Food Colorants 251

its ability to absorb and deposit them. The compounds, which have been isolatedonly from animal tissues, mainly xanthophylls, are the result of metabolic changes,generally oxidative, in the ingested carotenoids. Crude vegetable oils contain caro-tenoids, but bleaching and hydrogenation leads to almost complete degradation ofthese pigments. Particularly rich in carotenoids (0.05 to 0.2%) is crude palm oilcontaining mainly α-carotene and β-carotenes. Egg yolk contains only xanthophylls,mainly lutein, zeaxanthin, and cryptoxanthin (0.3 to 8.0 mg/kg).

9.2.3 CAROTENOIDS USED AS FOOD COLORANTS

The most commonly used natural carotenoid extracts for foodstuffs are annatto,paprika, and saffron. Many other sources, including alfalfa, carrot, tomato, citruspeel, and palm oil, are also utilized.

Annatto [E 160(b)] is the orange-yellow, oil-soluble natural pigment extractedfrom the pericarp of the seed of the Bixa orellana L. tree. The major coloringcomponent of this extract is the diapocarotenoid bixin (Formula 9.11), Several otherpigments, mainly degradation products of bixin, are also present, including trans-bixin, norbixin, and trans-norbixin. Bixin is a methyl ester of a dibasic fatty acid,which on treatment with alkalis is hydrolyzed to water-soluble norbixin. Two typesof annatto are therefore available: the extract in oil containing bixin, and a powdered,water-soluble form containing norbixin.

Paprika oleoresin [E 160(c)] is the orange-red, oil-soluble extract from sweetred peppers (Capsicum annum). The major coloring compounds are xanthophylls:capsanthin (Formula 9.8), capsorubin as their dilaurate esters, and β-carotene. Thepresence of characteristic flavoring and spicy pungency components limits applica-tion of this extract in foodstuffs.

Raw, unrefined palm oil contains 0.05 to 0.2% carotenoids with α- and β-carotenes,in a ratio of 2:3, as the main constituents. It is particularly used as a colorant formargarine.

Saffron, an extract of flowers of Crocus sativus, contains the water-solublepigment crocin, the digentiobioside of apocarotenic acid (crocetin), as well as zeax-anthin, β-carotene, and characteristic flavoring compounds. The yellow color of thispigment is attractive in beverages, cakes, and other bakery products. However, useof this colorant is restricted by its high price.

Carrot extracts (E 160a), carrot oil, and related plant extracts are also availableon the market. Their main components are β- and α-carotenes (Formulas 9.1 and9.2, respectively). Processes for the commercial extraction of carotene from carrotshave been developed. Purified crystalline products contain 20% α-carotene and 80%β-carotene. They may be used for coloring fat-based products, such as dispersionof microcrystals in oil.

Individual carotenoid compounds—β-carotene, β-apo-8′-carotenal (Formula9.10), β-apo-8′-carotenoic acid ethyl ester, and canthaxanthin (Formula 9.9)—aresynthesized for use as food colorants for edible fats and oils. Their properties aregiven in Table 9.2. The carotenoid pigments, in combination with surface activeagents, are also available as microemulsions for coloring foods with a high watercontent.



9.2.4 PHYSICAL AND CHEMICAL PROPERTIES

Carotenoids are extremely lipophilic compounds that are almost insoluble in water.In an aqueous medium they tend to form aggregates or adhere to surfaces. They aresoluble in nonpolar organic solvents such as hexane, halogenated hydrocarbons, ortetrahydrofurane. In oils their solubility is rather low, particularly in a pure crystallinestate. In the organism they are located in cellular membranes or in lipophilic com-partments. In some plants hydroxylated carotenoids are esterified with various fattyacids, which makes them even more lipophilic.

With their large number of conjugated double bonds, the carotenoids contain areactive electron-rich system, which is susceptible to reaction with electrophiliccompounds. This structure is responsible for the high sensitivity of carotenoids tooxygen and light. The central chain of conjugated double bonds can be oxidativelycleaved at various points, giving rise to a family of apocarotenoids.

Most carotenoids (but not vitamin A) are singlet oxygen quenchers. Singletoxygen 1O2, interacts with the carotenoid to give triplet states of both molecules.The energy of an excited carotenoid is dissipated through vibrational interactionwith the solvent to recover the ground state. Carotenoids are the most efficientnaturally occurring quenchers of singlet oxygen. They may also participate in thepropagation step of the oxidation process as chain-breaking antioxidants that scav-enge reactive peroxyl radicals. However, carotenoids serve this function best at lowoxygen tensions. At higher oxygen levels, a carotenoid intermediate radical mightadd oxygen to form carotenoid peroxyl radicals such as Car-OO, which could actas prooxidants, initiating the process of lipid peroxidation [Berg et al., 2000].

On the other side, radicals and peroxides, occurring in food as a result of lipidoxidation, accelerate oxidative degradation of carotenoid pigments, which leads toformation of epoxides located at the β-ionone ring, β-apo-carotenones, and β-apo-carotenals of different chain lengths. Lipoxygenase involved in the decay of vege-table matter may also cause the destruction of carotenoids. Antioxidants, includingascorbic acid, and their derivatives, tocopherols, and polyphenolics are used tosuppress this oxidative degradation.

TABLE 9.2Properties of Carotenoids Used as Food Colorants

Carotenoid Color

Solubility[g/100 cm3] w 20°C

λλλλmax

Vitamin Aactivity (IU/mg)Oils Ethanol

β-Carotene yellow 0.05–0.08 0.01 455–456 1.67β-Apo-8′-carotenic

acid ethyl esteryellow to orange 0.7 0.1 448–450 1.2

β-Apo-8′-carotenal orange to red 0.7–1.5 0.1 460–462 1.2Canthaxanthin red 0.005 0.01 468–472 0

Source: Adapted from Klaui, H. and Bauernfeind, J.C., Carotenoids as Food Colors in Carotenoids asColorants and Vitamin A Precursors, CRC Press, Inc., Boca Raton, FL, 1981.


Food Colorants 253

Due to oxidative degradation of carotenoids, aroma compounds are also formed,including β-ionone with an odor threshold value of 14 ng/g in water. The formationof β-ionone in dehydrated carrots causes an undesirable off-flavor with an odor likeviolets. Unsaturated ketones derived from carotenoid degradation are readily furtheroxidized. The stability of carotenoids in frozen and heat-sterilized foods is quitehigh, but it is poor in dehydrated products, unless the products are packaged in inertgas. Dehydrated carrots fade rapidly.

9.2.5 BIOLOGICAL ACTIVITY

A wide range of carotenoid chemical reactivity makes it obvious that they can exertbioprotective effects through a variety of mechanisms. According to Berg et al.(2000), carotenoids have diverse biological functions and actions, the most importantof which are summarized below:

Provitamin A activity β-carotene, α-carotene, β-cryptoxanthinAntioxidant All carotenoidsCell communication β-carotene, canthaxanthin, cryptoxanthinImmune function enhancers β-caroteneUV skin protection β-carotene, lycopeneMacula protection Lutein, zeaxanthin

Some carotenoids are precursors of a separate class of bioactive compounds, theretinoids, which can regulate cell growth and differentiation in various cell typesthrough interaction with ligand-dependent transcription factors. But of the 600 car-otenoids that have been identified, only about 30 are believed to have some provi-tamin A activity, especially β-carotene. One mole of β-carotene can theoretically beconverted, by cleavage of the C 15 = C 15′ double bond, to yield two moles ofretinol (Reaction 9.1). However, the physiological efficiency of this process appearsto be only 50%. The observed average efficiency of intestinal β-carotene absorptionis only two thirds of the total content. Thus, a factor of 1/6 is used to calculate theretinol equivalent (RE) from β-carotene, but only 1/12 from the other provitamin Acarotenoids in food (Combs, 1992).

Pathological processes, including cancer and cardiovascular disease, are com-monly associated with an oxidative stress condition. Carotenoids have a considerablyhigh antioxidant activity, which has most often been the focus of its role in preventingdisease initiation and propagation.

Excessive production of oxygen radical species and particularly hydroxyl radi-cals, can affect lipid cell membranes to produce lipid peroxides and reactive oxygenspecies (ROS), which are linked to a variety of degenerative diseases and accelerationof the aging process. Carotenoids are membranal pigments: apolar carotenes areimmersed in membranes and show limited mobility, whereas polar xanthophylls havea variable position and mobility in membranes. As can be deduced, carotenes havehigh antioxidant activity against radicals generated inside the membranes. The struc-ture of carotenoids, in particular the length of the polyene chain, significantlyinfluences its antioxidant properties.



Different methods have been used for determination of antioxidant activity. Oneof the frequently used methods is based on the ability to quench the colored 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid) radical (ABTS). The comparison ofantioxidant activity of some carotenoids, determined with this method and calculatedas the Trolox equivalent of antioxidant capacity (TEAC), is given in Table 9.3.

Carotenoids have also been reported to have immunomodulatory effects, suchas a reduction in UV-induced immunosuppression, and an increase in natural killer(NK) cell activity after dietary supplementation with β-carotene. Cell-to-cell com-munication by gap junction is another important physiological function, playing arole in morphogenesis and cell differentiation. Epidemiological studies have alsoshown an inverse association between carotenoid intake and the risk of cataract andage-related macular degeneration. Lutein and zeaxanthin are the principal compo-nents of macular pigments. Their content can be modified by diet modification orlutein supplementation.

Generally it was observed that people with low carotenoid intake or low bloodlevels, have an increased risk of degenerative diseases. In a number of these diseases,free radical damage plays a role in the pathophysiology of the disease. Earlier studiesfocused mainly on the protective effects of β-carotene and lycopene against prostate

REACTION 9.1 Formation of vitamin A from β-carotene.

15

15'

CHO

CH2OH

Retinal

Retinol

retinal aldehyde

reductase

alcohol

dehydrogenase


Food Colorants 255

and lung cancer, but there is as yet no definitive proof for a causal relationship, norfor a beneficial antioxidant effect of carotenoids.

The cancer-preventing effect of β-carotene has been investigated in severalintervention trials. However, in only one of these studies, the so-called Linxianstudy, a protective effect was found for a combined β-carotene, vitamin E, andselenium supplementation. In other studies no protective effect was found, and insome cases a higher risk of lung cancer has been observed after high doses ofβ-carotene. Therefore the conclusion from these studies is that high-dose supple-ments of β-carotene are contradicted mainly for heavy smokers, but could have abeneficial effect on individuals with a poor baseline carotenoid status. Probably adiet rich in high-carotenoid-containing fruits and vegetables is more efficaciousthan individual carotenoid compounds, because it represents a lower, regular intakeof various constituents with several mechanisms of action, as opposed to a highintake of one constituent with limited functions (Berg et al., 2000).

9.3 CHLOROPHYLL

Chlorophyll is the most widely distributed natural plant pigment. It is known thatchlorophyll has existed on Earth for at least 2.6 billion years. Over that period,innumerable plant species have come and gone, while the constitution and action ofchlorophyll has remained, despite the powerful forces of natural selection. Its annualproduction is estimated to be 1.2 billion tons worldwide, of which one quarter isterrestrial and the rest of marine origin (Humphrey, 2004). In higher plants and algae,except the blue-greens, chlorophyll is found in chloroplasts, while in blue-greenalgae and photosynthetic bacteria, it is located on intracellular lamellae. In livingplant tissues, in chloroplasts, chlorophyll is complexed with polypeptides, phospho-lipids, and tocopherols and held within hydrophobic membranes.

Chlorophylls are derivatives of dihydroporphirin chelated with a centrally locatedmagnesium ion (Formula 9.12). They are diesters: one carbonyl group is esterified

TABLE 9.3Antioxidant Activities of Some Carotenoids

TEAC (mM)

Lycopene 2.9β-Cryptoxanthin 2.0β-Carotene 1.9Lutein 1.5Zeaxanthin 1.4α-Carotene 1.3

TEAC = Trolox equivalent antioxidant capacity.

Source: Adapted from Rice-Evans, C., Sampson, J., Bram-ley, P.M., and Holoway, D.E., Free Rad. Res., 26, 381, 1997.



with methanol and the other with a C20 monounsaturated isoprenoid alcohol–phytol,which make the chlorophyll molecule hydrophobic. There are two chlorophylls: a(blue-green) and b (yellow-green), occurring in plants in a ratio about 3:1. Chloro-phyll b differs from chlorophyll a in that the methyl group on C3 is replaced withan aldehyde.

This is a stable ring-shaped molecule around which electrons are free to migrate.Because the electrons move freely, the ring has the potential to gain or lose electronseasily, and thus to provide energized electrons to other molecules. This is thefundamental process by which chlorophyll captures the energy of sunlight. In plantphotosynthesis, incoming light is absorbed by chlorophyll and other accessory pig-ments in the antenna complexes of photosystems I and II. The antenna pigments arepredominantly chlorophyll a, chlorophyll b, and carotenoids. Each antenna complex

FORMULA 9.12

O

OCCH2CH2

H

1 2

3

4

6 5

8

7N

N

Mg

N

N

R

CH2CH3

CH3 CH CH2

O

H

C O

Chlorophyll a R= —CH3Chlorophyll b R= —CHO

CH3OCH3

H3C


Food Colorants 257

has between 250 and 400 pigment molecules, and the energy they absorb is shuttledby resonance energy transfer to specialized chlorophyll a at the reaction center ofeach photosystem. When either of the two chlorophyll a molecules at the reactioncenter absorb energy, an electron is excited and transferred to an electron-acceptormolecule, leaving an electron hole in the donor chlorophyll. The resulting chemicalenergy is then captured in the form of ATP (adenosine triphosphate) and reducednicotinamide adenine diphosphate (NADPH) and is ultimately used to convert CO2

to carbohydrates. Chlorophyll a is common to all eucariotic photosynthetic organ-isms, and due to its role in the reaction center, it is essential for photosynthesis. Theaccessory pigments, such as chlorophyll b and carotenoids, are not essential.

Typical leaf material contains about 2.5 mg/g total chlorophylls, 0.3 mg/g xan-thophylls, and 0.15 mg/g carotenes (Humphrey, 1980). In many fruits, chlorophyllis present in the unripe state, and gradually disappears during ripening as the yellowand red carotenoids take over.

Fruit and vegetable quality and freshness are strongly associated with color.Chlorophyll pigments are unstable and may be used as an indicator of health andripeness of different plant material as well as of processing condition. Chlorophylldegradation may occur within a few or over several weeks. The degradation processis strongly dependent on the pH and temperature. The most important transforma-tions of chlorophylls are:

• Easy loss of Mg in dilute acids or replacement of Mg by other divalentmetals

• Hydrolysis of the phytyl ester in dilute alkalis or transesterification bylower alcohols

• Hydrolysis of the methyl ester and cleavage of the isocyclic ring instronger alkalis

Removal of Mg gives olive-brown pheophytin a and b. Replacing Mg by Fe orSn ions yields grayish-brown compounds, while Cu or Zn ions retain the green color.Upon removal of the phytyl group by hydrolysis in dilute alkali, or by the action ofchlorophyllase, green chlorophyllin are formed. Removal of Mg and the phytyl groupresults in olive-brown pheophorbide formation (Figure 9.1).

Chlorophylls and pheophytins are lipophilic due to the presence of the phytolgroup, while chlorophyllins and pheophorbids without phytol are hydrophilic. Thecopper complexes of both pheophytin and pheophorbid have the metal firmly bound;it is not liberated even by the action of concentrated hydrochloric acid, and notremoved to any appreciable extent, on metabolism; thus it is acceptable for thecoloration of foodstuffs.

The chlorophyll preparations for the food colorant market are mainly obtainedfrom alfalfa (Medicago sativa) and nettles (Urtica dioica). Brown seaweeds, whichare the commercial source of alginates, are also an interesting source of chlorophyllbecause as in single-cell phytoplankton, they contain chlorophyll c, which is morestable than chlorophyll a and chlorophyll b (Delgado-Vargas and Paredez-López,2003). Acetone, methanol, ethanol, and chlorinated solvents are used as extracting



vehicles. The yield of extraction is around 20% in which chlorophylls, pheophytins,and other degradation products are included.

Chlorophyll and its metabolites—pheophytin, pyropheophytin, pheophorbide,as well as chlorophyllin—have demonstrated in vitro antimutagenic and anticarci-nogenic effects against some substances known or suspected to cause cancer, suchas polycyclic aromatic hydrocarbons found in smoke, heterocyclic amines foundin cooked meats, as well as aflatoxin B1. The mechanism of these activities ofchlorophyll and chlorophyllin are unknown, but it is speculated that one possiblemechanism is the formation of complexes between the mutagen or carcinogen andthe chlorophyll or chlorophyllin through strong interaction between their planarunsaturated cyclic rings. Such complexes would effectively inactivate the mutagenor carcinogen.

Chlorophylls are approved in Codex legislation as food additives for differentapplications. However, the Na and K salts of Cu-chlorophylls are approved by theU.S. Food and Drug Administration (FDA) only in preparation of dentifrices anddrugs, but not as food additives (Delgado-Vargas and Paredez-López, 2003). Theoil-soluble chlorophylls, as well as the water-soluble chlorophyllins, a semisyntheticmixture of Na and Cu salts derived from chlorophyll, have good stability in lightand heat, and moderate stability with respect to both acid and alkalis. Their appli-cation as food colors is in canned products, confectionery, soups, and dairy products.

FIGURE 9.1 Transformation of chlorophyll pigments.

Mg2+

phytolchlorophilase

PHEOPHYTIN(olive-green)

acid

Mg2+phytol

alkali

methanol

CHLOROPHYLL(green)

phytol

acid

Mg2+

acid

phytol

alkali

CHLOROPHYLLIN(green)

CHLOROPHYLID (green)

PHEOPHORBIDE(olive-brown)


Food Colorants 259

9.4 HEME PIGMENTS

Heme is a metal-containing cofactor that consists of an Fe atom contained in thecenter of a large heterocyclic ring called porphyrin. There are three biologicallyimportant kinds of heme called a, b, c. The proteins that contain heme a or c arecytochromes, and the most abundant heme b is present in both hemoglobin andmyoglobin. The main function of heme is the retention of O2 and delivery of it forenzymatic reaction. Heme is made up of four heterocyclic pyrrole rings connectedat their corners by methylene bridges. The four nitrogen atoms bind the Fe(II) orFe(III). However, only Fe(II) can bind O2. Each myoglobin molecule contains oneheme prosthetic group inserted into a hydrophobic pocket. Myoglobin is a mono-meric heme protein found mainly in muscle tissue, where it serves as an intracellularstorage site for oxygen. During periods of oxygen deprivation oxymyoglobin releasesits bound oxygen, which is then used for metabolic purposes.

Hemoglobin consists of four protein chains, each about the size of a myoglobinmolecule, which fold to give a structure that looks very similar to myoglobin. Thushemoglobin has four separate heme groups that can bind a molecule of O2 each. Thecooperative interaction between binding sites makes hemoglobin an unusually effi-cient oxygen-transport protein. It binds oxygen in the pulmonary vasculature andreleases it in tissues, where the situations are reversed.

Myoglobin is the main pigment of meat. In fresh meat, in the presence of oxygen,the reversible reaction of myoglobin (Mb) with oxygen occurs and bright red-coloredoxymyoglobin (MbO) is formed. Due to oxidation of Fe(II) the brownish metmyo-globin (MMb) is formed.

Oxymyoglobin and myoglobin exist in a state of equilibrium with oxygen. Theratio of these pigments depends on oxygen pressure. In meat there is a slow oxidationof heme pigments, mainly myoglobin to metmyoglobin, which cannot bind oxygen.

Heating of meat results in the denaturation of the globin linked to iron, as wellas in the oxidation of iron to the ferric state, and formation of different brown hemepigments named hemichrome.

In the curing of meats, the heme pigments react with the nitrite of the curingmixture and the red colored nitrite–heme complex nitrosomyoglobin is formed, butit is not particulary stable. More stable is the pigment with a denatured globin portion,called nitrosylhemochrome, found in meat heated at a temperature of more than65°C. The reactions of heme pigments in meat and meat products are summarizedin Figure 9.2.

In the presence of thiol compounds as a reducing agent, in the reversible reactionmyoglobin may form a green sulfmyoglobin. Other reducing agents, such as ascor-bate, lead to the formation of cholemyoglobin. This reaction is irreversible.

REACTION 9.2 Basic transformations of myoglobin.

MbO2Mb MMb

red purplish red brownish



A potential source of red and brown heme pigments is animal blood and itsdehydrated protein extracts, which mainly consist of hemoglobin; these may be usedas red and brown colorings in meat products. However, in most countries, their useas food coloring agents is not permitted.

9.5 ANTHOCYANINS

9.5.1 OCCURRENCE AND STRUCTURE

Anthocyanins are among the most important groups of plant pigments. They arepresent in almost all higher plants and are the dominant pigments in many fruits andflowers, giving them red, violet, or blue color. They play a definite role in attractinganimals in pollination and seed dispersal. They may also have a role in the mechanismof plant resistance to insect attack.

Anthocyanins are part of the very large and widespread group of plant constit-uents known as flavonoids, which possess the same C6–C3–C6 basic skeleton. Theyare glycosides of polyhydroxy and polymetoxy derivatives of 2-phenylbenzopyry-lium or flavylium cation (Formula 9.13). Differences between individual anthocya-nins include (1) the number of hydroxyl groups in the molecule; (2) the degree ofmethylation of these hydroxyl groups; (3) the nature, number, and the position of

FIGURE 9.2 Transformation of myoglobin in meat. Mb = myoglobin, MMb = metmyoglo-bin, MbO2 = oxymioglobin, MbNO = nitroxymioglobin, dMMb = denatured metmyoblobin.

FORMULA 9.13

dMMb MMb MMbNO brownish brownish bright red

heating NO

heating oxidation reduction reduction

MbO2 Mb MbNO dMbNO red purplish red bright red bright red

heatingNO-O2

+O2

O

OHOH

HO

OH

R3'

R5'

35

7

3'

5'

+2

46

8

4'

6'

2'


Food Colorants 261

glycosylation; (4) and the nature and number of aromatic or aliphatic acids attachedto the glucosyl residue. From about 20 known naturally occurring anhocyanidins,only six occur most frequently in plants (Table 9.4). Substitution of the hydroxyland methoxyl groups affects the color of the anthocyanins. An increase in the numberof hydroxyl groups tends to deepen the color to a more bluish shade. An increasein the number of metoxyl groups increases redness.

Because of the possibility of various ose and acid substitutions at differentpositions, the number of anthocyanins is 15 to 20 times greater than the numberof anthocyanidins (Mazza and Miniati, 1993) The ose molecules most commonlybonded to anthocyanidins are glucose, galactose, rhamnose, arabinose, and di- andtrisaccharides, formed by combinations of these four monosaccharides. The mostcommon classes of anthocyanins are 3-monosides, 3-biosides, 3,5-diglycosides,3,7-diglycosides; however, glycosylation of the 3′-, 4′-, and 5′-hydroxyl group isalso possible. The composition and content of anthocyanins in fruits are verydiversified (Table 9.5).

TABLE 9.4Naturally Occurring Anthocyanidins

Antocyanidin R3’ R5’ λmax[nm] Color

Pelargonidin (Pg) H H 520 orangeCyanidin (Cy) OH H 535 orange-redPeonidin (Pn) OCH3 H 532 orange-redDelphinidin (Dp) OH OH 546 bluish-redPetunidin (Pt) OCH3 OH 543 bluish-redMalvidin (Mv) OCH3 OCH3 542 bluish-red

TABLE 9.5Anthocyanins in Some Fruits—Composition and Content

Fruits Main anthocyaninsTotal anthocyanins

(mg/100 g)

Blackberry Cy 3-Glu, Cy 3 Rut 83–326Bilberry Dp 3-Gal and Arab, Mv 3-Gal and Arab, Pt 3-Gal and Arab 250–490Black current Cy 3-Rut and Glu, Dp 3-Rut and Glu 250Chokeberry Cy 3-Gal, Cy 3-Arab, Cy 3-Glu, Cy 3-Xyl 520–800Cranberry Cy 3-Gal and Arab, Pn 3-Gal and Arab 78Strawberry Pg 3-Glu and Cy 3-Glu 7–30Red grapes Mv, Dp, Pt, Pn 3-Glucosides, 3-acetylglucosides, and

3 p-coumarylglucosides30–750

Source: Adapted from Macheix, J.J., Fleuriet A., and Billot J., Fruit Phenolics, CRC Press Inc., BocaRaton, FL, 1990; and Wang H., Coa G., and Prior R.L., J. Agric. Food Chem. 45, 304, 1997.



9.5.2 CHEMICAL PROPERTIES

In aqueous media, most of the natural anthocyanins behave like pH indicators: redat low pH, bluish at intermediate pH, and colorless at high pH. According toBrouillard (1982), in acidic and neutral media, four anthocyanin structures exist inequilibrium: the red flavylium cation (AH+), blue or red quinonoidal base (A),colorless carbinol pseudobase (B), and colorless chalcone (C) (Reaction 9.3).

The pH of the medium plays a particularly important role in the equilibriumbetween these different anthocyanin forms, and consequently in color modification.In a strongly acid solution, at a pH below 2, the red cation AH+ is the dominantform. As the pH is increased, a rapid proton loss occurs to yield the red or bluequinoidal base A, usually existing in two forms (Figure 9.3). On standing, a furtherreaction occurs, that is, hydration of flavylium cation AH+ to yield a colorless carbinolpseudobase B. Relative amounts of form AH+, A, B, and C at equilibrium vary withboth pH and the structure of anthocyanins. For the common anthocyanin 3-glycosidesor 3,5-diglycosides, the principal product formed on raising the pH above 3 is thecolorless carbinol pseudobase B (Figure 9.3). At this pH, however, small amountsof the blue quinoidal base and the colorless chalcones are also present and increasewith increasing pH. Between pH 4 and 6, very little color remains because theamounts of the colored forms AH+ and A are very small (Reaction 9.3). By varyingthe substitution pattern of the flavylium ring, anthocyanidin, which exists primarilyin the colored form, can be prepared (Jacobucci and Sweeny, 1983), but suchanthocyanin compounds are not found in plants.

The color of anthocyanin-containing media depends on different factors. Themost important are structure and concentration of anthocyanin pigments; pH; pres-ence of copigments and metallic ions, which influence the color shade; as well astemperature, presence of oxygen, phenoloxidase, ascorbic acid, and sulfur dioxide,which influence the anthocyanins’ degradation rate and color stability.

REACTION 9.3 Structural anthocyanin transformations in aqueous media.

OO

OH

O Gl

OH

O

OH

OH

HO

GlO

OHO

OH

OH

HO

GlO

OH

O

OH

O

HO

GlO OH

HOO

OHGl

O

OH

O+

OH

OH

HO

GlO

A

B

C

-H++H+ +H+

-H+

AH+: Flavylium cation

A: Quinonoidal bases

- H+/H2O

B: Carbinol pseudobase CE: E - Chalcone

CZ: Z-Chalcone


Food Colorants 263

The structure of the anthocyanin molecule has a marked effect on color intensityand stability. The increase of the number of hydroxyl groups in the B-ring shifts theabsorption maximum to longer wavelengths, and the color changes from orange tobluish-red. Methoxyl groups replacing hydroxyl groups reverse this trend. Thehydroxyl group at C-3 is particularly significant because it shifts the color fromyellow-orange to red. The same hydroxyl, however, destabilizes the molecule; the3-deoxyanthocyanidins are much more stable than the other anthocyanidins. Simi-larly, the presence of a hydroxyl group at C-5 and substitution at C-4, both stabilizethe colored forms. Glycosylation also affects the stability of these pigments; thehalf-life of anthocyanidins is significantly lower than their corresponding 3-gluco-sides. Anthocyanins containing two or more aromatic acyl groups, such as cinerarinor zebrinin, are stable in neutral or weakly acidic media. This is possibly a result ofhydrogen bonding between phenolic hydroxyl groups in anthocyanidins and aromaticacids. Brouillard (1981) observed that diacylated anthocyanins are stabilized bysandwich-type stacking caused by hydrophobic interaction between the anthocyani-din ring and the two aromatic acyl groups.

The increase in anthocyanin concentration results in an increase in absorbanceat λmax, which is greater than expected according to the Beer-Lambert law. It isprobably connected with anthocyanins’ self-association.

Intermolecular copigmentation of anthocyanins with other flavonoids, somephenolic acids, alkaloids, and other compounds, including anthocyanins them-selves, increases the color intensity (hyperchromic effect) and causes a shift in thewavelength (batochromic shifts), giving purple to blue colors. The intensity of thecopigmentation effect depends upon several factors, including type and concentra-tion of anthocyanins and copigments, pH, and temperature of the solvent (Brouil-lard et al., 1991). The pH value for maximum copigmentation effect is about 3.5,and may vary slightly depending on the pigment–copigment system. Color inten-sification by copigmentation increases with an increasing ratio of copigment toanthocyanins. Increasing temperature strongly reduces the color-intensifying effect.

FIGURE 9.3 Distribution of anthocyanin structures as a function of pH.

0

50

100

1 2 3 4 5 6

AH+

B

CA%

of t

otal

pH



The copigmentation phenomenon is widespread in nature, and also occurs in fruitand vegetable products, such as juices and wines.

In the presence of metals, anthocyanins can form purplish-blue or slate-graypigments called lakes. This reaction may induce color changes if fruit products,during processing or storage, are in contact with metals such as Sn, Al, or Fe.

9.5.3 BIOLOGICAL ACTIVITY

Anthocyanins are natural colorants that belong to the large family of phenoliccompounds—flavonoids. They are known to display different pharmacological andbiological effects, such as those that are vasoprotective, antiinflammatory, and radio-protective, as well as prevention of cholesterol-induced arteriosclerosis and heartdisease. However, the most important properties of anthocyanins seem to be theiractivity as potent free-radical scavengers and powerful chain-breaking antioxidants.Similar to other flavonoids, they can react with ROS and lipid peroxyl radicals, andinhibit lipid peroxidation at the early stage. It is very important because in vivo lipidperoxidation has been implicated as the primary cause of coronary heart disease,arteriosclerosis, cancer, and aging. The antioxidant effectiveness of anthocyaninpigments is structure dependent, but for the main aglicons, it is about two timeshigher than that of vitamins C and E.

Recently increasing attention has been focused on anthocyanins as naturalantioxidants because of their ubiquitous presence in plant products, particularly infruits, juices, and red wine. The daily intake of anthocyanins in humans is as highas 200 mg/day. Despite a relatively high potential intake in humans, the physiolog-ical impact of the anthocyanins is not well recognized because of the lack of purecompounds and their low stability. Anthocyanin-enriched materials, of differentpurity, from the skin of grapes and other by-products of wine and juice manufac-turing are produced in different countries. One of the better known, commerciallyavailable anthocyanin concentrates is extract of Vacciniun myrtillus—bilberry(VMA), which contains largely glycosides of delphinidin and cyanidin, and is usedto treat various microcirculation diseases resulting from capillary fragility (Wanget al., 1997). Red wine, much more than white wine and grape juice, is alsorecognized as a rich source of natural antioxidants. It has been demonstrated thatred wine has superoxide radical-scavenging potential, and is able to effectivelyinhibit oxidation of low-density lipoprotein in vitro and in vivo. However, in allthese products, not only anthocyanins but also other phenolic antioxidants, such asflavonols, and flavanols are present and may influence the biological and antioxidantactivity. Therefore the consumption of fruits rich in anthocyanins and red wineappears to be a good means of impeding the lipid oxidation process responsiblefor different diseases and aging.

9.5.4 STABILITY OF ANTHOCYANINS IN FOOD

Anthocyanins appear to have low stability in all products manufactured from fruits.This limits the use of these pigments as food colorants. Two main groups of factorsare responsible for the stability of anthocyanin color in fruits during processing:


Food Colorants 265

• Initial composition of the fruit, with regard to anthocyanins and otherconstituents including enzymatic systems

• Processing factors such as temperature, light, and the presence of oxygen

Several enzymes and, in particular, phenolases, peroxidases, and β-glucosi-dases, can decrease the quality of the initial product during the extraction of fruitjuices or the preparation of processed products, leading to browning and to loss ofcolor by enzymatic degradation of anthocyanins. Anthocyanins themselves are notgood substrates for o-diphenol oxidase, but they are instead oxidized by the chlo-rogenic acid ↔ chlorogenoquinone redox shuttle phenomena. Thus enzymatic oxi-dation of chlorogenic acid may by combined with nonenzymatic oxidation andpolymerization of anthocyanins. The same phenomenon has been observed in thepresence of catechins.

Ascorbic acid may have a protective effect with regard to anthocyanins becauseit reduces the o-quinones formed before their polymerization. However, ascorbicacid as well as products of its degradation increase the anthocyanin degradation rate.

Sulfur dioxide, widely used in fruit processing at concentrations as low as 30mg/kg, inhibits the enzymatic degradation of anthocyanins. At moderate concentra-tions on the order of 500 to 2000 mg/kg, it forms a colorless SO2–anthocyanincomplex. This is a reversible reaction: after removal of SO2 the color turns red again.

Regardless of the favorable action of high temperature on the blockage ofenzymatic activities, anthocyanins are readily destroyed by heat during processingand storage. A short-time, high-temperature process was recommended for bestpigment retention. For instance, in red fruit juices heated 12 min at 100°C, antho-cyanin losses appear to be negligible.

The reactions described above can proceed at different rates and can bring aboutdifferent changes in color depending on the composition of food products. Mostfrequently the red color slowly turns brown. However, in correctly processed andstored fruits, the color changes are so slow that they only slightly affect consumerappreciation of fruit products.

Anthocyanin concentrate may be used as a food colorant at a pH less than 4.

9.6 BETALAINS

Betalains, a class of water-soluble red-violet or yellow pigments, occur only in 10families of the order Caryophyllales (old name for Centrospermae) and some fungispecies, such as Amanita muscaria. The presence of betalain in plants is mutuallyexclusive of occurrence of anthocyanins, which are more widely distributed in theplant kingdom.

About 50 betalains have been identified, which are found in different plant organsand are accumulated in cell vacuoles, mainly in epidermal and subepidermal tissues.However, only red beetroot (Beta vulgaris L. Chenopodiaceae), cactus fruits (generaOpuntia and Hylocereus) are the only edible source containing these pigments.

Betalains consist of red-violet betacyanins (λmax ~ 540 nm) and yellow betaxanthin(λmax ~ 480 nm). The betanin (Formula 9.14), glucoside of betanidin, is the mainbetalain pigment in red beetroot and accounts for 75 to 95% of total pigments in red



beets. The other red pigments are isobetanin (C-15 epimer of betanin), prebetanin,and isoprebetanin. The latter two are sulfate monoesters of betanin and isobetanin,respectively. Unlike anthocyanins, betanins cannot be hydrolyzed to aglycone byacid hydrolysis without degradation. The major yellow pigments are vulgaxanthin Iand II (Formula 9.15). High betalain content in beetroot, on average 1% of the totalsolids, makes these vegetables a valuable source of food colorants.

Cactus fruits from the genera Opuntia and Hylocereus are the other edible sourcesof betalain pigments. There are important differences in the chemical composition andvisual appearance of these fruits. The color shade of the red juice of Opuntia ficus-indicacv. Rossa is similar to that of beet preparations, the juice from Opuntia ficus-indica cv.Gialla displays a yellow tonality, whereas the juice from Hylocereus polyrhizus is

FORMULA 9.14

FORMULA 9.15

N COOH

H

HOOC

H

+

HON COO-

HGlO

N COOH

H

+

HO N COO-

HGlO

H

HOOC

Betanin Isobetanin

+

NH

HOOC

H

COOH

NH

RO

COO-

R = —NH2 vulgaxanthin IR = —OH vulgaxanthin II


Food Colorants 267

characterized by purplish hues. Betacyanin contents in these juices are 74, 1.3, and525 mg/dm3, respectively, whereas betaxantins amounted to 36, 48, and 5.3 mg/dm3

(Stintzing, 2003).The other source of betalains is the family Amarantahaceae recognized as a rich

source of diverse and unique betacyanins. They consist of six simple (nonacetylated)and ten acetylated betacyanins, including eight amaranthine-type pigments, six gom-phrenin-type pigments, and only two betanin-type pigments. Total betacyanin pig-ment content ranges from 0.08 to 1.36 mg/g of fresh weight. The main pigment inthis family is amaranthine (betanidin 5-O-β-glucuronosylglucosides), whose contentaverages about 91% of total betacyanins. The effect of acyl groups on the colorintensity and stability of betacyanin pigments is not clear (Cai et al., 2001).

The color stability of betanin solution is strongly influenced by pH and heating.Betanin is stable at pH 4 to 6, but thermostability is greatest between pH 4 and5. As a result of betanin degradation, cyclo-DOPA and betalamic acid are formed(Reaction 9.4). This reaction is reversible (Czapski, 1985). Light and air have adegrading effect on betanin. These effects are cumulative, but some protectionmay be offered by antioxidants such as ascorbic acid. Small amounts of metalions increase the rate of betanin degradation. Therefore, a chelating agent canstabilize the color. Many protein systems present in food products also have someprotective effect.

In the last 10 years, several studies have presented data showing that red beetis a good source of antioxidants. In a test of linoleate peroxidation by cytochromec, betanin exhibits ten times higher antioxidant activity than tocopherol, and threetimes higher than catechin. These compounds are cationized, which increases theiraffinity to membranes, a great beneficial attribute for antioxidants (Kanner, 2001).

Beetroot red (E162), available as liquid beetroot concentrate and as beetrootconcentrate powders, is suitable for products of relatively short shelf life, whichdo not undergo severe heat treatment, for example, meat and soybean proteinproducts, ice cream, and gelatin desserts. Betalains possess high molar extinctioncoefficients and their coloring power is comparable to that of synthetic colorants.Compared with the latter, betalains are not toxic, nor do they cause allergicreactions.

REACTION 9.4 Degradation of betanin.

+

N COOH

H

HOOC

H

GlO

HO N

H

COO-

GlO

HON

H

COOH

H

N COOH

H

HOOC

H

OH

H2O

H2O

+

-

+

Betanin Cyclodopa glucoside Betalamic acid



9.7 QUINONE PIGMENTS

These pigments are widely distributed. They are the major yellow, red, and browncoloring materials of roots, wood, and bark. They also occur at high levels in certaininsects. The largest group is that of anthraquinone pigments. The most importantqininoid pigments commercially available for use in foodstuffs are cochineal andcochineal carmine.

Cochineal (E 120) is the red coloring matter extracted from the dried bodies offemale insects of the species Dactylopius coccus Costa or Coccus cacti L. Theseinsects are cultivated on cactus plants in Peru, Equador, Guatemala, and Mexico.

The major pigment of cochineal is polyhydroxyanthraquinone C-glycoside, car-minic acid (Formula 9.16), which may be present at up to 20% of the dry weightof the mature insects. Cochineal extract or carminic acid is rarely used as a coloringmaterial for food, but are usually offered in the form of their lakes. Aluminumcomplexes (lakes) can be prepared with various ratios of cochineal and Al varyingfrom 8:1 to 2:1, having corresponding shades from pale yellow to violet.

Cochineal carmine is insoluble in cold water, dilute acids, and alcohol, andslightly soluble in alkali giving a purplish-red solution. The shade becomes bluer athigher pH. Cochineal carmine is stable in light and heat, but the stability in thepresence of SO2 is poor. In powdered form this pigment can be used for coloringvarious instant foodstuffs in alkaline solutions, and in ammonia for coloring differentfoods, including baked products, yogurts, soups, desserts, confectionery, and syrups.It is currently the most robust red colorant among the authorized natural colorants,and its affinity for proteins means that it is an excellent colorant for dairy products,though it has the disadvantage of high production costs.

9.8 TURMERIC AND CURCUMIN (E 100)

Turmeric is native to South and Southeast Asia. It is cultivated in China, India, SouthAmerica, and the East Indies. The annual production is more than 240,000 tons,about 90% of which is produced and consumed in India. Turmeric belongs to theCurcuma genus of the Zingiberaceae family. Several species of Curcuma are known,but only the Curcuma longa L. represents the turmeric of commercial importance(Delgado-Vargas, 2003).

Turmeric is important as a spice and coloring agent. References to it can befound in ancient Indian Vedic texts. The yellow color is the principal functional

FORMULA 9.16

HOOC

HO

CH3 O

O

OH

Gl

OH

OH


Food Colorants 269

property of turmeric. The main compounds involved in color are curcumin (Formula9.17), demethoxycurcumin, and bisdemethoxycurcumin. The commercial forms areturmeric powder and oleoresin, as well as purified curcumin—a yellow, crystalline,odorless powder. Turmeric and curcumin are insoluble in water but soluble in alkalis,alcohols, and glacial acetic acid, and are used mainly as food color and secondarilyas a spice.

The use of turmeric depends on the food item and the part of world. Generallyturmeric powder is used in mustard paste and curry powder as a colorant and foraroma. Oleoresin is added to mayonnaise, to breading of fish and potato croquettes,and to nonalcoholic beverages. However, curcumin is added to products whereturmeric is incompatible, such as cheese, butter, ice cream, and some beverages. Allturmeric pigments have good heat stability, but they are light sensitive.

Curcumin is used not only as a food colorant and spice, but also as a health-promoting food supplement. It has been shown that curcumin has a wide range oftherapeutic actions: it protects against free radical damage because it is a veryeffective antioxidant; it reduces inflammation by lowering histamine levels; it pro-tects the liver from different toxic compounds; and it keeps platelets from clumpingtogether, which may protect against atherosclerosis.

9.9 RIBOFLAVIN (E 101)

Riboflavin (vitamin B2, Formula 9.18) is a yellow pigment present in many productsof plant and animal origin. Milk and yeast are the best sources of riboflavin.

It is an orange-yellow crystalline powder, intensely bitter tasting, which is veryslightly soluble in water and ethanol, affording a bright green-yellow fluorescent

FORMULA 9.17

FORMULA 9.18

HO

O

OHCurcumin

O

H3CO OCH3

N

N N

Riboflavin

O

NH

O

(HCOH)3

CH2OH

CH2

H3C

H3C



solution. Riboflavin is stable under acid conditions, but unstable in alkaline solutionand when exposed to light. Reduction produces a colorless leuco form, but color isregenerated again in contact with air.

Riboflavin-5′-phosphate sodium salt is much more soluble in water than unes-terified riboflavin, and is not so intensely bitter. It is one of the physiologicallyactive forms of vitamin B2. It is rather more unstable to light than riboflavin. Bothforms can be used as coloring and as an enriching food additive to cereal, dressing,and cheese.

9.10 CARAMEL (E 150)

Caramels are the brown to brown-black viscous liquids or hygroscopic powders resultingfrom carefully controlled heat treatment of different carbohydrate sources, such as dex-trose, invert sugar, malt syrup, or lactose in the presence of a selected accelerator. A goodsource must have high levels of glucose because caramelization occurs only via themonosaccharide. Heating induces several chemical reactions resulting in the generationof a complex mixture of polymeric substances (>3000 Da) and low-molecular-weightcompounds (<1000 Da) (Delgado-Vargas and Paredez-López, 2003). The compositionand coloring power of caramel depend on the type of raw material and processingconditions. Both Maillard and caramelization reactions are involved, and the commer-cial products are extremely complex in composition.

Four classes of caramels are recognized depending on the accelerator used: burntsugar, caustic, ammonia, and ammonium sulfite. The first is used mainly as a flavoringadditive, and the other as food colorants. Caramel is the colorant soluble in waterand insoluble in organic solvents. It not only imparts color but also has importantfunctional properties: it stabilizes colloidal systems and prevents the haze formationin beers, facilitating the dispersion of water-insoluble components; inhibits enzymaticbrowning; and retards the flavor changes and preserves the color of beverages exposedto light. The U.S. Food and Drug Administration (FDA) permits the use of caramelcolor in food in general. Over 80% of the caramel produced in the United States isused to color soft drinks, particularly colas and root beers.

9.11 MELANOIDINS

Melanoidins represent an important group of brown pigments formed as a consequenceof food processing. Formation of this pigment is favored by heat treatment and includesa number of complex reactions, mainly Maillard reactions. It is a type of nonenzymaticbrowning that involves the reaction of simple sugars and amino acids or proteins. Theybegin to occur at lower temperatures and at higher dilutions than caramelization, andoccur in most food upon heating and also take place in the human body.

The Maillard reaction has three basic phases:

1. The initial reaction is the condensation of the carbonyl group of reducingsugars with a free amino acid group, which loses a molecule of water, toform N-substituted glycosylamine.


Food Colorants 271

2. The unstable glycosylamine undergoes the Amadori rearrangement toform ketosamines.

3. The ketosamine can then react in different ways: One is simply dehydra-tion (loss of 2 H2O molecules) into reductones and dehydroreductones,which are powerful antioxidants. A second is the production of short-chain hydrolytic fission products, such as diacetyl, acetol, and pyruval-dehyde. A third path is the Schiff’s base or furfural path. This involvesthe loss of 3 H2O molecules and then a reaction with amino acids andwater. All these products react further with amino acids to form the brownnitrogenous polymers and copolymers called melanoidins (Nicoli andManzocco, 2002).

Because the Maillard reaction produces water, a high water activity environmentinhibits the reaction. In practice, this reaction occurs most rapidly at intermediateaw (0.5 to 0.8). The temperature increase or time of heating results in increases incolor development. Pentoses react more readily than hexoses, which in turn are morereactive than disaccharides (e.g., lactose). Different amino acids produce differentamounts of browning. Of all amino acids, lysine results in the most color in theMaillard reaction due to its free ε-amino group. Therefore, foods containing proteinrich in lysine, such as milk, are likely to brown readily.

Much of the color that develops when foods are heated is due to the occurrenceof melanoidins which, depending on their molecular weight, may be divided intotwo classes: low-molecular weight below 1000 Da, consisting of up to four linkedrings, and high-molecular weight up to 150,000 Da (Nicoli and Manzocco, 2002).Melanoidins formed in the advanced steps of the Maillard reaction are responsiblefor a great variety of changes in food quality attributes and stability. The mostimportant are browning, typical flavor formation, viscosity and water activityincrease, enzyme activity inhibition, antimicrobial activity, and lipid and pigmentoxidation slowdown. Typical effects of Maillard reactions may be observed aschanges in the color of dried and condensed milk, browning of bread into toast, aswell as the color of beer, chocolate, or maple syrup.

Most of the functional and technological properties attributed to melanoidinstrace their origin to the ability of these compounds to act as antioxidants able to actas chain breakers, reducing compounds, and metal chelators. However, food sub-jected to severe heat treatment may generate, via Maillard reaction, low but effectivelevels of mutagenic and carcinogenic amines.

9.12 MELANINS

Melanins are water-insoluble polymers of various types of phenolic and indoliccompounds, mainly derived from the tyrosine enzymatic oxidation. In general theyare not homopolymers, and form large and complex macromolecules. Melanins areresponsible for the black, gray, and brown colors of animals, plants, and microorgan-isms, where they are usually complexed with protein and often with carbohydrates.

Because melanins are the aggregates of smaller component molecules, thereare a number of differing types of melanins with differing proportions and bonding



patterns of these component molecules. Eumelanin is the most abundant melaninin animals and humans, and is responsible for the black and brown colors of skinand hair. This is the melanin form most likely deficient in albinism. In mammals,eumelanins are accumulated with proteins and metals, such as Fe, Cu, or Zn. Inseeds, spores, and fungi, another group of melanins—allomelanins (quinons)—ispresent.

Melanins form but also scavenge free radicals; they dissipate energy, and thusprovide protection from the damaging effects of radiation, electronic energy, andsunlight. They are able to bind metals and consequently, if any metal is toxic to theorganism, melanins can prevent its entry into cells. In conclusion, melanins are notimportant for normal growth and development, but very important for the survivalof many organisms.

9.13 SYNTHETIC ORGANIC COLORS

Until the mid-19th century all dyes were obtained from plant or animal extracts.The textile industry used natural pigments, such as cochineal, turmeric, wood mad-der, or henna. In 1856, H. Perkin established the first factory of organic syntheticdyes to produce mauve. A few years later the discovery of diazotization and acoupling reaction by Peter Griess was the next major breakthrough for developmentof the color industry. In the 19th century, synthetic organic dyes were developed,creating a more economical and wider range of colorants. Since then their qualityhas been improved due to extensive research and development. The economic impor-tance of the color industry is clearly reflected in the large number of synthesizedcompounds; as many as 700 colorants are currently available.

Toward the end of the 19th century, when synthetic colors were first adopted foruse on a large scale, they were hailed as a significant technological breakthrough. Theterm synthetic was associated with the idea of progress and synthetic colorants wereactually considered safer in food than the naturals, as they were tinctorially muchstronger and hence a smaller quantity was needed to achieve a specific colored effect.

Synthetic colorants were used in foods, medicines, and cosmetics, but throughthe years their importance diminished. This retrenchment of synthetic colorantsstarted about five decades ago. All synthetic food components suffered severe crit-icism, including synthetic additives and particularly food pigments. Color additiveswere one of the first man-made products regulated by law. Today, all food coloradditives are carefully regulated by federal authorities to ensure that foods are safeto eat and accurately labeled.

Government regulation of food colorants is complex, and consequently legis-lation differs around the world. As an example, some colorants are permitted in theUnited States and by the World Health Organization (WHO): alura red, brilliantblue, fast green, but not by the European Union (EU); others are permitted by theEU and WHO: carmoisine, ponceau 4R, patent blue V, but not in the United States;still other pigments have wide acceptability around the world, such as sunset yellow,tartrazine, indigotine and erythrosin (Delgado-Vargas and Paredez-López, 2003).

The main certifiable synthetic colorants, permitted for use in foods, drugs, andcosmetics (FD&C), according to their chemical structure, are:


Food Colorants 273

1. Azo dyes: a. Tartrazine (E 102); C.I. food yellow 4; bright orange-yellow powder;

freely soluble in waterb. Sunset yellow (E 110); C.I. food yellow 3; FD&C yellow No 6; orange-

red crystals soluble in water, slightly soluble in ethanol c. Alura red (E 129); C.I. food red 17; FD&C red No. 40; monoazo

compound; soluble in water and in 50% alcohol 2. Triarylmethane dyes:

a. Brilliant blue (E 133); C.I. food blue 2; FD&C blue No. 1; reddish-violetpowder or granules with a metallic luster; soluble in water and ethanol

b. Fast green; C.I. food green 3; FD&C green No. 3; red to brown-violetpowder or crystals; soluble in water, slightly soluble in ethanol

3. Indigoid dyes: a. Indigotin (E 132); C.I. food blue 1; FD&C blue No. 2; dark blue powder

with coppery luster; sensitive to light and oxidizing agents; soluble inwater, slightly soluble in alcohol, insoluble in organic solvent

4. Xanthene dyes:a. Erythrosine (E 127); C.I. food red 14; FD&C red No. 3; brown powder

soluble in water to cherry red solution (Delgado-Vargas and Paredez-López, 2003)

They can also be divided into water-soluble, oil-soluble, insoluble (pigment),and surface-marking colors. Water solubility is conferred on many dyes by intro-ducing to the molecule at least one salt-forming group. The most common is thesulfonic acid group, but carboxylic acid residues can also be used. These dyes areusually isolated as sodium salts. They have colored anions and are known as anionicdyes. The other dyes containing basic groups, such as –NH2, –NH–CH3, or –N(CH3)2,form water-soluble salts with acids. These are the cationic dyes and their coloredion is positively charged. If both acidic and basic groups are present, an internal saltis formed. Oil-soluble or solvent-soluble colorants lack salt-forming groups.

Precipitation of water-soluble colors, with Al, Ca, or Mg salts (generally withAl), forms water-insoluble lakes. Lakes may be prepared from all classes of water-soluble food colors, and they are one of the most important groups of food colorpigments.

The stability of synthetic colors in food processing conditions depends uponproduct composition, temperature, and time of exposure. Generally they are resistantto boiling and baking, but light has a destructive effect on all such colors. Certifiablesynthetic color additives are preferred over natural additives for their coloring ability.They need to be added in smaller quantities, are more stable, provide a wide rangeof hues, and do not impart undesirable flavors to foods. The use of synthetic colorsis the most reliable and economical method of coloring those products that havelittle or no natural color present, such as dessert powders, dairy-based desserts,beverages, dry powder drinks, candy and confectionery products, table jellies, andsugar confectionery.



REFERENCES

Barua, A.B. et al., Vitamin A and Carotenoids, in Modern Chromatographic Analysis ofVitamins, A.P. De Leenheer, W.E. Lambert, and J.F., Van Bocxlaer, Eds., MarcelDekker, Inc., New York, 2000, p. 8.

Berg, H. et al., The potential for improvement of carotenoid levels in food and the likelysystematic effect. J. Sci. Food Agric., 80, 880, 2000.

Brouillard, R., Origin of exceptional color stability of the Zebrina anthocyanin, Phytochem-istry, 20, 143, 1981.

Brouillard, R., Chemical structure of anthocyanins, in Anthocyanins as Food Colours, P.Markakis, Ed., Academic Press, New York, 1982, p. 1.

Brouillard, R. et al., pH and solvent effects on the copigmentation reaction of Malvin bypolyphenols, purine and pyrimidine derivatives, J. Chem. Soc. Perkin Trans. 2, 1235,1991.

Cai, Y. et al., Identification and distribution of simple and acetylated betacyanins in Amaran-thaceae, J. Agric. Food Chem., 49, 1971, 2001.

Combs, G.F., Jr., The Vitamins—Fundamental Aspects in Nutrition and Health, AcademicPress, Inc., San Diego, CA, 1992, 121.

Czapski, J. The effect of heating conditions on losses and regeneration of betacyanins, Z.Lebensm. Unters. Forsch., 180, 21, 1985.

Delgado-Vargas, F. and Paredes-Lopez, O., Natural Colorants for Food and NutraceuticalUses, CRC Press, Boca Raton, FL, 2003.

Humphrey, A.M., Chlorophyll, Food Chem., 5, 57, 1980.Humphrey, A.M., Chlorophyll as a color and functional ingredient, J. Food Sci. 69, 425, 2004.Jacobucci, G.A. and Sweeny J.G., The chemistry of anthocyanins, anthocyanidins and related

flavylium salts, Tetrahedron, 39, 3005, 1983.Kanner, J. et al., Betalains—A new class of dietary cationized antioxidants, J. Agric. Food

Chem., 49, 5178, 2001.Klaui, H. and Bauernfeind, J.C., Carotenoids as food colours, in Carotenoids as Colourants

and Vitamin A Precursors, Academic Press, New York, 1981, p. 47.Macheix, J.J., Fleuriet, A., and Billot, J., Fruits Phenolics, CRC Press, Boca Raton, FL, 1990,

p. 239.Mazza, G. and Miniati, E. Anthocyanins in Fruits Vegetables and Grains, CRC Press, Boca

Raton, FL, 1993, p. 1.Muller, H., Determination of the carotenoid content in selected vegetables and fruits by HPLC

and photodiode array detection, Z. Lebensm. Unters. Forsch. A 204, 88–94, 1997.Nicoli, M. and Manzacco, L., Functional and technological properties of food melanoidins,

International Congress on Pigments in Food, Lisbon 11–14, VI, 2002. Rice-Evans, C. et al., Why do we expect carotenoids to be antioxidants in vivo, Free Rad.

Res., 26, 381, 1997.Stintzing, F.C. et al., Evaluation of color properties and chemical quality parameters of cactus

juices, Eur. Food Res. Technol., 216, 303, 2003.Wang, H. et al., Oxygen radical absorbing capacity of anthocyanins,. J. Agric. Food Chem.,

45, 304, 1997.


275

10 Food Allergens

Barbara Wróblewska

CONTENTS

10.1 Introduction................................................................................................ 27510.2 Causes of Allergies .................................................................................... 276

10.2.1 Atopy ........................................................................................... 27610.2.2 Exposure to Allergens ................................................................. 27610.2.3 Environmental Factors ................................................................ 276

10.3 Mechanisms of Allergic Reactions to Food.............................................. 27710.4 Allergen Terminology................................................................................ 27810.5 Basic Food Allergens................................................................................. 278

10.5.1 Cow’s Milk Allergens ................................................................. 27810.5.2 Egg Allergens .............................................................................. 28010.5.3 Fish Allergens.............................................................................. 28110.5.4 Crustacea Allergens .................................................................... 28110.5.5 Nut Allergens............................................................................... 28210.5.6 Peanut (Arachis hypogaea) Allergens......................................... 28310.5.7 Soy (Glycine max) Allergens ...................................................... 28410.5.8 Wheat Allergens (Triticum aestivum) ......................................... 285

10.6 Other Plant Materials as Sources of Food Allergens................................ 28510.7 Food Additives........................................................................................... 28610.8 Effect of Processing on Allergenicity of Foods........................................ 28710.9 Allergen Cross-Reactions .......................................................................... 28910.10 Antibiotic Contamination in Food ............................................................ 291Glossary of Basic Terms Used in Immunology and Allergology ........................ 291References.............................................................................................................. 292

10.1 INTRODUCTION

Allergy is a very expansive disease. According to data obtained by the EuropeanAcademy of Clinical Allergology and Immunology, 35% of the total populationdisplays symptoms of allergy. A significant increase in allergy incidence concerns:

• Seasonal allergic inflammation of nasal mucosa • Atopic bronchial asthma• Atopic skin inflammation (especially in the female population in the form

of allergy to nickel—over 25% of patients)



A considerable increase in the incidence of allergic diseases has been recentlyattributed to the so-called hygienic hypothesis, which says that a small number ofbacterial infections and exposure to bacterial endotoxins in early childhood are heldresponsible for directing immunological reactions toward allergy phenotypes. Chil-dren from large families with a low standard of living are at low risk for developmentof allergic diseases.

The inhabitants of highly developed and industrialized countries are moreexposed to allergens of dust, mold, and domestic animals because they spend moretime in air-conditioned rooms (Jahnz-Różyk, 2004). The most common allergenicagents are pollens (49%), house dust (19%), chemicals (17%), food (17%), animalhair (16%), drugs (16%), nickel (13%), molds (6%), and latex (2%).

Recently, particular attention has been paid to an increasing occurrence of foodallergies, that is, an abnormal reaction of the organism with immunological back-ground caused by consumption of food(s) or food additives. A number of allergensources have been found making the so-called big eight of food allergens, whichaccount for 90% of all IgE-dependent food allergies, namely, peanuts, nuts, milk,egg, soy, fish, crustaceans, and wheat.

10.2 CAUSES OF ALLERGIES

10.2.1 ATOPY

The mechanisms of atopy, heredity, localization, and sequence of genes responsiblefor allergies have not been fully recognized yet. The probability of inheritingallergies when both parents are allergic is 60 to 70%; when only one parent isallergic, it is 30 to 40%; there is also a 10 to 15% chance that a child of nonallergicparents will be allergic. Genetic factors are estimated to be responsible for approx-imately 50% of all allergy cases. Because no changes were found within the humangenome in the last century, a lot of attention is presently being paid to recognizingenvironmental factors.

10.2.2 EXPOSURE TO ALLERGENS

A necessary condition for hypersensitive reactions is contact with an allergen. Themost common allergens are pollens, molds, house dust, mites and their excreta, animalepidermis, and insect venom. The degree of allergic symptoms depends mainly onindividual sensitivity. Allergic response is caused by proteins and glycoproteins con-tained in pollen particles. Combining allergens with specific antibodies activatesmastocytes which, as a result of lysis, excrete mediators such as histamine, leuco-trienes, and prostaglandins, which are responsible for the clinical picture of disease.

10.2.3 ENVIRONMENTAL FACTORS

An increase in allergy incidence is related to nonspecific factors with adjuvantcharacter. These factors are:


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• Ozone: This has an irritating effect on the respiratory tract and conjuctivalmucosa. An increase in the amount of ozone is caused by exhaust fumes,industrial fumes and, indoors, by copiers and laser printers. Ozone causesso-called oxygen stress, that is, the condition of an elevated concentrationof free radicals, which are responsible for destroying cells and structuressignificant for functioning of the organism.

• Formic aldehyde: This is released by paints, glues, synthetic carpets, andchipboards, causing damage to the ciliary apparatus of the mucosa respon-sible for clearing the respiratory tract of pollen, bacteria, viruses, and othersubstances.

• SO2, NO, NO2, CO2: These compounds are released during combustionof coal, gas, and engine fuels. In a humid environment they turn intosulfuric and nitric acid. They have a damaging effect on the respiratorytract mucosa, and by irritating nerve endings in the bronchi, cause inflam-mations. An increase in the number of people living in the vicinity ofhighways and suffering from dust diseases has been observed.

• Tobacco smoke: Both in the case of active and passive smokers, this bringsabout changes in the structure of the mucosa cilia of the respiratory tract,thus depriving it of the ciliary apparatus, which is directly related tocausing allergies. Tobacco smoke is also claimed to be responsible foroxygen stress.

• Respiratory tract infections: Frequent bronchial infections in childhoodare a cause of allergic responses at later ages. Asthma is likely to developat a later age in 80% of people who had bronchitis as children.

• Household chemicals: Detergents, washing powders, liquid cleaners, bathsoaps, and cosmetics, as well as alcohol and UV radiation can also be acause of oxygen stress.

Also, changes in lifestyle, mainly hygienic conditions, ways and styles of feeding(e.g., bottle nursing from birth, abrupt introduction of a vegetarian diet), as well asfrequent infections combined with inappropriately taken drugs are significant.

10.3 MECHANISMS OF ALLERGIC REACTIONS TO FOOD

There are four basic mechanisms of inducing allergy:

• Type I: Anaphylactic and atopic reactions • Type II: Cytotoxic and cytolytic reactions • Type III: Arthus reactions induced by immunological complexes • Type IV: Delayed cell reactions

Food allergies are most often caused by the type I mechanism, which refers toan allergic reaction occurring immediately following contact with an allergen. Pro-tein allergens are hydrolyzed in the stomach and intestine to peptides and amino



acids, which are recognized by the immunological system. So far no threshold valuefor the molecular weight decisive for the allergenic character of the formed peptideshas been established.

Following penetration into an antigen-presenting cell (APC), for instance, adendritic cell, allergens undergo proteolysis. Peptides formed during hydrolysis arebound by the major histocompatibility complex (MHC) II, and are presented onthe cell surface. By means of T cell receptors (TCR), T-lymphocytes recognize suchcomplexes. Activated lymphatic T-cells initiate B-lymphocytes to produce IgEantibodies by interleukins. In case of the next contact of the organism with previ-ously recognized allergens, immunoglobulin IgE binds to the surface of the mastcells through a high-affinity IgE receptor (FC&RI), which is followed by a releaseof allergic reaction mediators: leucotrienes, prostaglandins, and histamines. As aresult of such reactions, itching rashes, erythema, urticaria, papular rashes, or otherforms of allergic response occur.

10.4 ALLERGEN TERMINOLOGY

Allergens are most frequently proteins or glycoproteins capable of inducing reac-tions of the organism, which depend on IgE. A system of naming newly isolatedand characterized allergens has been established. The first three letters of a newallergen are given according to the taxonomic name of the species from which ithas been extracted. Next, there is the first letter of the kind of a given species andArabic number indicating the order of registering the new allergen with the AllergenNomenclature Subcommittee of the World Health Organization/International Unionof Immunological Societies (WHO/IUIS). For example, milk allergens, which orig-inate from domestic cattle (Bos domesticus), are named Bos d 4 (α-lactalbumin),Bos d 5 (β-lactoglobulin), or Bos d 8 (caseins). In many cases it is unnecessary tospecify the isoallergen or variant, and the corresponding suffixes may be deleted,such as Bet v 1 represents any Bet v 1 allergen (main allergen of birch) and Bet v1.0101 represents a variant number 1 of isoallergen Bet v 1. When a given name isidentical to that of an already existing one, it is necessary to add another letter tothe first part of the name (e.g., Ves v 5—major venom allergen from Vespula vulgaris,and Ves vi 5—the allergen of Vespula vidua). Furthermore, it must be establishedwhether the chemical compound that is to be designated as an allergen poses a threatto patients suffering from hypersensitivity. Labeling an allergen as dominating orless significant depends on the number of patients in the studied populations havingIgE antibodies appropriate for the compound tested (Table 10.1). The informationconcerning allergens is collected and made available in databases on the Internet.

10.5 BASIC FOOD ALLERGENS

10.5.1 COW’S MILK ALLERGENS

Cow’s milk contains about 30 to 35 g proteins per liter, of which caseins make 80%and whey proteins make 20%. Homologs of cow’s milk caseins occurring in the


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TABLE 10.1Selected Food Allergens

Allergen sourcesystematic andoriginal name

Allergenname Biochemical name

MW (kDa)SDS-PAGE

C: cDNAP: peptidesequence

Gadus callariascod (muscle)

Gad c 1 Allergen M 12 C

Salmo salarAtlantic salmon (muscle)

Sal s 1 Parvalbumin 12 C

Bos domesticuscow’s (milk)

Bos d 4 α-lactalbumin 14.2 C

Bos d 5 β-lactoglobulin 18.3 CBos d 6 Albumin bovine sera 67 CBos d 7 Immunoglobulin 160Bos d 8 Casein 20–30

Apium graveolenscelery

Api g 1 Homologue: Bet v 1 16 C

Api g 4 ProfilinApi g 5 55/58 P

Daucus carotacarrot

Dau c 1 Homologue: Bet v 1 16 C

Dau c 4 Profilin CMalus domesticaapple

Mal d 1 homologue: Bet v 1 C

Mal d 2 Thaumatin-like proteins CMal d 3 Nonspecific lipid transfer protein 9 CMal d 4 Profilin 14.4 C

Arachis hypogaeapeanut

Ara h 1 Vicilin 63.5 C

Ara h 2 2S Albumin 17 CAra h 3 Glycinin 60 CAra h 4 Glycinin 37 CAra h 5 Profilin 15 CAra h 6 2S Albumin 15 CAra h 7 2S Albumin 15 CAra h 8 Bet v 1 family 17 C

Lycopersicon esculentumtomato

Lyc e 1 Profilin 14 C

Lyc e 2 Beta-fructofuranosidase 50 CLyc e 3 Nonspecific lipid transfer protein 6 C

Solanum tuberosumpotato

Sola t 1 Patatin 43 P

Sola t 2 Cathepsin D inhibitor 21 PSola t 3 Cysteine protease inhibitor 21 PSola t 4 Aspartic protease inhibitor 16+4 P



milk of other animals, such as goats, sheep, or mares, are identical in 80 to 90% ofchemical structure; therefore, introducing these kinds of milk into diets of allergicpatients seems pointless (Wal, 2001). European Society of Pediatric Allergy andClinical Immunology (ESPACI) and European Society for Pediatric Gastroenterol-ogy and Nutrition (ESPGHAN) experts present a unequivocal opinion in this regardas they forbid applying both milk of a species other than cow and so-called partlyhydrolyzed formulas in cases of allergic responses (Høst et al., 1999). People allergicto casein are most frequently sensitive to all four fractions of this protein. Theimmunological defense of the organism manifested by forming anticasein antibodies(IgE) is related to the occurrence of homologous sequences of amino acids, inparticular cross-reacting epitopes. One of the epitopes with an immunoreactivecharacter resistant to digestive processes is the phosphorylated region, present withinα S1-, α S2-, and β-casein. Spuergin et al. (1997) characterized three peptidesequences (AA: 19–30, 86–103, and 141–150) of α-casein, which reacted with theserum collected from 15 allergic patients. These peptides were localized in thehydrophobic regions of the molecules and became accessible for antibodies onlyafter casein denaturation.

Among whey proteins, the β-lactoglobulin (β-lg) (Bos d 5) and α-lactalbumin(α-la) (Bos d 4) are the most potent allergens. β-lg occurs in cow’s milk in twogenetic forms: A and B, whose mutations differ in positions 64 and 118. Form Acontains the residues of aspartic acid and valine, while form B contains glycine andalanine. One molecule contains 2 disulfide bonds and 3 free thiol groups. Such astructure permits interaction with casein during thermal processes. β-lg is relativelyresistant to acid hydrolysis and protease action; therefore, it remains largely unhy-drolyzed during transport through the digestive tract, mainly the intestinal mucosa.β-lg belongs to a lypocaline family, whose characteristic features include bindingand transfer of hydrophobic ligands and retinol, as well as high allergenic potential.Taking even small amounts of antigen in the form of, for example, selected hydro-lyzed β-lg fractions, may activate tolerance of the organism.

The α-la of cow’s milk reveals a high structural affinity to the α-la of humanorigin. Studies on animals (Hopp and Woods, 1982) showed that the polypeptideloop between amino acid residues (60–80):S–S:(91–96) was the strongest antigenicregion. Amino acid sequences capable of binding IgE are also localized in thestrongly hydrophobic part of the α-la particle, for example, between 99 and 108amino acid residues, as well as in the region of amino acid sequences 17 to 58 and108 to 123. The degree of affinity of these regions to human α-la is, respectively,81 and 87%.

10.5.2 EGG ALLERGENS

The majority of allergies to egg concern children aged 4 to 5 years, or up to 10 yearsof age. The egg white (~10%) is the most significant source of allergens, of whichovotransferrin, ovomucoid, ovalbumin, and lysozyme are the strongest. Generally,100% of the examined patients allergic to hen’s eggs display a positive response to


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the presence of ovoalbumin (Langeland, 1983a,b). The data concerning lysozymes arenot unequivocal. Allergens isolated from the hen egg yolk are apoviteline of lipoproteinfraction and α-livetin, which may cause allergic responses through the respiratory tract(Poulsen et al., 2001). Among less potent allergens are ovomucin and phosvitin.

Allergy to egg may also be an occupational disease of egg processing industryworkers. Therefore, environmental monitoring is recommended to check the levelof egg allergens in processing plants, both in the places of direct contact in thetechnological lines and in the adjacent office facilities (Zanoni et al., 2002).

10.5.3 FISH ALLERGENS

Allergy to fish occurs more often in countries with the highest fish consumption(Norway, Japan) than elsewhere.

Allergy induced by fish consumption played a historic role in the pioneer studieson opaque organism reactions. It was the protein isolated from fish muscle tissue thatwas used by Prausnitz and Kustner to prove the existence of an agent in blood serumthat was years later defined as immunoglobulin IgE. The protein was the first allergenwhose amino acid sequence was characterized and called Cod M, thus giving rise tousing new terminology for allergens. In later terminology the name was altered to Gadc 1 (Gadus is the first part of the Latin species name of cod) and classified asparvalbumin. The proteins belonging to this group control the movement of calciumions to and from the cell. Their presence was found in the muscles of fish (0.05 to0.1%) and reptiles. The presence of protein structurally similar to the Gad c 1 structurewas confirmed in the muscle tissue of pike and carp. The molecular weight of Gad c1 is 12.3 kDa. The allergen consists of 113 amino acid residues and one glucosemolecule. At least five regions of binding IgE were demonstrated in Gad c 1 structure(Elsayed and Apold, 1983) with the arginin residue in position 75 playing the dominantrole. The glucose moiety localized next to Cys-18 had no influence on the allergenicityof the whole compound. Trypsin hydrolysis made it possible to reveal a very stronglyallergenic region (AA: 33 to 44) and some regions with lower allergenic potential (AA:88 to 96). Among the less important fish allergens are Ag-17-cod, protamine sulfateand a protein with molecular weight 63 kDa (Mata et al., 1994). Also isolated was anallergen from fresh cod extract with molecular weight 41 kDa, which is a homolog ofaldehyde-phosphate dehydrogenase (Das Dores et al., 2002).

10.5.4 CRUSTACEA ALLERGENS

Shrimp allergens are the best characterized group. The first ones (Antigens I and II)were isolated and described by Hoffman et al. (1981). Antigen I is a dimer with amolecular weight of 45 kDa, comprising 189 amino acid residues and 0.5% saccha-rides. It was isolated from both raw shrimps and chitinous-calcium exoskeletons.Antigen II was extracted from boiled shrimps, as acidic thermally stable glycoproteinwith a molecular weight of 38 kDa comprising 341 amino acid residues and 4%carbohydrates. In later studies, Nagpal et al. (1989) described two allergens, which



were labelled SA-I (molecular weight 8.2 kDa) and SA-II (molecular weight34 kDa), and in 1992 allergens Pen a 1 and Pen i 1, both with a molecular mass of36 kDa, were isolated from boiled shrimps (Panaeus aztecus) and (Panaeus indicus),respectively. On comparing the amino acid composition of allergens and their highhomology it was obvious that Pen a 1, Antigen II and SA-II were tropomyosin. Itis estimated that consumption of 1 ÷ 2 medium-sized shrimps can induce an ana-phylactic reaction in allergic patients.

Tropomyosin has also been determined as a crayfish allergen, Pan s 1 (Panulirusstimpsoni), and a lobster allergen, Hom a 1 (Homarus americanus). Both theseproteins were cloned, their sequences were studied, and it was found that they werehomologous to shrimp allergen Pen a 1 (Stanley and Bannon, 1999).

Hypersensitivity to crab allergens is mainly observed in populations occupation-ally involved in crustacea processing. IgE-dependent reactions concern mainlyextracts obtained during boiling crabs, and not during contact with the raw material.Allergenic proteins with molecular weights of 37 to 42 kDa were isolated from crabbroth or from boiled crabmeat extract.

10.5.5 NUT ALLERGENS

This group comprises nuts growing on trees in different climatic zones, includingalmonds, Brazil nuts, hazelnuts, and pistachio nuts.

The most common cause of allergies to nuts in Europe is the walnut (Corylusavallena). The first and main allergen, isolated by Hirschwehr and coworkers(1992), was Cor a 1 with a molecular weight of 17 kDa, which reacted with IgEpresent in the serum of all patients allergic to this nut species. Another recognizedallergen, with a molecular weight of 14 kDa, is pollen prophyline, which inducedpositive responses in 16% of patients studied. Pasterello’s group studies (2002)confirmed the existence of two allergens with molecular masses of 18 kDa and14 kDa, which turned out to be homologs of birch pollen allergens Bet v 1 andBet v 2, respectively. Other allergens were also found: (1) with a molecular weightof 9 kDa, most likely belonging to a group of lipid-transfer proteins (LTP), (2) witha molecular weight of 32 kDa, belonging to 2S albumins, with an atomic mass of35 kDa, described as a legumin, and (3) with a molecular weight of 47 kDa, aglycoprotein. Beyer et al. (2002) isolated a protein from walnut tree pollen with amolecular weight of 40 kDa, which was identified as Cor a 9 and classified as aglobulin of the 11S type. It was the first isolated walnut allergen whose detailedamino acid sequence was described, and whose cDNA library was established basedon oligonucleotides characteristics.

On the basis of the current data, a recombined allergen Cor a 1.04 with a low levelof IgE binding was established and proposed as a basic component of specific immu-notherapy in walnut protein allergy (Luttkopf et al., 2002). Determining the structureof its epitopes made it possible to find their affinity to peanut allergen Ara h 3 and soyallergen, which can be helpful in predicting cross-reactions (Beyer et al., 2002).

The main allergens of almonds are two proteins identified as albumin 2S, struc-turally homologous to walnut allergen Jug r 1, and gamma conglutin, 60% homol-ogous to gamma conglutin of lupine seeds, Lupinus albus, and protein 7S of soy,


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Glycine max. Both almond allergens cross-reacted with allergens of walnut andhazelnut allergens (Poltonieri et al., 2002).

The strongest allergen of Brazil nuts is an albumin protein with a molecularweight 8 kDa, that is, Ber e 1, with a high methionine content, consisting of twosubunits. On analyzing the IgE of patients allergic to nuts, it was found that theirserum also reacted with proteins with molecular weights of 25 and 58 kDa, bothallergens of minor clinical significance (Pastorello et al., 1998).

Wang et al. (2002) isolated the main allergen in cashews (Anacardium occiden-tal), labeled Ana o 1, with a molecular weight of 50 kDa. Its structure was foundto contain 11 linear epitopes, of which 3 were immunodominating. Ana o 1 is aprotein from a viciline family. In 2003, the same authors discovered the next majorcashew allergen, Ana o 2, which belongs to the legume family.

A characteristic feature of allergens of nuts growing on trees, and of many seeds,is that their main proteins potent for inducing allergic responses are legumins andalbumins 2S. This may be a valuable tip in further studies on identification of plantallergens and the occurrence of cross-reactions and it is, consequently, related tocorrect clinical diagnosing of allergies.

10.5.6 PEANUT (ARACHIS HYPOGAEA) ALLERGENS

The frequency of allergy to peanuts is 0.5 ÷ 0.7% of the total population. This kindof allergy is the most common in the United States (approximately 2 millionpatients), and in Europe (Great Britain, Holland, and France). Allergy to peanuts ispractically not observed in Germany, although studies in this direction indicate theoccurrence of allergy symptoms in patients of allergological clinics (Lepp et al.,2002). In Saudi Arabia, about 20% of allergic patients suffer from this kind ofhypersensitivity. Allergy to peanuts is the most common form of this disease andaccounts for numerous cases of anaphylaxis.

About 7 to 10% of total peanut proteins contain substances of confirmed aller-genic character. The two best-known, isolated, and characterized allergens are pro-teins Ara h 1 and Ara h 2. The former has a molecular weight of 65 kDa and contains4 immunodominating epitopes and 20 determinants of lower immunological signif-icance, and is completely resistant to high temperatures. The latter, Ara h 2, has amolecular weight of 17 kDa, while yet another, Ara h 3, is a homolog of an 11Sprotein. Recently isolated allergens occurring in peanuts were consecutively labeledas Ara h 4 (36 kDa), Ara h 5 (14 kDa), Ara h 6 (16 kDa), Ara h 7 (14.5 kDa), andAra h 8 (17 kDa).

Pons et al. (2002) isolated a peanut allergen belonging to an oleosine familyof low-molecular proteins (18 kDa) taking part in forming peanut mastocytes. Onanalyzing the serum of patients allergic to peanuts using immunoblotting, thestrongest reactions toward proteins were found for those with molecular weights ofabout 34, 50, and 68 kDa. These proteins are most likely oleosine oligomers. IgE-dependent reactions were stronger in the case of extracts obtained from roastedpeanuts than from raw ones.

So far the threshold dose of peanut protein capable of stimulating the organismto fight the allergen has not been determined. In some patients a dose of 100 µg of



peanut protein can provoke an allergic response, while in others this kind of reactionoccurs following ingestion of more than 50 mg of peanuts (Keck-Gassenmeier etal., 1999). In practice, the only effective protection measure for patients allergic topeanuts is avoiding contact with the potential allergen. This is often difficult becausevarious food products may be contaminated by trace amounts of peanut proteinsduring processing. Such contaminants are of course not specified on the productlabel. This also applies to beverages and cosmetics. Allergies may be triggered notonly by direct consumption, but also by inhalation. Wearing a bracelet indicatingpeanuts as a possible cause of anaphylactic shock and carrying a first-aid kit con-taining epinephrine for immediate administration are presently considered the bestprotection measures (Dutan and Rance, 2001). Symptoms of allergy occur almostimmediately from the immunological system, and the digestive and respiratory tracts.A frequent result of contact with peanut allergens is asthma.

10.5.7 SOY (GLYCINE MAX) ALLERGENS

Soy is a popular protein source that is introduced early to the infant diet in the formof special formulas, especially in the case of infants displaying symptoms of allergyto cow’s milk. However, it has turned out that about 30% of children allergic to milkare also allergic to soy. Therefore, studies were undertaken on the immunoreactivityof soy proteins and other components.

The main soy allergens are Gly m Bd 28K, Gly m Bd 30k, and 11S glycinin.Gly m Bd 28K is a glycoprotein oligomer with a molecular weight of about 150kDa, belonging to the vicilin family. Gly m Bd 30k also occurs as a glycoproteinwith a molecular weight of more than 300 kDa. This allergen is also known as P34,a protein binding mastocyte. Gly m Bd 30k is a homolog of thiol protease from thepapain family, although most likely it is not a protease because its active cysteineregion has been mutated to glycine.

Seed storage globulin 11S (glycinin), with molecular weights of 20 kDa and 33kDa, occur in the form of an oligomer with a molecular weight of 322 kDa. Theepitopes of this allergen have been identified. Beardslee and coworkers (2000)pointed out two immunodominating determinants A (AA: 217 to 235) and B (253to 265). Xiang et al. (2002) characterized the shorter epitope G2A (AA: 219 to 233)as significant. The peptide consisting of section AA: 247 to 261 revealed weakbinding with IgE. Helm and his group (1998, 2000) described a soy allergen as aunit of glycinin G2, with a molecular weight of 21 kDa, with 11 epitopes in sixregions: 1 to 23, 57 to 111, 169 to 215, 249 to 271, 329 to 383, and 449 to 471.The epitope in regions 1 to 23 was stated as specific for soy, whereas the remainingones were capable of binding antibodies of patients allergic to peanuts.

The other allergens of smaller clinical significance were labeled as Gly m 3,Gly m 4, 2S albumin, and 7S globulin.

Seed storage proteins are thermally stable, resistant to proteolysis, and usuallyoccur as aggregates with molecular weights of more than 100,000 kDa. Denaturationof β-conglycinin requires a temperature over 75°C, and even then only partial changeof the secondary and tertiary structure is noticeable (Mills et al., 2001). The capability


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of forming aggregates and gels during heating is used in food technology, but theeffect of these processes on allergenicity has not yet been fully determined.

Allergens occurring in soy lecithin, commonly used as an emulsifier in foodprocessing, pharmaceutical technologies, and production of cosmetics, have alsobeen identified. Commercial lecithin consists mainly of phospholipids, althoughthe presence of proteins causing IgE-dependent reactions has also been confirmedwith molecular weights of 7, 12, 20, 39, and 57 kDa. The 12-kDa protein has beenidentified as a 2S albumin with a high content of methionine, and protein 20 kDaas the Kunitz trypsin inhibitor. The 39-kDa protein has not yet been recognized;hence, it could be a new allergen. Soy lecithin may also be a source of so-calledhidden allergens (Gu et al., 2001).

10.5.8 WHEAT ALLERGENS (TRITICUM AESTIVUM)

Allergies caused by contact with wheat concern mainly patients occupationallyexposed to cereal allergens, such as workers in cereal processing plants and bakeries.

The main allergen that may bring about anaphylaxis is Tri a 19 (omega-5 gliadin).This protein is held responsible for immediate allergic response in children. It wassuggested that omega 5-gliadin should be used during IgE tests, which would allowelimination of oral wheat provocation, which is indispensable in making diagnoses(Palosuo et al., 2001). Matsuo et al. (2004) determined 7 epitopes, of which aminoacid residues in positions Gln 1, Pro 4, Gln 5, Gln 6, and Gln 7 initiated reactionswith IgE.

Allergy to wheat should not be mistaken for diarrhea chylosa or celiac disease,which is a gluten enteropathy.

10.6 OTHER PLANT MATERIALS AS SOURCESOF FOOD ALLERGENS

Four basic allergens occurring in potato have been classified. Sol t 1 (Sola t 1 orpatatin), is a storage protein that plays a protective role against parasites. It wasfound to be highly homologous to Hev b 7—the latex allergen. Sol t 2 is a cysteineinhibitor of proteases, mainly catepsin D. This protein has a 32% affinity to theallergen of Lolium perenne grass pollen, Lol p 11, which accounts for the common-ness of cross-reactions between pollens of grass, potato, and some fruits. Sola t 3is a cysteine inhibitor of proteases, mainly papain and bromelain, represented bytwo isoforms: Sola t 3.0101 and Sola t 3.0102. Sol t 4 is an aspartyl inhibitor ofproteases acting specifically against trypsin and chymotrypsin (Seppälä et al., 2001).Protease inhibitors have particularly stable conformation, high resistance to heatingand digestive enzymes, and are therefore capable of reaching the intestine wherethey can induce allergic food reactions.

Allergy to tomatoes is estimated to affect 1.5 to 16% of the world’s population.The data on the tomato as a source of allergens are few. Often the issue is consideredin relation to allergies to grass pollen or latex. Tomato allergen Lyc e 1 belongs toa family of proteins called profilins, which are a direct cause of cross-reactions withbirch pollen, which belongs to the same family. Recently another tomato allergen,



namely, fructofuranosidase, has been identified and labeled as Lyc e 2 (Westphal etal., 2004). IgE-dependent allergic reactions caused by the presence of Lyc e 2 affectabout 17% of patients among those hypersensitive to tomato. The structure of glycaneis held responsible.

The most important celery allergen is Api g 1, consisting of two isoforms: Apig 1.0101 and Api g 1.0201. This allergen is a homolog of birch pollen Bet v 1.Therefore, allergies to celery are frequent in countries of northern and central Europe,where birch is a very common tree. A recombined derivative of celery allergen, Apig 4, has been identified. It is a profilin that shows a high structural affinity to Bet v2 of birch pollen. Celery allergens cross-react not only with birch pollen antigensbut also in 78% of cases with soy (Gly m 3), in 75% with barley and, to a lesserextent, with mugwort and spices. The glycoprotein with a molecular weight of about60 kDa has been isolated and classified as Api g 5.

The thermolabile chief carrot allergen, Dau c 1, is a homolog of birch pollenBet v 1. Carrot also contains profilin corresponding to allergen Bet v 2. In centralEurope about 25% of atopic patients hypersensitive to foods are allergic to carrots.In 1985 a disease syndrome called celery-carrot-birch-mugwort-spice was describedfor the first time. This refers to an associated kind of allergy that can be caused byconsuming celery, carrot, some spices, and by birch and mugwort pollen. Simulta-neously, occurrence of cross-reactions caused by allergens from the mentionedsources can be expected.

In spite of a general opinion that many people are allergic to strawberries orwild strawberries, no analytical studies aimed at indicating a chemical substanceresponsible for hypersensitivity to these fruits were undertaken until 2004. The lateststudies by Karlsson et al. (2004) showed that the most significant allergen was ahomolog of protein Bet v 1, the main birch pollen allergen.

10.7 FOOD ADDITIVES

So far there is no proof that chemical substances added to food can cause foodallergy. They may contribute to initiating pseudoallergic reactions, so it is recom-mended that they be avoided by patients suffering from different kinds of hypersen-sitivity. Table 10.2 presents the types of clinical symptoms that may occur as a resultof an organism’s reaction to substances added to foods. Among the additives initiatingallergic reactions are preservatives, sweeteners, dyes, and flavorings.

Benzoates, when consumed excessively, can cause hypersensitivity in patientssuffering from asthma and allergy, and in those hypersensitive to aspirin disorders,in the functioning of the digestive tract.

Earlier studies suggested that aspartame (E 951) could cause Quincke’s edema,swelling of the eyelids, palms, and feet, or lip reddening in hypersensitive patients. Asthe occurrence of such symptoms is extremely infrequent, studies are still not complete.

Allergic patients should avoid food products containing artificial azo-dyes such astartrasine (E 102), food yellow 3 (E 110), Ponceau 4R (E 124), or amaranth (E 123).

Sodium glutamate (E 627) is a substance extensively used in the cuisine ofthe Far East and in Asian countries; therefore, the symptoms of food intolerance


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often caused by this substance are called Chinese Restaurant Syndrome. Yet dataverification does not confirm the thesis that sodium glutamate is a strong allergen.Studies point to other ingredients of Asian cooking, such as shrimps, peanuts, nuts,and some spices, which are most likely responsible for allergic reactions.

10.8 EFFECT OF PROCESSING ON ALLERGENICITYOF FOODS

Allergenicity of many proteins can be lowered as a result of heating, microwave andultrasound treatment, gamma radiation, chemical reactions (cross-linking), as wellas biotechnological and enzymatic processes. The effectiveness of the treatmentsdepends on the type and amount of allergen to be eliminated. Drastic processparameters deteriorate the sensory value of the products and may be the cause ofinteractions leading to the formation of new allergens.

Aggregation reactions, a result of hydrophobic interactions, are an unavoidableconsequence of protein denaturation. Such interactions may lead to the loss oforiginal antigenicity of the native proteins, but also to the formation of new antigenicstructures. The effect of temperature is due to the destruction of the conformation

TABLE 10.2Influence of Food Additives on Clinical Symptoms

Food additives Undesirable clinical symptoms

Dye Azo Urticaria, vessel purpura, severity on atopic changes of skin, Quincke’s edema, anaphylactic reaction, dyspnea attack

Nonazo Urticaria, anaphylactic shockPreservatives and antioxidants

Sulfuric anhydride and sulfites

Dyspnea attack, urticaria, edema, angioneuric edema, shock reactions

Benzoate Urticaria, atopic contact dermatitis, severity of atopic changes on skin

Nitrate (III), nitrate (V), sorbic acid and its salts

Urticaria

ButylhydroxyanisoleButylhydroxytoluene

Urticaria, severity of atopic changes on skin

Substances thatenhance tasteand odor

Monosodium glutamate MSG syndrome

Aromas andpharmaceutical raw materials

Vanillin Severity of atopic changes on skin, urticaria Cinnamic aldehyde Severity of atopic changes on skin, atopic contact

dermatitis, urticariaEnzymes Papain

α-amylazeAnaphylactic shock, allergic rhinitis, dyspnea attack

Sweeteners Aspartame Urticaria, Quincke’s edema, anaphylactic reactions, behavioral perturbation, headache



of the epitopes, while linear epitopes remain mostly untouched. The latter, if notdigested in the digestive tract, are easily transferred to the intestine and may triggerfood allergies (Davis and Wiliams, 1998).

Also, chemical modifications of proteins might reduce allergenicity. The effectof acetylation and succinylation on immunoreactivity of milk whey proteins, that is,α-la and β-lg, has been confirmed. The capability of milk proteins to bind specificantibodies following their reaction with acetic and succinic anhydrides decreased byover 99% (Wróblewska, 1996).

One of the most effective ways of modifying immunoreactive properties offood components is making use of milk proteins’ capability to conjugate withpolyethylene glycol (PEG). PEG is a nontoxic component, approved by the U.S.Food and Drug Administration (FDA) for both internal and external medical use.Conjugation of α-la and β-lg with PEG was found useful in lowering their anti-genicity. The preparations were assessed using the enzyme-linked immunosorbentassay (ELISA) method for determining immunoreactivity of particular modifiedproteins (0.08% for α-la and 0.05% for β-lg compared to 100% for raw milk)(Wróblewska and Jędrychowski, 2002).

Studies on the influence of microorganisms on the human organism, and partic-ularly on the immunological system, stress the significance of microflora, especiallyof Lactobacillus, naturally occurring in the human intestine. The Lactobacilli addedto yogurts have an immunostimulating effect because they increase the productionof interferon-γ and lymphokines and stimulate the production of secretory immuno-globulin A (IgA). A simultaneous growth in the activity of phagocytes and lympho-cytes is observed.

On analyzing fermented milk beverages obtained using meso- and thermophilicstrains, a significant lowering of allergenic properties was observed in 95% of them(Wróblewska, 1996). The results demonstrating the effect of enzymes producedduring the growth of Lactobacillus delbrueckii ssp. bulgaricus appear significant asthis strain, along with Streptococcus salivarius ssp. thermophilus, is a componentof yogurt cultures.

Producing fermented beverages with lowered immunoreactive properties usingthe most beneficial bacteria strains seems to be the right way to obtain hypoallergenicformulas. These formulas must appear palatable, as this is a decisive element forthe product’s desirability among children, which is the population group most vul-nerable to allergies caused by cow’s milk. Fermented milk products have a pleasanttaste with a diversified acid note. Another advantage of fermentation is lowering thecontent of lactose, which is a frequent cause of food intolerance. The criteria ofpalatability should be taken into consideration during production of hypoallergenicformulas because children (who are their main consumers), usually are very sensitiveto the taste and odor of milk products.

Attempts are being made to decrease the allergenicity of proteins by partialhydrolysis. Enzymatic protein hydrolysis used for producing diet formulas usuallyproceeds in mild conditions (pH 6 to 8, temperature 40 to 60°C), which allowsminimization of noxious products. Moreover, the hydrolysates are soluble, morethermally stable, and have a higher resistance to slight acidity changes or metal ionsthan the proteins from which they were produced. During manufacturing, care must


Food Allergens 289

be taken in the process especially with respect to the selection of enzymes suitablefor the substrate and optimization of the process parameters.

The hydrolysates of whey proteins, casein and soy, have been practically put touse in the production of formulas for infants with atopy and with assimilation anddigestion disorders for immunological reasons. So far, no hypoallergenic formulahas been produced that is completely devoid of allergenic and immunogenic prop-erties while being satisfactorily tasty. Only mixtures of amino acids are completelynonallergenic. Other hypoallergenic products available on the market contain pro-teins capable of triggering allergy mechanisms (Høst and Halken, 2004). The aimof the current research is to find less allergenic or tolerogenic fractions in modifiedproducts so as to determine the possibility of proposing a type of milk proteinmodification that will ensure a complete elimination of allergenicity or will becapable of stimulating the organism to produce a spontaneous immunological barrier(Calvo and Gomez, 2002). It is already known that a mixture of peptides obtainedas a result of casein hydrolysis with a molecular weight below 500 kDa still hasallergenic properties.

Hydrolysate allergenicity can only be confirmed during clinical trials usingstandardized samples. The possibility of inducing allergic reactions varies depend-ing on the patient’s physical and mental state, allergen dose, and time necessaryfor stimulating the organism (Høst and Halken, 2004). Allergenicity of ready prod-ucts can also depend on the carbohydrates and lipids used for supplementation inspecial diets.

Hydrolysis should be followed by suitable unit operations aimed at obtainingthe most beneficial product. Such conditions are met by, for example, ultrafiltration,which permits elimination of nonhydrolyzed proteins that may still be present afterhydrolysis.

New products can be approved as milk-replacement preparations if they are welltolerated by 90% of the population with previously confirmed hypersensitivity tothe proteins that are the basic material for the production of the hydrolysate studied.

The effect of gamma radiation on the changes in food material has also beenobserved with respect to the antigenicity of bovine blood serum albumin (Kume andMatsuda, 1995) and β-lg (Byun et al., 2002). The authors state that antigenic struc-tures of conformation epitopes of bovine serum albumin are destroyed as a resultof radiation. However, it was also found that in the case of sequential structuresantigenicity could be greater (Byun et al., 2004). In studies on animals, antigenicityof allergenic proteins was observed to decrease under the action of applied gammaradiation. This observation is significant for allergens toward which no other tech-nological methods can be applied without losing their properties, such as egg (Leeet al., 2005).

10.9 ALLERGEN CROSS-REACTIONS

Cross-reactions were defined in the IUPAC (International Union of Pure and AppliedChemistry) Compendium of Chemical Terminology in 1994 as the capability of asubstance other than an analyte to bind a reacting substance, or vice versa, as the



capability of a substance other than a reagent to bind an analyte (IUPAC Compen-dium, 1994).

In the case of studying allergenicity in clinical diagnostics, cross-reaction deter-mines the occurrence of clinical symptoms in a patient displaying simultaneoushypersensitivity to food, inhalation, and contact allergens. Classic cross-reactionsproceed mainly between plant pollen and some fruits and vegetables, and they usuallyconcern patients with symptoms of allergic rhinitis and bronchial asthma (Cudowskaand Kaczmarski, 2003) (Table 10.3). Discovery of prolifins, carbohydrate determi-nants, and lipid transfer proteins (LPT) contributed to working out the pathogenicmechanisms of food allergy.

Within studies on the immunoreactive and allergenic properties of milk proteins,the most significant issue is the immunological affinity of the amino acid compositionof particular milk proteins, that is, alike or identical structure of epitopes.

The α-la and β-lg are the best-known milk proteins with allergenic properties.α-la has been shown to have an epitope in position Val 42-Glu 49, while for β-lg itwas fragment Pro 48-Glu 55. The spatial structure of these fragments is almostidentical. Observations of the spatial structure of other milk proteins (lactoferrin andlactoperoxidase) revealed the occurrence of structures with similar conformations,which seem to be responsible for binding IgE antibodies. By comparing them toepitopes α-la and β-lg, amino acid loops of lactoferrin and lactoperoxidase havebeen defined as their most likely determinants. Experimentally determined allergenic

TABLE 10.3Cross-Reactivity between Pollen and Food Allergens

Source of allergen Name of allergen Origin of cross-reactive allergen

Birch Bet v 1, Bet v 2 Beech, oak, alder, hazel, chestnut, kernel fruits, kiwi, carrot, celery, curry, banana, lychee, mango, orange, soy, paprika, pepper, coriander, tree nuts

Mugwort Art v 1–4 Ragweed, chamomile, sunflower, carrot, celery, anise seed, curry, kernel fruits, kiwi, mango, fennel, paprika, caraway, pepper, latex

Carrot Dau c 1, Dau c 4 Birch, mugwort, celery, mango, melon, cucumberCelery Api g 1, Api g 4,

Api g 5Birch, grasses, mugwort, ragweed, melon, cucumber, carrot, curry, mango, paprika, pepper, caraway, coriander

Tomato Lyc e 1–3 Birch, grasses, peanut, latex Kiwi Act c 1 Birch, grasses, latex, banana, avocado, tree nut,

sesame poppy seed Peanut Ara h 1–7 Mugwort, kernel fruits, pea, soy, tomato, latex Tree nut Jug r 1, Jug r 2,

Ber e 1Birch, hazel, mugwort, kiwi, sesame, poppy seed

Latex Hev b 1–13 Grasses, mugwort, ragweed, kernel fruits, banana, kiwi, mango, melon, papaya, avocado, potato, tomato, peanut, chestnut, rubber plant


Food Allergens 291

regions of lactoferrin are the N-fragment (Gly 83-Thr 90) and C-fragment (Leu 572-Lys 579) (Sharma et al., 2001).

Explaining the immunological affinity of milk from different animal species iscrucial. Special attention is focused on presenting the affinity between cow’s andgoat’s milk expressed by a high level of cross-reactions, that is, a high degree ofimmunological affinity of particular proteins. In practice, this means a possibility ofoccurring health complications when goat’s milk is used as a replacement productfor allergic patients (Calvani and Alessandri, 1998: Bellioni-Businco et al., 1999;Restani et al., 1999, 2002; Pessler, 2004).

10.10 ANTIBIOTIC CONTAMINATION IN FOOD

Sometimes unexpected pollution or unestimated additives, such as antibiotics,present in food can cause an allergy. Sensitization can be achieved unconsciouslyby drinking cow’s milk or eating meat of an animal with bovine mastitis earliertreated with penicillin. Allergy caused by penicillin is relatively frequent and isestimated at 1 to 10%.

Penicillin has very strong immunogenic properties. The benzyl penicillol (β-lactamand trazolidin rings) called the bigger determinant is responsible for 95% of mildallergic reactions. The most frequent symptoms of antibiotic allergies are urticaria,erythema, Quincke’s edema, and asthma. The observed 5% of anaphylactic reactionscan be caused by the side chain of penicillin, which is a small determinant of antibiotics.

Some groups of β-lactams antibiotics demonstrated strong cross-reactivity, suchas penicillin with cephalosporins, penicillin with carbapenems, and cephalosporinswith monobactams (Puchner and Zacharisen, 2002).

GLOSSARY OF BASIC TERMS USEDIN IMMUNOLOGY AND ALLERGOLOGY

allergen An antigen capable of inducing type I hypersensitivity with IgEinvolvement.

allergy An organism’s hypersensitivity resulting from contact with an allergenin which the immunological system is engaged.

antibody A protein from the immunoglobulin group produced by the organismand involved in regulatory reactions. Depending on the heavy chain struc-ture, five immunoglobulin classes are distinguished: IgA, IgD, IgE, IgG,and IgM.

antigen A particle that forms a specific bond/binding with an antibody or T-cellreceptor.

atopy The hereditary capability to excessively produce IgE antibodies. B-cell A lymphocyte maturing in secondary lymphatic organs, which on its

surface has immunoglobulins acting as receptors for an antigen.cross-reaction The capability of a substance other than an allergen to react with

an antibody specific to a given allergen as a result of an occurrence of thesame or similar epitopes.



epitope (antigenic determinant) Part of an antigen (fragment of an amino acidsequence) recognized by a section of an immunoglobulin chain, called aparatope, or by a cell receptor particle.

immunogenicity The capability of a substance to induce defensive reactions ofthe organism by producing antibodies or allergic T-cells.

immunological (antigenic) adjuvant A substance which, taken along with anantigen, increases the immunological response to the antigen.

immunoreactivity The capability of producing a reaction between an antigenand an antibody.

paratope A fragment of immunoglobulin forming a bond or binding with anepitope.

T-cell A lymphocyte from the bone marrow, which transfers to the thymus as itdevelops, and reacts with an antigen via a specific receptor.

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295

11 Flavor Compoundsin Foods

Bonnie Sun-Pan, Jen-Min Kuo, and Chung-May Wu

CONTENTS

11.1 Sources of Flavors in Foods...................................................................... 29611.1.1 Flavors Formed Naturally in Plants............................................ 296

11.1.1.1 Spices and Herbs ......................................................... 29611.1.1.2 Fruits ............................................................................ 29611.1.1.3 Vegetables .................................................................... 297

11.1.2 Flavors Produced by Microbes or Enzymes............................... 29711.1.3 Flavor Produced by Heating or Cooking.................................... 29811.1.4 Flavors from Flavorants .............................................................. 298

11.2 Molecular Structure and Odor of Flavor Compounds.............................. 29811.2.1 Volatility and Intensity of Aroma Compounds........................... 29811.2.2 Flavor Compounds and Their Odors .......................................... 299

11.3 Changes in Flavor during Food Storage and Processing.......................... 30011.3.1 Changes Due to Nature of Flavor Compounds .......................... 30011.3.2 Changes Due to Continuing Aroma Biogenesis......................... 30111.3.3 Changes Due to Tissue Disruption or Enzyme Reactions ......... 301

11.3.3.1 Introduction.................................................................. 30111.3.3.2 Alliums......................................................................... 30111.3.3.3 Brassicas ...................................................................... 30311.3.3.4 Mushrooms .................................................................. 30311.3.3.5 Formation of Green-Grassy Notes in Disrupted

Tissues.......................................................................... 30411.3.3.6 Glycosides as Flavor Precursors ................................. 304

11.3.4 The Maillard Reaction and Flavor.............................................. 30511.3.5 The Role of Lipid Oxidation ...................................................... 30611.3.6 Interaction of Lipids in the Maillard Reaction........................... 30811.3.7 Extrusion ..................................................................................... 30811.3.8 Concentration and Other Processes ............................................ 30911.3.9 Changes Due to Food Storage .................................................... 309

11.4 Use of Flavors in the Food Industry ......................................................... 30911.4.1 Functional Properties of Flavor Compounds.............................. 30911.4.2 Collection or Production of Flavoring Materials ....................... 310

11.4.2.1 Natural Flavor Materials.............................................. 31011.4.2.2 Organic Chemicals Used in Flavorings ...................... 312



11.4.3 Flavor Manufacturing.................................................................. 31211.4.3.1 Flavor Compounding ................................................... 31211.4.3.2 Process Flavor.............................................................. 313

11.5 Biotechnological Production of Flavors.................................................... 31411.5.1 Microbial Production of Flavor Compounds.............................. 31411.5.2 Enzymatic Generation of Flavor Compounds ............................ 31511.5.3 Recombinant DNA Technology for Flavor Formations ............. 317

11.6 Applications of Flavors ............................................................................. 318References.............................................................................................................. 318

11.1 SOURCES OF FLAVORS IN FOODS

11.1.1 FLAVORS FORMED NATURALLY IN PLANTS

11.1.1.1 Spices and Herbs

Since antiquity, spices, herbs, and condiments have been considered virtually indis-pensable in the culinary arts. They have been used to flavor foods and beverages theworld over. Spices can be grouped according to the parts of the plant used: leaves(bay, laurel), fruits (allspice, anise, capsicum, caraway, coriander, cumin, dill, fennel,paprika, pepper), arils (mace), stigmas (saffron), flowers (safflower), seeds (carda-mom, celery seed, fenugreek, mustard, poppy, sesame), barks (cassia, cinnamon),buds (clove, scallion), roots (horseradish, lovage), and rhizomes (ginger, turmeric).Most of the spices and herbs contain volatile oils, called essential oils, which areresponsible for the characteristic aroma. Some spices (capsicum, ginger, mustard,pepper, horseradish) are pungent, while paprika, saffron, safflower, and turmeric arevalued for their colors. Many spices have some antioxidant activities (Chen et al.,1999; Lee et al., 2005; Teissedre and Waterhouse, 2000). Rosemary and sage areparticularly pronounced in antioxidant effects. Cloves, cinnamon, mustard seed, andgarlic have antimicrobial activities (Badee et al., 2003; Firouzi et al., 1998). Somespices have physiological and medicinal effects. Spiced foods contain substancesthat affect the salivary glands (Pruthi, 1980).

11.1.1.2 Fruits

In fruits, the ester formation is regulated by alcohol acyltransferase (AAT), alcoholdehydrogenase (ADH), and lipoxygenase (LOX). The changes in AAT activity duringripening and postharvest storage of apple show a pattern concomitant with ethyleneregulation, while ADH and LOX seem to be independent of ethylene modulation.Volatile production and accumulation depend on the availability of precursors. Fattyacids and amino acids play important roles in the generation of aldelydes, alcohols,and acids for ester formation (Defilippi et al., 2005).

Fruits, especially climacteric fruits such as the tomato, subject to stresses suchas low or high temperatures and low oxygen levels, are generally low in volatiles.Reductions in hexanal, hexenal, hexenol, and 6-methyl-5-hepten-2-one, and increasesin ethanol and acetaldehyde are stress induced. Preformed volatile compounds, such


Flavor Compounds in Foods 297

as isobutylthiazol and methylbutanal/ol are also subjected to changes resulting frompostharvest storage conditions (Boukobza and Taylor, 2002).

Volatiles of C6–C12 alkanals and alkenals emitted from plants inhibit fungalinfection, for example, in corn (Zeringue et al., 1996), soybean (Boué et al., 2005),and tomato. Different cultivars of fruit can vary significantly in volatile compositionsand sensory properties. For melons, the major difference lies in the content of esters,particularly sulfur-containing esters and straight 9-carbon chain compounds of alde-hydes and alcohols (Kourkoutas et al., 2006).

Citrus fruits contain peel oil, from which the essence oil is obtained duringconcentration of the juice process. Citrus oils are characterized by a high percentageof terpene hydrocarbons (limonene, C10H16), which contribute little to aroma. Theunique characteristics of limonene are its relative insolubility in dilute alcohol, andits susceptibility to oxidation, causing off-flavor production. If the monoterpenes areremoved, the resulting oil is called terpene-free or terpeneless oil. Aldehydes, esters,and alcohols are the main contributors to the aromas of citrus oil. These compoundsare relatively polar and soluble in water; therefore, they are satisfactory for appli-cations in foods and beverages. Citrus essence oil is a complex mixture of aromavolatiles. Compounds present in high concentration may provide little or no aroma,whereas some aroma volatiles found at trace concentrations may have intense aromaactivity. The concentrations must exceed the detection threshold in order for theodorous compounds to be perceived. Using the gas chromatography-olfactometrytechnique, the most intense aromas in orange essence oil are produced by octanal,wine lactone, linalool, decanal, β-ionone, citronellal, and β-sinensal (Högnadöttirand Rouseff, 2003). The balance of volatiles determines the final aroma and flavoracceptance.

Fruits other than citrus contain much less volatile aromatic compounds andcannot form essential oil in distillates. However, their juices, juice concentrates,extracts of dehydrated fruits, and distillates (essence) can be used as flavorantsdirectly added to foods.

11.1.1.3 Vegetables

Vegetables contain some flavor compounds, the concentrations of which are mostlytoo low to obtain essential oils. In the tissues of some vegetables, volatile compoundsare enzymatically produced when they are disrupted. Vegetables have the function offlavoring only after their cells are disrupted or after being fried in oil. Vegetable flavorsare classified as savory flavors, while fruit flavors are classified as sweet flavors.

In addition to the three natural flavor categories, spices and herbs, fruits, andvegetables described above, natural products like tea, coffee beans, cocoa beans,flowers (e.g., rose, jasmine), peppermint, and balsam have flavoring properties(Arctander, 1960; Furia and Bellanca, 1975; Ka et al., 2005).

11.1.2 FLAVORS PRODUCED BY MICROBES OR ENZYMES

Fermentation has been known and commercially exploited for centuries. Productslike spirits, liqueurs, wine, beer, and other alcoholic beverages; vinegar; cheese and



yogurt; miso, soy sauce, and fermented bean curd; ham and sausage; fish sauce;curing of vanilla beans, tea, and cocoa; pickles and sauerkraut; dough, bread, andother bakery products have special flavor notes.

Biotechnology for the production of flavoring materials has been developed inthe past couple decades. The technology relating to the production of flavors includescell and tissue culture; microbial fermentation; and bioconversion of substrates usingwhole microbial cells, plant cells, or enzymes (Baldermann et al., 2005; Harlander,1994; Kringer and Berger, 1998; Kuo et al., 2006). For example, some importantaroma compounds of fruits are formed from enzymatic degradation of carotenoids(Baldermann et al., 2005); components of bread flavors originate from fermentationand thermal reactions involving the participation of amylases, proteases, and lipoxy-genase (Martinez-Anaya, 1996). In a model system, lipoxygenase (LOX) extractedfrom fish gill reacts with roe lipid and results in a pronounced increase in green andfresh fishlike flavor notes, but a very slight decrease in polyunsaturated fatty acids(PUFA) (Pan and Lin, 1999); fish oil and chicken fat modified with LOX yields anaroma more desirable than the untreated lipids with minor decrease in PUFA (Huand Pan, 2000; Ma et al., 2004 ); banana leaf LOX extract reacts with 18:2 or soybeanoil pretreated with bacterial lipase produces a green and melonlike aroma, whilewith 18:3 produces sweet, fruity, cucumberlike flavor notes (Kuo et al., 2006).

11.1.3 FLAVOR PRODUCED BY HEATING OR COOKING

After being cooked, the flavors of foods such as wheat, peanuts, and sesame arequite different from those of the raw materials. Flavor formation from flavor pre-cursors in the processed foods are primarily via Maillard reaction, caramelization,thermal degradation, and lipid-Maillard interactions.

11.1.4 FLAVORS FROM FLAVORANTS

Flavorings play essential roles in the production of a wide range of food productsversatile in aroma to meet consumer needs. In this regard, flavor manufacturersrequire expertise in flavor formulations, research, and technical services, while flavorusers need a fundamental knowledge of flavor applications.

11.2 MOLECULAR STRUCTURE AND ODOROF FLAVOR COMPOUNDS

11.2.1 VOLATILITY AND INTENSITY OF AROMA COMPOUNDS

The flavor of food is largely perceived as a result of the release of odorous com-pounds, usually present in trace amounts in foods, into the air, in the mouth, andthence to the olfactory epithelium in the nose. Therefore, an aroma compound mustbe volatile. The volatility is caused by the evaporation or rapid sublimation of anodoriferous substance. It is proportional to the vapor pressure of the substance, andinversely proportional to its molecular weight. The aroma in food consists of com-pounds of high, intermediate, and low volatility. For example, the aroma of a meatlike



process flavoring from hydrolyzed soy protein includes: hydrogen sulfide (cooked-egg odor) and methanethiol (rotten/garbage odor), which are of high volatility, while2-methyl-3-furanthiol (meaty), 3-mercapto-2-pentanone (catty, urine), 3-methylthi-olpropanal (potato) are of intermediate volatility. Maltol and furaneol both give aburnt sugar odor and are of low volatility (Wu and Cadwallader, 2002).

Other characteristics relate to aroma compounds, of which flavor intensity is themost important. Threshold is used most extensively for quantification of flavorintensity. Psychophysically, a threshold can be defined as the minimum concentrationof a stimulus that can be detected (absolute threshold), discriminated (just-noticeabledifference), or recognized (recognition threshold). In general, the detection thresh-olds are lower than the recognition thresholds, if the difficulty in measuring bothare comparable (Pangborn, 1981).

The relationship between the molecular structure of an aroma compound and itsthreshold is still unclear. Volatility of a compound may not relate to its threshold.For example, the threshold of ethanol (boiling point 78°C) is much higher thanoctanol (boiling point 195°C) or other homologous alcohols. Ethanol has highvolatility but low odor intensity. It is often used as a solvent in compounded flavors.In general, sulfur- or nitrogen-containing compounds and heterocyclic compoundshave very low threshold values. Thresholds of some odorous compounds in aqueoussystems can be found at (http://www.leffingwell.com/ odorthre.htm, http://www.lef-fingwell.com/ald1.htm). Some of those present in lipid systems have been reportedin the literature (Ma et al., 2004; Meynier et al., 1998).

An odor unit is defined as the ratio of concentration to threshold. This unitquantifies the contribution of a specific component or a fraction to the total odor ofa mixture; however, it says nothing about the odor quality of the final mixture, nordoes it imply anything about the relationship between the stimulus concentrationand the intensity of sensation above the threshold (Teranishi et al., 1981).

11.2.2 FLAVOR COMPOUNDS AND THEIR ODORS

The relationship between the molecular structure of a chemical compound and itsodor has been the subject of much research and conjecture. It is still not possible topredict the aromatic profile from the structure of a given chemical, nor is it possibleto assume changes in flavor profile based on molecular structure modification. Evenstereoisomers may differ in odor both qualitatively and quantitatively. Nevertheless,the relationship between structure and odor is summarized as follows.

Small molecules such as ethanol, propanol, and butanol among alcohols; ace-taldehyde and propionaldehyde among aldehydes; and acetic acid, propionic acid,and butyric acid among acids are highly volatile and exhibit pungent ethereal,diffusive, harsh, or chemical odor characteristics. Only in extreme dilution of thesecompounds will desirable odor become perceptible. Bigger molecules of alcohols,aldehydes, and acids are mild and desirable at 5°C to 10°C. Molecules containingalcohols, aldehydes, and acids reduce their volatility and odor intensities withincreases in molecular size at temperatures higher than 10°C.

Compounds containing functional groups, such as –OH, –CHO, –CO, and–COOH, play important roles in exporting the odors of the compound. Acid is sour,



aldehyde yields a fresh note, and ester is fruity. However, the elongated alkyl groupenhances the fatty or oily note. Ketones, having two alkyl groups attached to acarbonyl group, give a more fatty aroma than the corresponding aldehydes.

Ester has a fruity note. When the initial alkyl group in alcohol or acid or bothis relatively large in molecular size or with its own characteristic note, the resultingester maintains this note in addition to the fruity note. Examples are citronellylacetate, having the fresh rosy-fruity odor, inherits the rosy note from citronellol;bornyl acetate, having a sweet herbaceous-piney odor with a balsamic undertone,maintains the odor of borneol.

The boiling point of ethyl acetate is 77°C, and its molecular weight (MW) is88. Those of its reactant, ethyl alcohol, are 78°C and MW 46, while those of theacetic acid are 118°C and MW 60, respectively. Ester has a higher molecular weightbut a lower boiling point in comparison to the precursor alcohol and acid. Manyesters, such as ethyl acetate, have a fresh note. Aldehyde has a relatively low boilingpoint. For example, acetaldehyde has a MW of 44, while its boiling point is 21°C.Therefore, aldehydes are often used due to their fresh note. For example, decanalcontributes to the fresh note in orange aroma.

Lactones are cyclic compounds with an ester functional group. They have thecharacteristic ester notes: fruity, oily, and sweet. However, they are cyclic compoundsand have relatively high boiling points. γ-Undecalactone, with a peachlike aroma,has a boiling point of 297°C.

Heterocyclic compounds generally have very low thresholds. Thiazoles, thi-olanes, thiophenes, furans, pyrazines, and pyridines are normally present in largernumbers and higher concentrations in cooked, fermented, processed seafoods ormeat products than in fresh ones.

Essential oils, oleoresin, or other natural flavoring raw materials have manyvaluable trace components, which play important roles in aroma. These componentsare not commercially available now because of their complexity and low threshold.It is not feasible to use a complicated manufacturing process for the very smallamount needed. The only source available is the natural product.

11.3 CHANGES IN FLAVOR DURING FOOD STORAGE AND PROCESSING

11.3.1 CHANGES DUE TO NATURE OF FLAVOR COMPOUNDS

A volatile compound evaporates continuously even at room temperature. Highertemperature accelerates the evaporation. Some food ingredients, such as lipids andproteins, may trap flavor compounds to some extent and reduce their volatility.Different flavor compounds have different volatilities. An aged food may not onlylose its total flavor, but also change the proportions of its flavor components, resultingin a changed odor.

Many flavor compounds containing double bonds or aldehyde groups are sus-ceptible to oxidation, cleavage, polymerization, or interaction among components(Sinki et al., 1997). Alcohols can be oxidized to the corresponding aldehyde andthen to acid. Alcohol and acid can react and dehydrate to form esters. Esters can be



hydrolyzed to alcohol and acid at neutral or alkaline pH. Aldehyde and alcohol canbe dehydrated by catalysis to form hemiacetal while the reverse reaction can occurin acidic conditions or in water.

11.3.2 CHANGES DUE TO CONTINUING AROMA BIOGENESIS

The amount of secondary metabolites, such as aroma compounds produced by aplant during its life cycle, is a balance between formation and elimination. The twoopposing functions are directly controlled by two main groups of factors. Theintrinsic factors comprise all internal or hereditary properties (e.g., genotype andontogeny), while the extrinsic factors comprise all external or environmental prop-erties (e.g., pressure, wind, light, temperature, soil, water, nutrients). Therefore, aplant material or the essential oil may have a quite different flavor quality due toculture conditions and maturity (Nagy and Shaw, 1990; Lawrence, 1986). For exam-ple, during ripening of chili, hexanal (green aroma) and 2-isobutyl-3-methoxypyra-zine (grassy aroma) decrease significantly, while compounds contributing to sweet,fruity attributes, such as 2,3-butanedione, 3-carene, trans-2-hexenal, and linaloolincrease noticeably (Mazida et al., 2005). Butanol, 3-methylbutanol, benzyl alcohol,and α-terpineol develop in papaya relative to fruit ripeness (Almora et al., 2004).The typical flavor of climacteric fruit such as banana, peach, pear, and cherry is notpresent during early fruit formation, but develops fully during a rather short periodof ripening. During that time, minute quantities of lipid, carbohydrate, protein, andamino acids are enzymatically converted to volatile flavors (Reineccius, 1994a).During postharvest handling, the plant continues the biogenesis of aroma.

Even pruning of tea plants has an impact on the flavor of tea. LOX activity andtotal fatty acids and polyenoic acids decline while hexanal, hexenal, and pentanalincrease with postpruning time. Tea flavor constituents including linalool, phenylac-etaldehyde, geraniol, theaflavins and sensory flavor index all elevations with timefrom pruning (Ravichandran, 2004).

11.3.3 CHANGES DUE TO TISSUE DISRUPTION OR ENZYME REACTIONS

11.3.3.1 Introduction

Some food flavors are not present in the intact plant tissues, but are formed byenzymatic processes when the plants are cut or crushed. Under these circumstances,the cells are ruptured and the flavor precursors are released and exposed to enzymes.Unique examples of this kind of flavor formation are described below.

11.3.3.2 Alliums

Garlic, onion, shallot, green onion, and chive belong to the genus Allium. Membersof this genus contain volatile sulfur compounds, including thiols, sulfides, disulfides,trisulfides, and thiosulfinates (Block and Calvey, 1994; Kim et al., 2005; Yamaguchiand Wu, 1975; Yu and Wu, 1989).



The enzymatic flavor formation reaction of the genus Allium can be generalizedas follows:

2 RSOCH2CHNH2COOH → RSSOR + 2 NH3 + CH3COCOOH

where R is methyl, propyl, 1-propenyl, or allyl. The 1-propenyl compound has beenidentified as the lacrimator in onions. When garlic is chopped or crushed, allinase,an enzyme present in intact garlic, is activated and acts on alliin to produce allicin.Allicin (diallyl thiosulfinate) is the major thiosulfinate compound in garlic and theprincipal bioactive compound present in garlic extract. In common with all thiosul-finates, allicin readily forms diallyl disulfide and diallyl trisulfide at room tempera-ture. Addition of soybean oil in the process of garlic disruption can slow down theconversion of allicin (Kim et al., 1995).

The water extract of heat-treated garlic contains mainly alliin due to allinaseinactivation by heat. The major compounds present in aged garlic extract are S-allylcysteine and S-allylmercaptocysteine (Kim et al., 2005). Medicinal use of garlicoil is mostly prepared by hydrodistillation of raw garlic homogenate. Garlic oil isinsoluble in water, and therefore can be separated or extracted afterward. Garlicoil consists of the diallyl, allyl methyl, and dimethyl mono- to hexasulfides. Garlicoil from oil-macerated or ether extracted garlic homogenate contains the 2-vinyl-[4H]-1,3-dithiin and 3-vinyl-[4H]-1,2-dithiin, allyl sulfides and ajoenes (Kimbariset al., 2006).

When alliin and deoxyllin, two important nonvolatile flavor compounds of garlic,undergo thermal reaction with an aldehyde, that is, 2,4-decadienal in a model system,volatile compounds are generated in 3 categories: (1) degradation products of alliinmainly allyl alcohols, (2) degradation products of deoxyalliin mainly allyl sulfides,allyl mercaptan, allyl thioacetic acid, and dithiacyclopentane, and (3) interactionproducts such as hexylthiophenes, 2-pentylpyridine, 2-pentylbenzaldehyde, and 5-formyl-2-pentylthiophene (Yu et al., 1994).

Aroma of fresh leek alliums is dominated by sulfur-containing volatile com-pounds. The formation pathway is summarized (Krest et al., 2000; Nielsen et al.,2003) as follows:

nonvolatile precursors, such as S-alk(en)yl-cysteine

↓ allinase (EC 4.4.1.4)

sulfenic acids (highly reactive)

↓

thiosulfinates, (pungent odor)

thiopropanal-S-oxide

↓

monosulfides/polysulfides



Allium aroma is dominated by dipropyl disulfide and declines during frozenstorage (Nielsen et al., 2004). Frozen storage of unblanched leek shows respiration,the rate of which is affected by the degree of intactness of tissue or slice thickness.Thinner slices generate higher concentrations of total sulfur compounds and totalaldehydes. Extended storage periods cause reductions in total sulfur-containingvolatile compounds and inductions of aldehydes later produces off-flavor. Nitrogenpackaging improves the aroma profile of fresh leek in comparsion to the atmospheric-air packaging (Nielsen et al., 2004 ).

The lipoxygenase (LOX) pathway also participates in the aroma developmentin fresh alliums as follows:

polyunsaturated fatty acids containing

cis,cis-pentadiene structure

O2 ↓ LOX (EC 1.13.11.12)

hydroperoxides

↓ hydroperoxide lyase (HPL, EC 4.1.2)

aldehydes

↓ alcohol dehydrogenase (EC 1.1.1.1)

alcohols

The alcohols contribute to fresh leek aroma, but larger amounts of alcoholsproduce off-flavor.

11.3.3.3 Brassicas

The Brassicas of importance include turnips, rutabagas, mustards, and the cole corps:cabbage, broccoli, cauliflower, and Brussels sprouts. The production of isothiocy-anates in Brassicas occurs via an enzymatic reaction on specific glycosides. Someof the isothiocyanates, especially allylthiocyanate, are highly pungent and are mainlyresponsible for the odor of brown mustard, horseradish, cabbage, and other crucifers.Any process that destroys or inactivates enzymes in these plants will cause decreasesin aroma production, resulting in a less distinctive flavor. This is usually the casewhen Brassica foodstuffs are commercially preserved.

11.3.3.4 Mushrooms

1-Octen-3-ol occurs in many mushroom species (Zawirska-Wojtasiak, 2004). Itcontributes significantly to the flavor of edible mushrooms such as Agaricus campes-tris (Tressl et al., 1982), and Agaricus bisporus (Wurzenberger and Grosch, 1982;Chen and Wu, 1984). 1-Octen-3-ol is formed enzymatically from linoleic acid, themajor fatty acid in A. bisporus. Enzymes involved in the pathway of formation of



1-octen-3-ol include lipoxygenase, hydroperoxide lyase, and allene oxide synthases(Grechkin, 1998).

Shiitake (Lentinus edodes) is a kind of edible mushroom highly prized in Chinaand Japan. Volatile flavor compounds in young, immature, mature, and old growthstages of shiitake mushrooms include 1-octen-3-ol, 3-octanol, 3-octanone, and 4-octen-3-one (Cho et al., 2003). Due to the difficulties of postharvest storage, themushroom has been traditionally preserved in dried form. The differences betweenfresh shiitake and dried shiitake lie in the contents of eight-carbon compounds (i.e.,3-octanone, 1-octen-3-ol, 3-octanol, n-octanol, and cis-2-octen-l-ol) and sulfurouscompounds (i.e., dimethyl disulfide and dimethyl trisulfide). Fresh shiitake containsmore eight-carbon compounds than dried shiitake does, while dried shiitake con-tains more sulfurous compounds than the fresh (Chang et al., 1991). The formationof volatile shiitake is affected greatly by the pH during blending. 1-Octen-3-ol and2-octen-l-ol are predominantly formed at pH around 5.0 to 5.5, while the formationof sulfurous compounds, such as dimethyl disulfide and dimethyl trisulfide, isaround pH 7.0. Two enzymatic systems are probably responsible for the formationof eight-carbon and sulfurous compounds. One of the two enzymes likely occursin dried shiitake, yielding more volatile sulfur compounds than in fresh shiitake(Chen et al., 1984).

11.3.3.5 Formation of Green-Grassy Notes in Disrupted Tissues

Six-carbon compounds, such as hexanal, 3Z-, and 2E-hexenal occur at high concen-trations in ruptured tissue of apples, grapes, and tomatoes (Fabre and Goma., 1999;Hatanaka, 1993; Schreier and Lorenz, 1981). (Z)-3-Hexen-l-ol, (E)-2-hexenal, hex-anol, (E)-2-hexen-l-ol, and hexanal are formed in bell peppers (Capsicum annuunVar. grossum, Sendt) after tissue disruption (Buttery and Ling, 1992). These com-pounds, having green and fruity characteristics, are biosynthetically derived fromthe action of 13-lipoxygenase (Fukushige and Hildebrand, 2005; Hatanaka, 1993;Hornostaj and Robinson, 1999). For example, the sensory impression of hexanol hasa green flavor note; 2-hexenol gives green and fruity notes; 3-hexenol (leaf alcohol),green and fresh notes (Whitehead et al., 1995).

11.3.3.6 Glycosides as Flavor Precursors

There are two forms of monoterpene derivatives in grapes: free and glycosidic con-jugates. The free form consists of compounds with interesting flavor properties, suchas geraniol, nerol, linalool, linalool oxides, α-terpineol, citronellol, hotrienol, andflavorless polyhydroxylated compounds (polyols) which, under mild acid hydrolysis,can yield odorous volatiles. Glycosides of volatile compounds identified in plants andfruits are mainly O-β-D-glucosides or O-diglycosides. The flavorless glycosides con-sist of α-D-glucopyranosides and diglycosides; 6-O-α-L-arabinofuranosyl-β-D-glu-copyranosides, 6-O-α-L-rhamnopyranosyl–β-D-glucopyranosides (rutinosides), and6-O-β-D-apiofuranosyl-β-D-glucopyranosides of predominantly geraniol, nerol, andlinalool, together with monoterpenes, are present at a higher oxidation state than the



free forms (Osorioa et al., 2003; Sarry and Gunata, 2004). As most of the aglyconesof these compounds have interesting sensory properties, their glycosides make up apotential aroma reserve more abundant than their free counterparts. Upon either acidor enzyme hydrolysis, the glycoside-bound aglycones can be released to produceflavor (Sarry and Gunata, 2004; Wu and Liou, 1986). β-Glucosidase, the mostabundant glycosidase, is present with α-arabinosidase and α-rhamnosidase in grapesand berries of various cultivars. In addition, enzyme treatment of juice or wineincreases the concentrations of volatile monoterpene flavorants. Prolonged aging ofwine or its exposure to elevated temperatures increases the concentration of freevolatile monoterpenes through hydrolysis of glycosidic precursors in wine (Gunataet al., 1992; Straus et al., 1986). Some of the liberated aglycones may already beodorous, such as linalool, geraniol, and nerol, while some give rise to potent flavorcompounds, such as β-damascenone, vitispirane, and theaspirane by further enzy-matic or chemical transformations during fruit juice processing or leaf productsprocessing (Sarry and Gunata, 2004).

Other plants such as papaya (Schreier and Winterhalter, 1986), nectarine (Take-oka et al., 1992), and tea leaves (Lee et al., 1984; Kobyashi et al., 1992) also containglycosides as precursors of flavors.

11.3.4 THE MAILLARD REACTION AND FLAVOR

The Maillard reaction plays an important role in flavor development especially in meatand savory flavor (Aliani and Farmer, 2005; Buckholz, 1988). Products of the Maillardreaction are aldehydes, acids, sulfur compounds (e.g., hydrogen sulfide, methanethiol),nitrogen compounds (e.g., ammonia, amines), and heterocyclic compounds, such asfurans, pyrazines, pyrroles, pyridines, imidazoles, oxazoles, thiazoles, thiophenes, di-and trithiolanes, di- and trithianes, furanthiols, and so forth (Martins et al., 2001;Yaylayan, 2003). Higher temperatures result in production of more heterocyclic com-pounds, among which many have roasty, toasty, or caramel-like aromas.

Sugar, ascorbic acid, amino acids, thiamine (de Roos, 1992; Aliani and Farmer,2005; Ames and Hincelin, 1992, Guntert et al., 1992, 1994; Yoo and Ho, 1997), andpeptides (Ho et al., 1992; Izzo et al., 1992; de Kok and Rosing, 1994; Lu et al., 2005)are potential reactants of the Maillard reaction. They are present in most foods, sothe Maillard reaction occurs commonly when these foods are cooked. Among thereactants studied (thiamine, inosine 5′-monophosphate, ribose, ribose-5-phosphate,glucose, and glucose-6-phosphate), ribose seems to be the most important flavorprecursor for chicken aroma. Using gas chromatography (GC)-odor assessment andgas chromatography-mass spectrometry (GC-MS) show that ribose increases roastedand chicken aromas. The changes in aroma due to additional ribose are probablycaused by an elevated concentration of compounds such as 2-furanmethanethiol, 2-methyl-3-furanthiol, and 3-methylthiopropanal (Aliani and Farmer, 2005). Aroma ofmeatlike process flavor can be produced by model systems containing sugar such asglucose, ribose, or xylose and a sulfur-containing amino acids, such as cysteine(Farmer et al., 1989; Hofmann and Schieberle, 1995; Ho et al., 1992). It can also beformed by thermal reaction of mixtures of enzyme-hydrolyzed vegetable protein,



cysteine, D-xylose, or D-ribose (Mussinan and Katz, 1973; Wu et al., 2000, Wu andCadwallader, 2002).

Cooking conditions determine the aroma of cooked foods. For example, themajor volatiles of water-boiled duck meat are the common degradation products offatty acids, while roasted duck meat contains not only the volatiles found in rawduck meat, but also pyrazines, pyridines, thiazoles, and so forth (Wu and Liou, 1992),which are Maillard reaction products. The wax gourd (Benincasa hispida, Cogn), avegetable known as winter melon or gourd melon, is used to produce beverages,candy, or jam, which are popular in Taiwan. The flesh of the gourd melon is whitein color. The major volatile compounds of fresh gourds are (E)-2-hexenal, n-hexanal,and n-hexyl formate, while those present in wax gourd beverages are 2,5-dimeth-ylpyrazine, 2,6-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2-methyl pyrazine, and2-ethyl-5-methyl pyrazine. The pyrazine compounds not present in the fresh waxgourd are formed from the sugar added and the endogenous amino acids duringprocessing of the beverage (Wu et al., 1987). This is an example of changes in flavorof foods during processing where the Maillard reaction plays an important role.

Hundreds of patents have been granted worldwide for processes and reactionproducts based on Maillard technology applied to manufacturing meat and savoryflavors (Aliani and Farmer, 2005; Buckholz, 1988; Mottram and Salter, 1988; Ouwe-land et al., 1988).

In addition, Maillard reactions may produce mutagenic components, pigments,and antioxidants, which are discussed in other sections of this book.

11.3.5 THE ROLE OF LIPID OXIDATION

The oxidation products of lipids are volatile aldehydes, acids, and so forth. Therefore,lipids are one of the major sources of flavors in foods. For example, much of thedesirable flavors of vegetables such as tomatoes, cucumbers, mushrooms, and peas(Ho and Chen, 1994; Zawirska-Wojtasiak, 2004), fresh fish (Hsieh and Kinsella,1989), fish oil (Hu and Pan, 2000), and cooked shrimp (Kuo and Pan, 1991; Kuo etal., 1994), as well as many deep-fat fried foods such as French-fried potatoes (Salinaset al., 1994; Sanches-Silva et al., 2005) and fried chicken (Shi and Ho, 1994) arecontributed by lipid oxidation.

Lipid oxidation catalyzed by lipoxygenase produces secondary derivatives, suchas tetradecatrienone, which is a keynote compound of shrimp (Kuo and Pan, 1991).During storage, there is a decrease in linoleic acid proportional to the increases involatile compounds, expecially hexanal, pentanal, and pentanol. Lipoxygenase-3 inseeds is responsible for the oxidation of linoleic acid producing stale flavor of thestored rice. In cooked rice, glutinous rice develops more of these volatiles of staleflavor than nonglutinous rice (Suzuki et al., 1999).

The core flavor of mashed potatoes consists of naturally occurring and thermallygenerated compounds. These compounds arise mainly from the oxidation of fattyacids, especially highly unsaturated fatty acids, and from the degradation and inter-action of sugar-amino acids. The extent to which these reactions affect the flavor ofthe final product depends on the age of the raw materials, storage conditions, and



processing techniques. For example, both lipid oxidation and nonenzymatic brown-ing reactions increase with age of the raw potato (Salinas et al., 1994).

Garlic develops its aroma from enzymatic reactions, as described previously.When garlic slices are deep fried, microwave heated, or oven baked, the aromachanges contribute to a different kind of garlic flavor (Yu et al., 1993). A novelS-compound forms from the interaction of garlic and heated edible oil (Hsu et al.,1993; Kim et al., 2005). Alliin and deoxyalliin can react with 2,4-decadienal, whichis one of the major oxidation products of polyunsaturated fatty acids, to form aroma(Yu et al., 1994).

Deep-fat frying is a universal cooking method. Stir frying is common in somecuisines, especially in Chinese cooking. Volatile compounds in oils change afterdeep-fat frying or stir frying and subsequent storage (Wu and Chen, 1992). Soybeanoil heated at 200°C for 1 h, with or without the addition of water, forms volatilealdehydes during storage at 55°C. Total volatiles increase up to 1100-fold. However,aldehyde content decreases, while volatile acid content increases significantly. Theincrease in hexanoic acid produces a heavy acrid, acid, fatty, and rancid odor oftendescribed as a “sweat-like” odor, which is responsible for the rancid note. Additionof water to the deep frying oil tends to retard the formation of volatile compoundsduring deep frying. Freshly stir-fried Chinese food has a much better flavor qualitythan after it has aged. The main change in volatile constituents of stir-fried bellpeppers during aging is the production of volatile carbonyl compounds from autox-idative breakdown of unsaturated fatty acids (Wu et al., 1986).

Generally, the undesirable flavor qualities of food are associated more closely withlipids than with proteins and carbohydrates. Lipids are responsible for the rancidity oflipid-containing foods. The term warmed-over flavor (WOF) is used to describe therapid development of oxidized odor in cooked meat upon subsequent holding. Therancid or stale odor becomes apparent within 48 h, in contrast to the more slowlydeveloped rancidity that becomes evident only after frozen storage for a period ofmonths. Although WOF was first recognized in cooked meat, it also develops in rawground meat exposed to air. Overheating of meat protects it against WOF by producingMaillard reaction products possessing antioxidant activity (Pearson and Gray, 1983).

Flavor differences between meats and meat products are associated with lipidoxidation products. The major difference between the flavor of chicken broth andthat of beef broth is the abundance of 2,4-decadienal and γ-dodecalactone in chickenbroth (Shi and Ho, 1994). Both compounds are well-known lipid oxidation products.A total of 193 compounds have been reported in the flavor of chicken. Forty-one ofthem are lipid-derived aldehydes.

Recently, phospholipids, such as lecithin are classified in nutraceutical foods(Colbert, 1998). The off-flavor associated with lecithin produced in fermented dairyproducts include compounds of 2,4-nonadienal, 2,4-decadienal, and hydrogen per-oxide (Suriyaphan et al., 2001). Hexanal, 2,4-heptadienal, 2-pentenal, and 2-hexenalare major volatile compounds of oxidized pork phospholipids (Meynier et al., 1998).

The flavor chemistry of lipid foods has been reviewed and compiled elsewherein the last decade (Beltran et al., 2005; Ho and Chen, 1994; Ho and Hartman, 1994;Min and Smouse, 1989; Pan and Kuo, 1994; Shahidi and Cadwallader, 1997; Shahidi,1998; Shahidi and Ho, 1999).



11.3.6 INTERACTION OF LIPIDS IN THE MAILLARD REACTION

The Maillard reaction and the oxidation of lipids are two of the most importantreactions for the formation of aromas in cooked foods. Interactions between lipidoxidation and the Maillard reaction have received less attention, despite the factthat lipids, sugars, and amino acids exist in close proximity in most foods. Lipids,upon exposure to heat and oxygen, decompose into secondary products, includingalcohols, aldehydes, ketones, carboxylic acids, and hydrocarbons. Aldehydes andketones produce heterocyclic flavor compounds reacting with amines and aminoacids via Maillard-type reactions in cooked foods (Aliani and Farmer, 2005; Shiba-moto and Yeo, 1992). Lipid degradation products, such as 2,4-decadienal andhexanal, can interact with Maillard reaction intermediates to form long-chain alky-lpyrazines as well as other heterocyclic compounds (Farmer and Whitefield, 1993;Yaylayan, 2003).

11.3.7 EXTRUSION

Extrusion cooking is a process whereby low-moisture (10 to 30%) foodstuffs aresubjected to heat, pressure, and mechanical shearing for a short time (20 sec to 2min). Extrusion can have a significant effect on flavor and aroma profiles of foodproducts manufactured through this process, for example, extrusion of wheat flourproducts (Heinio et al., 2003; Hwang et al., 1994). Depending on the raw materialcomposition, flavor development during processing is an important consideration forproduct quality. Certain mechanisms, such as nonenzymatic browning and lipidoxidation, are considered to have significant implications in the flavor characteristicsof food products. Oxidation and volatility of flavor compounds are important factorsduring heating and extrusion cooking at different temperatures and moisture contents.Lipid oxidation products are the major compounds of aroma generation in theextrudates prepared from wheat flour at high moisture content and low die temper-ature. By lowering moisture content, lipid degradation compounds decrease and theMaillard reaction products dominate the flavor profile. The lipid oxidation productssignificantly increase during storage of the extrudate prepared at low moisturecontent and high die temperature (Villota and Hawkes, 1988).

Formulations with starch, starch-caseinate, in biscuit mix show that whenaroma compounds limonene, p-cymene, linalool, geraniol, terpenyl acetate, andβ-ionone are added in water emulsion, oil solution, capsules, or inclusion com-plexes in β-cyclodextrin can lose free volatiles up to more than 90% during theextrusion process (Sadafian and Crouzet, 1987). Flavor retention increases throughencapsulation of volatile compounds in natural or artificial inclusion complexes.Soy protein isolate (SPI) and hydrolyzed vegetable protein (HVP) are importantingredients in extruded foods. SPI is used for texturization and to increase theprotein content of foods, while HVP promotes cooked and roasted aromas throughMaillard reactions. The volatile aroma components from SPI and acid-hydrolyzedvegetable protein are different. Aliphatic aldehydes and ketones are mainly foundin SPI, whereas pyrazines and sulfur-containing compounds are dominant in HVP(Solina et al., 2005).



11.3.8 CONCENTRATION AND OTHER PROCESSES

Some foods undergo special treatment in processing, which may affect the compo-sition of volatile components. For example, in hybrid passion fruit, the presence ofabout 1 to 2% starch makes heat processing, such as pasteurization and concentration,impossible or impractical unless the starch is removed before processing. However,the step of removing starch and concentration causes loss of volatile compounds offruit juice (Kuo et al., 1985).

11.3.9 CHANGES DUE TO FOOD STORAGE

Food preservation is designed to prevent undesirable changes in food and foodproducts. However, flavor changes in food products during storage occur continu-ously, although the deterioration of flavor quality is not significant in most cases.

Nonenzymatic reactions that occur during processing and storage of food prod-ucts are detrimental if foods contain reducing compounds or if these compounds areproduced during storage as a result of oxidation, acid hydrolysis, enzymatic reac-tions, or physicochemical changes. Water mediates the nonenzymatic browningreaction by controlling the liquid phase viscosity; by dissolution, concentration, anddilution of reactants; and by effects on the reaction pathways due to activation energylimitations in dehydrated foods (Saltmarch et al., 1981).

Citrus juice can have off-flavor formation during processing and storage.Changes in volatile components in aseptically packaged orange juice duringstorage at room temperatures can be monitored. Quantities of desirable flavorcomponents decrease during storage, while amounts of undesirable components,α-terpineol and furfural, increase progressively with prolonged storage (Mosho-nas and Shaw, 1987).

The ultra-high temperature (UHT) processing of milk owes its commercialsuccess to the fact that the rate of microorganism destruction increases more rapidlywith temperature than do the rates of the accompanying changes in color and flavor.At very high processing temperatures, high sterility may be achieved with minimaladverse nutritional and chemical effects. However, UHT milk darkens in color duringstorage. This effect is noticeable after a few months of storage at 20°C. It becomesmore pronounced at higher temperature and longer storage time. The milk alsodeteriorates in taste. The ε-amino group of lysine in milk proteins may react exten-sively with lactose through Maillard reaction before milk develops a marked off-fla-vor, discoloration, or instability (Moller, 1981). Spray-dried whey also has theproblem of browning via Maillard reaction.

11.4 USE OF FLAVORS IN THE FOOD INDUSTRY

11.4.1 FUNCTIONAL PROPERTIES OF FLAVOR COMPOUNDS

Food flavorings are compounded from natural and synthetic aromatic substances.The compounded flavor may or may not be found in nature. The reasons for usingflavors in foods are as follows (Giese, 1994a,b):



• Flavors can be used to create a totally new taste. This does not happenvery often, but some new flavors have been enormously successful, suchas those used by Coca-Cola® or Pepsi Cola®.

• Flavoring ingredients may be used to enhance, extend, or increase thepotency of flavors already present.

• Processing operations such as heating may cause loss of flavor, whilesome flavors already present may need supplementation or strengthening.

• Flavor ingredients can simulate other more expensive flavors or replaceunavailable flavors.

• Flavors may be used to mask less desirable flavors, or to cover harshundesired tastes naturally present in some processed foods, but not to hidespoilage.

Flavor compounds provide not only sensory quality to food, but also otherfunctional properties including antioxidative activity, antimicrobial activity, andhealth-promoting functions. Volatiles of C6–C12 alkanals and alkenals emitted fromplants are inhibitory to fungal infection, such as com (Zeringue et al., 1996), soybean(Boué et al., 2005), and tomato. Essential oil of clove, nutmeg (Dorman et al., 2000),Terminalia catappa leaves (Maua et al., 2003; Wang et al., 2000), and aroma extractsof dried soybean, kidney beans, mung bean, and adzuki bean (Lee and Shibamoto,2000; Lee and Lee, 2005), in addition to extracts of several members of the Alliumfamily (Yin and Cheng, 1998), and 23 essential oils isolated from various spices andherbs (Teissedre and Waterhouse, 2000) all have antioxidative activities. Eugenol,thymol, carvacrol, and 4-allylphenol from leaves of basil and thyme also show strongantioxidant activities (Lee et al., 2005). The aroma compounds may be used toprevent lipid oxidation in food preparation or food processing with health-relatedproperties that are based on their antioxidative activity (Ka et al., 2005).

Antimicrobial activities are found in essential oils of various herbs, spices (Badeeet al., 2003; Firouzi et al., 1998), onion, garlic (Block, 1985), mustard, and horse-radish (Delaquis and Mazza, 1995). Ninety-three different commercial essential oilshave activity against 20 Listeria monocytogenes strains in vitro, which correlate withthe chemical composition of each oil. Essential oils containing a high percentage ofmonoterpenes, eugenol, cinnamaldehyde, thymol (Lis-Balchin and Deans, 1997),and isothiocyanates show strong antimicrobial activity (Badee et al., 2003; Delaquisand Mazza, 1995). Flavors with antimicrobial activity can be a useful adjunct tofood preservation systems. Moreover, antimutagenic, anticarcinogenic, and antiplate-let activities are found in sulfur-containing compounds of several Allium members(Chen et al., 1999; Kimbaris et al., 2006).

11.4.2 COLLECTION OR PRODUCTION OF FLAVORING MATERIALS

11.4.2.1 Natural Flavor Materials

The sources, names, characteristics, and major flavor components of natural flavoringmaterials such as spice, herb, and so forth have been summarized in several books(Arctander, 1960; Furia and Bellanka, 1975; Reineccius, 1994b). A large number of



constituents in natural flavor materials are not flavor compounds. These nonflavorcompounds have to be removed to produce concentrated flavorants. The predominantmethods for reaching this purpose are distillation and extraction.

The essential oils are the distilled fraction of aromatic plants. Most often theyare steam distilled. These oils, which are primarily responsible for the characteristicaroma of the plant material, are generally complex mixtures of organic compounds.For example, during the concentration of citrus juices, a layer of essential oil isformed in the condensate. This oil is called essence oil. Terpenes can be removedfrom both essential oil and essence oil to obtain folded oil. Some fruit or vegetabledistillates containing flavor compounds are called essences. Although no essentialoil layer is obtained from vegetable distillate, it is used as a flavoring raw material.Citrus peels rich in essential oils are expressed to get cold-pressed oils.

Simultaneous distillation–extraction requires heating of the sample to collectvolatile compounds of a wide range of volatility in an organic solvent. Sometimesit may lead to odorous artifacts.

Extraction by vacuum hydrodistillation does not require heating at high temper-atures. It extracts compounds at high and low boiling points. It is particularly suitablefor analyses of volatile compounds of new products, such as fresh oysters (Pennarunet al., 2002) and fresh seaweed (Le Pape et al., 2002). The one disadvantage is thatsome volatile compounds may be lost or modified during the concentration followingthe vacuum hydrodistillation (Stephan et al., 2000).

Dynamic headspace extraction is generally performed at temperatures close tothat of vacuum hydrodistillation without heating. The extraction is only done toobtain the volatile compounds of low boiling points for analyses of materials suchas honey (Radovic et al., 2001), oysters (Piveteau et al., 2000), kombu (a dried ediblebrown algae, Takahashi et al., 2002), and edible red algae (Le Pape et al., 2004).

The nonvolatile flavoring constituents of aroma plants are recoverable by extrac-tion. Essential oils do not contain hydrophilic flavoring components, antioxidants,or pigments. The selection of solvent is limited depending on its toxicity, regardlessof whether it remains in the final product. Two kinds of solvents are used: (1) apolar solvent such as ethyl alcohol; for example, vanillin is soluble in ethyl alcohol,which is used as the solvent to prepare vanilla bean extract; (2) a nonpolar solventsuch as petroleum ether. Most aroma compounds are oil soluble. Therefore, petro-leum ether is used as the solvent to extract plant aroma. The extract after removalof the solvent is called concrete. Concrete may contain large portions of wax andfatty acids, and is further purified by ethyl alcohol extraction. The product is calledabsolute. The nonvolatile flavoring constituents of herbs and spices are recoverableby extraction. In practice, a solvent is chosen that dissolves both the essential oiland the nonvolatiles present. The resulting solvent-free product is known as oleo-resin. A disadvantage of the oleoresins is that they are very viscous and thick, makingthem difficult to handle or to mix in processing operations. Several products havebeen developed into extractives, which are convenient to use and avoid handlingproblems. Extractives can be dispersed in salt, dextrose, or other carriers to createdry soluble spices. They may also be dispersed in fats to make fat-based solublespices. Emulsification of extracts with starches and gums followed by spray drying



produces encapsulated spices. Solubilization of extracts with glycerol, isopropylalcohol, and propylene glycol produces liquid-soluble spices (Giese, 1994b).

11.4.2.2 Organic Chemicals Used in Flavorings

Organic chemicals used in flavorings include hydrocarbons, such as limonene,pinene, ocimene, α-phellandrene, β-caryophyllene; alcohols, such as hexanol,cis-3-hexen-l-ol, geraniol, citronellol, eugenol, 1-menthol; aldehydes, such as ace-taldehyde, hexanal, 2,4-decadienal, citral, vanillin; ketones, such as diacetyl, ionone,nootkatone; acids, such as acetic acid, butyric acid, pyroligenious acid; esters, suchas ethyl acetate, linalyl acetate, ethyl phenyl acetate, methyl dihydrojasmonate;lactones, such as γ-nonalactone, δ-decalactone, γ-undecalactone; hemiacetals, suchas acetaldehyde diethylacetal, citral dimethyl acetal; ethers, such as diphenyl oxide,rose oxide; nitrogen-containing compounds, such as trimethylamine; sulfur-contain-ing compounds, such as dimethylsulfide, thiolactic acid, allyl disulfide; and hetero-cyclic compounds, such as furans, pyrazines, pyridines, thiazoles. The names, chem-ical structures, physical and sensory properties, and uses have been summarized inseveral books (Arctander, 1969; Furia and Bellanca, 1975; Reineccius, 1994b). Manyorganic chemicals used in flavorings are produced by synthetic methods and arecommercially available. More and more natural compounds are used in flavoringsdue to the increasing demand for natural flavorings. They are produced or preparedby isolation of the compound from natural sources or by biotechnological methods.

Thousands of flavoring raw materials may form a flavor. A large number of theflavoring raw materials are supplied by different manufacturers and stocked. There-fore, a strict quality control system for flavoring raw materials as well as the productsis very important.

11.4.3 FLAVOR MANUFACTURING

11.4.3.1 Flavor Compounding

From thousands of raw material flavors, twenty to fifty items are commonly selectedand mixed in different ratios to blend a flavor as flavor compounding, which is atype of formulation. The raw material may be organic chemicals, essential oils,extracts, oleoresins, or processed flavors. Knowledge of their nature, physical andsensory properties, and applications are needed by flavorists. The professional prac-tice of flavor compounding requires at least 3 to 5 years of training.

How a flavor is formulated and modified using strawberry flavor as an exampleis shown in Table 11.1. The characteristic notes of strawberry are fruity, sweet,green, and a little bit oily and sour. Ethyl butyrate and methyl cinnamate havefruity notes, cis-3-hexen-l-ol, and ethyl hexanoate are green, benzaldehyde and2,5-methyl-4-hydroxy-3 (2H) furanone are sweet, butyric acid is sour, and γ-unde-calactone is oily-fruity. Formula 1 was originally designed for use in cake mix.Therefore, compounds with low boiling points, such as ethyl acetate, were notused. The solvent used was propylene glycol, which has a boiling point of 187.3°C.So the flavor compound was heat stable. However, the application of this flavor incake resulted in a sensory characteristic that was pineapple-like, but not strawberry.



Pineapple is oily, fruity, and sweet. Because butter, sugar, and milk were among theingredients in the cake mix, oily or fatty and sweet notes were derived from thoseingredients. Therefore, Formula 1 has to be modified by reducing the amount ofγ-undecalactone, benzaldehyde, and 2,5-dimethyl-4-hydroxy-3 (2H) furanone, andincreasing the amount of ethyl butyrate, ethyl hexanoate, cis-hexen-l-ol, butyric acid,and methyl cinnamate. The application test showed that Formula 2 gave the cake astrawberry flavor, although this modified flavor did not smell like strawberry beforeapplication in the cake.

11.4.3.2 Process Flavor

Process flavors include reaction flavors, fat flavors, hydrolysates, autolysates, enzymemodified flavors, and so forth. Production of dairy flavor by enzyme modificationof butterfat is an example (Lee et al., 1986; Manley, 1994), while meat flavorproduced by enzymatic reactions has a much longer history.

Raw meat has little flavor. Characteristic meat flavor varies with the species ofanimal, temperature, and type of cooking. Both water-soluble and lipid-solublefractions of meat contribute to meat flavor. The water-soluble components includeprecursors, which upon heating are converted to volatile compounds described as“meaty.” Many desirable meat flavor volatiles are synthesized by heating water-sol-uble precursors, such as amino acids and carbohydrates. The Maillard reaction,including formation of Strecker degradation compounds, and interactions betweenaldehydes, hydrogen sulfide, and ammonia, is important in the formation of thevolatile compounds of meat flavor. In addition, other kinds of flavors formed duringcooking can also be obtained from heat processing. The contingency is the avail-ability of the precursors, which may be too expensive to be isolated from naturalraw materials or to be synthesized.

The most practical way to characterize process flavorings is by their startingmaterials and processing conditions, because the resulting composition of volatilesis extremely complex comparable to the composition of cooked foods. Process

TABLE 11.1Formulas of Strawberry Flavors for Cake Mix

Compound Formula 1 Formula 2

Ethyl butyrate 1.5 3.0Ethyl hexanoate 1.0 2.0cis-3-Hexen-1-ol 1.0 2.0Benzaldehyde 0.3 0.2Butyric acid 0.4 0.82,5-Dimethyl-4-hydroxy-3(2H) furanone 2.0 1.0Methyl cinnamate 1.3 2.0γ-Undecalactone 0.9 0.5Propylene glycol 91.6 88.5Total 100.0 100.0



flavorings are produced every day by housewives in kitchens, by food industriesduring food processing, and by the flavor industry. The International Organizationof the Flavor Industry (IOFI) has guidelines for the production and labeling ofprocess flavorings (IOFI, 1990). Some key points are that reactants are strictlyappointed; flavorings, flavoring substances, flavor enhancers, and process flavoradjuncts shall be added only after processing is completed; and the processingconditions should not exceed 15 min at 180°C or proportionately longer at lowertemperatures, while the pH should not exceed 8.0.

Process flavors are very successful in some cases, but also unsuccessful in manycases. Natural flavor materials, such as meat extract or aromatic chemicals, may beadded to process flavors to enrich some notes or to increase the overall intensity.

11.5 BIOTECHNOLOGICAL PRODUCTIONOF FLAVORS

11.5.1 MICROBIAL PRODUCTION OF FLAVOR COMPOUNDS

Food products labeled as containing “all natural flavors” have a much higher marketprice than similar products containing artificial flavors. This preference has led to astrong upsurge in the requirement for natural flavor. The increasing demand for theseflavor compounds now exceeds their supply from traditional sources. This has moti-vated research efforts toward finding alternative natural ways of obtaining naturalflavor compounds. In the United States, the Code of Federal Regulations states thatproducts produced or modified by living cells or their components, includingenzymes, can be designated as “natural.” Thus, the use of biotechnological methodsusing microorganisms, enzymes, and recombinant DNA is a promising, economical,and environmentally friendly process for generating natural flavor compounds.

Traditional fermentation using microbial activity is commonly used for theproduction of nonvolatile flavor compounds, such as acidulants, amino acids, andnucleotides. The formation of volatile flavor compounds via microbial fermentationon an industrial scale is still in its infancy. Although more than 100 aroma compoundsmay be produced microbially, only a few of them are produced on an industrialscale. The reason for that is probably due to the transformation efficiency, cost ofthe processes used, and our ignorance of their biosynthetic pathways. Nevertheless,the exploitation of microbial production of food flavors has proved to be successfulin some cases. For example, the production of γ-decalactone by microbial biosyn-thetic pathways led to a price decrease from $20,000/kg to $1,200/kg (in U.S.dollars). Generally, the production of lactone can be performed from a precursor ofhydroxy fatty acids, followed by β-oxidation from yeast bioconversion (Benedettiet al., 2001). Most of the hydroxy fatty acids are found in very small amounts fromnatural sources, and the only inexpensive natural precursor is ricinoleic acid, themajor fatty acid of castor oil. Due to the few natural sources of these fatty acidprecursors, the most common processes have been developed from fatty acids bymicrobial biotransformation (Hou, 1995). Another way to obtain hydroxy fatty acidsis from the action of lipoxygenase (LOX). However, there has been only limitedresearch on using LOX to produce lactone (Gill and Valivety, 1997).



Vanillin, perhaps the most important aroma compound, occurs in the bean ofVanilla planifolia. At present, only 0.2% of this compound is extracted from beans,and the remainder is produced synthetically in the world flavor market. Thus, theproduction of vanillin via microbial transformation has been the most extensivelyinvestigated. Figure 11.1 shows some of the possible routes for the production ofnatural vanillin by microorganisms. The transformation of the natural stilbeneisorhapontin to vanillin is catalyzed by a microbial stilbene dioxygenase. This pro-cess has led to many patents (Hadegorn and Kaphammer, 1994; Walton et al., 2003).Alternative precursors are eugenol (Washisu et al., 1993) or isoeugenol. (Shimoniet al., 2000). However, the bioconversion produces relatively low yields. Direct useof ferulic acid to produce vanillin is probably the most promising approach due tothe fact that this precursor is a constituent of various grasses and crops, and is aproduct of the microbial oxidation of lignin (Martínez-Cuesta et al., 2005). Anexample is the use of sugar beet pulp or maize bran as the source of ferulic acid toproduce vanillin using two fungi (Bonnin et al., 2001). However, its recovery as apure precursor is difficult, and the existence of numerous side reactions may explainthe low bioconversion yields.

Benzaldehyde is the second most important material after vanillin. It is used asan ingredient in cherry and other natural fruit flavors in the flavor industry. Naturalbenzaldehyde is generally extracted from fruit kernels such as apricots. Nowadays,the fermentation of natural substrates, such as phenylalanine, offers an alternativeroute for the biosynthesis of natural benzaldehyde (Moller et al., 1998). However,production via the fermentation process can become commercially acceptable onlyif sufficient yields can be obtained.

Other applications of microorganisms used in the production of flavor com-pounds are listed in Table 11.2.

11.5.2 ENZYMATIC GENERATION OF FLAVOR COMPOUNDS

More than 3000 enzymes have been described in the literature, but only 20 areavailable for use in commercial processes (Armstrong and Brown, 1994). Lipases,esterases, lipoxygenases, glycosidases, proteases, and nucleases are the major enzymesused for flavor generation. Among them, lipases seem to be the most important enzymein commercial use. The lipase-catalyzed reaction includes hydrolysis, esterification,and transesterification. The reaction can be performed not only in aqueous systems,but also in organic solvents (Jaegar et al., 1994). Based on these features, lipases arebeing increasingly used to synthesize “natural” flavoring materials such as esters(Abbas and Comeau, 2003; Langrand et al., 1990). The (S)-2-methylbutanoic acid

FIGURE 11.1 Proposed pathways for the biosynthesis of vanillin.

EugenolConiferaldehyde

Ferulic acidVanillin

Isoeugenol

microbial stilbene dioxygenase

maize bran/sugarbeet pulp

Isorhapontin



methyl ester, which is known as a major flavor of apple and strawberry, is synthesizedusing lipase in organic media. Low-molecular-weight esters (LMWE) are flavoringagents for fruit-based products. Screening 27 commercial lipases shows that enzymesfrom Candida cylindracea, Pseudomonas fluorescens, and Mucor miehei (immobi-lized) promote synthesis of LMWE in nonaqueous systems (Rodriguez-Nogales etal., 2005; Welsh et al., 1990). LMWEs have also been produced using plant seedlinglipase (Liaquat and Apenten, 2000). The catalysis efficiencies among microbiallipases are different (Kwon et al., 2000). Isoamyl acetate, one of the most employedflavor compounds in the industry, can be produced by using immobilized lipase(Krishna et al., 2001).

Green note is present in a wide variety of fresh leaves, vegetables, and fruits.The characteristic aroma compounds responsible for green note include trans- andcis-2-hexenol, trans- and cis-3-hexenol (leaf alcohol), hexanol, hexanal, and cis-2-hexenal (Whitehead et al., 1995). These compounds are biosynthetically producedusing lipoxygenase (LOX) pathway enzymes (Fabre and Goma, 1999; Fukushigeand Hildebrand, 2005; Hatanaka, 1993; Németha et al., 2004). The commercialprocesses for generating green odor compounds have been established using LOXpathway enzymes as shown in Figure 11.2. Four major enzymes, that is, lipolyticenzyme, LOX, hydroperoxide lyase (HPLS), and alcohol dehydrogenase (using bakeryeast as a source) are involved in the formation of green odor compounds (Hatanaka,1993). Lipids such as commercial soybean oil or vegetable oil are hydrolyzed tofatty acids by lipolytic enzymes. Fatty acid 13-hydroperoxides are formed from theaction of a specific LOX, and then cleaved by HPLS into C6-green odor compounds.Among the various sources containing LOX activity, soybean (Gardner, 1989), pea(Chen and Whitaker, 1986), tomato (Riley et al., 1996), potato (Galliard and Phillips,1971), cucumber (Hornostaj and Robinson, 1999), almond (Santino, et al., 2005),banana leaf (Kuo et al., 2006), green algae (Hu and Pan, 2000; Kuo et al., 1991),and microorganisms (Bisakowski et al., 1997) are worth mentioning. HPLS is notavailable in commercial sources. It is used as vegetable homogenate. Mung beanseedlings (Rehbock et al., 1998) and guava (Tijet et al., 2000) show higher HPLS

TABLE 11.2Aroma Compounds Produced by Microorganisms

Aroma compounds Microorganisms Reference

Acetic acid bacteria Sharpell and Stemann, 1979Diacetyl bacteria Cheetham, 1996Geosmin bacteria Pollak and Berger, 19962-Acetyl-pyrroline bacteria Romanczyk et al., 1995Lactone yeast Benedetti et al., 2001Linalool yeast Welsh, 1994Benzaldehyde fungi Fabre et al., 1996Vanillin fungi Bonnin et al., 20011-Octen-3-ol fungi Assaf et al., 1995; Zawirska-Wojtasiak, 2004Jasmonate fungi Miersch et al., 1993



activity than other sources in producing green odor. Natural 2(E)-hexenal is producedin two steps from hydrolyzed linseed oil, which contains the most linolenic acidamong the available natural sources. In the first step, 13-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13-HPOT) is formed from linolenic acid bysoybean LOX-1. In the second step, 13-HPOT is cleaved by green bell pepper HPLS(Németha et al., 2004). Continuous generation of green odor volatiles using a biore-actor immobilized with LOX and HPLS has been investigated (Cass et al., 2000).

11.5.3 RECOMBINANT DNA TECHNOLOGY FOR FLAVOR FORMATIONS

The application of recombinant DNA technology in the flavor industries is lessadvanced than in the pharmaceutical and food industries. However, this technologyseems to have the most potential in future research. Nowadays, recombinant DNAtechnology has been increasingly used in several areas, including production ofaroma chemicals, improved flavor profiles through genetic engineering, removal ofoff-flavor, and enzymatic formation of flavor aldehyde (Muheim, 1998).

Fermentative or enzymatic processes are used to produce many flavor compoundsas mentioned before. However, most of the above-mentioned techniques have onlybeen reasonably applied so far, due to the fact that the enzymes or aroma chemicalsare generally produced in very small amounts, making their recovery an expensiveendeavor. Recombinant DNA technology allows efficient production of enzymes orflavor compounds. A good example is the production of green note compounds viaLOX pathway enzymes. The LOX and HPLS are the determinant enzymes for theconversion of fatty acids into natural food flavor components. Due to those enzymesoccurring in natural sources at low levels with instability and difficulty in purification,considerable efforts have been made to clone LOX or HPLS for commercial uses

FIGURE 11.2 Formation of green odor compounds via lipoxygenase pathway enzymes.

lipid

linolenic acid

l

linoleic acid

ipolytic enzyme

lipoxygenase

lipid

lipolytic enzyme

oxygenaselip13-hydroperoxide13-hydroperoxide

Hexanal

hexanol

hydroperoxide lyase

alcoholdehydrogenase


isomerase


cis-3-hexenals

cis-3-hexenoltrans-2-hexenalhexanol

hydroperoxide lyase


alcohol


dehydrogenase

trans-2-hexenol



in the production of natural flavor components. Many plant LOX from soybean seed(Steczko et al., 1991), pea seed (Hughes et al., 1998), potato tuber (Royo et al.,1996), and HPLS from tomato (Atwal et al., 2005), guava (Tijet et al., 2000), alfalfa(Noordermeer et al., 2000), and pepper (Bourel et al., 2004; Matsui et al., 1996)have been cloned and expressed in E. coli or yeast. The green note compoundsproduced from such recombinant yeast cells bearing the enzyme genes are identicalto those from the native enzymes. In addition, flavor compounds (such as leafalcohol) have been produced in larger amounts using such recombinant enzymes,than using native enzymes.

11.6 APPLICATIONS OF FLAVORS

Choosing the right type and dosage of flavor and adding the flavor at the right stepin food processing are important to flavor applications. A flavor can be admired onlyafter suitable application. Due to different application conditions, flavors are madeto have different characteristics, such as solubility in water or oil, and heat stability.There is no general rule for flavor applications. Flavor users need some basicknowledge of flavor, food chemistry, and processing technology, in order to handleflavor applications properly. For example, citral is the key compound of lemon flavor.If the flavor has undergone thermal treatment severe enough to let it be oxidized,then the hemiacetal formed causes changes in flavor. Limonene is a major constituentin citrus oils. It has to be removed to prevent off-flavor production in food processingand storage. The extent of evaporation loss of each flavor ingredient is different infood processing. Some food components, such as starch, lipids, and proteins, cantrap flavor compounds and reduce their volatilities. Some foods have their ownflavors or flavor production. Therefore, modification of flavor formulas is needed toaccommodate the flavor identity of different processed foods. Studies on flavorapplication for each food product are required to find the right strength, right form,and right step. Technical support for flavor users are standard services provided byflavor manufacturers.

Flavors can be used to develop new food products. At the 1995 IFT Food EXPO,flavor manufacturers created unique berry flavors that do not exist naturally and aremore exciting than natural aroma products. Mayberry, pepperberry, juneberry, moun-tainberry, bugleberry, and bellberry were all fabricated by creative flavor manufac-turers (Sloan, 1995). Creating new flavors for innovative flavor applications is achallenge to the flavor industry, thus leading to the development of newer and high-quality food products for quality living.

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329

12 Interactions of Food Components

Zdzisław E. Sikorski and Norman F. Haard

CONTENTS

12.1 Introduction................................................................................................ 33012.2 Water–Protein Interactions ........................................................................ 331

12.2.1 Water in Proteinaceous Structures .............................................. 33112.2.2 Antifreeze Proteins—Ice Crystal Interactions ............................ 33312.2.3 Protein–Protein and Protein–Polysaccharide Interactions.......... 334

12.3 Water–Lipid and Protein–Lipid Interactions............................................. 33512.4 Polysaccharide Interactions in Food Systems........................................... 336

12.4.1 Polysaccharide–Water Ions–Polysaccharide Interactions........... 33612.4.2 Polysaccharide–Lipid Interactions .............................................. 337

12.5 The Effect of Interactions on the Color of Foods .................................... 33812.5.1 Changes in Meat Pigments ......................................................... 33812.5.2 Changes of Chlorophylls............................................................. 33912.5.3 Interactions of Carotenoids ......................................................... 34012.5.4 Interactions of Other Pigments ................................................... 34112.5.5 Browning and Formation of Black Spots ................................... 341

12.6 Interactions Affecting Food Flavor ........................................................... 34212.6.1 Introduction ................................................................................. 34212.6.2 Hydrolytic, Oxidative, and Pyrolytic Reactions......................... 343

12.7 Changes in Texture of Foods .................................................................... 34412.7.1 Introduction ................................................................................. 34412.7.2 Rigor Mortis and Tenderization of Meat.................................... 34412.7.3 Texture Changes in Frozen Fish ................................................. 34612.7.4 Cross-Linking in Gels and Films................................................ 34612.7.5 Interactions in the Dough............................................................ 34712.7.6 Lipids and the Texture of Foods................................................. 348

12.8 Interactions and the Nutritive Value of Foods .......................................... 34912.8.1 Introduction ................................................................................. 34912.8.2 Changes in Digestibility.............................................................. 34912.8.3 Changes in Biological Value....................................................... 350

12.9 The Effect of Interactions on the Safety of Foods ................................... 35112.10 Final Remarks............................................................................................ 352References.............................................................................................................. 353



12.1 INTRODUCTION

Foods contain a multitude of various compounds. The components of foods formthe structural elements of different tissues in living plants and animals, serve asreserve material and sources of energy, and perform numerous other biochemicalfunctions. In natural or processed foods they are present in solutions or as a varietyof structures, stabilized by covalent bonds, hydrogen bridges, ionic forces, andhydrophobic interactions.

Foods can be classified as (1) intact tissue systems, such as fruits and vegetables,cereals, pulses, eggs, meats; (2) disrupted tissue systems, such as ground meat,purees, flour, tomato catsup; (3) noncellular or disrupted cellular systems, such asmilk, fruit juices, honey, oils; or (4) combinations thereof (Haard, 1995).

The interactions of components are of a physicochemical, biochemical, or chem-ical nature. The physicochemical interactions are responsible for the formation ofemulsions, foams, and also partially gels. Thus they have great impact on the sensoryquality of various food commodities and on the resistance to shearing, as well ason the flow of the material in the processing equipment. Chemical and biochemicalreactions in fresh and processed foodstuffs may affect all quality attributes includingthe color, appearance, flavor, aroma, texture, biological value, nutritional properties,safety, and processing suitability of foods. Thus they may improve the edible char-acteristics or in other cases delimit storage life.

It is not always clear whether interactions or reactions occurring in foodstuffs arenonenzymic, enzyme catalyzed, or result from microbial metabolism. For example,the development of bad odor in coleslaw prepared with mayonnaise and sour creamwas first attributed to microbial spoilage. However, study of the problem revealed thatendogenous enzymes in the cabbage were activated by the anaerobic conditions in thepackage and were responsible for the formation of offensively smelling volatiles. Ingeneral, enzyme-catalyzed reactions are of primary importance in untreated and intactplant and animal tissues, while nonenzymic reactions predominate in properly pro-cessed foodstuffs. There are, however, some exceptions to this general rule. In addition,enzymatic and nonenzymatic reactions may act in concert. For example, a color changeof the surface of red meats is caused by nonenzymatic reactions of desirable oxygen-ation and subsequent undesirable oxidation of myoglobin (see Section 12.5.1). How-ever, enzymatic reactions involved with respiration at the tissue surface can loweroxygen concentration and indirectly promote oxidation of myoglobin (MbFe(II)).Some tissues also contain the enzyme metmyoglobin (MbFe(III)) reductase, whichcan reverse the nonenzymic oxidation of MbFe(II). Likewise, texture deterioration thatoccurs during frozen storage of fish of certain species is primarily caused by thenonenzymic denaturation and aggregation of the major muscle protein myosin. How-ever, protein denaturation and cross-linking may be accelerated by the action of trim-ethylamine oxide demethylase, which liberates reactive formaldehyde or by phospho-lipase, which forms interactive free fatty acids (see Section 12.7.3).

The chemical and biochemical reactivity of the components in food systems iscontrolled by their chemical character (Table 12.1), compartmentalization in thetissues, the activity of enzymes and different enzyme inhibitors, and predominantlyby environmental conditions of storage and processing. The most chemically reactive


Interactions of Food Components 331

food components are reducing sugars and other carbonyl compounds, thiol groupsin proteins, ionizable and heterocyclic amino acid (AA) residues, polyenoic lipidsand pigments that are readily oxidizable, phenolics, terpenes, and radicals. Theconcentration of some of these compounds changes during storage of foods due tothe activity of endogenous enzymes. Some reactive groups are hidden in the structureof food polymers and thereby are not always accessible to interactions; other poten-tial substrates are separated by cellular compartmentalization in the intact tissues.Initially masked or protected food components may later interact as a consequenceof food storage and processing conditions. Maillard reactions, some enzyme-cata-lyzed reactions, and various oxidation processes are examples of important interac-tions that are facilitated by tissue disruption or processing.

The interactions in many foods may be affected by physical changes associatedwith handling of the raw material that leads to damage of the structure, like rupturingof the cell membranes in fish muscle, fruits, and vegetables by large ice crystalsformed in the intracellular spaces during slow freezing, selective cell breakage undercontrolled conditions of milling, which results in disruption of the endosperm cellsof the wheat grain, or disintegration of the muscle architecture during comminutingof meat in sausage manufacturing. Different interactions of food components mayalso be enhanced by high hydrostatic pressure of the order of 100 to 600 Mpa.Biochemical reactions in living tissues can also be a function of physiologicalprocesses related to ontogeny (e.g., fruit ripening and plant senescence) as well asstress and biological strain (e.g., chilling injury of plants or pale, soft, exudativecondition in myosystems). Moreover, the chemical reactivity and quantity of tissuecomponents that interact postharvest may be influenced by preharvest factors suchas the age or nutrition of the plant or animal and the plant cultivar or animal breed.

12.2 WATER–PROTEIN INTERACTIONS

12.2.1 WATER IN PROTEINACEOUS STRUCTURES

Water in foods serves as the solvent of numerous solutes and is normally thecontinuous, dispersing phase of solid components. In dehydrated foods, with loweredwater activity, the mobile and dispersing phase may be lipid. Water is not only the

TABLE 12.1Reactive Groups of Food Components

–SH –S–S––NH2 –NH–C(=NH)NH2 –CONH2

–OH –CHO R2C=O1O2

•O2– •OH H2O2 RO• ROO• ROOH ArO• ArOO•

–COOH –O–SO3H –O–PO3H2

–CH=CH– –CH=CH–CH2–CH=CH–CH3–(CH2)n–CH2– and other hydrophobic groups Ca2+ Mg2+ Fe2+ Fe3+ Cu2+ Co2+ Zn2+ and other cationsAdditives: NO• NO2

• O=N–OOH O=N–OO–



solvent in which reactions occur. It participates directly in many reactions such asprotein folding, protein–protein interactions, protein condensation, and hydrolysis.Proteins may occur in foods in solution or as fibrils, filaments, sheets, and particles.Fibrils, filaments, and sheets build the structure of cross-striated muscle (see Chapter2). Wheat gluten forms fibrils and strands, with starch granules adhering to theprotein matrix. The caseins of milk form polydisperse, spherical particles, from about25 nm to about 600 nm in diameter. Almost all seeds contain protein granules, alsocalled protein bodies or aleurone grains. These bodies, generally spherical, differ insize, depending on the species and variety of the cereal or legume. In corn, theaverage diameter is about 2 µm, in sorghum it ranges from 0.5 to 3 µm; in broadbeans 1 to 5 µm, in peanuts 2 to 10 µm, and in soybeans 2 to 20 µm. The proteinbodies of soybeans are nestled within a spongelike cytoplasmic protein network andare accompanied there by spherical lipid droplets (Heertje 1993).

Interactions with water are crucial for the conformation of proteins in moist foodsystems due to hydrogen bonding, hydrophobic and van der Waals interactions, andthe influence on ionic bridges. Water participates in hydrogen bonds with polar sidechains of proteins (Figure 12.1) and may form highly ordered water molecules ascages around hydrophobic residues. Because the latter is energetically unfavorabledue to the entropy decrease, the formation of amphipathic proteins into stablestructures, such as micelles or protein bodies, is closely related to water–proteininteractions. They are thus responsible for the stability of proteinaceous structuresin many foods, such as the functional properties of proteins in meat and fish productsand the casein micelles. Water is also an important reactant in which it is eliminated(condensation) or added (hydrolysis).

Water is the most abundant substance in living systems, making up more than70% of the weight of most tissues. Its amount in the tissues of slaughter animals,fish, shellfish, and mollusks is from about 3 to 7 times higher than that of proteins.Water can be held in meat mainly due to entrapment within the microstructure oftissues and because of various direct interactions with protein molecules. This prop-erty of muscle foods is known as water-holding capacity or water-binding potential.Any treatments, additives, and biochemical processes that result in loosening of the

FIGURE 12.1 Water molecules forming hydrogen bonds with amino acid residues.

C C N

R

H

H

C C

O

H

RN

H

CC

O

HR

NC O

HOHO

H

HO

H

H

H C HR

NH

C OCN

R

HH

CC

O

CN

H R

H

O

CN C

C

O O

CN

H

N

O

H

O

H

O H

H

NCC

H

R

O

O

C



myofibrillar structure by enhancing the mutual repelling of the fibrils, strands, andsheets increase the water-binding potential. Thus the factors affecting the retentionof water in meat systems, and thereby the juiciness of the products, include thecharacteristics of the proteins, the pH in the meat, the concentration of postmortemmetabolites, which accumulate in the process of tenderization, the presence ofadditives, mainly saccharides, salt, and phosphates, as well as changes in proteinsdue to freezing, frozen storage, mincing, and cooking.

The effect of the state of the proteins on the water-holding capacity can beobserved in pale, soft, exudative pork (PSE) and similar disorders in other foodmyosystems such as poultry and burned tuna meat. The PSE condition occurs dueto denaturation of muscle proteins at comparatively high temperatures and low pHin the carcass after slaughter. Burned tuna meat, instead of being red, translucent,and firm as prime quality tuna, is pale, exudative, soft, and slightly sour. It occursespecially frequently in hand-line-hooked fish that fought only a few minutes duringthe catch, but rarely in specimens that were subjected to destruction of the spinalcolumn or brain immediately after capture. According to the hypothesis of Watsonet al. (1988), high stress of the animal during hooking and fierce fighting causes alarge increase in concentration of the hormones norepinephrine and epinephrine inthe blood. These catecholamines potentiate muscle proteolysis by calpains in post-mortem muscle. Because the catecholamines in tuna are rapidly metabolized andhave a half-life of about 30 minutes, their concentration in the blood of long-line-caught fish after several hours in water is not high enough to affect the proteolysisof muscle proteins. The rapid lowering of pH in muscles is due to the decompart-mentalization of Ca+2 and its interaction with Ca+2-ATPase. The hydrolysis of ATPresults in H+ formation and a lowering of pH. Brain destruction decreases the rateof postmortem dephosphorylation of ATP in the muscles, and thus prevents rapidleaking of Ca2+ from the sarcoplasmic reticulum into the sarcoplasm. At a lowconcentration of Ca2+ there is no activation of calpains or myosin ATPase.

12.2.2 ANTIFREEZE PROTEINS—ICE CRYSTAL INTERACTIONS

In frozen foods most of the water, about 80% at a temperature of about –30°C, ispresent as ice crystals. The size and distribution of the crystals depend, for example,in ice cream, primarily on the rate of freezing and on shearing forces during proc-essing.

Some proteins interact with the water molecules in ice crystals. The blood offishes inhabiting polar oceans, as well as the blood of some insects, contains specificproteins, known as antifreeze proteins (AFP) and antifreeze glycoproteins (AFGP).The AFP are responsible for the noncolligative depression of the freezing point ofthe blood serum of these animals. Both the AFP and AFGP are preferentially boundto the ice lattice and retard the growth of the crystals.

Different mechanisms of the specific binding of AFP to the ice lattice have beenproposed, whereby the contribution of the hydrogen bonds, entropic effects, and vander Waals interactions has been postulated (Chao et al. 1997). The winter flounderAFP contains 4 ice-binding regions grouped along the polypeptide chain so that theyare compatible with the ice surface topology (Cheng and Merz 1997). Therefore the



AA residues in all regions simultaneously form 5 to 6 hydrogen bonds each withthe water molecules in the crystals. The proper sequence of the AA residues is alsonecessary for the van der Waals interactions and the hydrophobic effect to participatein the binding of the protein.

12.2.3 PROTEIN–PROTEIN AND PROTEIN–POLYSACCHARIDE INTERACTIONS

Most proteins with a molecular weight greater than 100 KDa have multiple subunits,identical or different. In nature, multimeric proteins can have from two to hundredsof subunits. Some protein assemblages, such as collagen, serve primarily a structuralrole in living systems. In other cases multimeric proteins have enhanced binding orcatalytic properties, such as the binding of oxygen to multimeric hemoglobin. Inliving systems, protein folding and protein–protein interactions may be spontaneousor may require the assistance of specialized proteins called molecular chaperones.As well, disulfide bond formation and the cis-trans isomerization of proline peptidebonds are catalyzed by specific proteins. In food systems, protein–protein interac-tions are normally facilitated by changes in environmental conditions, notably tem-perature and pH, by chemical cross-linking agents like formaldehyde, or by theactivity of enzymes, such as chymosin or transglutaminase.

There are numerous proteins in particular food commodities. Differing in AAcomposition and sequence and in molecular weight they behave differently in foodsystems. In some proteins the distribution of hydrophilic and hydrophobic segmentsof the polypeptide chains is such that it favors hydrogen bonds, ionic interactions,and hydrophobic associations of the molecules after their deconformation (Table12.2). Interactions of various reactive groups and hydrophobic patches exposed onthe surface of the molecules lead to formation of various intermolecular links, whichresult in changes in protein hydration, solubility, viscosity of solutions, film forma-tion, gelling, and adsorption on the interface between aqueous and lipid phases.Examples of important food protein aggregations and interactions include the for-mation of cheese curds (aggregated, destabilized casein micelles) from acidification

TABLE 12.2Characteristic Sequences in Some Food Proteins

Protein Characteristic sequences

αs1-casein –SerP–Ile–SerP–SerP–SerP–Glu–β-casein –SerP–Leu–SerP–SerP–SerP–Glu–Collagen –Gly–Pro–Hyp–α-Gliadin –(His)3–(Gln)6–Pro–Ser2–Gln–Val–Ser–Tyr–(Gln)2–Phosvitin –Asp–(SerP)6–Arg–Asp–

–Ser–Asn–Ser–Gly–(SerP)8–Arg–Ser–Winter flounderantifreeze glycoprotein –Asp–Thr–Ala–Ser–Asp–Ala6–Leu–Thr–Ala2–Asn–Ala–Lys–Ala3–Glu–

Leu–Thr–Ala2–Asn–Ala7–Thr–Ala–Arg–



of milk or chymosin catalyzed hydrolysis of κ-casein, interaction and cross-linkingof myosin causing loss of succulence of texture in fish of some species, aggregationof albumin proteins in heated egg, and development of gluten from the wheat proteinsgliadin and glutenin.

Many foods contain substantial amounts of glycoproteins, that is, proteins linkedto various monosaccharide or oligosaccharide moieties via O-glycosidic or N-gly-cosidic bonds. The saccharides are bound to the residues of hydroxyamino acids orasparagine. The best known food glycoproteins include κ-casein, ovoglycoprotein,ovomucoid, ovomucin, avidin, ovoinhibitor, ovalbumin, and some collagens, espe-cially fish collagens. The saccharide moieties may participate in covalent interchaincross-links and affect many functional properties of the glycoproteins, such as theirtensile strength, solubility, viscosity in solutions, ability of interactions with otherfood components, and biological activity.

Some proteins participate in interactions with polysaccharides. This may causeprecipitation or stabilization of the proteins even in the presence of precipitatingagents. The neutral polysaccharides like locust bean gum and guar gum, as well asmost saccharide polyanions, including gum Arabic, carboxymethylcellulose, andpectins do not prevent precipitation of casein micelles and αS1-casein by Ca2+ at pH6.8, while carrageenans, especially κ-carrageenan, form stable complexes with thisprotein (Phillips and Williams 1995). From NMR studies it can be deduced that bothhydrogen bonds and van der Waals contacts confer stability on the interaction ofwheat germ agglutinin with oligosaccharides (Espinosa et al. 2000). Under appro-priate conditions, polysaccharides may interact with proteins via Maillard reactionsand Strecker degradation forming products with reduced nutritional value, changedcolor and flavor, and altered texture.

12.3 WATER–LIPID AND PROTEIN–LIPID INTERACTIONS

In water-in-oil emulsion-type products, such as butter, margarine, and fat spreads,water occurs as droplets. In butter, the diameter of the droplets is below 10 µm, inspreads below 5 µm. Water droplets have a structure-forming role in such foods.Depending on their size and distribution they affect the rheological and sensoryproperties, as well as bacterial stability of the products. The stabilization of thedroplets is due to interactions with solid fat particles, monoacylglycerols and phos-pholipids, or proteins that accumulate at the water–lipid interface. The coalescenceof the droplets is prevented by interactions with fat lamellae.

In milk and dairy commodities, in mayonnaise, salad dressings, ice cream, andcomminuted meat and fish products, the lipids form globules dispersed in the aqueousphase. In fresh and in ultrafiltered milk the globules, 0.5 to 10 µm and 1 to 18 µmin diameter, respectively, are separated from each other and from the aqueousmedium by phospholipid-proteinaceous membranes, about 10 nm thick. On thesurface of these membranes are adsorbed lipoprotein particles. In whipped creamand ice cream the fat globules are assembled on the interface of the aqueous dis-persing phase/air bubbles. In conventionally produced ice cream the fat droplets are



about 1 µm in diameter. High-pressure homogenization, at about 2000 bar, makesit possible to reduce the size of the droplets to 0.3 µm and thus increase the perceptionof creaminess. In fresh cheese the globules are surrounded by a protein shell anddistributed in a protein network. In butter the globules are in a matrix of free fat.The core of these globules is formed by liquid fat that is covered by fat crystals.The softening temperature of butter depends on the proportion of fluid fat to fatcrystals. In mayonnaise the oil droplets form a honeycomb structure stabilized withinthe dispersing aqueous phase by the egg yolk phospholipids, and probably by lipidcrystals at the surface.

Amphiphilic lipids form micelles or lamellar phases, when present in the aqueousenvironment in concentration above the critical value. Different types of micelleshave various shapes, depending on the surface area of the polar groups and thevolume and length of the hydrophobic chains. The lipids may assemble into hexag-onal micelles. In one type of micelle, the polar heads of the amphiphilic lipidsprotrude to the surface, while the hydrocarbon chains are directed axially towardthe center of the cylinder. In another type of structure, the polar groups point to theaxis forming a hydrophilic channel inside, with the hydrophobic tails stretchingoutside. Lipidous lamellar liquid crystalline phases self-assemble by stacking ofstretched lipid bilayers separated by water lamellae. In wheat flour lipids, a transitionfrom the micellar to the lamellar phase takes place on hydration (Marion et al. 1998).

Noncovalent lipid protein complexes in foodstuffs are known to alter texture,for example, in the interaction of fatty acids and protein in frozen fish and in wheatdough gluten development. Wheat flour contains some low-molecular-weight pro-teins rich in cysteine and basic AA residues, which in the native state spontaneouslybind lipids or lipid aggregates. The thionins, ligoline, lipid transfer proteins, andpuroindolines belong here. They facilitate the spreading of monolayers of lipids atthe air–water interfaces, thus acting as very efficient foam stabilizers. Interactionsof puroindoline-a with lysophosphatidylcholine have a synergistic effect on foamstability (Marion et al. 1998). Moreover, degradation products of fatty acid hydro-peroxides (aldehydes, alcohols, acids, epoxides, ketones, cyclic fatty acid monomers,dimers, polymers) can interact with proteins in the Maillard reaction.

Fiber formation during high-moisture extrusion can be enhanced by the additionof some polysaccharides, such as starch, soy arabinogalactans, or maltodextrins inthe food mix before extrusion. During extrusion, polysaccharides form a separatephase, which appears to enhance protein aggregation in the direction of flow andreduce it in the direction perpendicular to extrusion (Akdogan 1999).

12.4 POLYSACCHARIDE INTERACTIONS IN FOOD SYSTEMS

12.4.1 POLYSACCHARIDE–WATER IONS–POLYSACCHARIDE INTERACTIONS

Interactions of polysaccharides with each other, ions, proteins, and lipids affect thewater holding, gelling, film forming, viscosity, crystal growth, and stabilization offoams and lipid emulsions. Thus they contribute to the sensory properties of meat



and fish products, milk gels, custards, creams, ice cream, salad dressings, cakes,bakery fillings, jam, marmalade, jelly, fruit drinks, and instant beverages, as well asto the tensile strength and barrier properties of biodegradable films and coatings.

Starch granules are abundant in many raw plant tissues and in processed foods.In most food products the granules occur in different modified forms due to variousstages of gelatinization and swelling, depending on the botanical variety of the starchyfood material, the water availability, and conditions of heating during processing.

The hydration of neutral polysaccharide networks depends on their affinity tothe solvent. The immobilization of water molecules is, to a large extent, due tohydrogen bonding with the hydrophilic groups of the hydrocolloids. In a polyelec-trolyte gel, like that of pectins, the swelling is increased by the effect of the coun-terions that accumulate within the network to neutralize the electrical charge of thepolymer. However, cross-linking of the pectins via Ca2+-mediated ionic bonds maydecrease swelling by counteracting expansion of the network. Polyanionic hydro-colloids interact with cationic counterions. Depending on the kind of anionic groupof the polysaccharide—CO2

–, PO32–, or SO4

–—and the properties of the cations thecounterions are bound together electrostatically with their coordinated water dipolesor after displacement of the hydration shell. Thus the effects of binding of variouscations to different anionic hydrocolloids, such as carboxymethylcellulose or carra-geenan, must be considered in selecting gum additives in food systems.

The interactions of water with many plant hydrocolloids (gums) generally donot lead to gelling. Instead, very viscous, pseudoplastic solutions are formed, evenat a low concentration of polysaccharides, due to the large molecular size of thepolymers. The rheological behavior of solutions of neutral gums is pH-stable, whilethe viscosity of solutions of polymers with ionizable groups depends significantlyon the pH of the system (Ramsden 2004).

Cryoprotective agents have been studied for use in food processing. A series ofmono-, oligo-, and polysaccharides; di- and polyalcohols; hydroxymonocarboxylicacids; and di- and tricarboxylic acids and highly branched oligosaccharides havebeen used as cryoprotectants (Auh et al. 2003). The physical mechanisms of cryo-protection by saccharides are not fully understood. It has been suggested that cryo-protectant activity is related to the replacement of the main hydration shell of polarhead groups of membrane phospholipids by hydroxyl groups of sugars. In otherwords, sugars serve as water substitutes when the hydration shell of a protein isremoved. All sugars interact strongly with water and participate in the water latticethrough hydrogen bonds. This presumably leads to the formation of extended regionsof hydrogen-bonded structured water in the vicinity of the cryoprotective molecule,which in turn may stabilize the surface hydration of proteins and protect them fromfreeze injury. The interaction of saccharide molecules with water is an essentialprocess in the cryopreservation mechanism, but no real evidence of this hydrationprocess has yet been produced.

12.4.2 POLYSACCHARIDE–LIPID INTERACTIONS

In aqueous systems these interactions are due to hydrophobic effects, like in bindingof a monoacylglycerol molecule by amylose. Accommodating the hydrophobic



saturated fatty acid residue inside the amylose helix forms an amylose–lipid com-plex. Heating increases the degree of binding of lipids by amylose, as in doughduring bread baking. Amphiphilic lipid–amylose interactions decrease the rate ofbread staling. The amylose–lipid complexes may also form gels. The rheologicalproperties of these gels depend on the concentration and type of lipids, and on thecrystalline form of the complexes (Eliasson 1998).

Interactions with lipids are responsible for the emulsifying properties of somehydrocolloids, such as gum Arabic. This polysaccharide, although having largemolecules, forms water solutions of comparatively low viscosity. This is becausegum Arabic also contains among its components a high molecular mass arabinoga-lactan–protein complex, in which the polypeptide chain is probably located at theperiphery of the macromolecule. The surface hydrophobicity of the polypeptideenables the adsorption of the gum on the oil–water interface. Denaturation of theproteinaceous moiety by heating leads to loss in the emulsifying efficiency of thehydrocolloid.

Because the polysaccharide–lipid interactions affect the rheological propertiesof the system, they may be utilized in formulating low-calorie emulsion-type foods.An emulsion of 20% soybean oil in a water solution containing 1 to 1.5% micro-crystalline cellulose behaves like a 65% pure oil emulsion with respect to viscosity,flow properties, and stability (Phillips and Williams 1995).

12.5 THE EFFECT OF INTERACTIONS ON THE COLOR OF FOODS

12.5.1 CHANGES IN MEAT PIGMENTS

The color of meat of slaughter animals, fish, mollusks, and crustaceans is affectedmainly by the contents and the chemical state of hemoproteins, predominantlyMbFe(II) and to a lesser degree hemoglobin, cytochromes, and hemocyanin; thepink to red color of the flesh of salmonid fishes is due to carotenoids. The contentof hemoproteins depends on the species, age, sex, nutrition, and living conditionsof the animal, the type of muscle and muscle fibers, and on the efficiency of bloodremoval during slaughtering. The natural, cherry-red color of beef meat on the fresh-cut surface is due to reduced forms of the muscle hemoproteins. Oxygenation ofMbFe(II), hemoglobin, and cytochromes at high partial pressure of oxygen leads tothe formation of light cherry-red pigments. Oxymyoglobin (MbFe(II)02) oxidizesslowly to brown MbFe(III), which undergoes further oxidation by H2O2, which maybe generated due to bacterial metabolism, to the strong oxidant ferrylmyoglobin(MbFe(IV)=O). Oxidation of thiol groups in meat proteins may lead to cross-linking.MbFe(IV)=O decomposes slowly or reacts with another molecule of MbFe(II) toyield MbFe(III):

MbFe(IV)=O → MbFe(III)

MbFe(IV)=O + MbFe(II) → 2 MbFe(III)



A green discoloration in meat is due to sulfmyoglobin MbFe(III)SH, but otherfactors may also influence the color.

In order to stabilize the desirable light-red color of fresh meat, appropriatepackaging of the cuts or various antioxidants may be used. Different carotenoids areable to decrease the rate of oxidation of MbFe(II)02 and to reduce MbFe(IV)=O,thus increasing the color stability of meat (Mortensen and Skibsted 2000). Alsoα-tocopherol is known to stabilize MbFe(II)02 in beef. It acts most probably bydelaying the generation of primary lipid peroxides and the release of their water-soluble free-radical breakdown products, predominantly unsaturated aldehydes orHO ⋅ radicals. It may also increase the potential of reduction of MbFe(III) in meat,thereby stabilizing the desirable color (Faustman and Wang 2000). Dietary supple-mentation of livestock with vitamin E is effective in delaying lipid oxidation anddiscoloration of fresh beef.

The desirable color of fresh meat can also be obtained by the formation of thebright cherry-red carboxymyoglobin. Storing of the meat cuts in modified atmo-sphere packages in the presence of about 0.5% CO has been approved in variousforms in different countries.

In order to prevent browning due to cooking, as well as to develop the desirablecured-meat flavor and inhibit the outgrowth of Clostridium botulinum spores inpasteurized products, NaNO3 and NaNO2 are used for curing. NaNO3 in the curingbrine and in the meat undergoes reduction by bacterial enzymes to NaNO2, whichis reduced by the endogenous cytochrome oxidase further to NO. MbFe(II) can alsoreduce NO2 to NO. The interaction of NO with MbFe(III) in the presence of ascor-bate, thiol compounds, and NADH-flavins results in the formation of light-rednitrosyl myoglobin (MbFe(II)=NO). In heated cured meat, the color is due to thethermally stable nitrosyl hemochromogen (see Chapter 6).

In cooked poultry, the pinking of white meat is regarded as a defect that suggestsan undercooked and thus unsafe product. The possible causes of this defect may bethe presence of nitrosyl hemochrome formed due to an incidental nitrate or nitritecontamination, residues of undenatured MbFe(II) or MbFe(II)02, the occurrence ofreduced globin hemochromes in well-cooked products, and the formation of carbonmonoxide myoglobin. As little as 1 µg of NaNO2 in 1 g of meat is enough to developa pink color in cooked chicken and turkey. Other factors affecting the occurrence ofthe defect include the initial color of the raw material, as well as pH and theoxidation-reduction potential in the meat (Holownia et al. 2004).

12.5.2 CHANGES OF CHLOROPHYLLS

Chlorophylls are rather unstable pigments. Depending on the conditions of cookingand blanching of green vegetables, and particularly pH, temperature, cations, oxi-dizing agents, and enzyme activity, they turn into various green, olive-green, olive-brown, grayish-brown, or colorless compounds. Pigment synthesis and degradationin detached fruits and vegetables can be influenced by storage conditions, such aslight, temperature, and relative humidity, and by ethylene. Ethylene is formed inplant tissue during ripening, following wounding, or by exposure of the plant to airpollutants. By virtue of its hormone action, ethylene initiates the degradation of



chlorophyll in mature plant tissues like fruits and leafy vegetables. Normally pro-cedures are established to retard the process in vegetables (via low temperatures andmodified atmosphere storage) and promote it in ripening fruit (by ethylene expo-sure—degreening). The exact mechanism by which ethylene action leads to chloro-phyll degradation is not completely known. Chlorophyllase (EC 3.1.1.14) is a thy-lakoid membrane glycoprotein that catalyzes hydrolysis of the phytol side chain ofchlorophyll (no change in color), although it appears that hydrolysis is followed byoxidative degradation of the tetrapyrrole involving a peroxidase (chlorophyll:H2O2

oxidoreductase). The participation of different lipoxygenase isoenzymes has alsobeen implicated in chlorophyll and carotenoid degradation in growing plants andseeds. Conditions of low pH and high temperature favor the conversion of chloro-phylls to their corresponding pheophytins, which results in olive-brown discolorationof canned green vegetables (see Chapter 9).

12.5.3 INTERACTIONS OF CAROTENOIDS

Carotenoid pigments in vegetables and fruits are mixtures of up to several dozencompounds. They easily undergo oxidation in the presence of air and light, but ifproperly protected they resist oxidation even at cooking temperatures. Oxidation ofcarotenoids during storage and processing of foods causes loss of the characteristiccolor of many commodities. A typical example is the appearance of brown discol-oration on stored red peppers due to oxidation of capsanthin. Oxidation of caro-tenoids may also lead to the development of aroma compounds. Heat, light, or diluteacid conditions cause isomerization of all-trans-carotenoids to various cis isomers.The reaction results in loss of vitamin A activity and changes in color. The directionof color change (red or blue shift) differs with the carotenoid. In some cases, suchas canning of pineapple, the orange hue is intensified.

The skin of many fish and the shell of marine crustaceans may have vivid yellow,orange, red, purple, blue, silver, or green colors. The major components of thepigments are various carotenoids and the products of their interactions with proteins.In marine organisms the complexes are predominantly formed due to noncovalentinteractions of astaxanthin and canthaxanthin or their derivatives with proteins,glycoproteins, phosphorylated glycoproteins, glycolipoproteins, and lipoproteins.Numerous structures and colors of carotenoproteins are known due to the diversityof interacting molecules (Zagalsky et al. 1990). The colors fade during storage ofthe caught fish, especially in direct, bright light, because the carotenoprotein com-plexes dissociate.

The major carotenoid component in the carapace of lobster is the red astaxanthin,but in the live animal it is present in the form of a blue carotenoprotein crustacyanin.However, boiling the blue or blue-gray crustacean denatures the protein and releasesfree astaxanthin, whereby the color of the carapace turns bright red.

Carotenoids are also responsible for the pink flesh pigmentation of salmonidfish, mainly astaxanthin and canthaxanthin. Oxidation of these pigments, especiallyin the presence of oxidized lipids, is responsible for the loss of the appealing colorof the flesh. Bleaching of the surface of cold smoked salmon fillets may also be dueto leaking of the pigments in processing, in the course of salting and rinsing. Dietary



supplementation with carotenoids is effective in improving the pigmentation of themeat of reared salmonid fishes. In marine animals, other pigments also contributeto color and iridescence, such as the brown-black melanins and melanoproteins andblue-purple indigoins.

12.5.4 INTERACTIONS OF OTHER PIGMENTS

The red, violet, or blue color of anthocyanins present in fruits and flowers dependson their structure and on the pH in aqueous media. Hydrophobic interactions andhydrogen bonds between different anthocyanins and with some phenolic acids andalkaloids affect the color and its intensity in various plants. The stability of antho-cyanins in food products is rather low and depends on their structure. They aresusceptible to enzyme-catalyzed degradation, to light, heat, oxygen, pH, and ascorbicacid. Various anthocyanins differ significantly in stability. In stored, heat-treatedfruits and juices the color may gradually diminish or turn brown due to polymer-ization reactions. In the presence of Al, Fe, and Sn, purplish-blue or slate-graypigments may be formed in processed fruits (see Chapter 9). The addition of SO2

to the 4-position of anthocyanins forms a bisulfite derivative and causes decoloriza-tion. In processed foods (e.g., during storage of frozen tissue that has not beenblanched) anthocyanins can be co-oxidized by degradation products resulting frompolyphenol oxidase or peroxidase-catalyzed oxidation of phenolic compounds.Decolorization of anthocyanins normally occurs following deglycosylation, and thisis catalyzed by endogenous or fungal glycosidases called anthocyanases.

Betalains, occurring predominantly in red beets and in some mushrooms as red-violet betacyanins and yellow betaxanthins, are sensitive to heat degradation in acidfoods and to oxidation. The major pigment of red beets, betanin, turns into colorlessand yellow compounds due to hydrolysis. The rate of degradation of betalainsincreases in the presence of metal ions.

12.5.5 BROWNING AND FORMATION OF BLACK SPOTS

Browning reactions are important because they affect the color, flavor, safety, andnutritional value of processed fruits and vegetables. Maillard browning is promoted bya high concentration of reactants (reducing sugars, amino acids/proteins), high temper-ature, and alkaline pH. Reducing sugars differ in their reactivity in this reaction, e.g.,glycoaldehyde > glyceraldehyde >> xylose >> glucose > fructose. Phosphorylated sug-ars, such as those that sometimes accumulate in postmortem myosystems, are generallymuch more reactive than their nonphosphorylated counterparts. An example of qualityloss due to the participation of phosphorylated sugars in Maillard browning is orangediscoloration of canned tuna.

The compounds formed in the early stages of Maillard reactions are essentiallycolorless or slightly yellowish. They are called premelanoidins—precursors ofbrown-colored melanoidins. The colorless products of molecular mass below 1000Da are produced mainly by reactions with free AA. Brown-colored melanoidins aregenerated predominantly by polymerization of intermediary products and by theirreactions with proteins. Another possible way is the formation of covalent bonds by



reaction of low-molecular-weight chromophor structures with side chains of proteins,especially lysine, arginine, or cysteine residues. The lysine residue can react withfuran-2-carboxaldehyde.

The reactions of oxidized lipids with amino acids and proteins also result in theformation of brown compounds—so-called protein-lipid browning. The browningrate is particularly high in reactions with cysteine, methionine, and tryptophan. Thediscoloration is objectionable especially in white poultry or fish muscle, wherebrowning appears even under refrigeration or frozen storage.

The shells and surface layers of marine crustaceans, predominantly lobsters,shrimps, and crabs, lose their attractive color shortly after they are caught and developvery dark or black spots. The melanosis starts with enzymatic changes catalyzed bythe endogenous polyphenoloxidase (PPO) complex and is followed by nonenzymaticpolymerization. In the first step, tyrosine is oxidized in the presence of monophenoloxidase to dihydroxyphenylalanine (DOPA), while diphenoloxidase catalyzes theoxidation of DOPA. Polymerization of the DOPA chinone leads to high-molecular-weight black pigments (Figure 12.2). In the polymerization reactions of DOPA,cysteine, tyrosine, and lysine residues in proteins are also involved. Very extensiveblackening is regarded as a drastic quality deterioration of the crustaceans. Theformation of black spots can be prevented by reducing the quinone to the colorlessDOPA by different sulfiting agents in the form of dips shortly after catch, usuallyon the fishing boats. Bisulfite also acts as a competitive inhibitor of PPO. A numberof other reducing compounds, antioxidants, and enzyme inhibitors have been pro-posed (Kim, Marshall, and Wei 2000). Similar reactions also occur in potatoes,bananas, figs, apples, nuts, or cereal products.

12.6 INTERACTIONS AFFECTING FOOD FLAVOR

12.6.1 INTRODUCTION

The flavor of a food is related predominantly to the concentration of the volatileflavor compounds in the vapor phase above the product, which is affected by theconcentration of the aroma compounds in foodstuffs, their volatility at the giventemperature, and on their affinity to other components.

Proteins, lipids, and polysaccharides have the ability of binding flavors due tohydrophobic interaction and hydrogen, ionic, or covalent bonds, depending on thestructure of the compounds. This binding decreases the volatility of the molecules,thus affecting the rate of loss of flavor during storage of the products. The stabilityof the system depends on the pH and temperature of food. Binding of alcohols,aldehydes, ketones, and other volatile compounds by various proteins have beendescribed (Bakker 1995).

FIGURE 12.2 Black spot formation.

tyrosine DOPA o-quinone brown polymersPOO + O2 POO + O2 amino acids

coloredreducing additive

colorless



Hydrophobic aroma compounds accumulate predominantly in the lipid phase offoods, thus decreasing their partitioning into the aqueous and vapor phases. Theair–lipid partition coefficient for any hydrophobic flavor compound is affected at agiven temperature by the fatty acid composition of the lipid and the degree of fatdispersion.

Hydrocolloids decrease the perceived intensity of various flavors in food systems,not only by binding of the volatile compounds, but also due to lowering the diffusionrate due to their high viscosity. Another important factor is complexation. Formationof cyclodextrin molecular-inclusion complexes with aroma compounds may protectflavors against evaporation and inhibit their undesirable chemical degradation duringprocessing and storage due to interactions with other food components. The forma-tion of inclusion complexes is controlled by the concentration of the guest moleculesand the dimensional compatibility of the flavor compounds and the cyclodextrincavity. Complexation improves the stability of various flavors, but it may alsodecrease their release from the cyclodextrin trap during consumption of the products(Reineccius et al. 2004).

12.6.2 HYDROLYTIC, OXIDATIVE, AND PYROLYTIC REACTIONS

Hydrolytic reactions in foods are broad ranging. They are catalyzed by enzymes andalso by acid or alkaline conditions. Hydrolysis of triacyglycerols, so-called hydrolyticrancidity, leads to free fatty acids, which sometimes have a soapy flavor (e.g., lauricacid). Heating starch or other saccharides under mild acidic conditions leads tohydrolysis, for example, formation of invert sugar from sucrose. Phenolic glycosidesare hydrolyzed under mild alkaline conditions. Such reactions can influence thetexture and flavor of fruits and vegetables. Phenolic glycosides may also be hydro-lyzed by hydrolases such as anthocyanases.

Aging changes in bovine meat lead to the liberation of peptides and AAs andthus enhancement of flavor formation in the roasted meat. The increase in theconcentration of free AAs in tenderized meats is caused mainly by endogenousproteolytic enzymes from the lysosomes and by calpains. Electrical stimulation ofbeef carcasses may increase the rate of releasing of the enzymes from the lysosomes,and thus the formation of free AAs.

The secondary products of lipid oxidation, especially carbonyl compounds, reactwith AAs and proteins forming rancid off-flavors. Oxidation of polyenoic fatty acidsleads to 2,4-alkabienals and conjugated alkatrienals, which reacting with AAs gen-erate a fishy off-flavor. Terpenes present in the majority of foods of plant origin arealso easily oxidized on storage and heating. Oxidized terpenes and products of thedecomposition of the intermediary hydroperoxides, including radicals, react withAAs and proteins forming numerous off-flavor compounds, often resembling, intheir flavor, rotten or spoiled food. Oxidation of ascorbate in the presence of molec-ular O2 results in the loss of vitamin C activity, and may be coupled to other reactions,such as disulfide bond formation during gluten development, or conversion of ethanolto acetaldehyde in wine aging.

Pyrolytic reactions, especially at temperatures higher than 150°C, mainlybetween AA and saccharides, lead to the development of numerous flavor compounds



(see Chapter 11). Pyrrolidone carboxylic acid formed from glutamic acid duringthermoprocessing of canned vegetables is believed to contribute to acid-catalyzedpheophytin formation and off-flavor.

12.7 CHANGES IN TEXTURE OF FOODS

12.7.1 INTRODUCTION

These interactions occur in the original, intact raw materials of food, for example,in the muscles of slaughter animals and fish postmortem or in frozen fish, storedvegetables and fruits, as well as in processed systems, like comminuted sausagebatters, fish gels, dairy products, extrudates, polysaccharide gels, dough, and breadcrumbs. They comprise predominantly the formation of various cross-links, due tocovalent bonds, hydrogen bonding, electrostatic forces, and hydrophobic interac-tions. These cross-links may decrease the quality of proteinaceous foods by inducingtoughness, for example, in some frozen stored fish, or are necessary for developingthe desirable, rheological properties of many products, such as the ability of a proteinto form disulfide bridges in extrudates when heated to high temperatures at lowmoisture content, which is a prerequisite to yielding high-quality products. The cross-linking may be induced by endogenous or added enzymes, different metabolites ofenzymatic reactions, chemical additives, or various processing factors, includingshearing and heating. On the other hand, endogenous hydrolases may decrease theeffect of cross-linking by degrading the polymers. In stored apples, enzymaticdegradation of the pectin structural material is responsible for a gradual decrease inthe firmness of the tissue due to the loss of binding between the adjacent cell walls.In fresh apples, the shear forces cause cutting of the tissue across the cells, whichresults in juicy texture, while apples after prolonged storage have a floury texture.In various food gels, a high content of uniform starch particles, 1 to 2 µm in diameter,Ca-pectinate, about 40 µm in diameter, or microcrystalline cellulose particles, isresponsible for fat mimetic properties.

Other interactions affecting food texture are those that facilitate the formationand stabilize different emulsions and foams. Air and other gases occur in manyfoods, such as the interstitial space of fruits and vegetables (e.g., apple tissue maycontain more than 25% air by volume), and as bubbles in ice cream, whipped cream,bread, and beer foam. In bread or cake the gas bubbles are dispersed in a solidmatrix, for example, of starch and protein, while in a fluid such as whipped cream,stabilization is due to a protein film and fat globules.

Thus, the texture of the products depends significantly on various interactionsof food components and additives and is affected by the processing parameters,predominantly time and temperature of heating, pH, shearing forces, and the presenceof metal ions.

12.7.2 RIGOR MORTIS AND TENDERIZATION OF MEAT

The postmortem changes in the rheological properties of muscle tissues, that is, thedevelopment of rigor mortis, gradual diminishing of the stiffness, and tenderization,



although triggered by biochemical processes, involve physical and chemical inter-actions of the muscle proteins.

The stiffness of the fish or meat is a physical manifestation of interactionsbetween the myosin heads in the thick microfibrils in the sarcomeres and the activecenters of the actin molecules in the thin microfibrils. These chemical interactionsare the consequences of earlier biochemical events. The concentration of Ca2+ in thesarcoplasm increases about 10 times with the time postmortem until the onset ofstiffening. This is due to depletion of the reserves of phosphocreatine and ATP, andin consequence, lack of energy needed for pumping the ions against the concentrationgradient back into the sarcoplasmic reticulum. At a high enough concentration ofCa2+ in the sarcoplasm, conformational changes in the regulatory proteins occur,which unblock the reaction sites on the actin molecules for cross-linking.

In the flesh of fish belonging to several temperate and tropical species, a phe-nomenon known as cold shortening occurs. In these fish the prerigor state after deathis about twice shorter at 0°C than at 10°C, which is rather unusual. This is accom-panied by a correspondingly faster release of Ca2+ from the sarcoplasmic reticulumand a higher rate of degradation of ATP at 0°C than at 10°C. According to Ushio etal. (1991) this phenomenon is caused by the fact that at 0°C, the Ca2+ uptake abilityof the reticulum is decreased, which leads to an increase in the concentration of Ca2+

in the sarcoplasm and thus activation of the myofibrillar ATPase.During aging of the carcass, rigor mortis gradually disappears and the meat

becomes less tough or overly tender in the case of some fish myosystems. At atemperature of about 3°C to 5°C, the highest tenderness in beef appears after 2 to4 weeks, and in pork from 6 to 10 days—the toughness decreases to 50 to 60% ofthe initial value. In current models, formed on the basis of biochemical research, ithas been suggested that tenderization during postslaughter conditioning of meat isdue to a gradual degradation of the proteins of the myofibrils and of the cytoskeletalproteins. Changes in the collagens and of the proteoglycan component of the extra-cellular matrix may also be involved (Purslow et al. 2001). These changes resultfrom a concerted action of different endogenous enzymes—large-scale disassemblyof the original structural elements of the muscle fiber, followed by further degradationof the smaller components. The cleavage of several proteins should lead to weakeningof the cytoskeletal network and thus of the sarcomere structure, resulting in decreasedintegrity and tensile strength of the muscle fibers. The rate of these processes isaffected by temperature and other conditions in the meat, which are both the causeand effect of different enzymatic responses to the activity of endogenous enzymeinhibitors, changes in compartmentalization, concentration of Ca2+ in the sarcoplasm,and pH. These factors are not constant during the aging of meat. In the carcasses ofconventionally slaughtered animals, the temperature in the center of the thickest partdrops during cooling in industrial conditions to 5°C after about 2 days. After 15hours the pH in such beef carcasses is about 6.

These data and the properties of the endogenous proteolytic enzymes, as wellas the fact that most chemically evidenced proteolysis occurs only after the devel-opment of meat tenderness, may indicate that the endogenous cathepsins, m-calpain,and µ-calpain, cannot contribute significantly to meat tenderization in practicalconditions of carcass handling. The weakening of the structures of the myofibrils



and the desmin intermediate filaments may be due to Ca2+ that accumulates in thesarcoplasm postmortem. Ca2+ ions at a concentration of 0.1 mM disrupt the filamen-tous structure of titin, nebulin, and desmin. They also weaken the Z-disks in thesarcomeres by binding to the phospholipids, which are the main components of thematrix reinforcing the Z-disk structure (Kanawa et al. 2002, Ahn et al. 2003).

12.7.3 TEXTURE CHANGES IN FROZEN FISH

Prolonged storage of frozen fish, mainly belonging to the Gadidae family, may leadto increased toughness of the meat, loss in functional properties, and decreasedATPase activity of myosin. This is the result of cross-linking of myofibrillar proteinscaused by interactions of AA residues with different reactive components, predom-inantly with formaldehyde generated by endogenous trimethylamine oxide demeth-ylase, and with several products of lipid oxidation or hydrolysis. Oxidizing lipidsmay contribute to protein cross-linking via radical polymerization and due to reac-tions of secondary oxidation products. Natural antioxidants—mixtures of plantextracts—are effective in inhibiting undesirable changes in frozen fatty fish. Theloss in functional properties is especially severe in minced fish due to increasedcontact of the interacting components of the mince.

By removing from the minced fish the water-soluble proteins and nonproteinnitrogenous components, prooxidative compounds and ions, as well as most of thelipids, a concentrate of myofibrillar proteins, known as surimi, is produced. Surimiis more resistant to undesirable changes in functional properties than the originalfish mince. However, extracting of the water-soluble components results in about a30% loss of crude protein from the fish meat.

The changes in myofibrillar proteins in frozen fish can also be limited by addingdifferent cryoprotective compounds, decreasing the storage temperature, and pre-venting lipid oxidation in the mince. Among the most common cryoprotectors aresucrose, sorbitol, mannitol, alginates, polyphosphates, citrates, ascorbate, andsodium chloride in different, proprietary mixtures. Several AAs, hydroxy carboxylicacids, branched oligosaccharides, and adenosine nucleotides are effective in modelsystems and in surimi (Matsumoto and Noguchi 1992). AAs with a high negativenet charge are most effective in preventing freezing denaturation of fish myofibrillarproteins. Generally, the low molecular cryoprotectors contain more than one reactivegroup suitably spaced in the molecule.

12.7.4 CROSS-LINKING IN GELS AND FILMS

Food gels may be made from proteins and polysaccharides due to chemical orenzymatic interactions of the polymers, and in some cases involvement of endoge-nous or added low molecular compounds. After deconformation of the native mac-romolecules a three-dimensional structure is formed, which is capable of immobi-lizing large quantities of solvent, various solutes, and inert filling material. Theconcentration of the polymers may be as low as a few percent of the total mass ofsome gels. The cross-links buttressing the structure result from hydrophobic inter-actions between the exposed nonpolar fragments of the chains in the aqueous envi-ronment, hydrogen bonds, ionic forces, and various covalent bonds.



A typical polysaccharide gel is that formed by starch. Native granular starchabsorbs in aqueous media up to 30% of water, by weight. The swollen granulesheated in water lose their crystalline structure and turn into gelatinized starch, whichgradually forms a paste of high viscosity. Salt and some low-molecular-mass hydro-philic components, competing for available water molecules, increase the gelatini-zation temperature. Cooling of the paste leads to further increase in viscosity dueto the development of a network of the hydrated starch molecules and the mass turnsinto a gel. During prolonged storage of the paste or gel, the crystalline structure ofstarch gradually develops again in a process called retrogradation and some wateris exuded. The rate of retrogradation depends predominantly on the proportions ofamylose and amylopectin in the granules and the contents of lipids, as well as onthe temperature (Jane 2004).

The mutual interactions of different polysaccharides may lead to increasedviscosity of solutions and gelling. The extent of synergism is affected by the chemicalcomposition of the polymers. The gelling of agarose, carrageenan, and furcellarancan be enhanced in the presence of other, nongelling polysaccharides. κ-Carrageenanincreases the gelling of mixtures of locust bean gum and other galacto- and gluco-mannans in aqueous media. The mechanism of this synergism may involve hydrogenbonding between the two interacting polysaccharides, self-aggregation of the galac-tomannan chains in the presence of the carrageenan network, or mutual exclusionof incompatible polymers. In some cases, even a very small addition of a specificpolysaccharide, about 0.5% of the total polymer concentration, can have a significantsynergistic effect on gelling of the system. Interchain associations of alginates andpectins enhance the gelling of these polymer mixtures (Phillips and Williams 1995).

Reactions influencing the intracellular cement of fruits and vegetables may alsohave a profound effect on texture. Phytic or citric acids can sequester Ca2+ fromintracellular pectate and thereby cause softening of canned fruit. Likewise, thepresence of added Ca2+ or the enzyme-catalyzed deesterification of pectin leads tothe firming of cooked vegetables like green beans and potatoes.

Cross-linking is also involved in the preparation of edible films, which are usedfor foods because of their barrier properties, as carriers of ingredients and antimi-crobial agents, and as enzyme supports. Such films can be made of various proteinsor of proteins in mixtures with saccharides or lipids. Their properties depend on theproportions and characteristics of the components and on the interactions betweenthe polymers, cross-linking agents, and plasticizers. The procedures applied forpreparing films generally comprise in the first step deconformation of the polymermolecules by heating, shearing, or exposing thin layers of the interface with air. Atappropriate concentrations of the denatured polymer in the dope, cross-linking isinduced by evaporation of the solvent or shearing in a spinneret and immersing ina coagulating bath.

12.7.5 INTERACTIONS IN THE DOUGH

Protein cross-links and protein–lipid–polysaccharide interactions are necessary forthe development of the desirable texture of cereal-based baked goods. During mixingwith water and salt, the flour particles hydrate and the glutenin polymers disaggregate



and form a membrane network. During preparation of bread dough, after additionof water, the wheat endosperm particles very rapidly exude proteinaceous fibrils.These fibrils are formed from the gluten protein film at the air–water interface, whichis created due to interfacial forces. Interactions of the strands with each other leadto cross-linking of the flour particles and cohesion in the dough (Bushuk 1998).

The dough can be viewed as a viscoelastic, hydrated matrix of gluten proteinswith inserted starch granules and cell wall debris. Thus it may be regarded as a filledgel system. Intra- and interpeptide disulfide bonds play a key role in building therequired structure of the dough. In the presence of soluble compounds containingthiol groups, some of the –S–S– bridges originally present in the gluten proteins aremobile due to disulfide interchange reactions, and interchain cross-links can beformed. Oxidizing agents added to the dough improve the texture of bread bypromoting the formation of the cross-links. Baking increases the number of disulfidebridges, adding to the stability of the loaf structure. Other covalent bonds alsocontribute to the properties of the dough. Cross-linking may be caused by dehy-droascorbic acid and by the products of its thermal degradation, like methyl glyoxal,glyoxal, diacetyl, and threose, which interact with the lysine residue of proteins(Fayle et al. 2000). Other covalent cross-links can probably result from enzymaticformation of γ-butyraldehyde catalyzed by endogenous diamine oxidase and subse-quent addition to it of nucleophilic AA residues in the presence of Cu(II) ions(Chiarello et al. 1996). Hydrolysis of a number of peptide bonds in the gluten proteinsby endogenous or added enzymes leads to weakening of the structure.

The interactions between proteins and starch are also important (Friedman 1995).The structure of the dough is significantly affected by hydrogen bridges. Accordingto Levebvre et al. (2003) gluten forms a network due to aggregation of particlesprimarily under the effect of hydrogen bonds and hydrophobic interactions, with thedisulfide bridges probably not involved directly in the network formation. Althoughhydrogen bonds are much weaker than covalent bonds, they may be formed in largenumbers due to a high content of glutamine residues in cereal proteins and numeroushydroxyl groups in starch and pentosans. Interchange reactions of the hydrogenbonds under mechanical stress facilitate stress relaxation of molded dough. Hydro-phobic interactions in aqueous systems between the apolar AA residues of the flourproteins also contribute to buttressing the dough structure, especially during bakingof the loaf. Ionic repulsion–attraction forces, although not numerous because of arelatively small number of ionizable residues in cereal proteins, also play a role informing the desirable, porous structure of bread crumbs (Wrigley et al. 1998).

12.7.6 LIPIDS AND THE TEXTURE OF FOODS

Lipid droplets have a structure-forming role in oil-in-water emulsions like milk,cream, ice cream, cheese, dressing, mayonnaise, and comminuted meat products.These droplets are stabilized by interactions with proteins, lecithins, or varioussynthetic surfactants. The rheological properties of milk gels depend on the size ofthe fat droplets and the nature of the stabilizing agent. Low-molecular-weight sur-factants make the surface of the droplets smooth and noninteractive and the gelsweak. In gels stabilized by whey proteins, the fat globules are cross-linked due to



protein interactions. On standing of chilled, natural milk, the lipoprotein membranesof fat globules interact with a serum protein, thereby forming clusters of globules.Clustering of the fat globules around the air bubbles contributes to the desirablecharacteristics of ice cream. In the cream-churning process, concentration of the fatglobules and spreading of the oil at the air–water interface occur, leading to sup-pression of the foam and formation of clumps of the water-in-oil emulsion of butter.

In various dairy products, margarine, shortenings, and chocolate, the fats occurin the form of crystals. Triacylglycerols crystallize in at least four forms: sub-α, α,β′, and β, differing in stability and melting temperatures, as well as in their effecton the creaming and rheological properties of the products. Transformations of thepolymorphic forms of cocoa butter are thought to be responsible for the developmentof the undesirable appearance of white or gray powdery surface deposits and lossof gloss of stored chocolate, known as bloom. Spherulites with random arrangementof fat crystals can be responsible for the sandy texture of various foods.

As discussed in Section 12.7.3, lipids and lipid oxidation and hydrolysis productsalso influence texture by interacting with proteins.

12.8 INTERACTIONS AND THE NUTRITIVE VALUEOF FOODS

12.8.1 INTRODUCTION

The nutritive value of foods may be affected primarily by interactions among pro-teins, proteins and saccharides, proteins and lipids, and with oxidizing agents. Therate of these interactions increases with the temperature applied in food processingand cookery. However, their effects also occur during prolonged storage even inrefrigerated and frozen products. The nutritive value may decrease due to loss indigestibility or chemical modifications making the nutrients unavailable for thehuman organism.

12.8.2 CHANGES IN DIGESTIBILITY

The digestibility of a protein depends, inter alia, on the structure of the protein.Tightening of the protein structure by additional cross-links that prevent unfoldingof the polypeptide chains may decrease the digestibility by making some peptidebonds, buried inside of the molecule, inaccessible to the enzymes.

Heating of foods may lead to cross-linking in proteins by isopeptide bondsbetween the ε-NH2 group of lysine and the β- and γ-carboxyl groups of aspartic andglutamic acid residues, respectively, or their amides. This may decrease the rate ofprotein hydrolysis.

Protein cross-linking may also be caused by interactions of amino groups withreducing saccharides or other compounds containing carbonyl groups. In foods theseare especially the secondary products of lipid oxidation and aldehydes contained insmoked commodities. As a result of a number of further reactions, different unsat-urated compounds are generated, which also interact with amines and AAs. Variouscross-linking reactions may be involved, including the formation of α-amino acid



amides and α-hydroxyacid amides (Büttner, Gerum, and Severin 1997). Cross-linking also occurs in food systems due to alkaline treatment (see Chapter 6).

12.8.3 CHANGES IN BIOLOGICAL VALUE

While the early products of the Maillard reaction can be utilized in the human organism,further changes gradually make the lysine residue nutritionally unavailable. This hap-pens mainly in the outer parts of baked, grilled, or fried products. In the center of abaked bread loaf, turkey, or chicken, the temperature is below 100°C and the loss oflysine is negligible. However, sterilization leads to a significant decrease of availablelysine in products rich in reducing sugars and high-value proteins. In various commer-cially produced enteral proteinaceous food formulas, the lysine availability is onlyabout 75% (Castillo et al. 2002). Significant loss of lysine has also been found incommercial infant formulas (Gonzales et al. 2003). The Maillard products are formedat a high rate, mainly in the outer parts of heated foods. However, in condensed milk,the reaction also proceeds at ambient temperature. Although the rate of reaction ismuch slower, the content of available lysine may drop by several percent due to monthsof storage. The decrease in availability of lysine can be followed by determining theunchanged AA residue, or by assaying some early Maillard reaction products, such asfurosine, Nε-carboxymethyllysine or pyrraline (Hartkopf and Erbersdobler 1995; Bel-itz, Grosch, and Schieberle 2001).

Thiamine and its decomposition products formed in heated foods may alsoparticipate in the Maillard reaction. In acid foods thiamine is stable, but decomposesreadily at alkaline pH. The average loss of thiamine due to boiling of eggs is about15%, curing of meat 20%, baking of bread 15 to 20%, cooking of vegetables 25 to40%; in different cooked, canned, roasted, and fried meat and fish products the lossranges from about 15 to 75%.

Interactions of dehydroalanine formed during heating in alkaline conditions (seeChapter 6) with the residues of other AAs lead to cross-linking, and in hydrolysatesof the heated protein a mixture of unnatural and D-forms of AAs can be found.However, the D-forms of AAs may be present in various foods not only due toheating in alkaline conditions. In the edible parts of some aquatic crustaceans andmollusks, D-alanine is synthesized by alanine racemase. Furthermore, fish saucesand fermented dairy products contain substantial amounts of D-alanine and D-aspartic acid of microbial origin. The interactions due to prolonged heating at alkalineconditions may decrease the nutritional value of proteins due to the loss of essentialAAs and lower absorption of the D-forms. Some products of the reactions inactivatemetalloenzymes, induce nephrocytomegaly in rats and kidney damage (Pearce andFriedman 1988; Friedman and Pearce 1989). A beneficial effect of heating underalkaline conditions is the release of nutritionally available niacin from grains. TheHopi Indians’ traditional procedure of cooking mature corn grain with alkaline woodashes hydrolyzed the ester linkages binding niacin to saccharides and turned thenutritionally unavailable polymer into free niacin (Wall and Carpenter 1988).

High-acid foods like lemon juice undergo nonoxidative degradation of ascorbicacid to 3-deoxypentulose 2-furaldehyde. The reaction appears to contribute to ascor-bate browning of these products as well as loss of vitamin C.



Several components of wood smoke, mainly phenols, aldehydes, and nitric oxide,may react with some AA residues in meat proteins during smoking and storage ofthe products. The results of various model experiments indicate that the contents ofSH and NH2 groups, as well as lysine availability decreases by from several up toabout 90% in cysteine solution in beef meat and fish due to smoking in variousconditions. Recent investigations have shown that in mild hot smoked fish, the loss ofavailable lysine is negligible (Kołodziejska et al. 2004).

Oxidation may damage AAs, decrease the digestibility of the protein by stablecross-linking, and lead to antinutritional degradation products. The rate of changesin proteins is controlled by the concentration of various endogenous and addedoxidizing agents, prooxidants, and sensitizers, such as chlorophyll, methylene blue,erythrosine, and riboflavin, antioxidants, temperature, and the sensitivity of variousAA residues. The effect also depends on the distribution of the reactive AA residuesin the polypeptide chains and on their availability for reaction with the oxidizingagent at the actual conformation of the protein. The oxidation of thiol groups innative proteins is less efficient than in denatured proteins. The number of surfacereactive SH groups in proteins increases due to heating in the temperature range of20 to 50°C (Sano et al. 1994). Oxidation of thiol groups in proteins by oxidizedlipids proceeds predominantly via the free radical mechanism.

Tryptophan undergoes autoxidation in acidic and alkaline solutions when heatedto 100°C or higher temperatures, in a free-radical reaction, whereby different deg-radation products appear (Friedman and Cuq, 1988). The destruction of tryptophanresidues in various proteins under conditions similar to those prevailing in foodcookery and sterilization is not higher than a few percent.

Oxidation of ascorbic acid with molecular O2 to form diketogulonic acid resultsin loss of vitamin C activity. Heating foods with thiamin at pH less than 6 resultsin cleavage of the methylene bridge of vitamin B1 to form pyrimidine and thiazoleand loss of vitamin activity. Likewise, the isomerization of all-trans carotenoids withprovitamin A activity favored by light, heat, and dilute acid conditions causes lossof vitamin A activity. Increased lipid oxidation from conversion of Fe2+ to Fe3+ andthe resulting reduced iron absorption or bioavailability is the consequence that canoccur when iron is oxidized.

12.9 THE EFFECT OF INTERACTIONS ON THE SAFETY OF FOODS

Heating may lead to the formation of N-nitroso compounds (NNC) in foods containinghigh amounts of nitrates or nitrites. About 90% of 300 tested NNCs have been reportedto be carcinogenic in animal experiments. Although NNCs are found mainly in heatedfoods, they are also formed in frozen stored products, albeit at a low rate. Additivescapable of binding the nitrosating agents, such as ascorbate and α-tocopherol, caneffectively retard the formation of NNCs. Several other compounds have also beenfound to be efficient inhibitors of N-nitrosation, such as long-chain acetals of ascorbicand erythorbic acids. Foods low in nitrates and amines contain usually up to 10 ngNNCs/g, while cured and heavily smoked meat and fish up to several hundred ng/g.In smoked foods, C-nitroso- and C-nitrophenols have also been found.



In potato chips, French fries, and processed cereals, fried beetroot, and friedspinach, different amounts of acrylamide may be found. The content of acrylamidein various foods increases with the time and temperature of heating, and may beabove the actual detection limit of about 10 ng/g (Food and Agriculture Organiza-tion/World Health Organization [FAO/WHO] 2002). It may be formed in Maillard-type reactions between glucose and asparagine, as well as by changes of AAs,acrolein, acrylic acid, and lactic acid at about 120°C. The contents of acrylamide inFrench fries can be significantly decreased by extracting the reducing sugars andasparagine from the surface of the cut potato (Biedermann-Brem et al. 2003).

About 20 different mutagenic and carcinogenic heterocyclic aromatic amineswere found in different heated foods. Their content in foods depends on the con-centration of substrates, enhancers and inhibitors, time and temperature of heating,water activity, and pH. Some of them are formed even at about 37 to 60°C, however,only after several weeks. In the presence of lipids, Fe2+, and Fe3+, the rate of reactionincreases, probably due to oxidation and the generation of radicals (Jägerstad et al.1998). Antioxidant spices—rosemary, thyme, sage, and garlic—as well as curingbrine, reduce the formation of several heterocyclic amines in fried meat (Murkovic,Steinberger, and Pfannhauser 1998). The total contents of these compounds inbroiled, fried, and grilled protein-rich products are generally on the order of severalng/g wet weight; however, in some commodities it may even reach 85ng/g (Tai, Lee,and Chen 2001). Interactions leading to the formation of these compounds arepresented in detail in Chapter 19.

Interactions of lipids and NaCl in heated foods may cause the formation ofcarcinogenic 3-monochloropropanediol. This compound has been detected in con-centrations on the order of 50 ng/g in bacon, salami, cured fish, bread, cakes, pastries,and cheeses, and up to 10 times more in malts. The rate of formation of 3-monochlo-ropropanediol increases with the temperature of food processing.

12.10 FINAL REMARKS

Interaction is the key word with respect to many aspects of the quality of industriallyprocessed and home-prepared foods. For the food technologist, it is not enough toknow the physical and chemical properties of individual food constituents. In orderto control the quality of various commodities, it is necessary to understand the effectof processing variables and storage conditions on the interactions of the differentcomponents in whole or disintegrated food materials. Although the results of numer-ous experiments carried out in model systems have already given insight into themechanisms of the interactions of many food constituents, more information mustbe obtained on these reactions in composite food systems. While studying physicaland chemical interactions in foods, it must be considered that ongoing biochemicalprocesses often rapidly modify the system and new components may emerge, whichcan play the role of reactive substrates or inhibitors that can change the reactionconditions and outcome.



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357

13 Main Food Additives

Adriaan Ruiter and Alphons G.J. Voragen

CONTENTS

13.1 Introduction................................................................................................ 35713.2 Classification.............................................................................................. 35813.3 Preservatives .............................................................................................. 359

13.3.1 Introduction ................................................................................. 35913.3.2 Sulfite........................................................................................... 36013.3.3 Nitrite........................................................................................... 36113.3.4 Sorbic Acid.................................................................................. 36113.3.5 Benzoic Acid ............................................................................... 362

13.4 Antioxidants............................................................................................... 36213.5 Flavorings, Colorants, and Sweeteners ..................................................... 36313.6 Stabilizers, Emulsifiers, and Thickening Agents ...................................... 36513.7 Clarifying Agents and Film Formers ........................................................ 36713.8 Acidulants .................................................................................................. 36713.9 Fat Substitutes and Fat Mimetics .............................................................. 36813.10 Prebiotics ................................................................................................... 369References.............................................................................................................. 371

13.1 INTRODUCTION

The addition of certain substances to foodstuffs was practiced in ancient times,mostly for improving preservation. Salt was added to perishable foodstuffs, such asmeat and fish, from prehistoric ages on. Smoke curing can also be considered as thefortuitous addition of constituents to food, as wood smoke contains a number ofcompounds that are absorbed by the food during the smoke-curing process or aredeposited onto the surface. These treatments not only prolong the shelf life of thefood, but also add to the flavor.

The preparation of any food product includes the addition of a number of ingredientsthat are not considered to be additives, but that clearly improve some properties of thefood, such as maintenance of quality, and are originally intended as such. Preparationof a marinade of sour wine or vinegar, for example, is a technique for preserving fish,which was known to the Romans, but acetic acid is not an additive in the strict senseof the word. In some cases, it is not so easy to determine whether the substance underconsideration is an additive. It is helpful, however, to keep in mind that an additive isintended as an aid, for some purpose or another, and not as an ingredient.



In 1955, the Joint Food and Agriculture Organization/World Health Organization(FAO/WHO) Expert Committee on Nutrition defined food additives as non-nutritivesubstances which are intentionally added mostly in small quantities, with the aim ofimproving the appearance, flavor, taste, composition, or shelf-life of foods. In a morerecent wording, these food additives are described as generally not intended as afoodstuff or as a characteristic ingredient which, irrespective of any nutritional value,are added, for any technological or sensoric reason, to a foodstuff during manufactur-ing, preparation, packaging, transport or storage, and from which it is expected thateither the substance itself or reaction or decomposition products become a permanentcomponent of the foodstuff or the raw material (van Dokkum, 1985; Kamsteeg andBaas, 1985). In the latter definition, the term improvement is not included, and theremaining presence of the compound, or reaction products from that compound, areincluded in the definition.

This may be the result of a shift in the attitude toward additives. The ranking,by the public, of actual food hazards, was almost inverse to that given by Wodicka(1977), in which microbiological and nutritional risks were listed at the top andfood additives in the last position of the ranking. To some extent, this inversionexists even today (Hall, 1999).

The preparation of any food product includes the addition of a number ofingredients that are not considered to be additives, but that clearly improve someproperties of the food, such as maintaining quality, taste, flavor, or texture, and areoriginally intended as such.

The origin of food additives remains a point of discussion. There is a continuingdemand, from the consumer’s side, for “natural” additives. No additive, however, iscompletely free of impurities. Products of chemical synthesis should be purified,eliminating starting materials and compounds resulting from side reactions. It mustbe stated that enzyme-catalyzed synthesis or modification more and more frequentlyreplaces purely chemical synthesis of modification.

“Natural” compounds should be purified as well in order to remove accompa-nying substances that have no significance in the final product. Generally speaking,purification is more difficult and more complicated for natural additives, as it isalso much more problematic to characterize the raw material, which may containa great many ill-defined compounds whose toxicity is largely unknown (Ruiter,1989). Feberwee (1989) points out that official legislation does not discriminatebetween safe natural and safe artificial food additives. The main difference in safetyevaluation between these two categories is our long experience with natural addi-tives (Lüthy, 1989).

13.2 CLASSIFICATION

Additives are most often listed and classified in the following categories:

• preservatives to extend the shelf life of foodstuffs• antioxidants to protect lipids in food from attack by oxygen• flavor enhancers to improve the perception of taste and flavors• sweeteners to replace sugars to provide a sweet taste to the product


Main Food Additives 359

• colorants to improve the appearance of a foodstuff • emulsifying agents to enable and maintain a fine partition between oils or

fats in water (or a partition of water between oils or fats), gas in liquid (foam)• thickening and gel-forming agents • clarifying agents• film formers• glazing agents• acidulants• fat substitutes and fat replacers• substances improving the nutritional value• pro- and prebiotics• many other substances such as anticlotting agents, moisteners, antifoam-

ing agents, flour improvers, leavening agents and baking powders, meltingsalts, stiffening agents, complexing agents, fillers, enzymes, and so on

With respect to the reactivity of additives, it is preferable to add another classi-fication, in which three groups can be distinguished:

• Substances that, simply by their presence, lead to the desired improvement.Most colorants, sweeteners, and some preservatives belong to this class. Manyof these additives do not display a strong reactivity toward other constituentsduring preparation and storage of the foodstuffs to which they are added.

• Substances that are added because of their reactivity toward undesirablecomponents already present or arising during manufacturing or storage,and that may be bound by these added substances. The reaction may bedirected toward these components themselves or toward their precursors.Antioxidants are an example of this category, as well as some peculiarsubstances reacting with matrix components to make desirable compo-nents such as flavor compounds.

• Food additives that participate in fortuitous reactions which, in some cases,may be undesirable.

This classification, however, also has its limits. First, there is hardly any additivethat does not take part in some chemical reaction. Furthermore, some food additivesmay also participate in unintentional reactions. Therefore, in this presentation, someadditives are discussed in an individual way, with emphasis on their reactivity withrespect to matrix compounds.

13.3 PRESERVATIVES

13.3.1 INTRODUCTION

Preservatives are added in order to protect a variety of foodstuffs against microbialspoilage. This protection is possible, in many cases, because of a chemical reactionbetween the preservative and the microorganisms. It may therefore be expected thatthese compounds show some reactivity toward food components as well.



Many preservatives are comparatively reactive compounds, such as sulfite,nitrite, and sorbic acid, but some of these show a moderate reactivity only (e.g.,benzoic acid). Sulfite, nitrite, sorbic acid, and benzoic acid are discussed below.

13.3.2 Sulfite

Disinfection by the vapors of burning sulfur is an old technique that was frequentlyused to decontaminate wine casks. Some sulfur dioxide was left in these vessels,preventing the wine from unwanted microbial infections.

At present, sulfites are used both as preservatives and as agents that stop brown-ing reactions. In food, the HSO3

– species predominates, while in dehydrated food,it is expected that S(IV) mainly exists as metabisulfite (S2O5

2– ), which is in equilib-rium with HSO3

– and SO32– (Wedzicha et al., 1991). Because of the nucleophilicity

of the sulfite ion, many reactions with food components are possible (Wedzicha,1991), one of which is a reversible addition to carbonyl compounds. It is suggestedthat the sulfite rather than the bisulfite ion acts as the nucleophilic agent (Wedzichaet al., 1991).

This reaction has many implications for foodstuffs. Aroma components possess-ing a carbonyl group, for example, become involatile and do not contribute anylonger to the overall flavor. Other nucleophilic reactions include the cleavage of S–Sbonds in proteins and addition to C=C bonds of α,β-unsaturated carbonyl com-pounds. Control of nonenzymatic browning is based on this latter reaction (McWeenyet al., 1974). A key intermediate of the Maillard reaction, that is, 3,4-deoxyhexulos-3-ene, is efficiently blocked by a fast reaction with sulfite, leading to the formationof 3,4-dideoxy-4-sulphohexosulose, which is much less reactive and in which sulfiteis irreversibly bound.

Ascorbic acid browning is also inhibited by the addition of sulfite (Wedzicha andMcWeeny, 1974). The same holds for polyphenol oxidase-catalyzed oxidation ofnatural phenols in fruit. The mechanism of the inhibition is by reaction of o-quinoneintermediates with sulfite, which leads to nonreactive sulfocatechols (Wedzicha, 1995).

An undesirable reaction of sulfite in food is the cleavage of thiamin by meansof an attack on the pyrimidin moiety (Zoltewicz et al., 1984). This was one of thereasons for a ban, in many countries, on the use of sulfite in meat. Another reasonis the preserving effect on the meat color, which makes stale meat look as if it werefresh. Sulfite, however, is unable to reduce metmyoglobin back to myoglobin (Wedz-icha and Mountfort, 1991).

An important reaction, in a quantitative respect, is the cleavage of disulfide bondsin meat proteins, in particular in lean meat.

The reducing capacities of sulfite should be emphasized as well. In fact, cleavageof S–S bonds by sulfite can be considered as a reduction. This property of sulfitemakes it useful as an additive to flour for biscuit making (Wedzicha, 1995). Thecleavage of disulfide bonds in wheat proteins speeds up and facilitates the productionof a satisfactory dough.

A quite different type of reaction, which also may occur in food, is that of reductionof azo dyes to colorless hydrazo compounds. As in the reaction with carbon com-pounds, the reactive species is SO3

2 – and not HSO3– (Wedzicha and Rumbelow, 1981).



13.3.3 Nitrite

lt has been known for a long time that small amounts of saltpeter (KNO3) are ableto cause a reddish discoloration of meat, which is characteristic for many meatproducts. In about 1900 it was established that it was not nitrate but its reductionproduct, nitrite, that was responsible for this color development.

Some decades later, nitrite was recognized as a potent inhibitor of microorgan-isms, including pathogens, in many meat products. In particular, the inhibition ofClostridium botulinum, with accompanying toxin formation, was established. Therole of nitrite in the characteristic cured meat flavor was not noticed until later.

The inhibitive action of nitrite is pH-dependent, which led to the assumptionthat undissociated nitrous acid was the active substance. This, however, is a hypo-thetical acid from which equimolar parts of H2O, NO and NO2 are formed. In the1960s it became clear that nitric oxide (NO) is the active species. Nitric oxide,however, can also be generated through the reduction of nitrite, for example, byascorbate or isoascorbate (Wirth, 1985).

Nitric oxide shows a strong binding to iron and is able to block iron atoms inbiologically active compounds important in cell metabolism, in particular in the outgrowthof germinated spores (Woods, Wood, and Gibbs, 1989). In meat products preserved withnitrite, NO binds to heme iron, thus forming nitrosomyoglobin (Giddings, 1977).

There is some evidence that inhibition of Cl. botulinum outgrowth in nitrite-curedmeat products is mainly due to the binding of iron in such a way that it is no longeravailable for outgrowth of Clostridium spores. This strong binding also explains theantioxidative properties of nitrite in these products (Grever and Ruiter, 2001).

Fat also is able to bind some nitric oxide. The amount incorporated in fat isconsiderably higher in unsaturated than in saturated lipids. Furthermore, nitrite isable to react with intermediates of the Maillard reaction, such as 3-deoxyosulose(Wedzicha and Wei Tian, 1989).

The reaction of nitrite with secondary or tertiary amines, though unimportant inquantitative respect, leads to N-nitroso compounds, which for a considerable part, arepotent carcinogens. These compounds may rearrange to form highly electrophilic diaz-onium ions that react with cellular nucleophiles, such as water, proteins, and nucleic acids.

Nitrate ingested with food or drinking water is partially reduced to nitrite in thebody, and contributes more than nitrite in meat products to the possible endogenousformation of N-nitroso compounds. A ban on the use of low amounts of nitrite asan additive is therefore not very rational, and deprives the consumer of a veryeffective guard against a number of pathogenic microorganisms, in particular Cl.botulinum, but also Cl. perfringens and Staphylococcus aureus.

Finally, nitrite may react in physiological concentrations and under gastric pHconditions with naturally occurring, as well as synthetic, antioxidants (Kalus et al.,1990). There are no indications for the formation of hazardous reaction conditionsfrom a mutagenicity viewpoint.

13.3.4 Sorbic Acid

The preserving properties of sorbic acid were recognized around 1940. During thelate 1940s and the 1950s, sorbic acid became available on a commercial scale,



resulting in its extensive use as a food preservative throughout the world (Sofos andBusta, 1983). As a straight-chain trans-trans dienoic fatty acid (FA)(CH3–CH=CH–CH=CH–COOH), it is susceptible to nucleophilic attack (Khandel-wal and Wedzicha, 1990b). The lowest electron density is associated with position3. Nucleophilic groups, e.g., the thiol group, however, may bind to the carbon atomat the 5 position as the terminal methyl group delocalizes the charge on that (Khan-delwal and Wedzicha, 1990a; Wedzicha, 1995). Sorbic acid inactivates several intra-cellular enzymes by this mechanism. It passes the cell wall in its undissociated formonly, which explains its low activity at higher pH values. The pKa value at 25°Camounts to 4.76. It is mainly active against yeasts, molds, and strictly aerobicbacteria. The toxicity toward mammals is low.

Sorbic acid is easily oxidized. This oxidation is accompanied by the developmentof a glyoxal-like flavor in sorbic acid preparations, and a brown color in a widevariety of model foods in which sorbic acid is included. Amino acids acceleratecolor development (Wedzicha et al., 1991).

13.3.5 Benzoic Acid

This compound is relatively stable during food processing and in food products. Itwas proposed in 1875 by H. Fleck as a replacement for salicylic acid, and can beconsidered as one of the first safe food preservatives. Like sorbic acid, it is mainlyactive against yeast and molds, but the growth of micrococci, E. coli, and manyother bacteria is retarded as well.

As is the case with sorbic acid, benzoic acid penetrates the cell wall in theundissociated form. As a consequence, it is active at lower pH values only (pKa at25°C = 4.19), and therefore serves as a preservative for sour products such as fruitjuices and jams. In shrimp preservation it is applied as a powder that is spread overthe shrimps, passes cell walls, and then ionizes in the intracellular fluid to yieldprotons that acidify the alkaline interior of the cell. The main cause of its activity,however, are biochemical effects (Eklund, 1980) such as inhibition of oxidativephosphorylation and of enzymes from the citric acid cycle (Chipley, 1983). Inmayonnaise preserved by benzoic acid, the undissociated acid is mainly present inthe lipid phase, which can be considered as a reservoir for the aqueous phase.

Benzoic acid is often combined with sorbic acid in order to reduce its peculiarflavor. It must be stated, however, that these are at least partly caused by impuritiesin the benzoic acid preparation used.

13.4 ANTIOXIDANTS

Antioxidants can be defined as “substances that, when present in low concentrationscompared to those of an oxidizable substrate, significantly delay or inhibit oxidationof that substrate” (Halliwell and Gutteridge, 1989).

Antioxidants are frequently added to unsaturated fats and oils in order to protectthem against oxidative deterioration. For this reason, they are also added to a varietyof food products containing unsaturated lipids. Antioxidants frequently applied areesters of gallic acid, butylated hydroxyanisole (BHA), butylated hydroxytoluene



(BHT), and tertiary butylhydroxyquinone (TBHQ). Of these, TBHQ is by far themost potent antioxidant. In both BHA and BHT, the butyl groups are also of a tertiarystructure (Figure 13.1).

Antioxidants are naturally present in many foodstuffs and are of great importanceas inactivators of radical formation. Some antioxidative enzyme systems are pro-duced in the human body and are supposed to play an important role in the cellulardefense against oxidative damage (Langseth, 1995).

Many of the antioxidants present in food function as terminators of chain reac-tions. A variety of compounds, such as phenols, aromatic amines, and conjugates,can function as chain-breaking antioxidants. They react with the chain-propagatingradical species, which results in the formation of radical species incapable of extract-ing hydrogen atoms from unsaturated lipids. These radicals may rapidly combinewith other radicals, or if a polyphenolic structure is present as in gallic acid esters,disproportionate into their original state and a quinoid form.

Because there are synergistic effects among antioxidants, commercial prepara-tions usually contain mixtures of them. As oxidative rancidity is strongly catalyzedby some heavy metal ions, in particular Cu++, antioxidant mixtures often containsequestrants (e.g., citric acid, EDTA) in order to complex these ions. Reductantssuch as ascorbic acid, which decrease the local concentration of oxygen, are alsoable to decrease the formation of peroxy radicals.

Fat oxidation by bacteria can be suppressed by the addition of preservatives suchas benzoic acid or sorbic acid.

13.5 FLAVORINGS, COLORANTS, AND SWEETENERS

Artificial flavorings are frequently added to a variety of foodstuffs. These prepara-tions mostly consist of a large number of different compounds, of which some show

FIGURE 13.1 Antioxodants. Top: from left to right, alkyl gallates, the two isomers of BHA.Bottom: from left to right, BHT and TBHQ.

OH

OHHO

COOR

C(CH3)3

C(CH3)3C(CH3)3(CH3)3C

CH3

C(CH3)3

OCH3 OCH3

OH OH

OH

OH

OH



a considerable reactivity. The way in which flavor components interact with the foodmatrix, and how this influences flavor perception, has been recently reviewed byBakker (1995).

Many interactions are of a purely chemical nature and may result from thepresence of aldehydes and their reactivity toward amino and thiol groups of proteins.Another frequently occurring type of interaction is the formation of hydrogen bondsbetween food compounds and polar flavor components such as alcohols. Starch andstarch-derived maltodextrins and β-cyclodextrin are able to form inclusion com-plexes with many flavor components. Many other interactions, although of greatinfluence on flavor perception, are of a physical nature and therefore are not men-tioned in this chapter.

Food additives such as dyes and sweeteners are not intended to react with matrixcompounds or to undergo other reactions. Some reactions may occur, however, anda few examples are given here.

As for food dyes, many of these are azo compounds, which implies the possibilityof reduction, for example, by the action of certain bacteria. The loss of color, inthese cases, is an indication of spoilage. Bisulfites are also able to reduce azo dyes(Wedzicha and Rumbelow, 1981).

Sweeteners have also been applied to foodstuffs for many years. Compoundssuch as saccharin, sodium cyclamate, aspartame, and several others are well knownand hardly need to be discussed here. In more recent times, sweeteners such assucralose and thaumatin have appeared on the scene. Sucralose is a sucrose derivativeobtained by chlorination by which the D-glucopyranosyl unit is converted to agalactopyranosyl unit chlorinated at position 4, while the fructofuranosyl unit ischlorinated at position 6. This results in an intense sweetness (600 times that ofsucrose) and a greater stability toward acids (Figure 13.2).

FIGURE 13.2 Sucralose.

CH2OH

Cl O

O

O

OH

OH

OH

HO

CH2Cl

CH2Cl



Thaumatin is a protein from katemfe fruit (Thaumatococcus danielli) with thestrongest sweetening properties hitherto known (2500× as sweet as sucrose). Theprotein is freely soluble in water, consists of 207 amino acids, shows a molecularweight of 22 kDa, and an isoelectric point (I.E.P.) of 11.5. The electrical charge inthe molecule is thought to be a major factor in its interaction with taste receptors(Boy, 1994). It synergizes with aspartame and with flavor enhancers such as 5'-nucleotides and sodium glutamate. Apart from this, it masks the aftertaste of sac-charin. It also masks metallic and bitter taste components.

Taste interactions between sweeteners are not uncommon. Another example isthe synergism between aspartame and acesulfame K. A 70%/30% blend, in somecases, yields a taste that cannot be distinguished from sugar. Acesulfam K alsoshortens the long, sweet aftertaste of sucralose (Meyer, 2001). Apart from tasteinteractions, some sweeteners also show synergism with flavors.

Some sweeteners may undergo chemical reactions, thereby losing their sweetproperties. The instability of saccharin at higher temperatures is well known. Apartfrom this, saccharin is subject to some degradation under acid conditions, yieldingsulfobenzoic acid, which has a disagreeable phenolic flavor. Another example isaspartame, which is the methyl ester of N-L-α-aspartyl-L-phenylalanine. Becauseof its nature, its stability in aqueous systems is limited. The maximum stability isbetween pH 3 and 5 and decreases at higher temperatures with a concomitant lossof sweetening power. The main degradation product is 3,6-dioxo-5-(phenylmethyl)-2-piperazinoacetic acid (Furda et al., 1975). Other decomposition products werelisted by Stamp and Labuza (l989), who added some novel components to this group.These all have in common a loss of sweet taste. Apart from this, aspartame showsremarkable reactivity toward a number of aldehydes that may be present in foodstuffsand contribute to flavor (Hussein et al., 1984; Cha and Ho, 1988), and thaumatinreacts with carrageenans if these are present (Ohashi et al., 1990).

Many unintended and sometimes unwanted reactions of artificial dyes, sweet-eners, and other additives with the food matrix are imaginable and should alwaysbe taken into consideration when the consequences of such additions to food arediscussed.

13.6 STABILIZERS, EMULSIFIERS, AND THICKENING AGENTS

The most important representatives of these compounds are polysaccharides, suchas starch and starch derivatives (α-1,4 D-glucans), cellulose and cellulose derivatives(β-1,4 D-glucans), plant extracts (pectins: α-1,4 D-galacturonans), seaweed extracts(carrageenan, agar, alginates), seed flour (guar and locust bean galactomannans,tamarind xyloglucans, konjac glucomannans), exudate gums (arabic, karaya, traga-canth), and microbial gums (xanthan, gellan, curdlan). Polymers are built of one ormore types of sugar residues, covalently attached in linear, linearly branched, andbranched structures. Their anomeric form (α/β), types of linkages (1,2; 1,3; 1,4;1,4,6; 1,3,6; etc.), presence of functional groups (carboxyl, phosphate, sulfate, esters,ethers), and molecular weight distribution determine their conformation in aqueous



systems (stiff/rodlike, random coil, helices), the intra- and intermolecular interactionsbetween molecules (dimerization, association, ionic interactions, hydrophobic inter-actions), and the interactions with other molecules (other polysaccharides, proteins,lipids). These interactions are the basis for viscous behavior, gelling, water binding,film forming, bulking, stabilizing, and emulsifying properties. Some polysaccharidesact synergistically in imparting these functions, such as locust bean gum and carra-geenan, locust bean gum and xanthan, and pectin and alginate. Important parametersfor applications are pH, heat and shear stability, syneresis properties, shelf life, andcompatibility with other food constituents. Derivatives of these polysaccharides withimproved functional properties are also used.

Emulsifiers are amphophilic compounds that concentrate at oil–water interfaces,causing a significant lowering of interfacial tension and a reduction in the energyneeded to form emulsions. They can be anionic, cationic, and nonionic compoundsthat have one or more of the following characteristics: surface active, viscosityenhancer, solid absorbent, or hydrophilic–lipophilic balance (HLB). They are addedto food emulsions to increase emulsion stability and to attain an acceptable shelflife. Polysaccharides are not surface-active agents, but rather macromolecular stabi-lizers that generally function through enhancement of viscosity and enveloping oildroplets in oil-in-water emulsions. The emulsifiers being used in food manufacturingwere categorized by Artz (1990) (see Table 13.1). Only lecithin is of natural origin.Its main source is the soybean, but it is also present in corn, sunflower, cottonseed,rapeseed, and eggs.

Emulsifiers stabilize emulsions in various ways. They reduce interfacial tensionand may form an interfacial film that prevents coalescence of droplets. In addition,ionic emulsifiers provide charged groups on the surface of the emulsion droplets,and thus increase repulsive forces between droplets. Emulsifiers can also form liquidcrystalline microstructures, such as micelles, at the interface of emulsion droplets.These are formed only at emulsifier concentrations larger than the critical micelle-forming concentration. These microstructures have a stabilizing effect.

TABLE 13.1Food Emulsifier Categories

Category Typical application

Lecithin (naturally occurring) and lecithin derivatives

—

Glycerol FA esters —Hydroxycarboxylic acid and FA esters Baking goods, margarineLactylate FA esters —Polyglycerol FA esters Baked goodsPolyethylene and propylene glycol FA esters O/W emulsionsEthoxylated derivatives of monoglycerides —Sorbitan FA esters Antistaling



Proteins also have a major influence on emulsion stability, although various typesof instability may occur: instability caused by coalescence or flocculation, and creamlayer formation caused by differences in density between oil droplets and the aqueousphase. In practice the latter problem is the most difficult to solve.

Creaming can be retarded by increasing the viscosity of the aqueous phase andby reducing the diameter of oil droplets or the density difference. The densitydifference between oil droplet and serum can be reduced by loading as much proteinas possible on the oil droplet surface. An open, random coil protein, such as sodiumcaseinate, is expected to yield less protein loaded on the oil surface than compactproteins do. It has been shown that open structure proteins, indeed, show a lowerequilibrium surface load than do compact structure proteins. (Zwijgers, 1992). Thehigher the equilibrium surface load, the better the emulsion-stabilizing protein.

In line with the foregoing, milk protein hydrolysates are useful foamers andemulgators. The foam- and emulsion-forming properties of milk peptides are evensuperior to those of intact milk proteins, as long as these show both charged andhydrophobic areas (Caessens, 1999).

The selection of emulsifiers for food preparations is mainly based on their HLBnumbers. This index expresses the hydrophile–lipophile balance, and is based onthe relative percentage of hydrophilic to lipophilic groups within the emulsifiermolecule. Lower HLB numbers indicate a more lipophilic emulsifier, while highernumbers indicate a more hydrophilic emulsifier. Emulsifiers with HLB numbersbetween 3 and 6 are best for water-in-oil emulsions and emulsifiers with numbersbetween 8 and 18 are best for oil-in-water emulsions.

13.7 CLARIFYING AGENTS AND FILM FORMERS

Clarifying agents or flocculants are used to eliminate turbidity or suspended particlesfrom liquids, such as chill haze in beer, precipitates in fruit juices and wines, or hazein oils. Often, they provide a nucleation site for suspended fine particles. Examplesof clarifying agents are lime in sugar juice clarification, pectic enzymes to breakdown pectins in fruit juices, and gelatin for clarification of fruit juices.

Film formers are used to coat a food by providing it with a protective layer andso make it more attractive in appearance or to increase its palatability. Film formersmay not impart flavor or mouth-feel of their own to the food. Examples are starchesto coat proteins to prevent Maillard reactions, mineral oils to seal pores of eggs, orsodium caseinate to encapsulate fat in whiteners.

13.8 ACIDULANTS

Food acidulants find their application, for the most part, in beverages and in fruit andvegetable processing. Apart from pH lowering, acidulants provide buffer capacity,impair sourness and tartness, enhance the effect of preservatives, and for some acid-ulants such as citric acid, prevent discoloration caused by trace metals (Seifert, 1992).



Besides acids, other components that produce or release acids are applied asacidulants, in particular when a slow release of acid is of importance. A well-known,slow-release acidulant is glucono-delta-lactone (GDL), which is used in bakery prod-ucts, dairy products, and in particular, meat products (Watine, 1995) (Figure 13.3).

The use of GDL in the maturation of dry sausages is well known. Duringpreparation of these sausages, GDL standardizes acidification, strongly reduces therisk of contamination, and improves the quality. GDL gradually lowers the pH to5.4, and after filling the sausage casings, the temperature is lowered to 0 to 4°C forsome hours. During this period the growth of starters and undesirable bacteria isinhibited. Then the temperature is increased so that fermentation can take placenormally.

13.9 FAT SUBSTITUTES AND FAT MIMETICS

Dietary fat contributes to the combined perception of mouth-feel, taste, and aroma.Fat also contributes to creaminess, appearance, palatability, texture, lubrication prop-erties of foods, and increases the feeling of satiety during meals. Apart from this, itis able to carry lipophilic flavor compounds and can act as a precursor for flavordevelopment. A very important characteristic of fat is its suitability for use in frying.

Due to their high caloric value, there is an increasing tendency to replace fatsand oils with components that are not calorific, but which can impart the sametechnological and sensory functionalities.

Two types of fat replacers can be distinguished—fat substitutes and fat mimetics.Fat substitutes are lipid- or fat-based macromolecules that physically and chemicallyresemble triacylglycerols (e.g., sucrose polyesters or alkyl glycoside polyesters). Fatmimetics are protein- or carbohydrate-based compounds that imitate organoleptic orphysical proteins or triacylglycerols (Voragen, 1998). Fat substitutes physically andchemically resemble fats and oils, as they are esters of polyols. They are stable atcooking and frying temperatures.

Many carbohydrate-based fat substitutes are mixtures of sucrose esters formedby chemical transesterification or interesterification of sucrose with one to eight fattyacids (FA). The class with six to eight FAs are called sucrose fatty acid polyesters.These molecules are too large to be broken down by intestinal lipase enzymes, andfor that reason, do not have any caloric value (Voragen, 1998).

FIGURE 13.3 D-Gluconolactone.

CH2OH

OH

OH

OH

O

O



Olestra, in this respect, is a promising compound. The stability of Olestra iscomparable to natural fats and even better. Olestra can be used as a frying medium,and appears to undergo slower oxidative and hydrolytic degradation (Lindley, 1996).The functionality and potential application of the sucrose FA polyesters is governedby the type of fatty esters used in the manufacturing process. The melting characterdepends on the length and the degree of saturation of the FA in a similar way. In theory,this melting character may be adjusted by the right choice of FA (Lindley, 1996).

Esters containing one to three fatty acids are called sucrose FA esters (SFEs).Unlike sucrose FA polyesters, the SFEs are easily hydrolyzed and absorbed by digestivelipases, and are thus caloric. SFEs containing five to seven free hydroxyl groups withone to three FA esters show both hydrophilic and lipophilic properties, and thereforehas excellent emulsifying and surface-active properties. In addition, they are effectivelubricants, anticaking agents, thinning agents, and antimicrobials (Voragen, 1998).

Other carbohydrates modified to fatty acid esters are sorbitol, trehalose, raffinose,and stachyose.

Saccharide-based fat mimetics differ strongly from fats and oils. Generally theyabsorb a substantial amount of water and are therefore not suitable for frying. Asthey can only carry water-soluble flavors, they lack the flavor of fats and oils. Inulinand starch hydrolysates (dextrose equivalent ~2) are striking examples of fat mimet-ics. The fat substitution is based upon its ability to stabilize water into a creamystructure that has a fatlike mouth-feel (Blomsma, 1997).

Most carbohydrate-based fat replacers are extracted from by-products rich incell wall polysaccharides. Polydextrose, which can be applied both as a carbohydrateand as a fat replacer, is obtained by vacuum thermal polymerization of glucose,using citric acid as a catalyst and sorbitol as a plasticizer (Voragen, 1998).

13.10 PREBIOTICS

In Chapter 12, it is stated that the use of prebiotics in association with useful probioticsmay be a worthwhile approach, as prebiotics may stimulate some probiotic strains.

The term prebiotic is derived from the Greek and can be translated as “prior tolife.” Generally, prebiotics comprise food additives that are barely or not digestedin the small intestine and end up in the colon where they, as already stated, maystimulate beneficial bacteria in the colon by serving as fermentation substrates.Promotion and regulation of health is the aim of these bioactive products, whichpresent an important trend in the present-day production of foods.

Nondigestible carbohydrates are the main representatives of this class of foodadditives. There are three main types of carbohydrates that are indigestible in the humansmall intestine, that is, nonstarch polysaccharides, resistant starch, and nondigestibleoligosaccharides (NDOs). As the average daily ingestion of the latter group is lowerthan the level considered as safe (not over 15 g/day), supplementation of NDOs couldbe beneficial (Voragen, 1998). These can be used in a wide range of processed foods,including dairy products, confectionery, bakery products, ready meals, breakfast cereals,and drinks. Other significant beneficial effects are (Van Haastrecht, 1995):



• replacement of sugar and fat• reduction of the number of unwanted bacteria in the colon• fiber enrichment of foods hitherto poor in fiber, such as white bread, dairy

products, and transparent drinks• prevention of tooth decay• regulation of lipid metabolism

Two specific groups of NDOs, which are commercially available, must be men-tioned, that is, fructo-oligosaccharides (FOS), obtained by transfructosylation ofsucrose using a β-D-fructosyltransferase or by hydrolysis of inulin by endo-inulinase;and galacto-oligosacharides (GOS) by transgalactosylation of lactose using β-galac-tosidase. Inulin is a virtually linear fructose polymer in which the fructose moleculesare linked by β-(2-1) bonds with an average degree of polymerization ranging from2 to 60 (average length of 10).

Most oligosaccharides have a moderate reducing power by which they are stillsubject to Maillard reactions when used in food to be heat processed. Fructo-oligo-saccharides of the GFn type, composed of fructofuranosyl residues and one terminal,nonreducing glucosyl residue as obtained by transfructosylation; lactosucrose andglycosylsucrose have no reducing power. Fructo-oligosaccharides obtained byhydrolysis of inulin can be of the GFn type or of the Fm type, the latter having areducing fructofuranosyl residue (Figure 13.4).

FIGURE 13.4 Two types of FOS (sucrose of comparison). (After van Haastrecht, J., Int.Food Ingredients, 1995.)

CH2OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OHOH

OH

OH

OH

HOHO

HO HOHO

O

O

O

O

O

O

O

O O

O

O

O

O

CH2OH

CH2OHCH2OH

CH2OH

CH2OH

CH2OH

CH2OH

CH2OH

CH2OH

n-1

Sucrose GFn Fm

m-2

CH2 CH2

CH2



At a pH less than 4 and treatments at elevated temperatures or prolonged storageat ambient conditions, oligosaccharides present in a food can be hydrolyzed resultingin the loss of nutritional and physicochemical properties. For fructo-oligosaccharidesit is reported that in a 10% solution of pH 3.5, less than 10% is hydrolyzed afterheat treatments of 10 s at 145°C, 5 min at 45°C, or 60 min at 70°C. After two daysat 30°C, less than 5% is hydrolyzed. The stability can greatly differ for the variousclasses of oligosaccharides depending on the sugar residues present, their ring formand anomeric configuration, and linkage types. Generally β-linkages are strongerthan α-linkages, hexoses are more strongly linked than pentoses and deoxysugars,and pyranoses are more strongly linked than furanoses.

Finally, resistant starches are dietary carbohydrates that have attracted increasedinterest on the part of food manufacturers due to the beneficial effects these mighthave on human health. It escapes digestion and absorption in the small intestine ofman and reaches the large bowel, where fermentation takes place. As a result, thepH in the colon is lowered and short-chain FAs are formed. They further increasefecal bulk and may protect against colon cancer, improve glucose tolerance, andlower blood lipid levels. The most important forms of resistant starch in the diet arebotanically encapsulated starch present in intact foods, starches with a B-type crys-talline structure present in unheated foods, starch retrogradated as a result of fullgelatinization and dispersion by processing, and thermally or chemically modifiedstarches (Voragen, 1998).

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Artz, W. Emulsifiers, in Food Additives, Bramen, A.L., Davidson, R.M., and Salminen S.,Eds., Marcel Dekker, New York, 1990, p. 347.

Bakker, J., Flavor interactions with the food matrix and their effects on perception, in Ingre-dient interactions—Effects on food quality, Goankar, A.G., Ed., Marcel Dekker, NewYork, 1995, p. 411.

Blomsma, C.A., Ingenious inulin, International Food Ingredients, 2, 22, 1997.Boy, C., Thaumatin: a taste-modifying protein, International Food Ingredients, 6, 23, 1994.Caessens, P.J.W.R., Vissers, S., Gruppen, H., and Voragen, A.G.J. β-Lactoglobulin hydrolysis.

1. Peptide composition and functional properties of hydrolysates obtained by theaction of plasmin, trypsin, and Staphylococcus aureus V9 protease, J. Agric. FoodChem., 47, 2973, 1999.

Cha, A.S. and Ho, C.T., Studies of the interaction between aspartame and flavor vanillin byhigh-performance liquid chromatography, J. Food Science, 53, 562, 1988.

Chipley, J.R., Sodium benzoate and benzoic acid, in Antimicrobials in Foods, Branen, A.L.and Davidson, P.M., Eds., Marcel Dekker, New York, 1983, p. 11.

Eklund, T., Inhibition of growth and uptake processes in bacteria by some food preservatives,J. Appl. Bacteriol., 48, 423, 1980.

Feberwee, A., Legal aspects of food additives of natural origin, in Proceedings of the Inter-national Symposium Food Additives of Natural Origin, Plovdiv, Bulgaria, 31 May–2June 1989, p. 22.

Furda, I., Malizia, P.D., Kolor, M.G., and Vernieri, P.J., Decomposition products of L-aspartyl-L-phenylalanine methyl ester in various food products and formulations, J. Agr. FoodChem., 23, 340, 1975.



Giddings, G.G., The basis of color in muscle foods, J. Food Science, 42, 288, 1977.Grever, A.B.G., Ruiter, A., Prevention of Clostridium botulinum outgrowth in heated and

hermetically sealed meat products by nitrite—A review, Eur. Food Res. Technol., 213,165, 2001.

Hall, R.L., 1999, Food safety: elusive goal and essential quest, IUFoST Founders Lecture,Sydney, October, Food Australia, 51, 12, 601, 1999.

Halliwell, B. and Gutteridge, J.M.C., Free Radicals in Biology and Medicine, 2nd ed, Clar-endon Press, Oxford, U.K., 1989.

Hussein, M.M., D’Amelia, R.P., Manz, A.L., Jacin, H., and Chen, W.-T.C., Determination ofreactivity of aspartame with flavor aldehydes by gas chromatography, HPLC andGPC, J. Food Science, 49, 520, 1984.

Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) ExpertCommittee on Nutrition, 4th report. WHO Technical Report Series, No. 97. WorldHealth Organization, Geneva, 1955, p. 29.

Kalus, W.H., Münzner, R., and Filby, W.G., Isolation and characterization of some productsof the BHA-nitrite reaction: examination of their mutagenicity, Food Additives andContaminants, 7, 223, 1990.

Kamsteeg, J. and Baas, M.I.A., E = Eetbaar (E = Edible), H.J.W. Becht, Amsterdam, 1985. Khandelwal, G.D. and Wedzicha, B.L., Derivatives of sorbic acid-thiol adducts, Food Chem-

istry, 37, 159, 1990a.Khandelwal, G.D. and Wedzicha, B.L., Nucleophilic reactions of sorbic acid, Food Additives

and Contaminants, 7, 685, 1990b.Ladikos, D. and Lougovois, V., Lipid oxidation in muscle foods: a review, Food Chemistry,

35, 295, 1990.Langseth, L., Oxidants, Antioxidants, and Disease Prevention, ILSI Europe Concise Mono-

graph series, ILSI Europe, Brussels, 1995.Lindley, M.G., Olestra: the ultimate in fat substitution? International Food Ingredients, 3, 35,

1996.Lüthy, J., Safety evaluation of natural food additives, in Proceedings of the International

Symposium Food Additives of Natural Origin, Plovdiv, Bulgaria, 31 May–2 June1989, pp. 35–40.

McWeeny, D.J., Knowles, M.E., and Hearne, J.F., The chemistry of non-enzymic browningin foods and its control by sulphur, J. Sci. Food Agric., 25, 735, 1974.

Meyer, S., Taste interactions of acesulfame potassium and other high intensity sweetenerswith fruit flavours in different food proteins, Abstracts, 2nd IUPAC InternationalSymposium on Sweeteners (2nd IUPAC-ISS), November 13–17, 2001, Hiroshima,Japan, pp. 55–56.

Möhler, K., Formation of curing pigments by chemical, biochemical or enzymatic reactions,in Proc. Int. Symp. Nitrite in Meat Products, Zeist, The Netherlands, Krol, B. andTinbergen, B.J., Eds., Pudoc, Wageningen, 1973, p. 13.

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Ruiter, A., Safety of food: the vision of the chemical food hygienist, in Food Science: BasicResearch for Technological Progress. Proceedings of the Symposium in Honour ofProfessor W. Pilnik, Wageningen, The Netherlands, 25 November 1988, Roozen, J.P.,Rombouts, F.M., and Voragen, A.G.J., Eds., 1989, p. 19.

Seifert, D., Functionality of food acidulants, International Food Ingredients, 3, 4, 1992.Sofos, J.N. and Busta, F.F., in Antimicrobials in foods, Branen, A.M. and Davidson, R.M.,

Eds., Marcel Dekker, New York, Basel, 1983, p. 141.



Stamp, J.A. and Labuza, T.R., Mass spectrometric determination of aspartame decompositionproducts: Evidence for β-isomer formation in solution, Food Additives and Contam-inants, 6, 397, 1989.

Van Dokkum, W., Additieven en contaminanten (additives and contaminants), Voeding in depraktijk, 6, 1, 1985.

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Voragen, A.G.J., Technological aspects of functional food-related carbohydrates, Trends inFood Science and Technology, 9, 328, 1998.

Watine, Ph., Glucono-delta-lactone: functional properties and applications, International FoodIngredients, 3, 39, 1995.

Wedzicha, B.L., Sulphur dioxide—the most versatile food additive? Chemistry in Britain,1030–1032, 1991.

Wedzicha, B.L., Interactions involving sulfites, sorbic acid, and benzoic acid, in IngredientInteractions—Effects on Food Quality, Goankar, A.G., Ed., Marcel Dekker, NewYork, Basel, 1995, p. 529.

Wedzicha, B.L., Bellion, I., and Goddard, S.J., Inhibition of browning by sulfites, in Nutri-tional and Toxicological Consequences of Food Processing, Friesman, M., Ed., Ple-num Press, New York, 1991, p. 217.

Wedzicha, B.L. and McWeeny, D.J., Non-enzymic browning of ascorbic acid and their inhi-bition. The production of 3-deoxy-4-sulphopentosulose in mixtures of ascorbic acid,glycine and bisulphite ion, J. Sci. Food Agric., 25, 577, 1974.

Wedzicha, B.L. and Mountfort, K.A., Reactivity of sulphur dioxide in comminuted meal,Food Chemistry, 39, 281, 1991.

Wedzicha, B.L., Rimmer, Y.L., and Khandelwal, G.D., Catalysis of Maillard browning bysorbic acid, Lebensmittel-Wiss. u.-Technol., 24, 278, 1991.

Wedzicha, B.L. and Rumbelow, S.J., The reaction of an azo food dye with hydrogen sulphiteions, J. Sci. Food Agric., 32, 699, 1981.

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14 Food Safety

Julie Miller Jones

CONTENTS

14.1 Introduction................................................................................................ 37514.2 Consumer Attitudes toward the Food Safety Problem ............................. 37614.3 Tests to Determine Food Safety ................................................................ 37814.4 Food Safety Concerns ............................................................................... 38014.5 Microbial Contamination of Food............................................................. 38114.6 Risk/Benefit as It Applies to Food............................................................ 38314.7 Nutritional Evaluation of Food Processing............................................... 384

14.7.1 Beneficial and Detrimental Effects ............................................. 38414.7.2 Effects on Vitamins ..................................................................... 38514.7.3 Effects on Minerals ..................................................................... 387

14.8 Newer and Novel Technologies................................................................. 38714.8.1 Irradiation .................................................................................... 38714.8.2 Biotechnology ............................................................................. 387

14.9 Additives .................................................................................................... 38814.10 Summary.................................................................................................... 389References.............................................................................................................. 389

14.1 INTRODUCTION

Safe food—it is what every individual expects in every mouthful and every govern-ment strives to provide for its populous. Because food is the object of our earliestpreferences and the subject of our strongest prejudices, food safety is a gut issue.Yet what seems on the surface to be both basic and imperative is not at all simpleand, in fact, is not achievable in the absolute sense. It is extremely hard for manyto accept even the idea that food is relatively—not absolutely—safe. What appearsto threaten food, threatens all in a very direct and visceral way.

An understanding of basic definitions concerning safety and toxicity is crucial.First all compounds, no matter how salutary, can be ingested in some manner orquantity that can be harmful—in other words, toxic. Toxicity is the capacity of asubstance to produce some adverse effect or harm. Even vital dietary componentssuch as water and vitamins can be consumed at toxic levels. Too much pure watercan cause renal shutdown; excessive vitamins can cause minor problems, such asflushing or nausea, or extremely adverse events such as liver damage, teratogenicity,



and death. The 1538 Paracelsus motto, “only the dose makes the poison” operatesfor food components (Jones 1992).

Second, what is safe for one is not safe for everyone. Individuals with allergies,inborn or acquired errors of metabolism, or certain diseases can ingest a food in ausual and customary manner and suffer adverse, and even, in rare instances, fataloutcomes. Thus foods that are safe for most are not safe for all.

Third, how the food is used or produced may alter its safety. Combinations ofa certain food and a drug or herb or in a bizarre or poor diet can render an otherwisesafe food harmful. In certain growing or production conditions, a food may acquirean unexpected contaminant such as a mycotoxin or a prion. Thus, what is usuallysafe harbors a masquerading toxin.

Because absolute safety is unattainable, relative safety is what is sought. Relativesafety is the probability that no harm will come when the food is consumed in a usualand customary manner. Even relative safety, when it comes to food, is a big orderbecause it requires constant diligence by all parties who come in contact with the food.Any glitch in the system, from the field to the table, can introduce a potential hazard.

The environment and the starting raw material must be optimal. There shouldnot be high levels of naturally occurring toxicants in the crop or animal itself. Safetymust be maintained by controlling conditions such that the possibility of contami-nation is minimized. Plant materials must be free of infestations, harmful residues,mold, and mycotoxins. Animal materials should not contain any veterinary drugresidues or any abnormal constituents transferred from feedstuffs. Metal particles,weed contaminants, or other incidental components from harvesting or processingmust be vigilantly prevented and tested. During transportation from the field to plantor market, carriers must handle the food to maintain its quality. Care must beexercised so that proper temperatures and moisture levels are maintained, and thatno contamination or pathogenic growth occurs during any point of the storage,shipping, and processing. Prevention of natural deterioration and further contami-nation is often done through packaging. Packaging materials and other appliedtechniques and handling procedures must not introduce risks of their own.

Once in the consumer’s hands, food must be cooked and handled properly inthe home or food service operation. This means food must not only be safe but thatit must provide the expected nutrients. Unfortunately, both the home and restaurantare sites for food mishandling.

Thus while maintenance of food safety from farm to fork is expected and takenfor granted, it actually requires both care and vigilance, and even with the utmostcare there is still an element of risk. While most scientists know that safe does notmean not risk free, this concept is extremely difficult to convey to consumers.

14.2 CONSUMER ATTITUDES TOWARD THE FOOD SAFETY PROBLEM

The difficulty in convincing consumers that no food is risk free stems from bothfood practices and attitudes. First, most consumers in industrialized nations are farremoved from the production and processing of food, and even the preparation of


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food. The modern strategy of procuring ready-prepared foods as take out or frozenpreprepared products means that many children rarely observe meals being prepared.Thus cooking in many homes has become a process of heating items to servingtemperature, usually in a microwave. In this scenario, safe preparation and foodhandling techniques may no longer be passed from generation to generation.

Second, consumer concerns about food may, in part, result from a concern abouttoday’s scientific and technological complexity, a lack of trust in big business andits advertising, and in government’s ability to protect food. For some, science hasbecome the problem, not the answer. Even for those with faith in the promise scienceholds, scientific complexity is confusing. Two experts espouse different assumptions;extrapolations and interpretations reach diametrically opposed positions often usingthe same data. Disagreement of scientists is not the only source of confusion. Mediasavvy, self-appointed experts or consumer groups’ spokespeople with easily under-stood messages, albeit specific agendas, add to the multiplicity of positions consum-ers hear. Messages from these sources are seemingly straightforward and demon-strative. These slanted messages do not necessarily present all sides of the data,unlike those from ethical scientists, and thus fail to communicate either conflictingevidence or flaws in the research. It becomes a nearly impossible task for theconsumer to sort through the cacophony of voices to find the truth. Groups thatposition themselves as anti-big business and technology and proconsumer and envi-ronment, whether justified or not, garner consumer trust. This is particularly true inareas where the fear factor is high or the technology is unfamiliar, such as irradiationand biotechnology. Oddly, consumer trust in such groups may be misplaced. Con-sumers often feel that the activist group is on their side and that those in governmentor industry must be espousing a position that furthers an agenda that is probusinessand not in the interest of the consumer. Yet activist groups often pick the mostegregious examples or cite studies that mainstream science discredits for use in apress release. This strategy not only wins them a primetime news story, but alsocreates consumer angst resulting in further contributions to their cause.

Recent scares, such as Sudan Red, dioxin, and mad cow disease, have fueledthe lack of trust in government food agencies whose charge it is to protect the public.The case of Sudan Red showed that illegal additives were regularly entering the foodsupply in countries where its use is prohibited. The dioxin example gave the publicjustification in their lack of trust because food authorities failed to inform the publicwhen they were first aware of potential dioxin contamination of feed and food. Madcow disease forced the government to reverse statements that a food or food com-ponent is safe, eroding public trust.

Communication about food may create misunderstanding. Direct conversationswith scientists often fail to enlighten consumers because scientists often pepper theinformation with jargon and complexities that only further confuse and frustrate.

Media stories about nutrition and food safety are often simplistic, boring, incom-plete, or biased. Media sound bites distill years of study and lengthy discussion intoa 60-second spot, and in the process omit important aspects that are key to a fullunderstanding of the study. A news item may be taken out of context, overgeneralizedor it may reflect the findings of a single study. In some cases the reported study isnot in agreement with the whole body of scientific literature, but this is not mentioned



in the story. Even if a study is accurately reported, frequently the news commentatorhas neither the required depth of understanding nor the time to interpret what aparticular finding means to someone who eats the food once a week.

Activist groups and advertisers may be selective about which studies they use.Studies that support their point of view, but do not fairly represent the full body ofknowledge, may be used by some groups and bloggers. Fear is heightened whenstatements about potential toxicity feature vulnerable groups such as children andpregnant women.

Inadequate scientific literacy also increases fear of chemicals and technology.Many consumers are unaware that human life has always entailed exposure tochemicals, and that everything ingested and inhaled is composed of chemicals.Elusive to most consumers is the fact that naturally occurring chemicals are abundantand many can be more toxic than synthetic ones. Most consumers believe theconverse—that natural chemicals are innocuous and synthetic ones are nefarious.Ironically, for food chemicals, much more is known about chemicals added to foodthan the chemicals inherently present in natural foodstuffs.

Adding to the fear of technology is a longing for a less complicated time betterknown as “the good old days.” This nostalgia fails to note the realities of the past,with shorter life spans, longer hours of preparation in the kitchen, limited availabilityof produce in the late winter months in the northern climes, and other detractorssuch as lack of food testing and regulation.

14.3 TESTS TO DETERMINE FOOD SAFETY

Human beings have always been intuitive toxicologists, relying on their senses ofsight, taste, and smell to detect harmful or unsafe food, water, and air. When asked,some consumers still feel that they are the prime determiners of food safety and tryto rely on themselves. Scientists and savvy consumers have come to recognize thatour senses are not adequate to assess the dangers inherent in exposure to a chemicalsubstance, especially one in which the ill effect is either cumulative or delayed. Thusthe sciences of toxicology and risk assessment have developed a protocol for testingand assessing the safety of foods and their constituents.

Toxicity tests are performed on all compounds used as intentional additives andpesticides (Dybing et al. 2002). Substances must first undergo acute toxicity tests,which use at least two species of experimental animals to determine the lethal dose50 (LD50)—the dose that kills half of the animals. If the substance is regarded asbeing of low toxicity, then metabolic tests are undertaken in several animal species.These tests track the fate of the compound in the body. If metabolites are formed,their fates and toxicity must also be determined. A variety of species are used totest for differences in metabolism and to determine which species will be mostsimilar to humans.

Subacute tests are then performed. These require the feeding of a range of dosesbelow the LD50 to at least two species for two to three months. A threshold or no-observed-adverse-effect level (NOAEL) is determined from the highest dose thatproduces no harm in the most sensitive species. Chronic tests follow. These involvefeeding a compound for a lifetime at doses 1000 to 100 times more than a human


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would likely ingest. These tests also involve two to three species. These tests notonly assess the health of the animal over its lifespan, but also determine if there areany reproductive or offspring abnormalities.

Safe levels for use are determined upon completion of testing and determinationof the NOAEL. The acceptable daily intake (ADI) is then determined by dividingthe NOAEL by a safety factor (Figure 11.1). The ADI is expressed as milligramsof the test substance per kilogram of body weight per day. The safety factor, often100, is arbitrary and may vary according to the test material and circumstances. Therationale for 100 is that if the average sensitivity of humans to a particular compoundis 10 times greater than for that of the most sensitive test animal, and if the mostsensitive humans are 10 times more sensitive than the test animals; the use of thefactor 10 × 10 = 100 would mean that the most sensitive individual could safelyingest the amount equivalent to the ADI.

The threshold of toxicological concern (TTC) may be determined when it is notpossible to set an ADI for constituents used in very small amounts, such as flavorcompounds. TTC refers to the establishment of a level of exposure for all chemicals,even if there are no chemical-specific toxicity data, below which there would be noappreciable risk to human health. The main advantage of the use of TTCs is that therisk of low exposures can be evaluated without the need for chemical-specific animaltoxicity data (Renwick 2005; Kroes et al. 2005). European Union (EU) countriesuse a decision tree approach (Szponer et al. 2003).

Carcinogens and prions need special treatment. Some scientists adhere to theidea that there is, for some carcinogens, no threshold or tolerance level, but currentlymore scientists believe that there is a threshold level and that with proper under-standing of the compound and proper animal experiments, this level can be deter-mined (Waddell 2005).

For carcinogens, the 100-fold safety factor may be inadequate, and factors ashigh as 5000 have been proposed. Tolerable daily intakes have been set for carcin-ogens in food (such as Aflatoxin B1) that are DNA adducts. Estimates of DNAadduct levels can be made by direct measurement or indirectly as a consequence oftheir presence, for example, by tumor formation in animal models or exposedpopulations epidemiologically (Jeffrey and Williams 2005).

Prions—misfolded proteins that lead to neurological disorders—also requirespecial precautions, partly because the mode of transmission is still under investi-gation. Research is needed to determine why some individuals are susceptible andothers may not be, and delineation of roles played by other cellular componentssuch as chaperone proteins. The current strategy is to keep prions out of the foodsupply rather than setting an ADI or TTC.

Novel foods also require special safety assessment protocols (Edwards 2005).A case-by-case approach is needed, which must be adapted to take into account thecharacteristics of the individual novel food. A thorough appraisal is required of theorigin, production, compositional analysis, nutritional characteristics, any previoushuman exposure, and the anticipated use of the food. The information should becompared with a traditional counterpart of the food, if this is available. In somecases, a conclusion about the safety of the food may be reached on the basis of thisinformation alone, whereas in other cases it will help to identify any nutritional or



toxicological testing that may be required to further investigate the safety of thefood. The importance of nutritional evaluation cannot be overemphasized. This isessential in order to avoid dietary imbalances that might lead to interpretationdifficulties, but also in the context of its use as food, and to assess the potentialimpact of the novel food on the human diet. The traditional approach used forchemicals with a large safety margin relative to the expected exposure cannot beapplied to novel foods. The assessment of safety in use should be based upon athorough knowledge of the composition of the food, evidence from nutritional,toxicological, and human studies, expected use of the food, and its expected con-sumption. Safety equates to a reasonable certainty that no harm will result fromintended uses under the anticipated conditions of consumption.

For all foods and components, the final step is to consider potential consumptionestimates of the foods or commodities that might contain the chemical or novel food.For microconstituents, this allows calculation of the maximum residue level (MRL).

Constant monitoring and reevaluation of these estimated intakes make certainthat estimates reflect real exposure (Caldas and Souza 2004). For some items accurateexposure estimates may require development of new sampling procedures to accu-rately assess food product consumption data for high-risk groups and greater con-sideration of food purchased away from home.

Even with the best scientific methods, extrapolations and judgments are requiredto infer human health risks from animal data. Basic toxicological concepts, assump-tions, and interpretations were found, according to a recent survey, to differ greatlybetween toxicologists and laypeople (Low et al. 2004), as well as differences amongtoxicologists working in industry, academia, and government. Toxicologists werefound to be sharply divided in their opinions about the ability to predict a chemical’seffect on human health on the basis of animal studies and other laboratory methodsbeing employed in lieu of animal tests (Ames and Gold 2000). This difference ofopinion creates public policy tensions. Renowned toxicologist Bruce Ames statedthat minimizing minuscule hypothetical risks may damage public health by divertingresources and distracting the public from major risks. In addition, some epidemiol-ogists state that the avoidance of possible risks from processes like irradiation maycontinue to allow hazards from foodborne disease that could be significantly reducedby use of irradiation.

14.4 FOOD SAFETY CONCERNS

Foodborne disease has always topped the list of food safety concerns for mostgovernment bodies around the world. Outbreaks due to Salmonella and other organ-isms such as Listeria and E. coli have placed foodborne disease at the top of the listof consumer food safety concerns (Kirk et al. 2002). This has not always been thecase. Chemicals and pesticides still manage to cause great concern. Scientific abilityto detect substances to the attogram level fuels consumer unease. In cases where theconsumer used to believe that the chemical was not in the food and is now there atvery small levels is disquieting for consumers, and consumers and scientists alikeare unable to interpret the effect, if any, of the small level of such a chemical.


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Bovine spongiform encephalopathy (BSE), foods produced by biotechnology—known to the consumer as genetically modified organisms (GMOs)—and food as avehicle for bioterrorism add to the growing list of consumer fears.

How a food safety concern is viewed varies by the perspective of the viewer.Scientific groups use documented hazards, known deaths, or cases of illness toextrapolate risks. Consumers judge risk not so much on documented hazard but onwhat they feel is a potential hazard or an unanswered or unanswerable questionabout the food safety risk assessment (Hansen et al. 2003). GMOs are cases wherescientists see that they offer no documentable hazard, but consumers feel they offertoo many unknowns. The European regulatory response, which invokes the precau-tionary principle, reflects this type of reasoning. This approach to food safety resultsin the failure to approve use of a foodstuff or a substance because of a lack of abilityto prove its safety, despite little or no good data proving it unsafe.

14.5 MICROBIAL CONTAMINATION OF FOOD

Pathogenic bacteria are responsible for the majority of food-related outbreaks.Opportunity for contamination exists at every stage in the food chain. Actual inci-dence of foodborne disease is unknown even in countries with fairly sophisticatedmonitoring systems because the number of cases is severely underreported. The mostrecent estimates from the Centers for Disease Control (CDC) in the United Statessuggest that there are 76 million illnesses and 5000 deaths each year from foodbornedisease (CDC 2003; Allos et al. 2004). Surprisingly, pathogens are actually deter-mined in under 20% of the illnesses. Of the known pathogens, Campylobacter jejuni,Salmonella species, Listeria, and Toxoplasma account for over 75% of foodborneillness. Staphylococcus aureus, Clostridium perfringens, Yersinia enterocolitica, aswell as the parasites giardia, cyclospora, and cryptosporidium are also problems,and much in the news for their potential to cause either very large or lethal outbreaks.E. coli, Listeria, and botulism are of significant concern because of the high degreeof morbidity and mortality (Yang, Angulo, and Altekruse 2000).

The increased incidence in all types of viral, bacterial, and parasitic infectionsis not only due to better reporting, detection, and surveillance, but also due tochanges in consumption patterns and perhaps shifts in weather patterns. People inmany Western nations buy more preprepared, prepackaged foods, demand out-of-season and exotic foods from all around the globe, utilize new technologies suchas modified atmosphere, demand food with less salt and fat, and use food serviceand delis more often. Coupled with these changes, consumers also desire feweradditives that might slow microbial growth. Greater pollution and changes in oceantemperatures affect the Gulf Stream as in the case of Vibrios, and this also meansmore foodborne disease. More dining out and deli food mean the possibility ofgreater contamination because a greater number of people are handling the foodand there are more potential steps for errors. All these trends can impact themicroorganisms and chemicals found in food.

An increase in numbers of those in vulnerable populations—the very young, thevery old, the chronically ill, and the immunocompromised—is a population factorincreasing the possibility of more foodborne disease. Increased lifespan and numbers



of transplant recipients and persons with conditions requiring immunosuppressantdrugs raise the number of people in at-risk groups.

Lack of knowledge of safe food preparation and storage also puts populationsat risk. A study by the U.S. Centers for Disease Control traced 77% of the microbialdisease outbreaks to food-service establishments, 20% to homes, and 3% to foodprocessing plants. From these data it can be seen that increased food safety programs,such as Hazard Analysis Critical Control Point (HACCP) or Longitudinally Inte-grated Safety Assurance (LISA), which are mandated in many parts of the world formanufactured foods, have reduced foodborne problems from commercially preparedfood. However, there needs to be a plan to reduce problems in food service and aneducation program to tell consumers about safe food handling and preparation.

Changes in consumer preference may also increase foodborne disease. Flavorpreference for a soft cheese made with unpasteurized milk or raw fish may increasethe risk of contracting Listeria, E. coli, or a parasite.

Price may also impact the choice of a specific food, and in turn be a choiceabout safety. Food safety is an income-elastic good. As incomes rise, so does thesizeable premium a consumer will pay for food that is perceived to be safer. Oddly,often those with more income and education engage in riskier food safety behaviors(Yang, Angulo, and Altekruse 2000). However, it must be pointed out that traditionalmarket mechanisms do not work directly with respect to food safety issues. First,food safety attributes are not readily determined at the point of purchase. Competingfood products are rarely appraised according to alternative levels of microbiologicalspoilage or the probability of contaminated cans. Second, a less safe product isusually not intentionally supplied. The problem often lies in accidental contaminationor a change in the process handling, shipping or storage, or product composition,such that microbial growth is allowed or contamination occurs.

Understandably consumers react viscerally to a dreaded outcome. If a suppliermakes a mistake, the company will be pilloried in all forms of media. One mistakein one can in a million (an undetectable number), which gives rise to a death, willnot only affect the sale of the particular product, but of all products of that type andby that manufacturer. Many companies fold in the face of such an adverse event.

To prevent such negative consequences, many industry programs are in placeworldwide. The requirements of the International Standards Organization (ISO),HACCP, and good manufacturing practice (GMP) mandate sanitary conditions forfood production in order to minimize chances for contamination. Other ISO programsset ingredient specifications to ensure that the raw materials incorporated into foodproducts meet required low levels of contamination.

New processing strategies for killing and detecting microbes are being perfected.Some use the new sciences of metabolomics and genomics. One such technique usesinfrared spectroscopy on light reflected from the surface of the food to producebiochemical “fingerprints” of any contaminating microorganisms and rapidly esti-mate their numbers. Such a technique changes the time needed to measure microbialcontamination on a carcass from hours to minutes, making food less risky byeliminating contaminated food before it enters the consumer food supply (Goodacreand Ellis 2005).


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Consumers are often unaware that there are long-term as well as short-term con-sequences of foodborne microbial disease. The acute effects are sometimes not theend of the illness. Several significant foodborne pathogens are capable of triggeringchronic disease, and even permanent tissue or organ destruction, probably via immunemechanisms. Arthritis, inflammatory bowel disease, hemolytic uremic syndrome, Guil-lain-Barré syndrome, and possibly several autoimmune disorders can be triggered byfoodborne pathogens or their toxins. More research is needed to fully understand themechanisms by which the immune system is inappropriately activated by these com-mon foodborne disease-causing agents (Smith et al. 2002). Long-term risks of micro-bial disease need to be better understood and communicated to consumers.

14.6 RISK/BENEFIT AS IT APPLIES TO FOOD

The risk/benefit concept is clear in many aspects of our lives. For instance, treatmentof disease has inherent and sometimes lethal risks, but benefits afforded are believedby most to outweigh the risks. Thus a vital risk is exchanged for a vital benefit.Physical activity may contribute to health (vital) and well-being (nonvital), but itmay pose a risk of injury (vital). However, the risk is voluntary and the athlete mayfeel in control. For food issues, the risk-benefit balance may be much less apparentto the consumer. Chemicals added to food inhibit microorganisms, but these maypose their own risks. Their presence in food is involuntary because in most casesconsumers did not choose their addition. Thus, consumers experience outragebecause they had no choice about whether to entertain the risk.

Furthermore, the risk-benefit equation is often quite difficult for consumersbecause of information imbalance. Frequently the consumer is informed of the risksof the use of a particular chemical or technology, but is not apprised of the benefits.For example, risks from pesticides with respect to potential increases in the numberof cancer cases or estrogenic effects, to reduction in the immune response, and toenvironmental concerns are frequent hot topics in the media. However, benefitsstating that pesticides reduce the potential of the lethal, naturally occurring carcin-ogen aflatoxin, reduce vector-borne disease or the number of bug parts and droppingsin food, decrease the amount of fossil fuel required to mechanically cultivate a field,and increase crop yield to feed a burgeoning global population only rarely make theheadlines or even the back pages of the news.

In the same manner, the risks of additives are often cited by various groups,while the benefits remain touted only in scientific journals to which the consumerdoes not have ready access. For instance, a preservative can have several importantbenefits. One, formation of a cancer-producing mycotoxin may be arrested by inhib-iting mold growth. Two, food costs can be reduced because staling and oxidationare retarded and less expensive packaging, transportation, and storage solutions arerequired. Three, food waste is reduced. Four, oxidized fats with their attendant healthrisks, are reduced. Five, preservatives make possible foods that meet consumerdesires with respect to convenience.

Looking at the risks and not the benefits of chemicals or technologies associatedwith food is like an accountant shearing a ledger in half and considering the liabilitieswithout the assets. However, even when consumers consider both risks and benefits, they



place more emphasis on the risk than the benefit. Activities or technologies that arejudged high in risk tend to be judged low in benefit, and vice versa. In situations wherethere is high risk, there tends to be a confounding of risk and benefit in people’s minds.Risk gurus Paul Slovic and his colleagues at Decision Research in Eugene, OR (Slovicet al. 2004), found that perceived risk is often not related to the probability of injury.

Communicating risk is about trust. Trust can be easily destroyed but is verydifficult to establish. Once there is an element of distrust, it fuels more distrust(Poortinga and Pidgeon 2005).

Risk communicators should be aware that risks people can choose to avoid, suchas skiing, give a sense of control. Furthermore, risks that are familiar and have beenaround a long time, such as Salmonella from potato salad or disease from smoking,are less disquieting to consumers and often minimized. Risks that are poorly tolerated(a) are involuntary, such as exposure to small amounts of pesticide residues in food,(b) have unknown effects, such as biotechnology-produced corn, or (c) have long-delayed effects such as are possible with prion exposure.

The framing of risks makes a tremendous difference in their acceptance. A studyreported in the New England Journal of Medicine showed that 44% of the respon-dents would select a procedure for lung cancer treatment when told they had a 68%chance of surviving, but only 18% would select the procedure if they were told theyhad a 32% chance of dying. It is no wonder that the consuming public has aninordinate fear of some food additives and pesticides because the information isalways reported in terms of increased risk rather than possible reduced risk due toless mold or bacterial growth.

In one study, the framing of a food risk had tremendous impact on risk accep-tance. Consumers in California were asked if they would accept food irradiation.The responses were varied but had strong negative leanings. When the same con-sumers were asked if they would accept irradiation as a way to use less pesticide orreduce a microbial hazard, irradiation acceptance increased (Bruhn 1999).

Consumers thus appear quite capable of understanding and using the risk/benefitconcept for making decisions about food choices. It is mandatory that all sides ofthe story get a hearing so that these informed choices can be made considering boththe risks and benefits.

The final word about risk/benefit is that the definition of the risks and the benefitsare not the same in all cases. The definition of benefit needs to be clearly defined.For some countries, the benefit is reduced postharvest food losses. The value of thisbenefit can vary with the country involved. Different weights may be assigned ifthere is a danger of a severe food shortage versus simply an increase in the cost ofthe substance. Thus, the science and art of risk assessment are difficult because eachstep in the process is value laden.

14.7 NUTRITIONAL EVALUATION OF FOOD PROCESSING

14.7.1 BENEFICIAL AND DETRIMENTAL EFFECTS

Food processing both maintains and destroys nutrients. All nutrients (except water)undergo either chemical or physical changes during processing that render them


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inactive or less bioavailable. This occurs for macro- as well as micronutrients. In afew cases, nutrients are even more available after processing. This seeming paradoxwill be explored.

For most foods, processing reduces the nutrient content when compared to thefreshly harvested item. However, processing makes food, and the nutrients within,available at a later date so the loss incurred must be compared with what wouldhappen to the diet if no processing was utilized. For some foods, processing makesthe nutrients more bioavailable as in the case of the carotenoid pigment lycopene.For some foods processing destroys toxic factors or antinutrients. Cassava, soybeans,and corn are all examples of important classes of food made either less toxic ormore nourishing through processing. Cyanide is removed from cassava with grindingand soaking, enzyme inhibitors and lectins are destroyed during the heating of thesoybean, and niacytin releases niacin when corn is processed with lime (CaO).

For macronutrients, heating of protein speeds the browning reaction, which inturn lowers the biological value of protein. Processing and heating of oils can reduceessential fatty acids, increase the degree of fat saturation, and introduce trans fatsinto the diet and release oxidation products making the fat have significant nutritionaldrawbacks. Pregelatinizing starch may allow faster absorption of blood glucose thanwould occur in the same food without having undergone such a treatment.

On the other hand, fat processing may reduce oxidation or allow for the additionof plant stanols in order to reduce cholesterol. Some processes increase the resistantstarch content of the food and may therefore increase the amount of dietary fiber inthe food.

14.7.2 EFFECTS ON VITAMINS

Vitamin loss during processing and cooking of various foodstuffs has been of interestto nutritionists, processors, and consumers. Vitamin C, the most labile vitamin, islost during canning, blanching, fresh or frozen storage, drying, and irradiation.Losses from a food can be 100% if the conditions of processing and storage are notcontrolled. For example, a study of processed mashed potatoes fortified with vitaminC shows the lability of this vitamin. Cumulative losses of vitamin C were 56% foradding the vitamin to freshly mashed potatoes, 82% for drum-dried potatoes, 82%for flakes stored 4.3 months at 25°C, and 96% for reconstituting mashed potatoesand holding them 30 min on a steam table. One serving (100 g) would contain 10ppm—2% of the adult Recommended Daily Allowance, (United States) (RDA). Amore stable isomer used to fortify the mashed potatoes would yield about 201 ppm(about 33% of the RDA of vitamin C per serving) (Wang et al. 1992).

Thiamin and folate, while not as labile as ascorbate, are easily lost duringprocessing. Thiamin is easily destroyed by heat and is extremely water soluble, somuch can be leached into liquids used during preparation of both meats and vege-tables. In addition to losses due to leaching, thiamin content can decrease markedlywhen subjected to basic pH. The two rings of the molecule split under alkalineconditions and this causes loss of all biological activity for humans. Thus, a quickbread with bicarbonate leavenings can lose up to 75% of the thiamine in the finished



baked product because of the synergistic effect of both heat and pH. Sulfites usedas preservatives will also cleave thiamin.

Folic acid is easily lost during storage of fresh vegetables at room temperatureand through many heat processes. Oxidative destruction of 50 to 95% of the folatecan occur with protracted cooking or canning. Currently several countries mandatefolate addition to cereal and flour products in order to prevent neural tube defectsand to reduce coronary disease and some cancers. Thus, the processed, fortifiedproduct will deliver folate. The addition of folate has had a dramatic effect in helpingto reduce neural tube defects.

Riboflavin is unstable in light and thus riboflavin-containing foods subjected toeither ultraviolet or visible light can show significant losses of riboflavin. Processingand packaging to minimize the effects of light are important measures to reduceriboflavin loss.

Pyridoxine (vitamin B6) losses in food are dependent on the temperature and onthe specific form of the vitamin. Thermal processing and low-moisture storage ofcertain foods results in reductive binding of pyridoxal to lysine in proteins makingit unavailable. Thus canning and drying losses can be substantial. Losses in cannedinfant formula are of particular concern because the formula may be an infant’s onlyfood source. During blanching there is not much measurable loss of vitamin B6

content, but recent studies have shown that loss of bioavailability or absorbabilitymay be significant. Most meats are a good source of vitamin B6, and luckily theylose little of it during preparation.

Fat-soluble vitamins show somewhat greater stability than water-soluble vita-mins. They are not as easily destroyed by normal cooking and are not leached intothe cooking water. However, vitamin A changes slowly from the all-trans form, themost biologically active form, to the cis form during canning and with long periodson a steam table. Carotenoids, currently valued for their antioxidant and possibleanticarcinogenic potential, also oxidize to some degree during heat treatment. Tra-ditional canning causes greater losses of vitamin A and carotenes than does high-temperature, short-time (HTST) processing (Rodriguez-Amaya 2003). Little lossoccurs in frozen blanched vegetables, but vitamin A and other carotenoids easilyoxidize in drying when no antibrowning additives such as sulfite are used.

Vitamin E is not very stable to heating followed by freezing, and is lost in millingof flour. Some vitamin E isomers are lost during the processing of oil. In manycountries, the vitamin D content of food is increased through fortification of dairyproducts. Vitamin K is not greatly affected by heat but is lost to light, so vitaminK-containing oils retain their vitamin content if stored in amber bottles.

Healthful phytochemicals are also affected by processing. Their effects can bechanged by processing as seen in the differences between green and black tea. Firingwhile the leaf is still green to make green tea retains more antioxidants than allowingthe leaf to wither and making black tea. The removal of the skin of the grape in themaking of white wine can change the number of phytochemicals. The use of sulfiteto prevent browning in the drying of golden raisins causes an increase in the anti-oxidant capacity of the raisin over those with no sulfite present.


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14.7.3 EFFECTS ON MINERALS

Minerals from meat and vegetables are leached into the cooking liquid and are lostto the consumer if the liquid is not ingested. In cereals, minerals are retained inthe bran and germ fragments of the grain and therefore lost to those who ingestonly refined grain products. However, even if the whole grain is ingested, thebioavailability of the mineral may be impaired because the mineral is tightly boundto the bran or the germ. Furthermore, some minerals may be released and mademore available during the cooking process while others become less bioavailable.Fermentation during the production of beer, wine, yogurt, and African tribal foodsaffects bioavailability of zinc and iron. Availability of iron in milk-based infantformula depends on whether iron is added before or after heat processing. Foodpackaging (e.g., tin cans) can alter food composition and thus potentially affectmineral bioavailability. Maillard browning has been reported to cause slightdecreases in zinc availability.

14.8 NEWER AND NOVEL TECHNOLOGIES

14.8.1 IRRADIATION

Treating fresh or frozen meats with ionizing radiation is an effective method ofreducing or eliminating foodborne human pathogens. Irradiation dose, processingtemperature, and packaging conditions strongly influence the results of irradiationtreatments on both microbiological and nutritional quality of meat. Radiation dosesup to 3.0 kGy have little effect on the vitamins in chicken or pork, but have verysubstantial effects on foodborne pathogens. Even vitamins, such as thiamin, whichare very sensitive to ionizing radiation, are not significantly affected by the U.S.Food and Drug Administration maximum approved radiation dose to control Tri-chinella, but at larger doses it is significantly affected (Fox et al. 1995).

14.8.2 BIOTECHNOLOGY

Biotechnology is viewed as a new process. In actuality, it is an extension of an oldprocess. Time-honored processes such as fermentation and plant and animal hus-bandry, employ biotechnology. However, in the last 20 years, the technology hastaken a giant step forward by using gene-splicing techniques to speed up and makethe process more precise. It allows incorporation of genes into tissues that wouldnot occur with normal breeding techniques.

Biotechnology has the potential to both increase and decrease available nutrientsin the same way that plant breeding can. Care must be exercised that the nutrientcontent of foods is not reduced when another attribute is engineered into the food.On the other hand, specific needed nutrients such as vitamin A or C can be bredinto the food.

There are several food safety concerns that must be addressed with geneticengineering. One is that natural toxic components might be increased. Breeding or



genetically engineering plants with natural herbicides or pesticides or with herbicideresistance has the potential of reducing pesticide use and intake. There is also thepotential of increasing a toxic component in the diet if many foods were developedto carry the same natural pesticide. Scientists must not be lured into the commonbelief that nature is benign and chemicals from the lab are noxious.

Allergenicity is another food safety concern surrounding foods produced bybiotechnology. The transfer of a protein into a food though biotechnology couldintroduce the offending protein into a food where the particular protein would notbe expected. However, authors of a recent review noted that the risk assessmentprocess to date is robust, and that no biotech proteins in allowed foods have beendocumented to cause allergic reactions.

Determining if food produced by biotechnology has adverse effects on theenvironment need more research. Currently, the ability of laboratories and regulatoryagencies to determine if a food has been genetically modified is limited. Furthermore,the accuracy varies for different food products. Unknowns about how certain bio-technology species will affect the balance of nature is also a concern. There is fearthat bioengineered products might become dominant strains.

14.9 ADDITIVES

Food additives can enhance or diminish the safety and nutritional quality of a food.By preventing oxidation of fat and vitamins, antioxidants can both enhance safetyand maintain nutritional quality. Antibrowning agents, such as sulfite, help retainphytochemicals, vitamins A and C, but lower the amount of thiamine, folate, andpyridoxal. Sorbic acid can prevent mold and possible mycotoxins but can formprotein adducts in the stomach, which affect the availability of the protein. VitaminsC and E react with nitrate to prevent the formation of nitrosamines. This reactionwill protect against the potential carcinogenicity of the nitrosamines, but uses thevitamins in the process. Phosphates have antimicrobial properties because of theirsequestering capacity. This capacity can have an antinutritional effect because ofmineral-binding ability. Vitamins themselves are added to fortify and enrich products,making the food have more nutrients than it might otherwise. On the other hand,highly fortified foods might erroneously lull consumers into thinking that one servingof the fortified food frees them from caring about other parts of the diet. Consumersneed to be educated that there is much more to food that is beneficial than just theadded vitamins and minerals.

New products that replace entire foods or macronutrients, such as fat replacers,must be evaluated both for their nutritional contribution and for their dietary impact.These foods may help some people reach needed dietary goals by reducing saturatedfat or total calories. They may also encourage overconsumption and fail to helpconsumers eat fewer calories. In some instances, additives that were intended foruse in micro amounts are now being used in macro quantities. They may exceed theADI and have effects that would make them unsafe at high levels of use.

Additives, like all food components, need to be looked at with risks and benefitsin mind. Contrary to popular belief, food additives can be regarded as the safest andmost studied constituents of our food supply. This is as it should be. Continuing


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surveillance of food additive intake and effects is a mandatory consequence of theiruse. Continuous assessment of their safety must be considered in light of changedusage patterns and new data. This constant vigilance can make consumers feel thatscience and technology is untrustworthy because they perceive that science changesits mind. In fact, just the reverse should be true. Because additive safety is constantlybeing challenged to ensure that only the most wholesome food products are on themarket, fear should be lessened. Substances should never be added to food withoutcareful analysis and a conservative approach to their use. A clear benefit to the enduser must be present before a food additive should be allowed.

14.10 SUMMARY

Food safety requires continuous responsibility all along the food chain from the farmto the fork. This means that the farmer, transporter, manufacturer, grocer, preparer,and consumer all have roles. Greater knowledge about potential risks and clearcommunication to the consumer of risks and benefits are critical. For the scientist,better understanding and monitoring of components of food and the prevention offood contamination are needed to produce a safe and nutritious food supply.

REFERENCES

Allos, B.M., Moore, M.R., Griffin, P.M., and Tauxe, R.V. 2004. Surveillance for sporadicfoodborne disease in the 21st century: the FoodNet perspective. Clin. Infect. Dis. 38(suppl 3).

Ames, B. and Gold, L.L. 2000. Paracelsus to parascience: the environmental cancer distrac-tion. Mutat. Res. 447 (1), 3.

Bruhn, C.M. 1999. Consumer perceptions and concerns about food contaminants. Adv. Exp.Med. Biol. 459, 1.

Caldas, E.D. and Souza, L.C. 2004. Chronic dietary risk for pesticide residues in food inBrazil: an update. Food Addit. Contam. 21 (11), 1057.

CDC (Centers for Disease Control). 2003. Preliminary FoodNet data on the incidence offoodborne illness—selected sites, United States. MMWR 3, 52, 340.

Chen, B.H., Peng, H.Y., and Chen, H.E. 1995. Changes of carotenoids, color, and vitamin Acontent during processing of carrot juice. J. Agric. Food Chem. 44 (7), 1912.

Dybing, E., Doe, J., Groten, J., Kleiner, J., O’Brien, J., Renwick, A.G., Schlatter, J., Steinberg,P., Tritscher, A., Walker, R., and Younes, M. 2002. Hazard characterisation of chem-icals in food and diet. Dose response, mechanisms and extrapolation issues. FoodChem Toxicol. 40(2–3), 237.

Edwards, G. 2005. Safety assessment of novel foods and strategies to determine their safetyin use. Toxicol Appl Pharmacol. 207 (2 Suppl), 623.

Fox, J.B.J., Lakritz, L., Hampson, J., Richardson, R., Ward, K., and Thayer, D.W. 1995.Gamma irradiation effects on thiamin and riboflavin in beef, lamb, pork, and turkey,J. Food Sci. 60 (3), 596.

Goodacre, R. and Ellis, D. 2005. http://nutraingredients.com/news/ng.asp?id=62772Hansen, J., Holm, L., Frewer, L., Robinson, P., and Sandoe, P. 2003. Beyond the knowledge

deficit: recent research into lay and expert attitudes to food risks. Appetite 41 (2), 111.



Jeffrey, A.M. and Williams, G.M. 2005. Risk assessment of DNA-reactive carcinogens infood. Toxicol Appl Pharmacol. 207 (2 Suppl), 628.

Jones, J.M. 1992. Food Safety. Eagan Press, St. Paul, MN.Kirk, S.F., Greenwood, D., Cade, J.E., and Pearman, A.D. 2002. Public perception of a range

of potential food risks in the United Kingdom. Appetite 38 (3), 189. Kroes, R., Kleiner, J., and Renwick, A. 2005. The threshold of toxicological concern concept

in risk assessment. Toxicol Sci. 86 (2), 226. Low, F., Lin, H.M., Gerrard, J.A., Cressey, P.J., and Shaw, I.C. 2004. Ranking the risk of

pesticide dietary intake. Pest Manag. Sci. 60 (9), 842.Occhipinti, S. and Siegal, M. 1994. Reasoning about food and contamination. J. Personality

and Social Psychology 66 (2), 243.Poortinga, W., Pidgeon, N.F. 2005. Trust in risk regulation: cause or consequence of the

acceptability of GM food? Risk Anal. 25 (1), 199. Renwick, A.G. 2005. Structure-based thresholds of toxicological concern-guidance for appli-

cation to substances present at low levels in the diet. Toxicol. Appl. Pharmacol. 207(2 Suppl), 585.

Rodriguez-Amaya, D.B. 2003. Food carotenoids: analysis, composition and alterations duringstorage and processing of foods. Forum Nutr. 56, 35.

Slovic, P., Finucane, M.L., Peters, E., and MacGregor, D.G. 2004. Risk as analysis and riskas feelings: some thoughts about affect, reason, risk, and rationality. Risk Anal. 24(2), 311.

Smith, J.L. 2002. Campylobacter jejuni infection during pregnancy: long-term consequencesof associated bacteremia, Guillain-Barré syndrome, and reactive arthritis. J Food Prot.65 (4), 696.

Szponar, L., Traczyk, I., Jarzębska, M., and Stachowska, E. 2003. Estimation of the intakeof food additives—methods applied in the European Union. Rocz Państw Zakl Hig.54 (4), 363.

U.S. Food and Drug Administration, Office of Food Additive Safety. Redbook 2000 Toxico-logical Principles for the Safety Assessment of Food Ingredients. July 2000; UpdatedOctober 2001, November 2003, and April 2004, http://www.cfsan.fda.gov/~red-book/red-toca.html, visited October 2005.

Waddell, W.J. 2005. Dose thresholds should exist for chemical carcinogens. Toxicol. Sci. 85(2), 1064.

Wang, X.Y., Kozempel, M.G., Hicks, K.B., and Seib, P.A. 1992. Vitamin C stability duringpreparation and storage of potato flakes and reconstituted mashed potatoes. J. FoodSci. 57 (5), 1136.

Yang, S., Angulo, F.J., and Altekruse, S.F. 2000. Evaluation of safe food-handling instructionson raw meat and poultry products. J. Food Prot. 63 (10), 1321.


391

15 Prebiotics

Bob Rastall

CONTENTS

15.1 The Gut Microflora and Health................................................................. 39215.2 Consequences for Health of Prebiotic Consumption................................ 394

15.2.1 Mineral Absorption ..................................................................... 39415.2.2 Resistance to Infection................................................................ 39515.2.3 Reduction in Cancer Risk ........................................................... 39515.2.4 Modulation of Blood Lipids ....................................................... 396

15.3 Methods for Testing of Prebiotics............................................................. 39715.4 Established Prebiotic Oligosaccharides..................................................... 398

15.4.1 Fructans ....................................................................................... 39815.4.1.1 Chemistry..................................................................... 39815.4.1.2 Prebiotic Activity ......................................................... 399

15.4.2 Galacto-Oligosaccharides............................................................ 39915.4.2.1 Chemistry..................................................................... 39915.4.2.2 Prebiotic Activity ......................................................... 399

15.4.3 Lactulose ..................................................................................... 40015.4.3.1 Chemistry..................................................................... 40015.4.3.2 Prebiotic Activity ......................................................... 401

15.5 Emerging Prebiotic Oligosaccharides ....................................................... 40115.5.1 Isomalto-Oligosaccharides .......................................................... 401

15.5.1.1 Chemistry..................................................................... 40115.5.1.2 Evidence for Prebiotic Claim...................................... 401

15.5.2 Soybean Oligosaccharides........................................................... 40215.5.3 Gentio-Oligosaccharides ............................................................. 40215.5.4 Xylo-Oligosaccharides ................................................................ 402

15.6 Novel Candidate Prebiotics ....................................................................... 40315.6.1 Pectic Oligosaccharides .............................................................. 40315.6.2 Novel Gluco-Oligosaccharides ................................................... 403

15.7 Future Perspectives .................................................................................... 40415.7.1 Targeted Prebiotics ...................................................................... 40415.7.2 Persistent Prebiotics .................................................................... 40515.7.3 Antiadhesive Activity .................................................................. 405

References.............................................................................................................. 406



15.1 THE GUT MICROFLORA AND HEALTH

The human digestive tract is home to a huge and diverse population of microorgan-isms, largely bacteria. The most densely colonized region is the colon (Figure 15.1)with around 1012 bacteria per gram of contents. Indeed, colonic bacteria account foraround 95% of the living cells in the human body. The colon is a highly reducinganaerobic environment and hence the most numerous bacterial groups present arestrict anaerobes such as bacteroides, bifidobacteria, and clostridia (Figure 15.2),although over 500 species are known. It is estimated that these species only representaround 60% of the true diversity of the colonic microbiota. This complex ecosystemis sustained by nutrients arriving from the small intestine and is very metabolicallyactive; it is believed that the colon represents the most metabolically active organin the body. Products of bacterial metabolism range from fatty acids, largely as aresult of the metabolism of saccharides to phenolic compounds derived from proteinbreakdown. The bacterial population also produces a wide range of enzymes thatfurther act on nutrients and primary products of metabolism.

Given the metabolic activity of the colon, it should not come as a surprise thatthe colonic microbiota exert an influence on the health of the host. The state of thecolonic microbiota can have an impact on both acute and chronic disorders. In orderfor an invading pathogenic bacterium to initiate infection, it must first establish itselfto some degree in the gut. Many of the predominant bacteria that are resident in thegut, notably the bifidobacteria and lactobacilli, produce a range of antimicrobialcompounds that render the colon a more hostile place for pathogens. This, coupledwith competition for cellular receptor sites and nutrients, forms a barrier effectagainst acute infections. Some normal residents of the gut, for instance, Clostridiumdifficile, can cause acute pathological effects. Their numbers are usually maintainedat a low level due to competition with the normal flora. In the event of a disturbancein the balance of the normal flora, however, such as occurs after prolonged antibioticusage, C. difficile can multiply and cause antibiotic-associated diarrhea.

FIGURE 15.1 The bacterial population of the gastrointestinal tract.

The stomach has very low bacterialnumbers of about 103 ml–1 Typical organisms: H. pylori

The small intestine has much higher numbers of about 104 ml–1– 106 ml–1

Typical organisms: Lactobacillus sp.,Gram-positive cocci

The colon has the largest and most diverse bacterial load of about 1012 ml–1

Typical organisms: Bacteroides spp.,Clostridium spp., Bifidobacterium spp.,Lactobacillus spp., Eubacterium spp., Gram- positive cocci


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The colonic microflora has also been implicated in chronic gut disorders. It isestablished that many dietary components, including proteins, are metabolized tocarcinogens and tumor promoters. Conversely, butyric acid, produced by clostridiaand eubacteria from carbohydrate breakdown, acts a fuel for colonocytes, maintainscolonic epithelial integrity, and stimulates apoptosis in cancer cells. The gut florahas also been implicated in the etiology of ulcerative colitis (UC). It is known thatso-called germ-free rodents are immune from experimentally triggered UC. It is alsothe case that certain bacteria, for instance, the sulfate-reducing Desulfovibrio desul-furicans, which produces acutely toxic H2S, may associate with cell surfaces andtrigger the inflammatory response characteristic of UC.

Given the impact of the gut flora on human health, there has been much interestin the idea of modulating this ecosystem to promote health. Historically this has beenattempted by the administration of live, probiotic bacteria. This approach has a longhistory and there have been many successful human and animal trials of probiotics,although not all trials have shown positive results. Two major concerns with probioticsare the problems of maintaining viability in the food product, and questions over theirsurvival and activity during passage through the gastrointestinal (GI) tract.

An attractive alternative to probiotics is the use of prebiotics. The prebioticapproach targets the intrinsic probiotics already resident in the gut, and as they arecarbohydrate in nature, they can be formulated into a wide variety of foods withminimal concerns over their integrity. In order to be considered as a prebiotic, asaccharide must fulfill certain criteria. The first of these is that it must be nondigestible

FIGURE 15.2 Bacterial groups in the colon.

Pseudomonads

Eubacteria, C. coccoides, C. leptum

Log No. per g feces11

2

Bifidobacteria

Lactobacilli

MetabolitesShort- chain fatty acids

VitaminsAntimicrobial compounds

Health implicationsInhibition of pathogens Colonization resistance

Immune modulation

Clostridum perfringens

Clostridum difficile

Sulphate reducers

Atopobium

Bacteroides

E. coli

Gram positive cocci

Veillonellae

Staphylococci

MetabolitesPhenolic compounds

CarcinogensToxins

Genotoxic enzymes

Health implicationsGastrointestinal infection

Colon cancer Ulcerative colitis



by humans. Many saccharides do fulfill this criterion (see below) and hence theytransfer to the colon largely intact. The second criterion is that they are selectivelyfermented by health-positive probiotic bacteria in the colon. With our current under-standing of the gut flora, these are species of Bifidobacterium, Lactobacillus, andEubacterium. However, not all species within these genera are equally health posi-tive, and the particular balance of species present in any given individual will dependon factors such as genotype and diet. It is also true that as understanding of thediversity and activities of the colonic microbiota increases, other bacteria may wellbe seen to be health positive.

The number of prebiotic food ingredients in the European Union (EU) and theUnited States is small (currently only inulin, fructo-oligosaccharides (FOS), andgalacto-oligosaccharides (GOS) are used in foods), although there is commercialinterest in increasing this number. A much wider number are used in Japan (Table15.1), thanks in part to the Japanese system of licensing ingredients as functionalfoods. Ingredients can obtain the status of foods for specified health use (FOSHU)and this has undoubtedly facilitated the development of the functional food marketin Japan. A wide range of nondigestible saccharides, generally oligosaccharides,have been proposed as prebiotics, with greater or lesser degrees of experimentalsupport for the claim. These are reviewed below.

15.2 CONSEQUENCES FOR HEALTH OF PREBIOTIC CONSUMPTION

15.2.1 MINERAL ABSORPTION

There is increasing evidence that consumption of prebiotics can increase absorptionof calcium from the gut [1]. Although the small intestine is the major site of mineral

TABLE 15.1Suppliers of Prebiotic Oligosaccharides

Oligosaccharide Major manufacturers Trade names

Fructo-oligosaccharides Meiji Food Materia (Japan)Beghin-Meiji (France)

MeioligoActilight

Inulin Orafti Active Food Ingredients (Belgium) RaftiloseGalacto-oligosaccharides Clasado (UK)

Friesland Foods (The Netherlands)Yakult (Japan)Nissin Sugar (Japan)

Bi2munoVivinal GOSOligomateCup-Oligo

Isomalto-oligosaccharides Showa Sangyo (Japan) Isomalto-900Lactulose Morinaga Milk (Japan)

Solvay (Germany)Soybean oligosaccharides The Calpis Food Industry Co. (Japan) Oligo-CCXylo-oligosaccharides Suntory Ltd (Japan) Xylo-oligoGentio-oligosaccharides Nihon Shokuhin Kako (Japan) Gentose


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absorption, the colon is responsible for a significant amount of the absorption ofcalcium from the diet, and there is clear and consistent evidence that this can beincreased by fructans intake [1].

15.2.2 RESISTANCE TO INFECTION

The gut microflora acts as a barrier to infection by foodborne pathogens by contrib-uting to colonization resistance [2]. Commensal organisms such as bifidobacteriaand lactobacilli can prevent colonization via several mechanisms. The acid secretedby these organisms as a result of carbohydrate fermentation reduces colonic pH,inhibiting pathogen growth. In addition it is known that bifidobacteria and lactobacillican produce inhibitory compounds against a range of gastrointestinal pathogens, atleast in vitro [3]. They also compete for nutrients and for receptors on intestinal cellsurfaces. It seems logical to expect, therefore, that prebiotic-induced increases inthe ratio of bifidobacteria and lactobacilli would enhance colonization resistance.

There are, however, very few studies designed to investigate inhibition of patho-gens by prebiotics. There have been some studies in rat models on the preventionof Salmonella enteritidis using lactulose [4, 5]. It was assumed by these researchersthat the acidification of the gut by lactulose fermentation reduced the growth of thepathogen. It was, however, also found that the acidification resulted in increasedtranslocation of pathogens from the gut [5]. Using mouse models, GOS has beenshown to be protective against Salmonella typhimurium [6] and FOS has been shownto protect against Listeria monocytogenes and Salmonella typhimurium [7]. Inhumans, Cummings et al. [8] have studied the effect of FOS on travelers’ diarrhea.A group of 244 healthy individuals who were traveling to areas of the world withincreased risk of diarrhea consumed 10 g of FOS or placebo in a double-blind study.They found that the group on FOS experienced a lower incidence of diarrhea (11.2%vs. 19.5%) but this difference was not statistically significant.

In a more recent study, Lewis et al. [9] studied the ability of FOS to protectelderly individuals against antibiotic-associated diarrhea (AAD). They fed eitherFOS or sucrose (placebo) at 12 g/day to a group of patients over the age of 65 whowere taking broad-spectrum antibiotics. The FOS-consuming group did not, how-ever, experience a lower incidence of AAD despite having elevated bifidobacterialnumbers.

15.2.3 REDUCTION IN CANCER RISK

There have been several studies on the reduction of cancer risk by consumption ofprebiotics. The primary focus has been on the reduction of genotoxicity. Severalassays for genotoxicity have been used, such as the Comet assay for DNA damage,assay of genotoxic enzyme activities, and of tumor-promoting metabolites.

One of the earliest studies was undertaken by Ito et al. [10] who fed GOS tohumans. They found decreases in nitroreductase (an enzyme known to activatecarcinogenic substances), isovaleric acid, and indole, which are products of proteo-lysis followed by deamination. These observations are consistent with the prebioticeffect, as bifidobacteria and lactobacilli produce much lower levels of genotoxic



enzymes than bacteroides and clostridia [11]. Later studies on GOS using an in vitromodel of the human gut (see below) showed an increase in the activities of nitrore-ductase and azoreductase [12]. These changes occurred very rapidly, however, anddid not correlate with bacterial population changes. The mechanism of the decreasein genotoxicity is thus still to be clarified.

In a human trial on lactulose, Tuohy et al. [13] found no effect on genotoxicityusing the Comet assay. A lack of any effect on cancer risk markers of lactulosefeeding was also seen by Bouhnik [14], who looked at levels of fecal bile acids andsterols. A positive impact on tumor-promoting metabolites and genotoxic enzymeswas, however, seen in studies by Terada et al. [15] and Balongue et al. [16]. A recentstudy by De Preter et al. [17] investigated the consequences of lactulose consumptionto toxic metabolites using stable isotope techniques, and these researchers found asignificant reduction on lactulose feeding.

While a reduction in cancer risk biomarkers is very likely desirable, it is notestablished that this will actually lead to a lower risk of developing cancer. Thereare, however, some studies on the ability of prebiotics to inhibit the development oftumors in rats and humans. Challa et al. [18] saw fewer aberrant crypt foci (aprecursor to the development of a tumor) when rats treated with azoxymethane werefed lactulose, and Rowland et al. [19] showed that lactulose could protect against1,2-dimethylhydrazine dihydrochloride-induced DNA damage in human flora-asso-ciated rats. A similar result has been seen with FOS [20]. A study in humans hasfound that lactulose can significantly reduce the chances of recurrence of colonicadenocarcinoma after removal by surgery [21].

Recently a large-scale human study was carried out on the effects of synbioticfeeding on human cancer risk. In this trial (the EU “Syncan” project, Van Loo andJonkers, [22]) a mixture of FOS and inulin (Synergy1, Orafti, Belgium) togetherwith the probiotics Bifidobacterium animalis and Lactobacillus rhamnosus were fedfor three months. The intervention took place in groups of 40 polypectomized andsurgically treated cancer patients in a double-blind parallel study (20 individuals pergroup) with maltodextrin as a placebo.

Consumption of the synbiotic resulted in a prebiotic effect with increases inbifidobacteria and lactobacilli with decreases in clostridia and coliforms. DNA dam-age assessed by Comet assay was reduced in the test groups [23] and the polypec-tomized patients also showed reduced cell proliferation compared to the controls.

15.2.4 MODULATION OF BLOOD LIPIDS

There is some evidence that prebiotics can influence lipid metabolism, although themechanisms are unknown at the present time. Studies have been performed inanimals and in humans. An early trial carried out in diabetic rats fed xylo-oligosac-charides (XOS) as a replacement for some of the saccharides in the diet [24]. Theseresearchers found decreases in blood cholesterol and triacylglycerols levels down tothose found in normal animals. Human studies have also shown that prebiotics inthe form of FOS can downregulate blood lipids in hyperlipidemic subjects [1, 25].They do not, however, seem to influence lipid levels in normal subjects [25].


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The mechanisms of these effects are not known with any certainty but mayinvolve inhibition of hepatic enzymes by propionate formed by the gut flora [26].

15.3 METHODS FOR TESTING OF PREBIOTICS

A prebiotic claim for a candidate oligosaccharide must, ultimately, be supported bya well-designed human volunteer trial, preferable with several doses being fed, andwith the microbiology being performed by modern molecular techniques. Such trialsare, however, expensive and time consuming. For that reason it is desirable to carryout in vitro experiments first, to identify likely candidates for human trials.

Pure cultures have been used in the past, but this approach does not give anyuseful information about how a dietary component will impact upon the complexmixed colonic microflora. Batch cultures with mixed fecal inocula can be used togive a first indication of the selectivity of the fermentation. Simple non-pH-controlledbatch cultures have been used in this regard [11], although a more physiologicallyrelevant testing method is the use of pH-controlled systems [28]. Recently, batchculture approaches have been scaled down to utilize only 7 mg of carbohydrate incultures in microcentrifuge tubes [29]. These microscale cultures allow testing ofscarce experimental saccharides and have been validated against pH-controlled cul-ture systems.

Batch culture testing can then be followed by more extensive evaluation in anin vitro model of the human colon. There are several such models in use around theworld. They vary in terms of their complexity and hence vary in the type of datathey provide. The Simulation of the Human Intestinal Microbial Ecosystem (SHIME)reactor [30], for instance, is a complex model that simulates most of the attributesof the human intestine with the exception of selective absorption of nutrients. Amore simple system is the three-stage chemostat model [31]. This only simulatesthe luminal microbiology of the colon, but it has been validated against the coloniccontents of sudden-death victims. This is a useful system to study prebiotic effectsand it allows an assessment of the persistence of the effect through the colon—something that cannot easily be done in humans.

Whichever system is used, modern DNA-based microbiology methods areneeded to obtain reliable information about the microflora changes that followfermentation of prebiotics. Traditional culture-based methods do not provide theselectivity needed [32] and biochemical characterization of the colonies growing onselective agar plates must always be carried out. In addition, much of the colonicmicroflora is believed to be uncultivable and will not be detected by selective media.Fortunately a range of DNA-based techniques are now available to characterize suchcomplex mixed cultures [33].

A widely used method for characterization of the gut flora in prebiotic experi-ments is fluorescent in situ hybridization (FISH). This involves the use of fluores-cently tagged DNA probes targeted at diagnostic regions of the 16S rRNA genes.These sequences can be specific for individual genera, groups of related bacteria, orindividual species. Most labs using this technique for prebiotic studies utilize genus-and group-specific probes. Fluorescently tagged bacteria are then counted either



microscopically [27] or by automated techniques such as image analysis [34] or flowcytometry [35].

Although FISH overcomes the problems of noncultivable bacteria and givesgood coverage, other techniques can be used to characterize the diversity of thebacterial population. One such technique is denaturing gradient gel electrophoresis(DGGE). DGGE involves a polymerase chain reaction (PCR) amplification of the16S rRNA genes followed by electrophoresis on a denaturing gradient gel. Ampl-icons from different species band at different positions on the gel giving a pictureof the diversity. Bands can be excised from gels and then sequenced to identify thespecies. Although DGGE is qualitative, the data can then be used to inform a FISHprobing strategy to obtain quantitative data.

A promising recent technique is quantitative PCR (qPCR). This involves the useof species-specific primers to generate quantitative data on bacterial populations[36]. The technique is not yet a mature technique as far as the gut flora is concernedbut as the number of available primers increases, the technique will gain in utility.

15.4 ESTABLISHED PREBIOTIC OLIGOSACCHARIDES

15.4.1 FRUCTANS

15.4.1.1 Chemistry

Fructan prebiotics can be divided into inulin extracted from plant sources or lowermolecular weight FOS. FOS are either derived from inulin by partial enzymatichydrolysis or are derived from sucrose by the transferase activity of β-fructofura-nosidase enzymes. The commercial source of inulin for functional food applicationis chicory root.

Chicory inulin is a linear chain of fructofuranose residues via linked β2 → 1linkages. A proportion of the chains terminates in a glucopyranosyl-residue linkedβ1 → 2 as in sucrose and is thus nonreducing. The molecular weight distribution ofplant-extracted inulin I is a disperse with a degree of polymerization (dp) of two toaround sixty. The average dp is 12 with approximately 10% of the molecular specieshaving a dp of two to five [37]. FOS derived from chicory inulin by hydrolysislargely terminates in fructofuranosyl residues but a low proportion terminates inglucosyl residues. The hydrolysis products have a dp range of two to seven with anaverage of four.

Chicory inulin and inulin-derived FOS are commercialized in Europe by OraftiNV (Tienen, Belgium) who manufacture a range of products with varying degreesof purity and varying technological properties. Of note is a product called Synergy,which is a mixture of high-molecular-weight inulin chains prepared by fractionationwith low-molecular-weight FOS.

FOS derived from sucrose all terminate in glucosyl residues and are thus non-reducing. The synthetic products have a dp of two to four with an average of 3.6.

Synthetic FOS are commercialized in Europe by Beghin Meiji (Thumeries,France).


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15.4.1.2 Prebiotic Activity

The fructans are, by a large margin, the most thoroughly researched of the prebiotics.There is an abundance of literature on the biological properties of fructan prebiotics,including some well-designed human trials using modern microbiological techniques.

In vitro studies have been carried out using pure bacterial cultures [3, 38] andusing mixed fecal inocula [39, 40]. These have established that inulin and FOS cansupport the growth of Bifidobacterium spp. and, to a lesser extent, Lactobacillusspp., in mixed fecal cultures at the expense of less desirable microbial genera suchas Bacteroides and Clostridium.

The conclusions of these investigations have been confirmed in several in vivotrials in animals and humans. Studies in gnotobiotic (“germ-free”) rats have showna consistent selection for bifidobacteria and lactobacilli [41–43]. Human trials usingtraditional microbiology techniques have been conducted with between 8 and 40subjects over periods ranging from 7 days to 3 weeks [37]. Generally these studieshave shown significant increases in bifidobacteria and several have shown decreasesin clostridia and bacteroides. More recently, molecular microbiological methods havebeen used, principally FISH. These have involved between 8 and 31 subjects for 2weeks to 2 months [44–46]. An increase in fecal bifidobacteria relative to othergroups was consistently seen.

15.4.2 GALACTO-OLIGOSACCHARIDES

15.4.2.1 Chemistry

GOS are synthesized by galactosyl transfer reactions catalyzed by β-galactosidaseutilizing lactose as a substrate. Lactose acts as both glycosyl donor and acceptor insuch reactions, and a complex series of oligosaccharides are built up with dp rangingfrom 2 to 8 [47]. The commercial products are not pure and they typically containaround 50% GOS with 38% glucose and 12% lactose on a weight basis. The principalcomponents are trisaccharides and tetrasaccharides and GOS contain mainly β1 → 4and β1 → 6 linkages with lesser amounts of β1 → 2 and β1 → 3 [48]. The precisecomposition in terms of dp and linkage depends upon the enzyme used in manufac-ture [48, 49].


GOS are considered as nondigestible oligosaccharides (excluding the glucose andlactose components) on the basis of in vitro tests, feeding studies in rats, and breathhydrogen tests in humans [50–52].

GOS have been fairly extensively studied in vitro and in vivo in animal andhuman feeding trials. It is, however, probable that the early studies used GOS withdifferent composition from later studies.

GOS generally display a consistent prebiotic effect in vitro in mixed fecal cultures.In comparative studies with other prebiotics using batch fecal cultures and molecularmicrobiological analyses, GOS displayed good selectivity for bifidobacteria and



decreased clostridial populations [27, 28, 53]. GOS has also shown good selectivityin three-stage models of the human colon, particularly for lactobacilli [12]. GOSalso reduced the β-glucosidase, β-glucuronidase, and arylsulphatase activities, con-sidered to be undesirable in the colon. Inulin had little effect.

The source of the enzyme used to manufacture the GOS can have an impact onthe selectivity, at least in pure culture. Rabiu et al. [48] used β-galactosidases froma panel of probiotic bacteria to synthesize GOS mixtures from lactose. These werethen tested for their ability to support the growth of the probiotic panel. The GOSmixtures had varying linkage profiles and supported different growth rates in theprobiotic bacteria. Significantly, most of the GOS mixtures supported higher growthrates in the producing organism than in the others. Although not yet shown in mixedculture, this raises the exciting possibility of targeting prebiotics at particular speciesof probiotics. This idea has been developed by Tzortzis et al. [53], who used wholecells of Bifidobacterium bifidum NCIMB 4117 to manufacture a novel GOS mixture.Tzortzis et al. [54] studied the prebiotic properties of the novel GOS in a three-stagemodel of the human colon. The GOS had a significant bifidogenic effect in vesselsone and two, modeling the ascending and transverse colon, respectively. The totalbacterial population and those of lactobacilli, bacteroides, and the Clostridium his-tolyticum group were also monitored, but no significant changes were seen. A feedingstudy was then carried out in pigs where a significant prebiotic effect was seen, toa higher level than with inulin. Using an in vitro cell culture system, the novel GOSalso inhibited the adhesion of strains of E. coli and Salmonella enterica serotypeTyphimurium. This GOS is now being commercialized by Clasado.

Trials of GOS in human volunteers have had mixed results. Early trials in Japan,using traditional, culture-based, microbiological methods showed prebiotic effects.Tanaka et al. [51] found elevated levels of bifidobacteria and a synergistic effect whenadministered with a Bifidobacterum breve strain. Ito et al. [10] also found a positiveprebiotic effect, with elevated bifidobacteria and lactobacilli with decreases in Bacteroi-des and Candida species. They also found decreased levels of the undesirable metab-olites, ammonia, indole, cresol, propionate, isobutyrate, valerate, and isovalerate.

More recent trials, however, which have used larger numbers of volunteers, havenot shown a bifidogenic effect [52, 55, 56]. Satokari et al. [56] used molecularmicrobiological methods. It is not clear why these latter studies did not show aprebiotic effect in humans. One possibility is that the starting levels of bifidobacteriain some of the more recent trials were rather high. It has been documented that thebifidogenic effect seen is inversely proportional to the initial level of bifidobacteria[57]. It is also likely that the earlier work conducted in Japan used different GOSpreparations than the more recent ones.

15.4.3 LACTULOSE

15.4.3.1 Chemistry

Most prebiotics are extracted from plant tissues or are manufactured using enzymes.Lactulose is an exception, however, in that it is produced by alkaline isomerizationof lactose to produce 4-O-β-galactopyranosyl-D-fructose, [58]. The commercial


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lactulose product is very pure and is available in crystalline and syrup formulations.These are currently targeted at medical applications, however, and no food-gradeform of lactulose currently exists on the market in the EU. Although lactulose hasa long history of use as a laxative and for treatment of hepatic encephalopathy, it isa prebiotic at sublaxative doses. It is used in Japan in foods and has FOSHU statusin several food vehicles.


Lactulose was actually one of the earliest recognized prebiotics and has been testedin vitro and in vivo. Rycroft et al. [27] carried out comparative studies in static batchcultures with microbiology by FISH and found that lactulose was selective forbifidobacteria. Palframan [28] then tested it in pH-controlled batch cultures andfound similar increases in bifidobacteria and decreases in bacteroides.

Lactulose also has good support for a prebiotic claim in human volunteer trials.Terada et al. [15] showed a significant increase in bifidobacteria with a reduction inbacteriodes, streptococci, Clostridium perfringens, and Enterobacteriaceae. Thiswas achieved with a very low dose of only 3 g per day. More recent work has usedmore satisfactory study designs (placebo-controlled and double-blinded) and haveshown consistent bifidogenic effects with reductions in less desirable bacterial groups[13, 14, 16]. A significant study is that of Tuohy et al. [13] who used FISH tocharacterize the microflora changes occurring in a human trial at 10 g per day. Theyfound significantly enhanced numbers of bifidobacteria and reduced numbers ofclostridia. It seems clear that lactulose can be considered a prebiotic, although it isnot used as such outside Japan.

15.5 EMERGING PREBIOTIC OLIGOSACCHARIDES

15.5.1 ISOMALTO-OLIGOSACCHARIDES

15.5.1.1 Chemistry

Isomalto-oligosaccharides (IMO) consist of oligosaccharides with (largely) α1-6linkages, although they also contain some panose (Glcα1-6Glcα1-4Glc). The molec-ular weight range is 2 to 6. They are commercial products manufactured from starchby a two-stage process [59]. This involves the use of α-amylase and β-amylase tohydrolyze starch to maltose and then transglycosylation catalyzed by α-glucosidase.This effectively converts the α1-4 linkages to α1-6 linkages.

IMO do not strictly qualify as prebiotics as they are partially metabolized bythe human small intestine [60]. Some of the ingested material, however, does reachthe colon where it has a selective metabolism.

15.5.1.2 Evidence for Prebiotic Claim

There is very little evidence to support a prebiotic claim for IMO. The human trialshave been small and poorly controlled [61, 62]. They have reported a prebiotic effectusing culture-based techniques.



In vitro work has supported a prebiotic claim for IMO. The comparative studyby Rycroft et al. [27], using fecal batch cultures and FISH to enumerate bacterialgroups, showed that IMO are selective for bifidobacteria. Palframan et al. [28] usedpH-controlled fecal batch cultures, also with FISH, and these authors also foundgood selectivity with IMO.

It is clear that good human trials with molecular microbiology techniques areoverdue to support a prebiotic claim for IMO.

15.5.2 SOYBEAN OLIGOSACCHARIDES

Soybean oligosaccharides (SOS) are extracted from soybean whey and consist pre-dominantly of raffinose and stachyose. They are manufactured commercially.

SOS have been investigated in small human feeding trials using culture-basedtechniques. Hayakawa et al. [63] fed 10 g per day to six volunteers for three weeksand demonstrated an increase in bifidobacteria and concomitant decrease inclostridia. Wada et al. [64] found similar results in a study of seven volunteers takinga low dose of 0.6 g/day for three weeks. They also reported decreases in genotoxicenzymes and in toxic bacterial metabolites. Hara et al. [65] saw an increase inbifidobacteria on feeding 1 to 2 g per day.

SOS have also been investigated in a comparative study by Rycroft et al. [27]who found that they increased populations of bifidobacteria but also produced atransient increase in bacteroides.

15.5.3 GENTIO-OLIGOSACCHARIDES

Gentio-oligosaccharides (GeOS) are β1-6 linked gluco-oligosaccharides with a dpranging from 2 to 7. They are commercialized as Gentose by Nippon Shokuhin Kakoin Japan. As they are β-linked they have a bitter taste, limiting their applications infood products.

Gentio-oligosaccharides are claimed to be prebiotic but there is very little pub-lished data to support this claim. One study has shown prebiotic potential in vitro[66]. Using a 24-h fecal batch culture system [66], GeOS were compared to FOSand other prebiotics. They resulted in the greatest increases in bifidobacteria, lacto-bacilli, and total bacteria, but also stimulated nonprobiotic bacteria to some extent.FOS, however, were more selective for probiotics.

More recently Sanz et al. [67] used the microscale batch culture system toexamine the selectivity of Gentose and fractionated Gentose. In this system, maximalprebiotic index was seen with the dp 3 fraction.

To date there have been no peer-reviewed articles on the prebiotic activity ofgentio-oligosaccharides in humans and consequently there is no information avail-able on health attributes.

15.5.4 XYLO-OLIGOSACCHARIDES

Xylo-oligosaccharides (XOS) are manufactured by enzymatic hydrolysis of xylan[59]. They are manufactured commercially from corncobs by Suntory in Japan. Thecommercial product is mainly dp 2-4 with linear β1-4 linked chains.


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There is in vitro evidence that XOS are selectively fermented by bifidobacteria,although some of this is using pure culture techniques [68, 69]. Data in mixed culturecomes from the Rycroft comparative study [27], which showed that XOS werestrongly selective for bifidobacteria but with lesser selectivity for bacteroides.

There is very little data from human feeding trials to support the prebiotic statusof XOS. Okazaki et al. [70] carried out a study on nine volunteers who consumed5 g per day for three weeks. The bifidobacterial population was selectively increased.

There is no human study so far using molecular techniques.

15.6 NOVEL CANDIDATE PREBIOTICS

15.6.1 PECTIC OLIGOSACCHARIDES

Pectins represent an interesting and abundant resource of complex oligosaccharides.Oligosaccharides can be made from pectins by enzymatic hydrolysis [71] or by acidextraction of orange peel [72]. Pectic oligosaccharides (POS) made by enzymatichydrolysis of citrus and apple pectins have been evaluated as candidate prebioticsin vitro [73]. In pure culture, Bifidobacterium angulatum, B. infantis, and B. ado-lescentis displayed better growth on POS I than on the highly methylated citruspectin (HMP) they were derived from. B. pseudolongum and B. adolescentis grewfaster on POS II than on the lower methylated apple pectin from which they werederived. In fecal batch cultures, higher PI values were seen with lower degrees ofmethylation (PI24-HMP = –0.11, PI24-LMP = 0.033; PI24-POS I = 0.071 and PI24-POS II =0.092). The lower molecular weight oligosaccharides resulted in higher PI valuesthan the parent pectins.

POS prepared from orange peel by acid extraction [72] have also been evaluatedfor their prebiotic activity [74]. These POS contain glucose in addition to rhamnoga-lacturonan and xylogalacturonan pectic oligosaccharides. In fecal batch cultures POSincreased bifidobacteria and eubacteria; however, they had a lower prebiotic indexthan did FOS. POS produced higher levels of butyrate than did FOS [74].

As they can be cheaply manufactured from agricultural waste materials, POSrepresent an exciting new development in prebiotics. They have been found to havemore than one functionality, as they also inhibit binding of pathogens and toxins tohuman cells (see below). POS have not yet been evaluated in a human trial.

15.6.2 NOVEL GLUCO-OLIGOSACCHARIDES

IMO and GeOS are, of course, gluco-oligosaccharides. There is, however, scope formanufacturing other forms of gluco-oligosaccharide as candidate prebiotics.α-Linked gluco-oligosaccharides (GlOS) can be made using the transfer reactionsof several enzymes. One enzyme, dextran dextrinase (EC 2.4.1.2), produced byGluconobacter oxydans, catalyzes the transfer of glucose from the nonreducing endof maltodextrin chains onto other maltodextrins to build up α1-6 linked oligodextranchains. They can be manufactured using a whole-cell approach in bioreactors [75]and they have been evaluated using fecal batch cultures [76] and three-stage gutmodels [77]. GlOS increased numbers of bifidobacteria and lactobacilli within 24 h



in batch cultures. Bacteroides, clostridial, and eubacterial populations were slightlydecreased by 48 h. The same materials resulted in increases in numbers of bifido-bacteria and lactobacilli in all three vessels of the gut model system, representingthe proximal, transverse, and distal colonic areas. The prebiotic indices of the gluco-oligosaccharides were 2.29, 4.23, and 2.74 in V1, V2, and V3, respectively.

The bacterium Leuconostoc mesenteroides produces a range of enzymes thatproduce dextrans from sucrose [78]. L. mesenteroides NRRL B-21297 produces analternansucrase enzyme that can transfer glucose from sucrose onto acceptor oli-gosaccharides including maltose and gentio-oligosaccharides. In this way a seriesof oligosaccharides can be produced with α1-6 and α1-3 linkages preceding eitherα1-4 or β1-6 linkages. These novel molecules have been evaluated as prebioticoligosaccharides in vitro. Using maltose as an acceptor, oligosaccharides were syn-thesized and then size fractionated. Oligosaccharide fractions with dp3, dp4, dp5,dp5.7, dp6.7, and dp7.4 were obtained and tested using pure cultures and a microscalefecal batch-culture system. In pure culture, most of the bacteria tested failed to growwell on dp6.7 and dp7.4 fractions and grew best on dp3 [79]. In mixed fecal culture,dp3 resulted in the highest prebiotic effect, followed by dp4 and dp6.7. dp7.4 wasnot selectively fermented [80].

With gentio-oligosaccharides as acceptor, the fractionation resulted in dp3, dp4,dp6, and a fraction of dp 6-8. The dp 4 fraction displayed the highest PI [80].

15.7 FUTURE PERSPECTIVES

15.7.1 TARGETED PREBIOTICS

With the advent of nutrigenomics and its promise of individually targeted nutrition,the generation of prebiotics targeted at particular probiotics is an interesting prospect.Virtually all of the fermentation studies to date have characterized the microflorachanges at the genus level only. This is due to the technical difficulty of characterizingthe complex colonic ecosystem at a species level. New DNA-based techniques toachieve this are now becoming available.

Two approaches can be considered in the generation of targeted prebiotics. Oneis the screening of as wide a range of candidate prebiotics as possible and usingtechniques such as quantitative PCR and DGGE to examine the resultant bacterialpopulations. An alternative is to utilize the enzymes of the target probiotics them-selves for the synthesis of oligosaccharides. These enzymes should produce oligosac-charide mixtures that would be more readily metabolized by the producing organism,resulting in higher selectivity. Early results with this method are encouraging andnovel GOS mixtures have been produced this way [48]. Many of these mixturesresulted in higher growth rates for the producing species in pure culture, althoughthe selectivity has not yet been described in mixed culture systems.

Targeted prebiotics could be developed for several applications:

1. Targeted at particular probiotics. Synbiotics could be developed on pro-biotics with well-defined health benefits; synbiotic forms of these probi-


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otics with targeted prebiotics should have better survival and subsequentcolonization in the gut.

2. Infant formula foods. Bifidobacteria are thought to predominate in the gutflora of breast-fed infants, while the gut flora of a formula-fed infant moreclosely resembles that of an adult. There would seem to be a clear appli-cation for prebiotics targeted at the bifidobacterial species present inbreast-fed infants.

3. Functional foods for the elderly. Above the age of 55 to 60 years, bifido-bacterial counts in the colon decrease relative to those of younger adults.The development of functional foods targeted at the elderly was initiatedin the EU-funded Crownalife project, part of the ProEUhealth cluster. Arange of probiotics, many of them novel species, have been obtained fromelderly individuals. These have then been used to develop targeted preb-iotics [81]. The aim was increased resistance to gastrointestinal pathogensincluding E. coli, Campylobacter jejuni, and Clostridium difficile.

15.7.2 PERSISTENT PREBIOTICS

The majority of chronic gut diseases arise in the distal colon and spread proximally,the most important of which are colonic cancer and ulcerative colitis. Many of themetabolites elaborated by the nonprobiotic flora in the colon are genotoxic or aretumor promoters [82, 83]. Although the precise role of these compounds in coloncancer is still not understood, it would seem sensible to reduce their levels in thecolon. There is a growing body of evidence that prebiotics have a protective effectagainst the development of colon cancer [11].

The precise mechanism behind the development of ulcerative colitis is still notclear but there is evidence that colonic bacteria, particularly the sulfate reducers suchas Desulfovibrio desulfuricans, are involved in the etiology of the disease [84, 85].

If a prebiotic is to have a protective effect, it must reach the distal colon andinfluence the microflora there. An approach to increasing persistence is regulationof the molecular weight distribution of the prebiotics. Most prebiotics are of relativelylow molecular weight with the exception of inulin. High-molecular-weight polysac-charides are not generally prebiotic but are nonselectively fermented. It seems thatselectivity decreases with increasing molecular weight, consequently developmentof persistent prebiotics will be a compromise between persistence and selectivity.The most persistent of the current prebiotics is Synergie II, manufactured by Orafti(Tienen, Belgium). This is an inulin and FOS mixture with controlled chain-lengthdistribution. The long-chain inulin exerts a prebiotic effect in more distal colonicregions and the lower-molecular-weight FOS is rapidly fermented in the proximalcolon. This might be a more generic means of developing persistent prebioticsderived from polysaccharides.

15.7.3 ANTIADHESIVE ACTIVITY

The first step in the pathogenesis of bacteria and viruses is often adhesion to a hostcell surface. Specific cell surface oligosaccharides often act as receptors for the



pathogen [86]. There is thus potential for the development of receptor-active oli-gosaccharides for incorporation into foods as decoy agents [87].

Recent data suggest that several agents might have antiadhesive activity.Caseinoglycomacropeptide (CGMP) is a commercial product from Arla foods.CGMP has three glycosylation sites and a heterogeneous array of complex oligosac-charide structures. CGMP has been found to protect Chinese Hamster Ovary (CHO)cells from the effects of E. coli heat labile enterotoxins LT-I and LT-II and choleratoxin at concentrations as low as 20 ppm [88]. Feeding CGMP at 1 mg per dayprotected mice from LT-I and LT-II. More recently, CGMP was found to protectinfant rhesus monkeys from E. coli–induced diarrhea [89]. The mechanism wasbelieved to be antiadhesive. Recently, CGMP was found to inhibit the binding ofthree VTEC E. coli strains to human HT-29 cells by 31 to 51% relative to the controlvalue [90]. Enteropathogenic (EPEC) strains were also inhibited, albeit with pro-nounced serotype variation, from 4 to 87% of the control value. CGMP inhibitedadhesion at low concentrations with IC50 values from 0.12 to 1.06 mg ml–1.

Pectic oligosaccharides have also been evaluated for their antiadhesive activity.Enzymatically generated pectic oligosaccharides have been found to inhibit adhesionof uropathogenic E. coli to uroepithelial cells in vitro [91] and to protect HT-29 cellsin vitro [92].

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51. Tanaka, R. et al. Effects of administration of TOS and Bifidobacterium breve 4006on the human fecal flora. Bifidobacteria Microflora 2, 17, 1983.

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54. Tzortzis, G., et al. A novel galactooligosaccharide mixture increases the bifidobacte-rial population numbers in a continuous in vitro fermentation system and in theproximal colonic contents of pigs in vivo. Journal of Nutrition 135, 1726, 2005.

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16 Probiotics in Food

Maria Bielecka

CONTENTS

16.1 Introduction................................................................................................ 41316.2 Scientific Basis of Probiotic Functionality and Justification

of Their Use............................................................................................... 41416.3 Probiotic Strain Selection.......................................................................... 41516.4 Probiotic Effects ........................................................................................ 417

16.4.1 Introduction ................................................................................. 41716.4.2 Intestinal Infections..................................................................... 41816.4.3 Immune Stimulation.................................................................... 41816.4.4 Effect on Nonspecific Immune Responses ................................. 41816.4.5 Effect on Specific Immune Responses ....................................... 41916.4.6 Factors That Influence the Efficacy of Lactic Acid Bacteria ..... 42016.4.7 The Future ................................................................................... 420

16.5 Probiotic Foods.......................................................................................... 42116.5.1 Food Products Containing Probiotics ......................................... 42116.5.2 Efficacy of Probiotic Products .................................................... 42116.5.3 Safety........................................................................................... 42216.5.4 Critical Questions Related to Probiotics .................................... 423

16.6 Recommendations for Future Research .................................................... 423References.............................................................................................................. 424

16.1 INTRODUCTION

The increasing consumer awareness that diet and health are linked is stimulatinginnovative development of novel products by the food industry. The new products,which should satisfy consumer needs, are functional foods containing probioticmicroorganisms with scientifically supported health claims for improving one’s stateof well-being and helping reduce the risk of diseases. Probiotic bacteria are used asthe active ingredient in functional foods most frequently as bioyogurt, and also asdietary adjuncts and health-related products. The health benefits attributed to probi-otic bacteria can be categorized as either nutritional benefits or therapeutic benefits.

The term probiotic is derived from the Greek language and means “for life.” Itwas first used by Lilly and Stillwell in 1965 to describe “substances secreted by onemicroorganism which stimulated the growth of another” (Lilly and Stillwell, 1965,



p. 362), and thus was contrasted with the term antibiotic. After several modifications,Schrezenmeir and de Vrese (2001) proposed the following definition of probiotics:“A preparation of or a product containing viable, defined microorganisms in sufficientnumbers, which alter the microflora (by implantation or colonization) in a compart-ment of the host and by that exert beneficial health effects in this host” (Schrezenmeirand de Vrese, 2001, p. 361). This definition confines the probiotic concept to effectsproduced by viable microorganisms, but is applicable independent of the probioticsite of action and route of administration. Therefore, this definition may include suchsites as the oral cavity, the intestines, the vagina, and the skin. In the case of probioticfoods, the health effect is usually based on alteration of the gastrointestinal micro-flora, and therefore based on survival during gastrointestinal transit.

Although there are numerous probiotic products for human consumption on themarket, there is a lot of skepticism regarding their beneficial effects. This has, inpart, been due to some reports of their positive health benefits, which have beenpublished with little scientific backup. Furthermore, many of the microorganismsincluded in these products are not viable and have not been selected either for specificbeneficial properties or for their ability to survive in the gastrointestinal tract. Ifhealth claims regarding probiotic bacteria are to be substantiated, it is imperative toestablish which strains have been used and from which source they have beenobtained.

16.2 SCIENTIFIC BASIS OF PROBIOTIC FUNCTIONALITY AND JUSTIFICATIONOF THEIR USE

The total mucosal surface area of the adult human gastrointestinal tract (GI) is upto 300 m2, making it the largest body area interacting with the environment. Thishuge surface would suggest a great capacity for effective absorption, and for defen-sive exclusion of infections, toxic, and allergenic material from the internal milieu.The gut-associated lymphoid tissue (GALT) makes the GI tract the largest lymphoidor immune organ in the human body. It has been estimated that there are approxi-mately 1010 immunoglobulin (antibody)-producing cells per meter of small bowel,thus accounting for ~80% of all immunoglobulin-producing cells in the body (Targanand Shanahan, 1994).

Sterile at birth, the GI tract rapidly acquires a commensal enteric microfloraresulting in the creation of a complex intestinal ecosystem. This microflora adds anadditional competitive component to the body’s defensive capability through com-petitive exclusion. It is also essential for the maturation of GALT. The balance ofthis commensal microflora may be altered by physiological changes in endogenousacid and bile secretion, by diet and bowel movements, by colonization by pathogens,by liver or kidney diseases, pernicious anemia, cancer, radiation, oral use of antibi-otics or immunosuppressive agents, by surgical operations of the GI tract, immunedisorders, and emotional stress. Many of these parameters are influenced by age,particularly in the late decades of life.


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It is estimated that the intestines of humans contain ~1014 viable bacteria cells,which is about 10 times more than that of all eukaryotic cells in the human body(Mitsuoka, 1996). The intestinal microflora of healthy humans is confined to thedistal ileum and the colon. It consists of over 400 species belonging to about 40genera. Most of them are by nature anaerobic, and some of them attain high popu-lation levels. Approximately 30 to 40 bacterial species constitute 99% of the culti-vable fecal microflora of a healthy human. Some of these are present in populationsof at least 1010 to 1011 colony-forming units (cfu) per gram of feces (wet weight).These live bacteria make up approximately 30% of the fecal mass. Fecal microflorais a representative of the microflora of the colon. While the numerically predominantgenera of bacteria detected in the feces of different individuals are the same, thereis variation in the occurrence and population size of bacterial species.

Gibson (1998) assigned the gut bacteria into three groups: beneficial (Lactoba-cillus, Bifidobacterium), harmful (Pseudomonas aeruginosa, Staphylococcus,Clostridium), and opportunistic (Enterobacteriaceae, Eubacterium, Bacteroides). Inhealthy subjects, they are well balanced and the beneficial bacteria dominate. Ben-eficial bacteria play useful roles in nutrition and the prevention of disease. Theyproduce essential nutrients such as vitamins and organic acids, which are absorbedfrom the intestines and utilized by the gut epithelium and by vital organs such asthe liver. Organic acids also suppress the growth of pathogens in the intestines. Otherintestinal bacteria produce substances that are harmful to the host, such as toxinsand carcinogens. When harmful bacteria dominate in the intestines, essential nutri-ents are not produced and the level of harmful substances rises. These compoundsmay not have an immediate detrimental effect on the host, but they are thought tobe contributing factors to aging, promoting cancer, liver and kidney diseases, hyper-tension and arteriosclerosis, and reduced immunity. Little is known regarding whichintestinal bacteria are responsible for these effects.

The normal balance of intestinal flora may be maintained or restored to normalfrom an unbalanced state by a well-balanced diet or by oral bacteriotherapy. Oralbacteriotherapy, using intestinal strains of lactic acid bacteria (LAB), such as Lac-tobacillus and Bifidobacterium, can restore normal intestinal balance and producebeneficial effects.

16.3 PROBIOTIC STRAIN SELECTION

Although progress in probiotic research has been achieved, not all of the availableprobiotic bacteria on the market have adequate scientific documentation. If nutri-tional and health benefits are to be derived from products containing probioticbacteria, it is desirable to understand the mechanisms by which these benefits arederived and use those strains demonstrating the most promise in this regard. Con-sequently, it is necessary to establish rational criteria for screening and selection ofcandidate microorganisms, and also to evaluate the efficacy of the selected strainsor the food products in well-controlled human dietetic or clinical trials.

As a result of food industry collaboration with scientists and clinicians, supportedby European Union-funded programs, the aim of promoting the generation and



dissemination of consensus, outlined criteria for the selection and assessment ofprobiotic microorganisms are as follows. They must

• be of human origin (controversial)• demonstrate nonpathogenic behavior• exhibit resistance to technological processes (i.e., viability and activity in

delivery vehicles)• prove resistance to gastric acid and bile• adhere to gut epithelial tissue• be able to persist in the gastrointestinal tract• possess antimicrobial activity• modulate immune responses• have the ability to influence metabolic activities (e.g., β-galactosidase

activity, vitamin production, cholesterol assimilation)

These requirements were further expanded by Berg (1998) and Salminen et al.(1996, 1998a), who stated that

• each potential probiotic strain should be documented• extrapolation of data even from closely related strains is not acceptable• only well-defined strains, products, and study populations should be used

in trials• where possible, all human studies should be randomized, double blinded,

and placebo controlled• results should be confirmed by independent research groups• preferentially, the study should be published in a peer-reviewed journal

In the development of probiotic foods, LAB strains such as Lactobacillus andBifidobacterium, have been most commonly used. This is primarily due to theperception that they are desirable members of the intestinal microflora colonizingintestines of newborns as one of the first genera (Goldin and Gorbach, 1992; Berg,1998). In addition, LAB have traditionally been used in the production of fermenteddairy products, and have been accorded generally recognized as safe (GRAS) status.

The requirement that the strains of probiotic bacteria used should be of humanorigin is based on the observation that only human strains can be adhesive and colonizethe human GI tract, which is the first step in promoting colonization resistance (Huisin’t Veld et al., 1994). It has been proposed that species specificity does occur, andfor strains to be beneficial to a particular host, they should be isolated from thatspecies. This is not well documented, and subsets of probiotic bacteria currentlyemployed in the dairy industry are not of human origin, and therefore do not meetthe criteria for selection as probiotic microbes acceptable for human consumption asoutlined above. The bifidobacterial strains isolated from market bio-yogurt wereidentified phenotypically and genetically as B. animalis (Roy et al., 1996; Roy andSirois, 2000; Bielecka et al., 2000a). The strains belonging to B. animalis speciessurvived well at low pH and bile, and adhered well to Caco-2 and HT-29 cell lines,as well as to colon epithelium of humans and rats (Bielecka et al., 2000b). Moreover,



either isolated from bioyogurts, rats and adults consuming bioyogurts, B. animalisstrains survived to the highest extent and were unusually homogeneous. Recently, aBifidobacterium strain, isolated from market bioyogurt, was classified as B. lactis—the new species genetically most similar to B. animalis (Meile et al., 1997). The nameof species B. lactis has been given due to the source of isolation and to justify its usein bioyogurt. Cai et al. (2000), comparing B. animalis JCM 1190T and B. lactisJCM 11602T, found their phenotypic and genetic similarity. B. animalis and B. lactiswere the most closely related species on the phylogenetic tree, and showed a highsimilarity in 16S rRNA sequences (98.8%). The levels of DNA–DNA hybridizationbetween the strains of B. lactis and B. animalis ranged from 85.5 to 92.3%, showingthat they represent a single species (a genospecies is characterized by a DNA–DNAsimilarity of more than 70% and 16S rRNA similarity of more than 95%). TheBifidobacterium species most often isolated from human colon of adults areB. catenulatum, B. longum, B. adolescentis, B. bifidum, and B. pseudocatenulatum,and rarely B. angulatum and B. animalis. Among species of Lactobacillus, isolatedfrom human feces and directly from intestinal homogenized mucosa as potentiallyadherent probiotic bacteria, the following were identified: L. paracasei, L. salivarius,L. acidophilus, L. crispatus, L. gasseri, L. reuteri, L. rhamnosus, and L. plantarum(Molin et al., 1993). No single dominant species was found among the isolates.

A critical criterion of selection is that the probiotic strain must be tolerated bythe immune system and should not provoke the formation of antibodies against theprobiotic. This latter property, in conjunction with the ability of some LAB tosurvive and colonize in the gut, has given rise to further applications, which involvetheir use as live vectors for oral immunization, that is, introducing antigens targetingthe GALT and aiming to induce a mucosal immune response (Marteau and Ram-baud, 1993).

Strain viability and maintenance of desirable characteristics during product man-ufacture and storage is also a necessity for probiotic strains. The ability to multiplyrapidly on relatively cheap nutrients is a distinct advantage, as ease of culturing isimportant for technical application of the strains. Strain survival depends on suchfactors as the final product pH, the presence of other microflora, the storage tem-perature, and the presence or absence of microbial inhibitors in the product. Exploi-tation of the latest biotechnological advances in culture production, preservation,and storage, should aid in maintaining high numbers of probiotic bacteria in products.

16.4 PROBIOTIC EFFECTS

16.4.1 INTRODUCTION

A number of benefits for the ingestion of probiotics have been reported. Accordingto Vaughan et al. (1999), the beneficial effects of probiotic strains demonstrated andproposed are the following:

• increased nutritional value (better digestibility, increased absorption ofminerals and vitamins)

• alleviation of lactose intolerance



• positive influence on intestinal flora• prevention of intestinal tract infections• improvement of the immune system• reduction of inflammatory reactions• prevention of cancer• antiallergenic activity• regulation of gut motility• reduction of serum cholesterol• prevention of osteoporosis• improved well-being

16.4.2 INTESTINAL INFECTIONS

The primary claim regarding probiotics is their beneficial influence on the intestinalecosystem, which in turn may provide protection against gastrointestinal infectionsand inflammatory bowel diseases. The desirable effects on human health includeantagonistic activity against pathogens, antiallergenic action, and other effects onthe immune system. Whereas some of these claims remain controversial, well-planned clinical trials increasingly support them for carefully selected probioticstrains (Ouwehand et al., 1999). Some probiotic strains have been selected as bac-tericidal against Salmonella, Escherichia coli, and Staphylococcus aureus in theassociated cultures with the pathogens (Bielecka et al., 1998). The strains increasedBifidobacterium population numbers in the colons of both healthy and Salmonella-infected rats, and protected the natural balance of intestinal microflora (Bielecka etal., 2002). Bouhnik et al. (1996) and Matsumoto et al. (2000) observed an increasein Bifidobacterium population numbers in feces of healthy volunteers after two weeksof bioyogurt consumption. Bifidobacterium and L. acidophilus administered in milkwere effective in reducing Candida and Clostridium difficile occurrence in feces(Corthier et al., 1985).

The mechanism by which protection is offered by these probiotics has not yetbeen fully established. However, one or more of the following are possible: compe-tition for nutrients, secretion of antimicrobial substances, blocking of adhesion sites,attenuation of virulence, blocking of toxin receptor sites, immune stimulation, andsuppression of toxin production.

16.4.3 IMMUNE STIMULATION

One of the most interesting aspects of probiotic supplementation is directed towardimmune responses. Orally administered probiotic strains of LAB exert a positiveimpact on nonspecific and specific host-immune responses. Nonspecific immuneresponses constitute the first line of host defense.

16.4.4 EFFECT ON NONSPECIFIC IMMUNE RESPONSES

The results of many experimental studies (Perdigon and Alvarez, 1992; Paubert-Braquet et al., 1995; De Petrino et al., 1995) have shown that the consumption ofcertain strains of LAB are able to enhance:



• phagocytic activity of peritoneal and pulmonary macrophages and bloodleukocytes

• secretion of lysosomal enzymes, reactive oxygen, nitrogen species, andmonokines by phagocytic cells

• in vivo clearance of colloidal carbon, an indicator of phagocyte functionof the reticuloendothelial system

Similar results have been reported for human subjects. Ingestion of fermentedmilk containing L. acidophilus LA1 or B. bifidum Bb12 for three weeks resulted inincreased phagocytic activity of peripheral blood leukocytes (Schiffrin et al., 1997).This increase in phagocytosis was coincidental with colonization by LAB.

The results of several animal studies indicate that the ability to enhance phago-cyte cell function is strain dependent. Structural differences in cell wall compositionof different LAB strains are suggested as being responsible for differences in efficacy.Furthermore, strains that are able to survive in the gastrointestinal tract, adhere tothe gut mucosa, and persist above a critical level are more efficient in stimulatingphagocytic cells (Schiffrin et al., 1997). The results of studies of a large number ofBifidobacterium strains belonging to different species showed a large diversity insurvival at low pH and bile, and in the adherence to the colon epithelium betweenstrains belonging to the same species, excluding B. animalis, in which the strainswere uncommonly homogeneous (Bielecka et al., 2000b). These and other resultsproved the importance of careful probiotic strains selection.

Recent studies by Gill (1998) have shown that the magnitude of response alsodepends on the dose of LAB. Mice receiving a milk-based diet containing 1011 cfuper day of L. rhamnosus HN001 for 10 days showed significantly greater phagocyticactivity than mice receiving 109 or 107 LAB.

16.4.5 EFFECT ON SPECIFIC IMMUNE RESPONSES

Specific immune responses can be classified into broad categories: humoral immu-nity (HI) and cellular-mediated immunity (CMI). HI is affected by antibodies pro-duced by plasma cells (mature B lymphocytes), which bind specifically to antigenicepitopes on the surface of pathogenic organisms, and with the aid of complementskill these pathogens. Specific classes of antibodies have specific functions. Forexample, IgA antibodies predominate at the mucosal surfaces and prevent adherenceof pathogens to the gut mucosa. IgG and IgM are involved in systemic neutralizationof bacterial toxins and promote phagocytosis by monocytes-macrophages viaopsonization. Antibodies are effective at neutralizing or eliminating extracellularpathogens and antigens.

CMI is mediated by T-lymphocytes. On exposure to antigen or pathogen T-lymphocytes of predetermined clones proliferate or produce cytokines. Throughthese cytokines, T-cells influence the activities of other immune cells, for example,augmenting the ability of macrophages to kill intracellular pathogens and tumorcells. In addition, subsets of T-cells act as helper cells (CD4+) for antibody produc-tion, as mediators of delayed type hypersensitivity (DTH), or as cytotoxic cells



(CD8+) against virus-infected cells and cancer cells. CMI is particularly effectiveagainst intracellular pathogens and tumor cells.

Several studies in experimental animals and humans have demonstrated theimmunostimulatory effects of lactic cultures on several aspects of humoral and cell-mediated immunity (Nader de Macias et al., 1992; Perdigon et al., 1990; Kaila etal., 1992). Whether the immunoenhancing effects of LAB are related to their immu-nogenicity is not known. It is well documented that mucosal antibodies prevent theadherence of pathogenic microorganisms to gut epithelium. It is likely, therefore,that antilactobacilli antibodies in gut secretions may interfere with the ability ofLAB to adhere and proliferate in the gut. If adherence to and colonization on gutepithelium are essential for LAB to exert beneficial immunoenhancing effects, itwill be desirable to select strains that do not elicit an immune response to themselves.

16.4.6 FACTORS THAT INFLUENCE THE EFFICACY OF LACTIC ACID BACTERIA

Several factors have an impact on the ability of LAB to influence immune function(Biedrzycka et al., 2003; Gill, 1998; De Petrino et al., 1995; Paubert-Braquet, 1995;Portier et al., 1993), for example:

• a large variation exists in the ability of LAB to affect the immune system• the effect of LAB on the immune system is dose dependent, with a higher

intake of LAB resulting in a superior response compared with a lowerintake

• live cultures are more efficient at enhancing certain aspects of immunefunction than the killed cultures

• lactic cultures delivered in fermented products induce a superior responsecompared to cultures given in unfermented products

There is no information on the efficacy of LAB in relation to host age, physio-logical status, and dietary intake. All these factors may have an important bearingon the ability of LAB to influence host responses.

16.4.7 THE FUTURE

There is now strong evidence that certain strains of LAB are endowed with thecapacity to stimulate both nonspecific and specific immune functions. This highlightsthe opportunities for the dairy and health food industries to develop novel, value-added immunity-enhancing food products. However, many significant gaps remainin our knowledge. Therefore, future studies should be directed at:

• demonstrating the relevance of immunomodulation to better health• defining the effective dose for each strain• elucidating the mechanisms by which LAB act on the immune system• identifying new strains that are able to inhabit desired anatomical sites in

the gut and modulate desired immune functions



16.5 PROBIOTIC FOODS

16.5.1 FOOD PRODUCTS CONTAINING PROBIOTICS

Products containing probiotics come in a variety of forms:

• Conventional foods: probiotic-containing yogurts, fluid milk, cottagecheese, consumed primarily for nutritional purposes, but also for probi-otic benefit.

• Food supplements or fermented milks: food formulations whose primarypurpose is to be the delivery vehicle for probiotic bacteria and theirfermentation end products, consumed for health effects, in monoculture(Yakult, Japan) or mixed cultures (Actimel, Danone; LC1, Nestle) andothers.

• Dietary supplements: capsules and other formats designed to be taken byhealthy individuals to improve health.

In the development of probiotic-containing food products, the product conceptmust take into account the desire of consumers for health-enhancing foods, but notat the expense of taste, convenience, and the pleasurable nature of the product. Theability to communicate messages on health to the consumer is extremely important,for example, consumers can be told that the product promotes gastrointestinal healthor supports the body’s natural immune function, but the claims must be scientificallyaccurate and in accordance with actual legislation, which may differ from countryto country. In addition to communications on the health benefits of probiotic prod-ucts, another important area of communication is on viable count or active ingredi-ents in probiotic products. However, statements as to content and counts of bacteriain commercial products are frequently not accurate, and may claim the presence ofcertain genera, species, or strain of probiotic, but rarely include any messages onprobiotic potency. In countries where there is no legislation requiring this type oflabeling, this leaves the consumer unable to make an informed choice based on aguaranteed probiotic content of a food product. In the long run, the failure of theindustry to self-regulate in this regard may be its undoing. Consumers, unable toidentify a potent product, may find a specific probiotic product ineffective and turnaway from the entire product category.

16.5.2 EFFICACY OF PROBIOTIC PRODUCTS

Probiotic consumption can be justified only if a health effect or reduction in risk ofdisease can be realized by the consumer. Substantiation of health effects is a chal-lenge the probiotic industry is now facing, in large part because of the high costassociated with conducting controlled clinical or epidemiological evaluations. Thesehealth effects are believed to be genera, species, and strain specific. In many casescontrolled studies have not been done, however; a lack of proof has a great influenceon the use of health claims. There is no motivation for companies or consumers to



invest in technology supported mainly by testimonial, inference, or supposition.Therefore, the clinical evaluation of commercial products must be conducted and isessential for labeling health claims. The few probiotic strains whose effect in humanhealth is supported by clinical evaluations have a legitimate marketing advantage,even though the strains may be no more effective than other, untested bacteria.

Salminen et al. (1996) and Fonden et al. (2000) listed the variables that must becontrolled for clinical evaluation of probiotics:

• strain or combination of strains• growth conditions of strain• format for delivery (dried, supplemented in a food product, grown in a

food product)• consumption level of the active ingredient• test population• validated biomarkers in combination with the clinical end point• clinical protocol, with preference for blind, placebo-controlled formats

16.5.3 SAFETY

Although the safety of traditional lactic starter bacteria has never been in question,the more recent use of intestinal isolates of bacteria (bifidobacteria, intestinal lacto-bacilli, and enterococci) to be delivered as probiotics in high numbers to consumerswith potentially compromised health, has raised the question of safety. These intes-tinal isolates do not share the centuries-old tradition of being consumed as compo-nents of fermented dairy products. However, their presence in commercial productsover the past few decades has not given any reason for safety concern.

The safety of lactobacilli and bifidobacteria was the most recently reviewed bySalminen et al. (1998b). The general conclusion is that the pathogenic potential oflactobacilli and bifidobacteria is quite low. This is based on the prevalence of thesemicrobes in fermented foods, as commensal colonizers of the human body, and theconcomitant low level of infection attributed to them. A report from the LABIndustrial Platform (Guarner and Schaafsma, 1998) indicated that the overall risk ofinfection from LAB (excluding enterococci) is very low, but that L. rhamnosusdeserved particular surveillance due to the greater proportion of infections attributedto this species compared to infections by other lactobacilli. One commercial strainof L. rhamnosus GG, has been used repeatedly in clinical trials and human studies,including one involving enteral feeding of premature infants (Saxelin, 1997).

Regarding the safety of enterococci, the picture is less clear. Foods containingenterococci are consumed on a regular basis. However, safety reports seem to agreethat enterococci pose a greater threat than other LAB. Giraffa et al. (1997) concludedthat the enterococci, although a group of phenotypically heterogeneous organisms,display potential pathogenic properties. Adams and Marteau (1995) excluded entero-cocci from the LAB they regard as having low pathogenic potential. Enterococcihave been isolated from clinical infections: Enterococcus-mediated infections of thebiliary tract, the abdomen, burn wounds, surgical wounds, and many others (Jett etal., 1994). Often, enterococci are isolated as pure cultures from these infections,



showing their primary pathogenic nature (Aquirre and Collins, 1993). In the UnitedStates, vancomycin-resistant enterococci comprise a worrisome source of nosoco-mial infections. This forms a serious threat to humans, as it is known that genetransfer between enterococci and related organisms can occur rather easily. In thisrespect, transfer of the VanA gene from Enterococcus faecium to Staphylococcusaureus has already been observed in vitro (Noble et al., 1992). These observationsand contradictory characteristics of enterococci make the use of these microorgan-isms more and more questionable as food fermentation agents (Arthur et al., 1996).

16.5.4 CRITICAL QUESTIONS RELATED TO PROBIOTICS

Sanders and Huis in’t Veld (1999), in their extensive review, outlined the followingcritical issues related to probiotics:

• identification of physiologically relevant biomarkers, which can be usedto assess parameters of probiotic effectiveness in humans (strain, dose,strain growth, and delivery conditions.

• definition of active principle(s)• stability parameters for active principle(s)• resolution of taxonomy of probiotic species and development of user-

friendly methods for conducting speciation assessments• science-driven implementation of findings in commercial products• epidemiology and properly controlled human intervention studies to con-

firm probiotic efficacy

16.6 RECOMMENDATIONS FOR FUTURE RESEARCH

If probiotics are to be used to treat and prevent infection, the first studies to beundertaken must fully characterize (phenotypical, genotypical traits) the microor-ganisms that will be used. If they do not demonstrate antiinfective traits in vitro, itseems unlikely that they can be efficacious in humans. A scientific rationale for theselection of the best species or strain for use as probiotics is not possible withoutmore information on the mechanisms by which probiotics exert their beneficialeffects in vivo.

The benefits of probiotics are often demonstrated under defined experimentallaboratory conditions, but these beneficial effects fail to materialize in clinical trials,often because the trials are not properly controlled or consist of too few subjects.Large, double-blind, clinical trials are essential for establishing the practical andscientific logic of the probiotic concept.

The full potential of therapeutic manipulation of the enteric flora with probioticsmay not be optimally realized until our understanding of the normal flora is complete.The interaction between the host and the commensal flora requires basic investigation.

Further information concerning the molecular basis of probiotic strains can havean impact on the development of strains with safe and effective novel probioticeffects. There is enormous potential for metabolic engineering as has already beendemonstrated for several lactic acid bacteria. Indigenous bacteria vectors, such as



Lactobacillus, might be considered safer than the Salmonella and virus vectorspresently being considered for these purposes.

The use of prebiotics in association with useful probiotics may be a worthwhileapproach, as the prebiotics preferentially stimulate some probiotic strains. Combi-nation of probiotic and prebiotics as synbiotics can also enhance probiotic effec-tiveness.

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17 Mood Food

Maria H. Borawska

CONTENTS

17.1 Introduction................................................................................................ 42717.2 Dietary Amino Acids and Neurotransmitters in the Brain ....................... 42717.3 Sweets and Brain Function ....................................................................... 42917.4 The Effect of Lipids on Mood .................................................................. 43117.5 Vitamins Improve Mood............................................................................ 43117.6 Selected Mineral Components Important for Mood................................. 433

17.6.1 Introduction ................................................................................. 43317.6.2 The Role of Zinc......................................................................... 43317.6.3 The Involvement of Copper ........................................................ 43417.6.4 The Importance of Iron............................................................... 43417.6.5 The Role of Selenium ................................................................. 434

17.7 Alcohol and Mood..................................................................................... 435References.............................................................................................................. 436

17.1 INTRODUCTION

There is much to be learned about the influence of food and nutrition on the centralnervous system. Human mood disturbance is associated with anorexia nervosa,bulimia nervosa, and binge eating. Many people eat more food when they arenervous, stressed, or depressed in order to, consciously or not, improve their mood—habitual or temporary states of feeling. In this chapter an attempt is made to showhow foods or their components influence human moods by acting on the centralnervous system (CNS).

This knowledge is necessary for the conscious control of human reactions tofood, which may affect mood.

17.2 DIETARY AMINO ACIDS AND NEUROTRANSMITTERS IN THE BRAIN

The brain is unique in the sense that it is a large complex of neural systems, all ofwhich must interact with completely functioning parts (especially limbic structure),which determine behavior. Mood is likely to result from the interaction of multiple,semiindependent, neural circuits working together in harmony. Additionally, complex



interactions between neural systems are required for maintenance of appetite, sleep,weight stabilization, and interest in sexual activity.

Several studies suggest that brain functions, including mood, respond to changesin nutrition. Neurotransmitters or neuromodulators are synthesized from compoundsthat are essential dietary constituents. In the brain there are important biogenicamines, crucial for mood: serotonin, norepinephrine (NE), and dopamine (DA), aswell as amino acids: gamma-aminobutyric acid (GABA), glutamic acid, glycine (co-agonist of glutamate), and N-methyl-D-aspartic acid (NMDA), and peptides—encephalins and endorphins. Serotonin, DA, and NE are formed from tryptophan ortyrosine, respectively. GABA is synthesized from glutamic acid and evokes effectssimilar to those of serotonin.

Serotonin is of key importance for human emotional behavior. The bulk oftryptophan (the precursor of serotonin) is in the blood with protein complexes, andonly 5% has free access to CNS transport. After a meal rich in saccharides, thesecretion of insulin increases, and as a consequence, the bioavailability of aminoacids from proteins, except for tryptophan increases. Tryptophan emerges above theother amino acids, and so more easily crosses the blood–brain barrier and the levelof serotonin increases. Most tryptophan is to be found in 100 g of the followingproducts: yellow cheese, 284 to 519 mg; meat and sausage, 90 to 385 mg; fish andtheir products, 73 to 325 mg; almonds, 310 mg; nuts, 36 to 275 mg; cereal products,1 to 402 mg; vegetables and their products, 5 to 798 mg; and fruit, 3 to 74 mg(Kunachowicz et al., 2005). Animal tissues and fruit (bananas, pineapples, plums,and nuts) contain serotonin, but it is taken into the blood platelets and does not crossthe brain barrier. Tryptophan can enhance mood, an effect seen most clearly inpatients who are mildly depressed (Moore et al., 2000),

Dopamine is one of the most intensively studied neurotransmitters in the braindue to its involvement in several mental and neurological disorders. Disturbances inthe dopaminergic system have been implicated in the etiology of mood disorders.There is a significant relationship between the dopamine D4 receptor gene and mooddisorders, especially major depression (Manki et al., 1996). In a high-protein meal(meat) there is more tyrosine than tryptophan, so it crosses the blood–brain barrierand increases the synthesis of DA. Other endogenous biogenic amines, tyramine andphenylethylamine (PEA), are also formed indirectly from tyrosine. These substancesact as sympathomimetic compounds, and at physiological doses they may act tointensify dopaminergic and noradrenergic neurotransmission. PEA is produced bybrain tissue and is rapidly metabolized by monoamine oxidase-β and aldehydedehydrogenase to phenylacetic acid, the major metabolite of PEA in the brain.Several studies have suggested that PEA is an important mood modulator and thatits deficit may contribute to pathogenesis of depression (Sabelli and Javaid, 1995).Tyramine and other biogenic amines (tryptamine, PEA, cadaverine, putrescine, his-tamine) appear in foods such as wines and beers (red wine to 25.4 and beer to 17.6mg per liter), chocolate, different types of cheese (especially blue cheese, whichcontains tyramine 12.9 mg and tryptamine 158.5 mg per 100 g wet extract), fer-mented meats (tyramine is found in a higher concentration than in other amines; indry sausage 10.2 to 50.6 mg or shrimp sauce 24 mg per 100 g) and fermentedvegetables (tyramine in soy sauce up to 466 and salted black beans up to 45 mg per


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100 g of the product) (Flick and Granata, 2005). The manufacturing process usedin fermentation can increase or decrease the concentration of biogenic amines in theend product. Storage time and temperature after manufacturing can also increase theamount of biogenic amines present, and their concentration in cheese could be usedas an indicator for freshness, the quality of the raw materials, and the manufacturingprocesses involved. Tyramine is deaminized by monoamine oxidase, and whenmonoamine oxidase inhibitors (MAOIs) are used as medication and foods high intyramine are ingested, a hypertensive crisis cheese syndrome can arise.

In interactions between neurotransmitters and their receptors, the regulation ofbehavioral complexity relies on second messenger signals within cells like G pro-teins, and on cell membrane receptors to relay information from the extracellularenvironment to the interior of the cell (Gould and Manji, 2002).

17.3 SWEETS AND BRAIN FUNCTION

Sweets constitute the group of food for which human beings have always had someinborn preferences because the majority of sweet fruits or edible parts of plantsfound in the natural environment are not poisonous. Even infants show a positivemimic reaction to sweet liquids (Blass and Hoffmeyer, 1991).

The group of sweets includes honey, candies, jellies and marmalades, candiedfruits, sweets made of cacao, seed pulp and caramel mass like halvah, sesame snaps,as well as biscuits, cakes, and ginger biscuits. Moreover, refined sugar is an importantingredient of numerous beverages, jams, puddings, and some candied fruits.

Saccharose and simple sugars derived from ingested sweets cause a suddenincrease of the glucose level in blood, and as a result, also that of insulin. The abilityof each carbohydrate to raise the glucose level in blood is defined as the glycemicindex. Glucose has been given an arbitrary value of 100, and other carbohydratesare given numbers relative to glucose. Glucose is taken up by cells in an insulin-independent way not only in the liver, but by other tissues such as brain and redblood cells, and in an insulin-dependent manner by cells in muscle and adiposetissue. Glucose is metabolized by all tissues of the body, with only 30% to 40% ofglucose intake being metabolized by the liver. The brain, which is completelydependent on glucose for its energy needs in normal dietary conditions, is capableof the complete oxidation of glucose to CO2 and H2O via glycolysis and the citricacid cycle. As the result of glycolysis, energy is conserved in the chemical form ofATP, with ATP synthesis both by the substrate-level phosphorylation and by electrontransport linked to oxidative phosphorylation. Moreover, insulin simplifies the proc-ess of transferring essential amino acids from blood into the cells, but tryptophanin the blood rises, which facilitates its penetration through the blood–brain barrierinto the CNS. It is the basic material in the synthesis of the serotonin neurotransmitterin the brain, and it has influence on human mood and the ability to calm down.

After an artificial sweetener (aspartame) in high doses activates the serotoninsystem in rats through the mechanism of impeding the presynaptic release of seroto-nin, the sensibility of its postsynaptic receptor rises accordingly. Aspartame has noeffect on mood in young people, but increases the number and severity of symptomsin patients with a history of depression (Walton, Hudak, and Green-Waite, 1993).



Even an oral dose of 25 g of glucose improves the ability to acquire newknowledge and remembering new information, whether taken before or during learn-ing. Glucose helps remembering and learning not only by delivering energy to thecells, but also through the insulin in the CNS it can directly influence insulin receptorsand their reaction with the central cholinergic system (Ragozzino et al., 1998). Thereis no correlation between a rise in the glucose level and memory. This correlationis not linear with respect to the acquired amount of sugar; larger amounts do notenhance this effect.

Products made of cacao grains, nut pulp (halvah), and almond pulp (nougat)contain a good deal of fat (especially halvah) and phosphorus, calcium, magnesium,and B vitamins. Chocolate made of cacao pulp, cacao fat, and sugar may containabout 300 active substances, depending on the additional ingredients (nuts, vanilla,milk powder, coffee, and cinnamon).

Polyphenols present in chocolate, especially flavonoids (mainly catechin), havefourfold stronger action as antioxidants than those in green tea (Stark, Bareuther,and Hofmann, 2005; Lee at al., 2003), but only in dark bitter chocolate, becausemilk additives decrease the bioavailability of polyphenols. Other rich sources offlavonoids include citrus fruits, berries, onions, parsley, legumes, and red wine. Theyhave a wide range of biological effects including antiinflammatory, antiallergenic,and anticancer activity.

Eating sweets causes release of endogenous opiates, which have not only ananalgesic effect, but also influence mental illness and mood. It has been proved thatgiving babies water with sugar stops their crying caused by a prick of a needle.Chocolate, chocolate bars, and other chocolate products play the most importantrole in the processes of addiction to sweets. Chocolate is not only a source of energyfrom carbohydrates and fats, but also contains many pharmacologically knownsubstances, such as the alkaloids salsolinol and xanthines. Salsolinol stimulates thedopaminergic receptors D2 and D3 and releases ß-endorphins. Xanthines, such astyramine and PEA, are commonly used for their effectiveness as mild stimulantsand bronchodilators. They are very dangerous when used concurrently with MAOIdrugs. Tyramine is also a known trigger of migraine attacks. Methylated xanthinederivatives include caffeine, theophylline, and theobromine. These alkaloids inhibitphosphodiesterase by evoking an increase in cAMP and cGMP, and block adenosineof A1 and A2 receptors. Adenosine is a modulator of CNS neurotransmission, andits modulation of dopamine transmission through A2A receptors has been implicatedin the effects of caffeine.

Other, lesser-known substances contained in chocolate are anandamide(derived from the Indian word ananda, meaning “bringing calm”) and its twoanalogs, which are natural agonists of the cannabinoid receptors, CB1 and CB2.They belong to the cannabinoids, whose central effects include disruption ofpsychomotor behavior, intoxication, stimulation of appetite, antinociceptiveactions (particularly against pain of neuropathic origin), antiemetic effects, short-term memory impairment, and a possible improvement of mood (we do not feelthat time is “running away”). The properties of cannabinoids that might be oftherapeutic use include analgesia, muscle relaxation, immunosuppression, antiin-flammation, antiallergenic effects, sedation, stimulation of appetite, antiemesis,


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lowering of intraocular pressure, bronchodilation, neuroprotection, and antineo-plastic effects. Anandamide does not cause the loss of the rat pheochromocytoma(PC-12) cell line (a useful model for neurobiological and neurochemical studies)in a relatively low concentration. However, a massive cell death occurred whencells were incubated with a higher concentration of anandamide (Sarker et al.,2000). Cannabinoids cause psychical relaxation and positive moods, that is, theeffects may, to some extent, be similar to those caused by small amounts of ethanol.They also increase hearing and seeing sensitivity. In an experiment conducted onanimals, it was proven that rats that chose sweet water also preferred drinkingalcohol. Under these circumstances the following questions arise: Is it possible tohave a similar correlation between ethanol and sweets in the case of people, andcan eating large amounts of sweets lead to alcohol addiction? These two questionsstill remain unanswered. Nevertheless, it has been proved that members of familieswith previous alcohol problems show a greater tendency to eat sweets.

It can be concluded that addiction to sweets, so difficult to fight against, alsodepends on a preference for all that tastes sweet, and this influences the CNS,improves mood, the ability to learn, and lowers aggression, but in large amounts,may evoke dependence and memory impairment. It is, however, safe to eat smallamounts of sweets, preferably 1 or 2 pieces of dark bitter chocolate with an estimatedlow glycemic index of 22.

17.4 THE EFFECT OF LIPIDS ON MOOD

Lipid molecules are important constituents of all living cells. Fats, especially unsat-urated fatty acids n-6 and n-3, are very significant in the diet because they build theneuron membranes. The brain and nervous tissue membrane lipids contain a partic-ularly high proportion of arachidonic acid (AA) and docosahexaenoic acid (DHA)and low concentrations of their 18-carbon precursors. The soft part of the brain andretina are very rich in DHA, which is essential for proper neural functioning andvision. Even small changes in the concentration of these acids or in the proportionof the main phospholipids, cholesterol and its esters, may have a considerableinfluence on the functioning and structure of the receptor tissues. AA and DHA,even though they act as renewed transmitters per se, play an important role inregulating and conducting signals (Marszalek and Lodish, 2005).

It is now known that two G-protein-coupled receptors, metabotropic glutamateand cannabinoid, are transsynaptically linked by a small lipid messenger, which hasprofound implications both for control of synaptic transmission and for new thera-peutic strategies. Some symptoms of schizophrenia can be related to disturbancesin the synthesis and release of the endogenous cannabinoid of anandamine,eicosanoid, an amide of AA and ethanoloamine.

17.5 VITAMINS IMPROVE MOOD

The most important group of vitamins, the lack of which is related to disability ofbrain functions, includes: B1, B6, folic acid, and B12.



In the counteraction of depression and impeding oxidative changes in brain lipidsand proteins, an important role is played by vitamins: C, E, and A.

Most of the vitamin B1 in the body is present as diphosphate (TDP)—90% withapproximately 10% as thiamine triphosphate (TTP) in the brain and other neuraltissues—and is connected with the cholinomimetic effects. The TTP form is involvedin nerve membrane function. Thiamine functions in prime interconversions of sugarphosphates and in decarboxylation reactions with energy production from α-ketoacids and their acyl-CoA derivatives, which are catabolically derived from carbohy-drates and amino acids (Stipanuk, 2000). Acetyl-CoA is an important precursor ofacetylcholine, demonstrating an obvious biochemical link between thiamine, as TDP,and the normal functioning of the CNS. Clinical signs of deficiency in vitamin B1

include the nervous symptoms (mental confusion, polyneuritis, peripheral paralysis,muscular weakness, ataxia, edema—wet beriberi, or muscle wasting—dry beriberi)and cardiovascular system symptoms (enlarged heart, tachycardia). Thiamine defi-ciency is extremely common among chronic alcoholics. Dietary thiamine for peoplein a large part of the world is obtained from either unrefined cereal grains or starchyroots and tubers. Other natural sources of thiamine are pork, nuts, and legumes.Thiamine is unstable in alkaline solution, and may be lost from cereals by refiningand overgrinding. In raw fish, shellfish, ferns, and some bacteria there are thiaminasesthat hydrolytically destroy the vitamin in the gastrointestinal tract. In addition, therealso exist heat-stable, antithiamine factors that are found in ferns, tea, betel nuts,and a large number of plants, vegetables, and in the tissue of some animals.

There are six major compounds of vitamin B6; pyridoxal (PL), pyridoxine (PN),pyridoxamine (PM); and their respective 5′-phosphate derivatives (PLP, PNP, PMP)as a coenzyme for more than 100 enzymes, which primarily include enzymesinvolved in amino acid metabolism and in glycogen metabolism. PLP is a cofactorfor decarboxylases, which are involved in neurotransmitter synthesis. The clinicalsymptoms of B6 deficiency from the CNS are depression and confusion, epileptiformconvulsions, and other clinical signs including inflammation of the tongue, lesionsof the lips and corners of the mouth, seborrheic dermatitis, and microcytic anemia.Rich sources of vitamin B6 are meat, fish, potatoes, bananas, and pulses. VitaminB6 compounds are sensitive to light and high temperature.

Both low folic acid (folate is used as a generic name for all these derivatives)and low vitamin B12 (cobalamin) status have been found in studies of depressedpatients, and a relation between depression and low levels of the two vitamins wasfound in studies of the general population of England (Coppen and Bolander-Gouaille, 2005). Folic acid is important in the synthesis of tetrahydrobiopterin,which is a cofactor for the hydroxylation of phenyalanine and tryptophan, and isthe rate-limiting step in the synthesis of DA, NA, and serotonin. There is noparticularly rich source of folate, with the exception of liver. Fresh vegetables areusually considered to be a good source of folate, but a high intake is not achievedin most diets. The bioavailability of food folate is less than 50%, and large lossescan occur during food preparation, such as heating and by leaching out. Cobalaminis involved in many aspects of physiological functioning, including red blood cellproduction, DNA, and myelin synthesis. Lower B12 levels have been noted to beassociated with increased overall psychological distress, with specific mood states,


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including depression, anxiety, and irritability (Baldewicz et al., 2000). Sources ofvitamin B12 are meats, fish, eggs and milk products. Fruits, vegetables, and grainproducts are devoid of vitamin B12, because only microorganisms can produce it.

17.6 SELECTED MINERAL COMPONENTS IMPORTANT FOR MOOD

17.6.1 INTRODUCTION

Depression is not a homogenous illness, and one of its primary symptoms is moodlowering. An antidepressant effect can be observed after patients are given micro-elements such as zinc, lithium, and rubidium. Lithium is used in the prevention ofdepression; it inhibits one of the serine-threonine kinases (GSK-3β) and evokeschanges in the expression of genes.

17.6.2 THE ROLE OF ZINC

Zinc is the critical nutrient for the development of the central nervous system. Someclinical investigations point to changes in the blood zinc level as a potential markerof depression (Nowak et al., 1999). The role of zinc in human nutrition has beenknown for a long time. Zinc is essential for normal fetal growth and development,and for milk production during lactation, and it is extremely necessary during thefirst year of life when the body is growing rapidly. This microelement is known toact on more than 200 enzymes by participating in their structure or in their catalyticand regulatory functions. It is a structural ion of biological membranes, closely relatedto protein synthesis. Zinc plays an important role in gene expression and endocrinefunction, and participates in DNA and RNA synthesis, and cell division. Zinc isintimately linked to bone metabolism, thus zinc has a positive influence on growthand development. The zinc concentration in bones is very high compared with othertissues, and it is considered an essential component of the calcified matrix. Zinc alsoenhances vitamin D effects on bone metabolism through the stimulation of DNAsynthesis in bone cells. This indicates that zinc in bones is very important duringstages of rapid growth and during development. The role of zinc as a therapeuticagent has also emerged. Studies have demonstrated that zinc supplementation provedto be beneficial in fighting infections within human populations. Zinc lozenges as atherapeutic agent reduce the duration of cold symptoms by 50%. Zinc reduces theincidence and duration of acute and chronic diarrhea and acute lower respiratory tractinfections in infants and children. It is an effective agent for the treatment and long-term management of Wilson’s disease. Zinc is particularly necessary in the develop-ment of the immune response. Studies on humans showed that zinc deficiency affectedthymic functions adversely, caused a shift from Th1 to Th2 function, and activatedmonocytes and macrophages. The role of zinc in this mechanism is especially impor-tant in food allergy, which is mainly a problem in infancy and early childhood.

The largest amounts of well-absorbable zinc is found in food of animal origin,particularly in oysters and meat. Vegetables and seeds have a lower bioavailabilityof Zn (see Chapter 4; Tables 4.1 and 4.3).



17.6.3 THE INVOLVEMENT OF COPPER

Zinc is connected with copper in its influence on the conversion of neurotransmittersin the brain. The serum copper concentration in depression is significantly higherthan in the control. Copper is involved in the function of several cellular enzymes;it is required for infant growth, host defense mechanisms, bone strength, red andwhite cell maturation, iron transport, cholesterol and glucose metabolism, myocardialcontractility, and brain development. The highest content of copper is in grainproducts, but the main sources of copper in diets are usually vegetables (see Chapter4, Tables 4.1 and 4.3).

17.6.4 THE IMPORTANCE OF IRON

Another microelement important for our health and mood is iron. It has several vitalfunctions in the body: as a carrier of oxygen from the lungs to the tissues, as atransport medium for electrons within the cells, and as an integral part of importantenzyme reactions in various tissues (Stipanuk, 2000). A very large number of iron-containing enzymes have been described, and they play key roles not only in oxygenand electron transport, but also as signal-controlling substances in some neurotrans-mitter systems in the brain, for example, the dopaminergic and serotonin systems.The mechanism of absorption depends on the kind of iron present in the diet—hemeiron and nonheme iron. On the mucosal cells there are two separate types of receptorsfor iron. The factors enhancing iron absorption are ascorbic acid, meat, fish, seafood,and certain organic acids. Heme iron in meat and meat products in the diet constitutesabout 1 to 2 mg, or 5 to 10%, of the daily iron intake in most industrialized countries(see Chapter 4, Tables 4.1 and 4.3).

Factors inhibiting iron absorption are phytates, iron-binding phenolic com-pounds, calcium, and soy protein.

17.6.5 THE ROLE OF SELENIUM

Selenium status also modifies mental functions. A low selenium status leads todepressed mood, while high dietary or supplementary selenium improves the mood(Benton and Cook, 1990). Low selenium status is associated with a significantlyincreased incidence of depression, anxiety, confusion, and hostility, and with senilityand cognitive decline in elderly people. Subjects with head or neck cancers, urinarytract cancers, or with rheumatoid arthritis also have a depleted concentration ofselenium in the blood (Borawska et al., 2004). The brain selenium level in Alzheimerpatients is only 60% of that in control groups. An accumulated toxic level of seleniumevokes severe selenosis, with such symptoms as malodorous breath, dermatitis, lossof hair, and neurological abnormalities.

In the human body, selenium is bound to a range of selenoproteins. However,only two of them are present in the plasma. Most of this element in the plasma,about 60 to 80%, is associated with selenoprotein P. This protein concentration isassociated with the selenium nutritional status in humans. Extracellular glutathioneperoxidase (GSHpx) is the other protein. It contains almost 30% of plasma selenium.This can be easily explained by selenium accumulation in plants in the form of


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organic compounds, originating in the inorganic selenite and selenate present infertilizers. The element is mostly built into the amino acid methionine, instead ofthe sulfur atom, resulting in selenomethionine formation, which can be nonspecifi-cally incorporated into human or animal proteins. Selenocysteine follows anothermetabolic pathway being utilized in the synthesis of selenoproteins. Inorganic formsof selenium, such as selenate and selenite, directly enter the pool from which allselenium is used for the synthesis of selenoproteins, and the excess is excreted. Thus,GSHpx is predominantly regulated by the levels of selenocysteine or inorganicselenium types. Most forms of selenium salts and organic selenocompounds areeasily absorbed from the gastrointestinal tract. More than 90% of selenomethionineis absorbed in the small intestine through the Na+-dependent neutral amino acidtransport system. Little is known about selenocysteine intestinal absorption. Bothselenocysteine and selenomethionine in particular have better bioavailability inhumans than inorganic selenium types, which are commonly used as seleniumsupplements. Selenium bioavailability from grains or vegetables, in which seleno-methionine is a predominant form, is as high as 85 to 100%, whereas from animalfood, containing mostly selenocysteine, it is about 50%.

The total body content of selenium (3 to 30 mg) varies according to the geochem-ical environment and dietary intake. Selenium concentration in food depends on itscontent in the soil from which the foods are derived. Selenium is present in soil atboth toxic (in the Dakotas) and deficient (such as in Ohio, New York, and Poland)levels. Natural food with a high protein content can be regarded as a rich source ofthis element. Several studies report poultry, liver, eggs, and fish to be abundant inselenium, in contrast to fruits and vegetables (except onions and garlic; from20 Se µg per kg in England to 44,800 in China), (see also Chapter 4, Tables 4.1 and4.3). Nevertheless, selenium content in foods can be considerably reduced by foodprocessing and depends on temperature and time (except fish and meat). The dryingof grain for 12 hours at 100°C brings about a 7 to 23% decrease in selenium. Boilingcauses greater (29 to 44%) selenium losses in mushrooms and asparagus, but doesnot influence the selenium level in cereals and rice.

17.7 ALCOHOL AND MOOD

Drinking alcohol, in most cases, is associated with inducing positive feelings likepleasure or a reduction of negative feelings like uncertainty and tension. However,sometimes it can lead to an increase in negative emotions or can have no directeffects on mood at all (Lloyd and Rogers, 1997). Alcohol affects many mental andperceptual processes and motor skills. Consumption of alcohol influences womento a greater degree than men, in part because the same amount of ethanol produceshigher blood alcohol concentrations in women. Alcohol appears to reduce the powerof a stressful emotional stimulus to alter mood. However, alcohol intake does notaffect each mood to the same degree. Alcohol may affect mood differently (fromelation to depression), and it depends on dosage and genetic predisposition. In smallamounts, alcohol appears to improve people's moods and may release inhibitionsthat will help them feel more sociable, making it a way to add to the fun in socialdrinking situations.



The beneficial effects of moderate alcohol consumption (especially red wine)include a possible reduced risk of coronary heart disease as well as stress amelio-ration. The polyphenols contained in red wine, more than white, inhibit oxidativestress and have a vasodilatory effect on the subject. On the other hand, alcoholoften evokes dependence, more quickly in women than in men. The lack of cross-aggregation between alcoholism and nonbipolar depression, the usually earlier onsetof depression as compared with alcoholism, and the clinical observation that depres-sion often remits after 2 to 4 weeks of abstinence from alcohol, are consistent withthe hypothesis that chronic alcohol intoxication can induce depression (Brown etal., 1995).

Chronic heavy alcohol consumption brings on deficiencies in vitamin B1, vitaminB6, β-carotene, vitamin E, and to a lesser extent vitamins A, B2, and C. It increasesthe risk of other nutritional deficiencies (protein/calorie) and adversely affects bothmacro- and micronutrients (Mg, Zn, Se) and certain cancers. The effects of alcoholintake on mood, behavior, and cognition may be partly mediated by biologicalchanges related to deficiency in these components of food. Most of the damagearises from the toxic effect of excess alcohol in many metabolic processes. Theimmediate euphoric and depressive effect of alcoholic beverages on mental functionis the following: after 1 pint of beer, 2 glasses of wine, or a double whiskey, inrelation to blood alcohol content (BAC) about 0.3 mg/cm3 (0.03 g/100 cm3 in theUnited States) the likelihood of having an accident increases (Garrow et al., 2000).After 2.5 pints of beer, 5 whiskies, or 5 glasses of wine, there is an increase incheerfulness, impaired judgment, a loosening of inhibitions, and a risk of losingone’s driver’s license if caught driving while intoxicated (BAC 0.08 g/100 cm3 inthe United States, Canada, several European countries, and New Zealand). At BACat 0.1g/100 cm3 and below 0.06 g/100 cm3, brake reaction time in a driving simulatoris slowed (Liquori et al., 1999). After drinking 6 pints of beer, half a bottle of spirits,or two bottles of wine, ethanol (BAC about 0.2 g/100 cm3) evokes stagger, doublevision, and loss of memory. After drinking one bottle of spirits, when BAC increasesto 0.5 g/100 cm3 and above, death is possible. Alcohol is directly toxic to the liver(alcoholic hepatitis and cirrhosis) and can lead to acute and chronic pancreatitis,cardiomyopathy, hypertension, arrhythmias, and cerebrovascular hemorrhage.

Intake of alcohol affects several different physiological functions, with negativeeffects on the cardiovascular system, cellular immunity, and hemostasis, and shouldbe consumed in only small doses (one to two glasses of wine per day).

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Blass, E.M. and Hoffmeyer, L.B. 1991. Sucrose as an analgesic for newborn infants, Pediatrics87:215–218.


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18 Food Componentsin the Protection of the Cardiovascular System

Piotr Siondalski and Wiesława Łysiak-Szydłowska

CONTENTS

18.1 Etiology of Atherosclerosis ....................................................................... 43918.2 Specific Impact of Components of the Diet ............................................. 442

18.2.1 Decrease of Energy Supply ........................................................ 44218.2.2 Dietary Control of Lipids............................................................ 44218.2.3 Protective Effect of Dietary Fiber and Antioxidants.................. 44518.2.4 Mineral Components ................................................................... 44618.2.5 Dietetic Modification of Homocystine Level ............................. 446

18.3 Cardioprotective Nutraceutics ................................................................... 44718.4 General Dietetic Recommendations for the Protection of the

Cardiovascular System .............................................................................. 44818.5 Diet, Lifestyle, and Cardiovascular Diseases............................................ 44818.6 Summary.................................................................................................... 448References.............................................................................................................. 449

18.1 ETIOLOGY OF ATHEROSCLEROSIS

Epidemiological data show that in industrialized countries with a high nationalincome the most important cause of death is cardiovascular disease (CVD), account-ing for 50% of all deaths. The mortality caused by CVD shows important differencesamong various countries. It is lowest in Japan, France, Spain, and Italy—from 50to 150 cases per 100,000 inhabitants between the ages of 40 and 69. The highestmortality from CVD is found in the countries of the former Soviet Union, the CzechRepublic, Hungary, and Ireland—from 400 to 500 deaths per 100,000 inhabitantsin the same age group (Benzie, 1999).

The following forms of atherosclerosis can be distinguished:

• ischemic heart disease (IHD)• cerebral vessel disease• peripheral blood vessel disease



The essence of these diseases is organ lesions caused by too little inflow ofnourishing, arterial blood. The principle of this phenomenon is decreased lumen ofarterial vessels, a result of wall thickening called atherosclerotic plaque. The appear-ance of atherosclerotic plaque is a long process, sometimes taking 30 to 40 years.The earliest evidence of this was found in young U.S. Army soldiers injured duringthe Korean War.

Injury of the vessel endothelium plays an important role in the formation of primaryatherosclerotic deformation. It is caused by inflammation and infiltration of the vesselwall by macrophages, proliferation of myocytes into the endothelium of blood vessels,and transport of oxidized lipoproteins to the cells, which build the blood vessels.Symptoms of atherosclerosis, as the effect of organ ischemia, appear when the diameterof a vessel supplying blood is decreased by more than 75% of the initial lumen. Thedeveloping plaque protrudes to the interior of the blood vessel and causes injury ofthe surrounding tissue followed by adhesion and aggregation of thrombocytes, forma-tion of thrombus, and usually sudden stenosis or occlusion of the vessel lumen.

The main component of atherosclerotic plaque is cholesterol. Therefore, itssynthesis, transport, and metabolism, as related to the general metabolism of lipids,are strictly connected with the formation of atherosclerosis and its symptoms. Theprocess of plaque formation is very complicated. There are also many nonlipid factorsthat have an impact on the integrity of the vessel wall (see Table 18.1). Among them,homocysteine, the balance of the oxidation state, anti- and prothrombotic factors,vasodilators, vasoconstrictors, and anti- and proinflammatory factors can be men-tioned. Actually, there are about 400 known factors in CVD. One of the mostpowerful risk factors characterized with the highest frequency is the metabolicsyndrome. Abdominal obesity, determined by waist-to-hip ratio (WHR), bloodhypertension, glycemia, and lipid impairment, are well-established components ofthe metabolic syndrome (see Table 18.2).

The frequency of metabolic syndrome in the American population is about 6.5%in 20-year-olds and 43.5% in persons over 60 years of age. In Poland, it is 39.5%in people over 60 years of age (Expert Panel, 2001).

CVD is one of the most common causes of hospitalization. Moreover, sudden deathsdue to CVD-related reasons very often occur in patients without any previous symptoms

TABLE 18.1Lipid and Nonlipid Risk Factors of Atherosclerosis

Lipid Nonlipid

Triacylglycerols HomocysteineRemnants lipoproteins Thrombotic factorsSmall, dense LDL Inflammation factorsLipoprotein (a) Glucose intoleranceMetabolic syndrome Metabolic syndrome

Source: From Liuton, M.R. and Fazio, S. 2003. Am. J. Cardiol. 92,19i–26i. With permission.


Food Components in the Protection of the Cardiovascular System 441

of illness. Two epidemiological unfavorable tendencies are also observed: higher inci-dence of disease in women and the appearance of CVD in younger age groups.

In general, CVD risk factors are divided into two main groups related to lifestyleand genetic predisposition as modifiable and nonmodifiable (Table 18.3). Amongthe factors, there are those strictly related to diet and lifestyle. It is suggested thatprevention concerning primary and secondary prophylaxis should be connected withmodification of lifestyle and generally should concern nutritional habits. Obesity,diabetes type II (insulin independent), and high blood pressure are in a large degreea consequence of dietary habits. This is confirmed in the results of epidemiologicalas well as clinical and experimental studies. It has also been proven that specificdiet factors can have a protective influence.

TABLE 18.2Diagnostic Criteria of Metabolic Syndrome

Parameter WHOa ATP IIIb

Fasting glucose [mmol/dm3] 5.6 6.1Triacylglycerols [mmol/dm3] 1.7 1.69HDL-C [mmol/dm3]

Men 0.9 <1.04Women 1.30 <1.29

BMI [kg/m2] >30 >30Waist circumference [cm]

Men >80 >102Women >94 >88

Blood pressure [mm Hg] >140/90 >130/85

a Data from International Diabetes Federation, 2005, Diabetes in Control. b Data from Ford, E.S., Giles, W.H., and Dietz, W.H. 2002. JAMA (Journal of the American MedicalAssociation) 28, 356–359.ATP III stands for The Third Report of the National Cholesterol Education Program Expert Panel onDetection, Evaluation, and Treatment of High Blood Cholesterol in Adults.

TABLE 18.3Main Risk Factors of Cardiovascular Diseases, Modified and Nonmodified

Modifiable Nonmodifiable

Atherogenic dietObesityLack of physical activityHypertensionDiabetes mellitusHigh LDL cholesterol

AgeMale sexGenetic traits



Balance studies of dietary intake, examined in different countries, allow estab-lishment of a positive correlation between mortality caused by cardiovascular pathol-ogies and consumption of saturated fatty acids, cholesterol, sugar, and animal proteins,and a negative correlation with the consumption of polysaccharides and vegetables.Further research showed a significantly lower mortality linked with higher consump-tion of fiber, full grain products, vitamin E, and carotene (Benzie, 2001).

An epidemiological study conducted in the 1980s, called the Seven CountryStudy, indicated the influence of diet on the prevention of cardiovascular incidentsas well as on their onset (Keys et al., 1986).

18.2 SPECIFIC IMPACT OF COMPONENTS OF THE DIET

18.2.1 DECREASE OF ENERGY SUPPLY

A one-year study of a group of 18 patients eating a lower-energy diet (26% protein,28% lipids, 46% carbohydrates, energy 4.7 to 8.2 MJ/d) compared to patients withan “All-American diet” (8.2 to 14.8 MJ/d, 18% protein, 32% lipids, 50% carbohy-drates) revealed a significant decrease of body/mass index (BMI), total cholesterol,LDL cholesterol, triacylglycerols (TG), and blood pressure. Moreover, a significantincrease in HDL cholesterol was observed (Fontana et al., 2004).

The epidemiological data also proved that excessive consumption of carbohy-drates—over 57% of the energy requirement for men and more than 59% forwomen—is correlated with lower levels of HDL, higher levels of BMI, and highervalues of TG. In the group with the highest carbohydrate intake, there was also thehighest consumption of monosaccharides (up to 50%) (Yang et al., 2002).

In the case of an isocaloric diet, replacement of 4% of the energy from saturatedfatty acids with carbohydrates reduced the risk of CVD by 5%, and substitutionof the energy from unsaturated fatty acids for carbohydrates increased that risk.Not only was the total supply important, but also the structure of the dietary intake.The ideal diet consists of 27% lipids, 59% carbohydrates, 55 g fiber/(55g/10.5 MJ)(Kraus, 2000).

18.2.2 DIETARY CONTROL OF LIPIDS

Epidemiological studies and interventional trials in past years proved that n-3 fattyacids, α-linolenic acid of plant origin, as well as eicosopentaenoic acid (EPA) anddocosahexaenoic acid (DHA) from sea fish, have protective properties for thecardiovascular system. Their consumption reduces the frequency of sudden heartdeath, hypertension, and general mortality caused by heart vessel factors (Kris-Etherton et al., 2002). About a 30% decrease in mortality caused by heart vesselfactors was observed when seafood was included in the diet at least twice a week(Hu et al., 2002).

Consumption of at least 1 g of α-linolenic acid daily was correlated with a 40%decrease in the risk of atherosclerosis symptoms, and this quantity of the acidconsumed revealed a reverse correlation with the frequency of CVD (Djousse etal., 2001).



It is presumed that polyunsaturated fatty acids (PUFA) have an effect on geneticexpression linked with the control of metabolism of fatty acids as follows:

• they decrease the expression of genes responsible for cholesterol and fattyacid synthesis, for example, steroylo-CoA desaturase

• they increase the expression of genes responsible for oxidation of fattyacids: palmitoyl-carnitine transferase and peroxysome proliferator acti-vated receptor L (PPAR L)

• they decrease lipid stores in the body (Olson, 2002; Clark, 2001)

Furthermore, PUFA are substrates for production of extremely importanteicosanoids—cytokines influencing the immune system. Fatty acids of the n-6 familycontribute to the synthesis of molecules having a proinflammatory character. Theyinduce an increase in body temperature, intensify the perception of pain, and createedemas due to an increase in the permeability of vessels. Fatty acids of the n-3family cause a general inhibition of metabolism of n-6 fatty acids and enhance theimmunological function. Moreover, the eicosanoids, as final products, do not havean inflammatory effect (see Figure 18.1). Consequently, after a dose of n-3 PUFA,the following effects can be observed (Connor, 2000):

• decrease in heart sensitivity to ventricular arrhythmias• decreased tendency for thrombosis• lower TG level before and after meals • slow development of atherosclerotic plaque• decreased expression of adhesion molecules

FIGURE 18.1 The balance of n-3 and n-6 fatty acids metabolism: COX = cyclooxygenase,LOX = lipoxygenase, PC = prostacyclins, PG = prostaglandins, TH = thromboxanes, LT =leukotrienes, EPA = eicosapentaenoic acid.

Arachidonic acid EPA

COX LOX COX

PC, LT-4 PG-3 LT-5PG-2,

TH

Proinflammatory and immune deregulation

Less inflammationsand immune regulation

LOX



• decrease in the level of growth factors originating from platelets• decreased NO-dependent inflammation markers • lower arterial hypertension

Monoenic fatty acids (MUFA) do not induce changes in the level of HDL andLDL; nevertheless they have a hypocholesterolemic effect (Hegsted et al., 1965).

PUFA as well as MUFA built into cellular membranes induce greater plasticityand better permeability, and also change the local environment of receptor proteins.This has a fundamental role during states of hypoxia. In an experimental study,restoration of 100% of heart function was noted in a group of animals on a diet richin PUFA, as compared to the period before hypoxia. In the case of a diet includingplant oils, only 75% restoration was observed (Demeison et al., 1993); see Table 18.4.

The side effects of PUFA consumption can be seen in doses greater than 3 g/day.Most often this involves a fishlike taste, and can cause an increase in LDL andintestinal disorders.

The results of interventional investigations suggest that the positive effect ofdietetic modification can be linked with lipoprotein E genotype (allele apo E4).Presently there is a lack of final conclusions concerning this problem (Rubin andBerglund, 2002).

A diet with a higher content of MUFA and PUFA induces an increase in HDLlevel, a decrease of LDL in serum, of adhesion molecule expression, and in sensitivityof LDL for oxidation, as well as more intensive production of NO. Lipid-solublevitamins, vitamin E, β-carotene, and vitamin C, act in a similar way. These substancesdecrease LDL oxidation when administered simultaneously in quantities of, respec-tively, 80 mg, 60 mg, and 1 g daily for 3 months. Application of vitamin E aloneproduced the same effect (Moreno and Mitjavila, 2003). It is assumed that theMediterranean diet has a protective effect on the cardiovascular system due to itshigh PUFA content (Bautista and Engler, 2005).

TABLE 18.4Recommendations Concerning the Intake of N-3 Fatty Acids

Population Recommendation

Patients without symptoms of CVD Eat various types of fish at least 2 times per week and plant oils rich in α-linolenic acid (flax seed oil)—should supply 0.3 to 0.5 g EPA + DHA

Patients with symptoms of CVD Eat 1 g EPA + DHA daily, optimally as fatty fishes. Pharmacological supplementation of n-3 fatty acids after physician’s consultation.

Patients needing TG lowering 2 to 4 g EPA + DHA/day in the form of supplements.

Source: From Kris-Etherton, P.M., Harns, W.S., and Appel, L.J. 2002. Circulation 106,2747–2757. With permission.



18.2.3 PROTECTIVE EFFECT OF DIETARY FIBER AND ANTIOXIDANTS

Dietary fiber plays a particular role in the organism, especially with respect todigestive tract functioning, but its role is not limited to these organs. It was observedthat dietary fiber supply and especially its soluble form, pectin, decreases the riskof coronary disease. In a group of patients with daily intake of more than 15 g ofdietary fiber/7.3 MJ the risk of coronary disease is reduced more than 11% (Bazzanoet al., 2003). The best sources of dietary fiber are grains, porridge, bran, oats, andguar gum.

The mechanism of action of dietary fiber is due to decreased absorption ofcholesterol and TG from the digestive tract, decreased glycemic index of food,increased insulin sensitivity, increased fibrinolytic activity, and increased choles-terol metabolism to bile acids. Recent investigations have shown a lower frequencyof heart attacks among people consuming large amounts of whole-meal bread(Jacobs et al., 2001).

Free radical generation in the organism leads to production of oxidized LDL,proteins, and nucleic acids, which can promote atherosclerosis development. Fruitsand vegetables are among the best sources of free radical scavengers.

Epidemiological investigations carried out in the United States over a 19-yearperiod involving a population of nearly 10,000 patients between the ages of 25 and75 have proven a reverse dependence between vegetable and fruit consumption andmortality caused by cardiovascular factors. Consumption of vegetables and fruits atleast three times a day (500 g) was linked with lower blood pressure, lower frequencyof strokes, lower mortality caused by CVD, and general mortality. This positive effectwas observed despite a higher consumption of energy, higher levels of total cholesterol,and a higher frequency of diabetes in the group with the higher consumption ofvegetables and fruits in comparison with the group with significantly lower consump-tion of vegetables and fruits. In the opinion of the above-cited authors, nutrients in thewhole diet, including vegetables and fruits, may have an additive and synergistic effectthat is difficult to achieve using supplements exclusively (Bazano et al., 2002).

In recent years there has been a lot of attention paid to natural polyphenols fromplants because of their cardioprotective effect (Table 18.5). These substances arewell absorbed from the digestive tract and consequently are in the micromolarconcentration in blood (see Table 18.5).

Wine consumption has provoked a lot of emotion since the discovery of the so-called French paradox. Despite high consumption of lipids in France, there is lowmortality caused by CVD. The cardioprotective effect of wine is linked with thepresence of resveratrol and other polyphenols. It has been proven experimentallythat the protective effect is due to higher production of NO as a result of increasedendothelial enzyme NO-synthase activity and increased LDL receptor expressionand decreased secretion of ApoB. This effect seems to be similar to the effect ofatorvastatine—a drug decreasing the plasma cholesterol level (Pal et al., 2003).

Polyphenols as wine components are also responsible for lower production of freeradicals. The alcoholic component decreases platelet aggregation and the level of fibrin-ogen in plasma (Wolin and Jones, 2001). Perhaps that is why Plato said, “No thingmore excellent, no more valuable than wine was ever granted to mankind by God.”



Polyphenols of soybean also have similar cardioprotective effects, as do soyproteins. Consumption of soy proteins in a quantity greater than 6 g/day caused adecrease in LDL level in a group of women in the pre- and postmenopausal period.The U.S. Food and Drug Administration proposes 25 g of soy protein per day as adiet supplement to improve patients’ lipid profiles (Rossel et al., 2004).

18.2.4 MINERAL COMPONENTS

Potassium, magnesium, and calcium cannot be omitted in the discussion of protectivecomponents in the diet. These elements have a direct impact on decreasing arterialblood pressure and the function of the endothelium among others, by modulatingthe volume of the vascular bed (Suter, 1999).

18.2.5 DIETETIC MODIFICATION OF HOMOCYSTINE LEVEL

Homocystine, an endogenous amino acid, has an important role among nonlipid riskfactors of CVD. The end product of its metabolism is methionine and cysteine. Theefficacy of this metabolic pathway depends on vitamins B6, B12, and folic acid.

Most often B-vitamin deficiency is responsible for an increased plasma level ofhomocystine, which causes protein cross-linking, including the proteins of the cellmembranes. Structurally altered proteins cause, among other effects, vessel endo-thelium dysfunction: increased thrombocyte aggregation, prothrombotic tendencies,

TABLE 18.5Interventional Studies Using Polyphenols

Source Substance Dose Effect

Gingko biloba quercitin 120 mg extract decrease of blood pressure, increase in fasting plasma insulin and C-peptide

Onion + tea quercitin 110 mg decrease of oxidative degradation of DNA

Soya protein food Genistein daidzein 86 mg decrease of oxidized LDL Supplement isoflavons 132 mg lower fasting insulin, decrease of insulin

resistance, lower LDLGreen tea catechins 8 cups increase of fatty acids oxidationGreen tea catechins 375 mg lower body mass and waist

circumference Cacao procyanidins 100 g chocolate increase of plasma antioxidative

potentialprocyanidins 200 mg lower P-selectin,

lower aggregation of plateletsRed wine procyanidins

anthocyaninsquercitin

375 mg lower LDL oxidation

Source: From Williamson, G. and Monach, C. 2005. Am. J. Clin. Nutr. 81, 243–255. With permission.



and intensified oxidation stress. They also stimulate proliferation of the smoothmuscles of the blood vessels. It is assumed that atherosclerosis observed amongvegetarians is significantly caused by a B12-vitamin deficit and as a consequence, anincreased level of homocystine (Patel and Lovelady, 1998). Inherited hyperho-mocystinemia with early symptoms of atherosclerosis, appearing as an enzymemetabolic defect, is very rarely seen.

Increased intake of products rich in folic acid leads to a decrease in the plasmahomocystine level. Examples of these products are berries, citrus fruits, and dark-green leafy vegetables (Silaste et al., 2003). In some countries there is a nationalpolicy of folic acid supplementation of the most often consumed products, such ascorn flakes or flour, for example, in the U.S. and the U.K. (Flood et al., 2001). Astandardized portion of enhanced alimentation covers 17% to 50% of the dailyrequirement for this microcomponent (Arens, 2001).

Deficiency of vitamin B12 is a very common pathology among patients over 60years of age because of atrophic changes of the mucous membrane of the stomachand impaired absorption of this vitamin (Carmel, 1997). A good example of a productrich in B12 is bovine liver, which contains about 100 µg of the vitamin/100 g.Significant quantities of vitamin B6 can be found in whole-grain products—about0.30 mg/100 g. Folic acid, as well as vitamin B6 are extremely thermolabile com-pounds. Preparation of meals leads to losses of these vitamins —approximately 40to 50% during cooking, frying, or baking (Southgate, 1993).

18.3 CARDIOPROTECTIVE NUTRACEUTICS

Many cardioprotective nutraceutics have been introduced on the market. However,clinical evidence of their activity concern only particular groups of patients and donot have a wider significance. Fitosterol supplements are used in the production ofmargarine and energizing bars (see Table 18.6).

TABLE 18.6Selected Cardioprotective Nutraceutics

Compound Function

Arginine Substrate for NO productionTaurine Osmolite, Ca canal activatorCo Q10 Coenzyme in mitochondrial chain reactionCarnitine Mitochondrial transport of fatty acidsN-acetylocysteine Free radicals’ scavengerCreatine Production of phosphocreatine—storing energyGlutathione AntioxidantSelenium Antioxidantβ-Sitosterol Hipolipemic

Source: From Saffi, A.M., Samela, C.A., and Stein, R.A. 2003. Car-diovasc. Rev. Rep. 24, 381–385. With permission.



18.4 GENERAL DIETETIC RECOMMENDATIONSFOR THE PROTECTION OF THE CARDIOVASCULAR SYSTEM

General recommendations for cardiovascular health include the following:

• Use a diet that meets your energy demand; it will decrease the risk ofmetabolic syndrome

• Use a diet regulating the metabolism of lipids in the body; n-3 fatty acidsdecrease the risk of symptoms of atherosclerosis

• Use a diet rich in fiber and antioxidants in order to increase the effective-ness of heart protection

• Use a diet with high levels of B vitamins and folic acid to prevent highlevels of homocystine

• Eat minimally processed products • Physical activity, adequate for age and ability, should be a permanent

element of lifestyle

18.5 DIET, LIFESTYLE, AND CARDIOVASCULAR DISEASES

Arteriosclerosis causes complex diseases, the most frequent form of which is IHD.There are two fundamental causes of diseases that can be revealed during anamnesis.One is genetic. Very often, similar diseases affect close relatives of the patient. Theother is a long period of inadequate nourishment consisting of excessive energyintake, too many fats and carbohydrates, and too much salt. These factors predisposethe patient to the so-called metabolic syndrome, and in consequence lead to ischemicmanifestations that result from decreased lumen of arterial vessels, caused by wallthickening called atherosclerotic plaque. Eliminating this cause would probably limitthe number of patients who require intense pharmacological and interventionisttreatment because of arteriosclerosis. Patients treated for CVD should be informedthat their faulty dietary habits and lifestyle must be changed. It seems that suchaction would improve the effectiveness and persistence of therapies applied.

18.6 SUMMARY

A typical Mediterranean diet reveals heart-protective properties. This has been con-firmed in epidemiologic studies (Lyon Diet Heart Study) as well as in dieteticintervention trials. The protective activity is linked with a high supply of n-3 fattyacids coming from fish and seafood, and high consumption of whole-grain products,as well as fruits and vegetables. Introduction of the Mediterranean diet and itsalternatives (Asian and African diets) results in HDL increase, a decrease of bodymass, insulin resistance, as well as glucose resistance, and decreases in total cho-lesterol and TG. The blood vessel endothelium is also improved and inflammationfactors are decreased.



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19

Mutagenic, Carcinogenic, and Chemopreventive Compounds in Foods

Agnieszka Bartoszek

CONTENTS

19.1 Introduction................................................................................................ 45219.2 The Role of Mutagens in Carcinogenesis................................................. 45319.3 Metabolic Activation and Formation of DNA Adducts

by Food Mutagens and Carcinogens......................................................... 45619.4 Tests for Mutagenicity and Carcinogenic Properties of Food

Components ............................................................................................... 45919.5 Foodborne Mutagens and Carcinogens..................................................... 461

19.5.1 Introduction ................................................................................. 46119.5.2 Mycotoxins .................................................................................. 46219.5.3 Nitrosamines................................................................................ 46219.5.4 Mutagens in Heat-Processed Foods............................................ 465

19.5.4.1 Heterocyclic Aromatic Amines ................................... 46519.5.4.2 Polycyclic Aromatic Hydrocarbons............................. 46719.5.4.3 Acrylamide and Furan................................................. 46819.5.4.4 Effect of Commercial Processing and Cooking

Techniques ................................................................... 46819.5.5 Mutagens in Tea, Coffee, and Alcoholic Beverages .................. 47019.5.6 Other Risk Factors ...................................................................... 471

19.6 Chemopreventive Food Components ........................................................ 47319.6.1 Anticarcinogenic Food Components........................................... 47419.6.2 Cancer Chemoprevention ............................................................ 477

19.7 Final Comment .......................................................................................... 478References.............................................................................................................. 480


452


19.1 INTRODUCTION

The factors and substances able to induce changes in the genetic code are calledmutagens. Those that can cause cancer, excluding genetic susceptibility, are calledcarcinogens. Such factors are omnipresent in the human environment; they can beof natural origin or be formed as a result of numerous chemical processes. To thesefactors belong a variety of synthetic chemicals, combustion products, water and airpollutants, sunlight and ionizing radiation, cigarette smoke, alcohol, and some foodcomponents. Factors such as specific occupational exposures or cigarette smokingare clearly high-risk conditions for cancer. Diet, as a prevailing environmentalvariable related to cancer risk, was first proposed by Doll and Peto based on epide-miological observations (Doll and Peto, 1981). Their conclusion was a turning pointin identifying not only causes, but paved the way to preventability of cancer (Colditzet al., 2006).

A fundamental observation in cancer epidemiology during the last century wasthat cancer incidence and mortality rates vary dramatically across the globe (Parkin,1998). In addition, rates of cancer among populations migrating from low- to high-incidence countries change markedly; in most cases they approximate the rates inthe new region within one to three generations. For instance, the replacement offoods of plant origin with foods of animal origin, notably meat products and dairyproducts, increases cancer incidence, especially the risk of breast, colon, prostate,and rectum cancers (Bingham, 1999). These cancers are virtually absent in thepopulations of some countries of the developing world and generally the overallcancer burden there is strikingly lower. Similar conclusions were inevitable in thecase of epidemiological studies on prostate cancer incidence and breast cancerincidence in migrants to the United States from Poland (Staszewski and Haenszel,1965) and Japan (Wynder et al., 1991), respectively. These cancers are relativelyinfrequent in the countries of origin, but in the investigated populations of immigrantsthey reached the level observed in the United States, even within one generation.Such lines of evidence indicate that the primary determinants of cancer rates are notgenetic factors, but rather environmental and lifestyle factors that could, in principle,be modified to reduce cancer risk.

It has been estimated that approximately 35% of cancer deaths in the UnitedStates and Europe are attributable to dietary habits (Peto, 2001), either throughingestion of compounds that initiate or promote cancer, or through the lack ofprotective substances (e.g., Manson and Benford, 1999). Not surprisingly, the pres-ence of potential mutagens and carcinogens, as well as anticarcinogenic substancesin foods have become of widespread interest.

There are several sources of food mutagens and carcinogens. Some are sub-stances naturally present in food or contaminating food products such as mycotoxins.Pollutants (e.g.. heavy metals or dioxins) and pesticides used in agriculture constitutean increasingly important group of environmental carcinogens found in food anddrinking water. Food additives, long regarded as negligible cancer risk factors, arecurrently drawing more concern as new data on their biological properties becomepublished. However, the majority of mutagens and carcinogens found in foods areformed during food processing, especially thermal processing. Because of invaluable



453

advantages such as increasing the shelf life of foods (which can then be economicallypriced), decreasing the risk of diseases caused by foodborne pathogens, improvingthe taste and nutritive value of food, and providing easy-to-prepare and time-savingconvenience foods, processing and heating of foods will always be a foundation ofthe food industry. Therefore, it is of utmost importance to establish what processesare responsible for mutagen and carcinogen formation in food, and to clarify theirinvolvement in the transformation of a normal cell into a cancerous one. This fieldof research is progressing rapidly, and it may be expected that the gathered knowl-edge should bring about new technologies that will combine the current benefits offood processing with minimizing the formation of harmful compounds. It shouldalso enable the elaboration of sound dietary recommendations aimed at diminishingcancer risk.

19.2 THE ROLE OF MUTAGENS IN CARCINOGENESIS

Transformation of a normal cell into a cancerous one manifests itself macroscop-ically as uncontrolled cellular growth, resulting in the formation of a tumor con-sisting of cells that do not differentiate into their specialized tissues, may metasta-size invading other sites of the body, and eventually lead to death of the organism.Recent developments in the area of molecular carcinogenesis demonstrated thatneoplastic transformation involves the accumulation of multiple genetic alterationsin critical cancer-related genes; therefore, cancer is often referred to as a diseaseof the genes (Sugano, 1999).

Cancer-associated genes are numerous and include oncogenes, tumor suppressorgenes, genes involved in the regulation of the cell cycle, development, DNA repair,drug metabolism, genes involved in immune response and angiogenesis, as well asother correlates of metastasis. There is evidence that certain alleles of these genescontribute to cancer susceptibility and are mutated in tumors. The alleles conferringincreased risk for cancer might require an environmental influence to have theireffect. Genetic background modifies the risk of disease for exposed individuals (riskmight be raised or lowered). In the majority of cases in which diet is involved inthe carcinogenic process, it is susceptibility genes that are thought to be most relevant(Dean, 1998; Sinha and Caporaso, 1999).

The process of carcinogenesis in humans (originally identified in animals),resulting in genetic alterations in cancer-related genes, proceeds in a multistagemanner over a long latent period (WCRF/AICR [World Cancer ResearchFund/American Institute for Cancer Research], 1997; Sugano, 1999). At the onsetof cancer development, two major stages can be distinguished: initiation and pro-motion. They are followed by the final stage of the carcinogenic process, known asprogression, which comprises the growth of the tumor and its spread to other bodyparts. Carcinogens responsible for the changes, which can lead to the conversion ofa healthy cell into a neoplastic cell, are divided into genotoxic and epigenetic (Taylor,1982). Genotoxic carcinogens, acting at the initiation stage, are those displayingtoxic, lethal, and heritable effects to karyotic and extrakaryotic genetic material ingerminal and somatic cells. Damage to genetic material may involve covalent mod-ification of nucleotides, as well as breakage, fusions, or impaired segregation of


454


chromosomes (WCRF/AICR, 1997; Luch, 2005). Most mutagenic and carcinogenicfood components are genotoxins.

For genotoxic foodborne carcinogens, DNA damage, especially covalent modi-fication, is crucially important, and without DNA adduct formation, such agents donot induce cancers (Swenberg et al., 1985). Not all DNA adducts are critical lesions;only those altering the important cancer-related genes by causing specific mutationsfollowing DNA replication are essential for neoplastic transformation. The cellbearing mutated gene(s) may be eliminated by various mechanisms protecting theorganism against development of abnormal cells, or it may persist within tissue.However, it needs to undergo cycles of cell duplication, involving epigenetic controlmechanisms, to generate a permanently altered, mutated cell that expresses preneo-plastic characteristics giving rise to a clone of initiated cells.

Such a clone of cells susceptible to cancerous growth, if exposed to one or morefactors (mostly epigenetic) called promoters, may proliferate to a definable focus ofpreneoplastic cells. This stage is known as promotion. Epigenetic factors operatingas promoters of cancer usually require high and sustained exposures. Their effects,unlike genotoxins, are reversible (Weisburger and Williams, 2000). To such factorsbelong many natural and man-made chemicals, including those present in food.Mechanisms of promotion are less well understood than those of genotoxin action,but they are thought to involve stimulation of cell proliferation, blockage of com-munication pathways between normal and mutated cells, and others. Also, partiallyreduced oxygen molecules such as hydroxyl radicals or superoxide radicals, oftenreferred to as oxygen radicals, act at the stage of promotion. They arise as a sideeffect of normal metabolism, and their formation is believed to underlie the cancer-promoting effect of high-protein and high-fat diets because these food constituentsare intensively metabolized. Oxygen radicals can bind to various cellular componentsincluding DNA; they were also shown to influence gene expression (Ames et al.,1993; Burcham, 1999).

The scenario of chemical carcinogenesis described above has been criticizedfor some time, as it is now clear that the genome in cancer cells becomes veryunstable not only due to mutations, but also because of the impairment of signalingpathways and systems of regulation of gene expression (Luch, 2005). The differencebetween genotoxic and epigenetic factors has become less obvious because thesame carcinogen may influence carcinogenic processes in several ways (Figure19.1). The heterocyclic aromatic amine denoted PhIP, formed during meat heating,may serve as a good example. This compound, once regarded as a typical genotoxininducing DNA adducts, was shown to also display estrogenic activity, and as aconsequence to promote hormone-dependent cancers, such as breast cancer (Lauberet al., 2004). Moreover, numerous genotoxins, apart from inducing genome damage,stimulate so-called oxidative stress associated with generation of reactive oxygenspecies (ROS), some of which belong to important signaling molecules whoseexcess may impair normal signaling pathways. As already mentioned, ROS are alsoDNA-damaging agents.

The most recently discovered epigenetic processes responsible for genome insta-bility, which may be induced by both nutrients and nonnutrients, influence geneexpression by modulating the methylation pattern of chromatin. These changes



455

involve both losses and gains of DNA methylation (in position 5 of cytosine in CpGdinucleotide) as well as altered patterns of histone modification. In the case of tumorcells, the hypomethylation of the genome as a whole is accompanied by hyperme-thylation of genes preventing tumor growth, mainly tumor-suppressor genes. Thishypomethylation and histone modification influence the chromatin structure andconsequently its functioning. While hypermethylation occurring in so-called CpG-islands, that is, transcription start sites of genes with high frequency of CpG dinu-cleotides, leads to epigenetic silencing of the gene. Most importantly, the methylationpattern is reproduced upon replication, meaning that it is heritable and that constantdonors of methylation groups are needed in a growing tissue. Folate and methionineare the major nutritional sources of methyl groups, and it has been suggested thatincreased cancer risk in the elderly may be associated with their reluctance toconsume meat products (Huang, 2002; Laird, 2003; Baylin and Ohm, 2006).

Until the end of the 20th century it was accepted that the differences in cancersusceptibility between individuals and populations resulted from polymorphisms ofgenes responsible for activation and detoxification of carcinogens to which thehuman organism may be exposed. It has now been recognized that many more genesmust be taken into account as being related to cancer. Consequently, nutritionresearch has shifted from epidemiology and physiology to molecular biology andgenetics, and moves toward genomics, transcriptomics, and metabolomics, or in thisparticular case, so-called nutrigenomics. Nutrigenomics is still more at the stage ofpromise than actual results, but its goal is clearly to study genomewide influences

FIGURE 19.1

Genotoxic and epigenetic effects of carcinogens. (From Luch, A.,

Nature Rev.Cancer

,

5,

113, 2005. With permission.)

CARCINOGEN EXPOSURE

METABOLISM

Genes

Cell cycleDNA repair

DifferentiationApoptosis

Non-genotoxic mechanisms:• inflammation

• immunosuppression

• reactive oxygen species

• receptor activating

• epigenetic silencing

Genotoxic mechanisms:

• DNA adducts

• chromosome breakage, fusion, deletion,

mis-segregation, non-disjunction

• aneuploidia

• hypermutability

• genomic instability

• loss of proliferation control

• resistance to apoptosis

Genomic damage Altered signal transduction

CANCER

excretion

cell


456


of nutrition, especially the risk of diet-related diseases to which cancer belongs(Muller and Kersten, 2003).

19.3 METABOLIC ACTIVATION AND FORMATIONOF DNA ADDUCTS BY FOOD MUTAGENSAND CARCINOGENS

The vast majority of carcinogens, such as those in foods, account for a large fractionof the human cancer burden, but do not possess mutagenic and carcinogenic prop-erties themselves. In order for these properties to be revealed, metabolic activationin an organism is required, which leads to the formation of electrophilic metabolitescapable of binding to nucleophilic centers in DNA. Therefore, in literature the namepromutagen or procarcinogen is often used to describe compounds that must beconverted by cellular enzymes into genotoxic mutagens and carcinogens.

Metabolic activation of carcinogens involves many enzymatic systems knownas phase I enzymes. The most important is cytochrome P450 complex, consistingof several different isoenzymes, which are particularly active in the liver. Otherenzymes include peroxidases, quinone reductases, epoxide hydrolases, sulfotrans-ferases, and others. Their variety reflects the diversity of chemical structures ofcompounds to which an organism is exposed. These may be harmful substances aswell as needed ones or even those indispensable for its proper functioning. Onecould argue that the activation of carcinogens is an undesirable side effect of met-abolic pathways, which were developed in the course of evolution most probably inorder to improve the utilization of nutrients and elimination of unwanted or harmfulsubstances.

Competing with enzymatic activation are detoxification processes involvingphase II enzymes. These enzymes catalyze the attachment of polar groups toincrease water solubility of normal metabolites as well as foreign compounds andthereby facilitate elimination. To the enzymes responsible for removal of mutagensand carcinogens belong most of all glutathione-S-transferases and glucuronyltrans-ferases; however, phase I enzymes are sometimes also involved in the initial stagesof detoxification. Activation and detoxification may run in parallel and be catalyzedby the same enzymatic system. For instance, epoxidation of benzo[a]pyrene bycytochrome P450 in position 7,8 results in the formation of a carcinogenic metab-olite, while in position 4,5 it produces an inactive derivative readily excreted fromthe organism. Some examples are given below of well-established metabolic acti-vation pathways for a few classes of mutagenic compounds found in food, alongwith the major products of reaction of their main toxic metabolites with DNA,more precisely with guanine, which is the preferred site of binding of electrophilicintermediates.

Metabolic activation of aflatoxin B

1

, belonging to the class of mycotoxins, iscatalyzed by cytochrome P450. The enzymatic conversion of this compound canfollow many pathways, however only the epoxidation in position 8,9 produces the



457

ultimate carcinogen. This metabolite binds to the N

7

position of guanine giving anunstable adduct 8,9-dihydro-8-(N

7

-guanyl)-9-hydroxy-aflatoxin B

1

, which eitherundergoes spontaneous depurination or rearrangement to a stable 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamide)-9-hydroxy-aflatoxin B

1

following the opening of the imidazole ring (Wakabayashi et al., 1991).

The formation of nitrosamines in the reaction of amines with nitrites under acidicconditions in the stomach can be considered as nonenzymatic activation of aminespresent in food. Nitrosamines undergo further metabolism, catalyzed enzymaticallyby cytochrome P450, involving hydroxylation (Anonymous, 1993).

The hydroxylated derivative is unstable, and in a series of spontaneous reactionsgives rise to methyl carbocation, which alkylates guanine in position O

6

, hence inthe site taking part in the formation of hydrogen bonds in DNA with a complementarybase—cytosine.

Metabolic activation of benzo[

a

]pyrene consists of three enzymatic reactions.

REACTION 19.1

REACTION 19.2

OO

O

O

OCH3

O

O

O

+ DNAO

O

OCH3

OO

O

N+

N

sugar

NH

NNH2

OOH

O

O

H3CO

OO

O

N

NH

sugar

NH

NNH2

O

O

H

OH

CH3

NHCH3

NO2- H+

CH3N

CH3

NO

OH2

cytochrome P450

CH3N

NO

OH

CH3N

CH3

NO

CH2OH+

CH3

N

N

OH - H2O

H2C N

N

CH3 N2

H+

H2C N

N


458


First, the formation of epoxide in position 7,8 is catalyzed by cytochrome P450;epoxidation in position 4,5 results in detoxification of this compound. Then, epoxidehydrolase converts the epoxide into 7,8-dihydrodiol, which is subsequently oxidizedto 7,8-diol-9,10-epoxide. The formation of four different diastereoisomers is feasible,among which anti-9,10-epoxide derived from (-)-7,8-dihydrodiol is by far the mostcarcinogenic (Dipple and Bigger, 1990). In DNA, this derivative reacts most fre-quently with guanine in a way that positions 10 of benzo[

a

]pyrene and N

7

of guaninebecome linked together.

Aromatic compounds substituted with amino groups, such as heterocyclic aro-matic amines present in protein food products, are usually activated by cytochromeP450 to hydroxylamines. This type of metabolism is observed in the case of 3-amino-1-methyl-5

H

-pyrido[4,3-

b

]indole (Trp-P-2).

REACTION 19.3

REACTION 19.4

bayregion

cytochrome P450

O

epoxidehydrolase

OH

OH

cytochrome P450

OH

OH

O

7,8-oxirene

7,8-dihydrodiol 7,8-dihydrodiol-9,10-epoxide

N

H

N

NH2

CH3

cytochrome P450

N

H

N

NH

CH3

OH

N

H

N

NH

O SO3

- SO42-

N

H

N

NH

O NH

unstable



459

After further spontaneous rearrangements, hydroxylamine derivatives produceelectrophilic intermediates, which are able to modify DNA bases (Sugimura andSato, 1983). The group of heterocyclic aromatic amines includes so many differentcompounds that a large variety of chemical structures of DNA adduction productsformed by them can be expected.

The first step leading to the activation of acrylamide to DNA binding species isepoxidation to glycidamide catalyzed by one of the cytochrome P450 isoenzymes—cytochrome CYP2E1—involved in the activation of numerous food carcinogens. Theepoxide moiety binds to nitrogen N3 of adenine or nitrogen N7 of guanine (Gha-nayem et al., 2005) as shown below.

It is now generally accepted that the enzymatic systems implicated in metabolismof carcinogens may be the reason for different susceptibilities of humans to cancer(van Iersel et al., 1999; Manson and Benford, 1999; Wolf, 2001). Therefore, thegenes coding enzymes responsible for biotransformation of carcinogens have beenincluded in the list of cancer-related genes. Importantly, many dietary compoundscan influence various phase I and II enzymes by induction or inhibition. For example,cytochrome P450 isoenzyme CYP1A2 (phase I) activity may be induced by poly-cyclic aromatic hydrocarbons in grilled and smoked foods, and inhibited by narin-genin in grapefruit. Similarly, phase II enzyme glutathione-S-transferases can beinduced by many nonnutrient phytochemicals, dietary lipids, and reactive oxygenspecies (Sinha and Caporaso, 1999). Current research attempts to relate geneticallycorrelated sensitivity and environmental exposures, including dietary impact, toindividual cancer risk.

19.4 TESTS FOR MUTAGENICITY AND CARCINOGENIC PROPERTIES OF FOOD COMPONENTS

Food products contain thousands of compounds—some of nutritive value, nonnutri-tive components and numerous additives, substances formed during processing, andpesticide residues. Their safety is required for human health protection includingcancer risk assessment. In order to evaluate the carcinogenicity of individual foodconstituents and their mixtures, often of unknown chemical structure, as well as the

REACTION 19.5

acrylamide

CH2

NH2

O

NH2

O

O

glycidamide

CYP 2E1 guanosine

N

N

N

N+

NH2

sugar

H2N

OH

ONH2


460


impact of cooking procedures, short-term reliable and inexpensive tests are neces-sary. Because cancer risk associated with chemical compounds is thought to stemmainly from their ability to induce mutations, mutagenicity is used in the assessmentof carcinogenic properties of food components. This ability can be detected with theaid of bacteria whose culturing is easy, quick, and economical. In the case of bacterialmutagenicity tests, it is assumed that the factors capable of damaging bacterial DNAcan interact in a similar way with DNA of higher organisms. The evaluation ofcarcinogenicity, that is, the ability of substances to induce cancers, is performedmainly in mice and rats.

The method most widely used to evaluate mutagenic activity is the Ames test(Ames et al., 1975). It utilizes mutant strains of

Salmonella

typhimurium

that areunable to synthesize histidine, and thus dependent on an outer source of this aminoacid. The back mutation in the appropriate gene makes the bacteria histidine inde-pendent. The frequency of back mutations increases in the presence of mutagenicfactors. To mimic metabolic activation of mutagens, typical for mammalian cells butoften absent in bacteria, microsomal fraction (usually isolated from rat liver) is addedconcomitantly with the substance studied. Currently, an array of

Salmonella

strainsis available that enables not only the evaluation of the overall mutagenic activity ofa given compound, but also the type of mutation it induces. Moreover, the techniquesof genetic manipulation offered by modern molecular biology allowed the construc-tion of bacterial strains expressing various animal and human gene-coding enzymesimplicated in the activation of chemical carcinogens. For instance, a strain of

Sal-monella typhimurium

expressing mammalian cytochrome CYP1A2, and NADPHcytochrome P450 reductase, thus two enzymes believed to be most important forthe metabolism of foodborne mutagens and carcinogens have been constructed(Aryal et al., 1999). Another

Salmonella typhimurium

strain, YG 1024, was engi-neered to overexpress O-acetylase, an enzyme catalyzing acetylation ofhydroxyamines formed from heterocyclic amines. This pathway is fairly specific, sostrain YG 1024 is recommended for the improved detection of the activity ofmutagenic amines arising during heat processing of meat (Yoxall et al., 2004).However, as pointed out by many researchers,

in vitro

mutagenicity tests are in somecases overly sensitive and may not reflect exposures and mechanisms of biologicalrelevance to humans. It is, therefore, generally accepted that mutagenic propertiesof a given compound detected in bacteria need to be assessed in appropriate

in vivo

assays (MacGregor et al., 2000). For instance, commercially available transgenicMutaMouse

®

and the BigBlue

TM

mouse and rat models, through use of the bacterialtransgene as the mutational target, assure metabolic conversion of a compound testedtypical for higher organisms. After exposition of a mutagenic substance in an animal,the bacterial transgene is recovered and the frequency of its mutation assayed in thenatural host, that is,

Escherichia coli

.To assess carcinogenicity, several doses of potential carcinogens are administered

to animals. The highest of them correlates to the maximum tolerated dose (MTD)that does not cause severe weight loss or other life-threatening signs of toxicity. Asa result of such studies, the lowest dose is determined at which carcinogenic effectsare still observed. The next level below that is assumed not to have a biologicaleffect—the so-called

no effect level

. This value, divided by a safety factor of either



461

100 or 1000, correcting for the difference in sensitivity between animals and humans,is considered the acceptable daily intake (Anonymous, 1988). Such studies areusually very lengthy, so shorter alternative carcinogenicity assays are being devel-oped. The new animal tumorigenesis models are designed with the aid of geneticengineering and are characterized by rapid development of tumors. In literature,there are described, for example, transgenic mouse models overexpressing oncogenec-myc (Ryu et al., 1999) or c-myc and tumor growth factor TGF

α

(Thorgeirsson etal., 1999), immunodeficient (SCID) mice (Salim et al., 1999), as well as knock outp53-deficient mice not expressing tumor suppressor p53 gene (Park et al., 1999).The recent study designed to evaluate if rodent models of colon carcinoma are goodpredictors of efficacy in humans suggests that rodent models roughly predict theeffect of anticarcinogens, with rat models being better than the mouse models used.However, the prediction may not be accurate for all agents (Corpet and Pierre, 2005).

The International Agency for Research on Cancer (IARC, 1982) and the U.S.Environmental Protection Agency (USEPA, 1984) in Europe and the United States,respectively, proposed a classification system for carcinogens based on scientificevidence. According to this classification, a factor is a “definite” carcinogen whenan association has been established between exposure and outcome. A factor isconsidered to be a “probable” carcinogen when the association is established, butchance, bias, and confounding cannot be ruled out with reasonable confidence.Finally, a factor is a “possible” carcinogen when available studies do not permit aconclusion of probable or definite association between exposure and outcome. Foodcarcinogens are found in all three classes of carcinogens.

Although neither

in vitro

mutagenicity tests nor carcinogenicity tests in animalscan fully reflect the consumer's health risk associated with a given chemical, theyplay an essential role because they enable the identification of those substances infoods that require detailed toxicological evaluation and whose consumption in largeramounts should be avoided. They are also useful in screening potential anticarcino-genic agents.

19.5 FOODBORNE MUTAGENS AND CARCINOGENS

19.5.1 I

NTRODUCTION

The idea that nutrition is an important factor in the risk of cancer is not new. Reportsfrom the 19th and 20th centuries, based on observations made during clinical prac-tice, often indicated diet as a risk factor. More recently, albeit already classic, Dolland Peto’s survey of epidemiological evidence pointed to links between meat con-sumption and an increased incidence of specific cancers (Doll and Peto, 1981). Themost recent epidemiological studies carried out in the United States (Chao et al.,2005) in which 150,000 people took part showed that the group who ate the mostprocessed meat had twice the risk of developing colon cancer compared with thosewho ate the least. Those who ate most red meat also had a 40% higher risk of gettingrectal cancer. Possible culprits identified so far in red meat (burgers, meatloaf, beef,liver, or pork) include iron, toxins formed during cooking, environmental pollutants,and preservatives.


462


In this chapter, mainly mutagens and carcinogens arising as a result of foodprocessing are described because these substances are believed on one hand torepresent one of the major dietary cancer risk factors, and on the other hand, thehealth hazard that can be readily reduced by changing food storage and preparationtechnologies. Most of these compounds are genotoxic carcinogens and were exten-sively reviewed elsewhere in this series (Bartoszek, 2006) along with the impact ofprocessing methods on their formation (Cross and Sinha, 2006).

A large group of potential mutagenic and carcinogenic substances of plantorigin was omitted, although humans may consume as much as a few grams ofthem daily (Ames and Gold, 1990). Potential plant mutagens and carcinogensbelong to a variety of classes of chemical compounds, such as hydrazine deriv-atives, flavonoids, alkenylbenzenes, pyrrolizidine alkaloids, phenolics, saponins,and many other known and unknown compounds. Plants produce these toxins toprotect themselves against fungi, insects, and animal predators. For example,cabbage contains at least forty-nine natural pesticides and their metabolites, afew of which were tested for carcinogenicity and mutagenicity; some of whichturned out positive. However, there is no evidence that plant-based food increasescancer risk. In contrast, epidemiological studies demonstrate that phytochemicalsfound in edible plants exhibit numerous activities preventing carcinogenesis.Therefore, their role will be discussed in a chapter concerning anticarcinogenicfood components.

19.5.2 M

YCOTOXINS

Mycotoxins are highly toxic compounds produced by molds, mostly in the genera

Aspergillus

,

Penicillium

, and

Fusarium

. They represent the most dangerous contam-ination, arising mainly during storage of numerous food commodities, such as cornor peanuts. Tropical and subtropical countries are particularly favorable locations formycotoxin production because of often poor food harvesting and storage practices.

The first three compounds belonging to the group of mycotoxin B

1

for whichcarcinogenic properties were demonstrated, included aflatoxins and sterigmatocystin,which induce liver cancers, and ochratoxin A, which is implicated in the developmentof kidney cancers in experimental animals (Wakabayashi et al., 1991).

Aflatoxin B

1

is the most carcinogenic mycotoxin, and based on available toxi-cological and epidemiological data, has been classified as a human hepatocarcinogen(IARC, 1987).

This list is, however, constantly growing as new carcinogenic mycotoxins areidentified. Most of them are DNA-damaging agents with the exception of fumonisinB

1

, the most frequent among fumonisins contaminating corn and other grain prod-ucts. Its mode of action involves apoptic necrosis, atrophy, and consequent abnormalregeneration of target organs (Dragan et al., 2001).

19.5.3 N

ITROSAMINES

A number of nitroso compounds, N-nitrosamines among them, are potent carcin-ogens. The carcinogenic nitrosamines most commonly found in protein foods are



463

N-nitroso-dimethylamine (DMN), N-nitroso-diethylamine (DEN), N-nitrosopyrroli-dine (NPYR), and N-nitrosopiperidine (NPIP).

These compounds supposedly increase the risk of colon, rectum, stomach, pan-creas, and bladder cancers. Nitrosamines are most prevalent in cured meats, but werealso detected in smoked fish, soy protein foods dried by direct flame, as well as in

FORMULA 19.1

FORMULA 19.2

fumonisin B1

CH3

CH3

CH3

OR

OR CH3 OH

OHOH

NH2

O

O

O

O O

OCH3

aflatoxin B1

R = C

O

CH2 CH CH2 COOH

COOH

NH

O

OH

Cl

OOCH3

O OH

ochratoxin A

N-nitrosodiethylamine (DEN) N-nitrosodimethylamine (DMN)

N-nitrosopyrrolidine (NPYR)

H5C2

N

H5C2

N

O

H3C

N

H3C

N

O

N

NO

N

NO

N-nitrosopiperidine (NPIP)


464


food-contact elastic nettings. Dietary surveys indicated weekly mean intakes of thesecompounds amounting to about 3

µ

g per person (Anonymous, 1988; Cassens, 1995).In addition, the precursors of nitrosamines, especially nitrate, are abundant in someleafy and root vegetables (Table 19.1).

Nitrate and nitrite are also formed endogenously in the human body. In mam-malian organisms, following enzymatic conversion of L-arginine, nitric oxide isproduced, which in turn may be converted to nitrite and nitrate (Hibbs et al., 1987).A portion of nitrate, either ingested or endogenously formed, carried out in the blood,is secreted by salivary glands into the oral cavity. Here nitrate can be reduced bymicrobial flora and swallowed. Hence, it ends up in the gastric environment, similarto nitrite ingested with food. Under the acidic conditions of the stomach, the nitro-sation of amines present in food by nitrite occurs, giving rise to N-nitrosamines.Animal studies suggest, however, that

in vivo

formation of nitrosamines does notoccur to a significant extent, and from a cancer risk perspective, preformed N-nitrosocompounds consumed in items such as cured meat or fish, are much more significant(WCRF/AICR, 1997).

The presence of nitrites has both positive and negative impacts on food safety.On one hand, in many countries, a correlation between stomach and liver cancers,induced probably by nitrosamines, and the amount of nitrites consumed is observed(Fine et al., 1982). On the other hand, nitrites inhibit the growth of

Clostridiumbotulinum

, thus reducing the risk of food contamination by botulinum toxins. More-over, under the acidic conditions of the stomach, where they are involved in theformation of carcinogenic nitrosamines, nitrites are capable of neutralizing carcin-ogens formed as a result of protein pyrolysis (Pariza, 1982).

TABLE 19.1The Most Important Sources of N-Nitrosamines and Their Precursors—Nitrites and Nitrates—in the Human Environment

Substance Source

N-nitrosamines Cured meat (especially bacon)Smoked fishSoy protein foods dried by direct flameSome alcoholic beveragesFood-contact elastic nettingsRubber baby-bottle nipplesCosmetics

Nitrites Cured meatBaked goods and cerealsVegetablesNitrate reduction

in vivo

Nitrates Drinking waterNatural constituent of beets, celery, lettuceNitrate fertilizer residues



465

Because the presence of nitrites is mainly a consequence of vegetable cultivationand food processing, changes in technology may lead to a considerable decrease inthe amounts of these compounds in food products, thereby diminishing the risk ofcancers induced by nitrosamines. Nonetheless, they are likely to remain a necessaryadditive of preserved foods because an alternative to nitrites as curing agents andmicrobiological preservatives has not been found so far. It has been learned though,that the formation of carcinogenic nitrosamines during thermal processing, for exam-ple frying of cured meats, can be largely inhibited by the addition of antioxidants,such as ascorbate and alpha-tocopherol. The addition of such compounds has nowbecome a standard procedure (Cassens, 1995).

19.5.4 M

UTAGENS

IN

H

EAT

-P

ROCESSED

F

OODS

In the 1960s, with the advent of experimental models of chemical carcinogenesisand the publication of the mutagenicity test by Ames (Ames et al., 1975), thedetection of specific chemical carcinogens in the human diet became possible. Thesurprising news was that cooking of proteinaceous food under normal cookingconditions promotes mutagenesis. Mutagens were found in grilled and fried meatand fish and methanol extracts of their charred parts, in smoke condensates producedwhile cooking these foods, and in heated, purified proteins as well as amino acids.Most of examined mutagens proved carcinogenic in mice and rats inducing cancersof various organs (Nagao, 1999).

The formation of mutagens in canned foods is also associated with sterilization,although temperatures applied in this case are relatively low: 110 to 120

°

C.Mutagenic substances produced during canning have not been characterized chem-ically so far (Krone et al., 1986). In the case of other protein foods, such as milk,cheese, eggs, or legumes, the presence of mutagenic substances was detected onlyafter thermal processing associated with a change of color resulting from burning(Robbana-Barnat et al., 1996).

19.5.4.1 Heterocyclic Aromatic Amines

Heterocyclic aromatic amines (HAAs) are formed during thermal processing of manykinds of foods, especially foods containing much protein. They may be associatedwith increased incidence of human tumors in the colon, breast, stomach, liver, andother organs. However, data gathered so far do not allow us to draw final conclusions(WCRF/AICR, 1997). On one hand, a comprehensive case-control study, designedto estimate HAA risk with respect to the background of common polymorphisms ingenes implicated in metabolism of these compounds, suggested that they do not playan important role in the etiology of colorectal cancer in humans (Sachse et al., 2002).On the other hand, the estimation of U.S. dietary exposures to HAA among differentgroups of people pointed to PhIP (see Formula 19.4), comprising two-thirds of thetotal HAA intake, as a risk factor in prostate cancer (Bogen and Keating, 2001).

Around 20 different food-derived HAAs have been isolated to date. The productsof amino acids and protein pyrolysis, whose chemical structures are given below,are produced at temperatures higher than 300

°

C. Therefore, they are detected mainly


466


in the surface layers of meat and fish subjected to open flame broiling. Thesecompounds are strong mutagens, though they usually are not very potent carcinogens(Sugimura and Sato, 1983; Nagao, 1999).

Another type of HAAs are generated in the dry crust of foods baked at 150 to200

°

C. These are derivatives of quinoline, quinoxaline, and pyridine formed in thereaction of creatine or creatinine with amino acids and sugars. All the reactants arethus natural constituents of meat. These HAAs, examples of whose chemical struc-tures are given below, are the strongest foodborne mutagens known and are carci-nogenic in rodents (Wakabayashi et al., 1991); the compound designated IQ wasshown to be carcinogenic in nonhuman primates (Adamson et al., 1994). They arefound in the crust of fried or broiled meat and fish, as well as in fried and bakedmeats and heated meat extracts (Krone et al., 1986).

Compounds PhIP and MeIQx are the most prevalent of the HAAs in the humandiet. Daily consumption may be as high as about 9 ng/kg/day (Bogen and Keating,2001). These amounts ingested by humans may not be sufficient to induce cancersby themselves. At least such a conclusion can be drawn from comparison with animalintakes. However, many environmental factors may be implicated in neoplastictransformation in man. HAAs may be one of these factors (Nagao, 1999), especiallytaking into account the recently discovered estrogenic activity of PhIP and thepostulated role of HAAs in the etiology of breast cancer (Lauber et al., 2004).

Because HAAs belong to the most abundant foodborne substances possiblyaffecting cancer risk, much research is devoted to clarifying their impact on tumorinduction. It was found that dietary polyenoic fat, such as corn oil used for fryingmeat patties, significantly enhances PhIP mammary carcinogenesis in rats, and it

FORMULA 19.3

FORMULA 19.4

N

NH

CH3

CH3

CH3NHCH3

NN

NH2

Trp-P-1 Glu-P-1

N

NH2

Phe-P-1

5-phenylpyridin-2-amine1,3,4-trimethyl-5H-pyrido[4,3-b]indole 6-methyl-5,9-dihydropyrido[3',2':4,5]imidazo[1,2-a]pyridin-2-ylamine

N

N

N

CH3

NH2

IQ

N

N

N

N

CH3

NH2

CH3

MeIQx

N N

N

NH2

CH3

PhIP

3-methyl-3H-imidazo[4,5-f]quinolin-2-amine

3,8-dimethyl-3H-imidazo[4,5-f]quinoxalin-2-ylamine

1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridin-2-amine



467

has been suggested that PhIP initiates the carcinogenic process while dietary fatserves as a promoter (Ghoshal et al., 1994). Of particular concern are the results ofexperiments performed in rats, which demonstrated that PhIP is passed via the liverto the breast and is secreted in the milk of lactating animals. The newborn pupsreceived a dose sufficient to induce tumors. Such a route of exposure may also existin other mammals, including humans. Moreover, it has been shown that enzymessecreted by human mammary glands are able to activate these compounds (Gor-lewska-Roberts et al., 2004). This would mean that humans are exposed to HAAsin foods continuously from early life, even

in utero

(Paulsen et al., 1999).

19.5.4.2 Polycyclic Aromatic Hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs), containing a system of condensed aro-matic rings, are formed as a result of incomplete combustion of organic matter. PAHsare associated with elevated risk of cancers in various tissues, especially skin andlung. It has been established that for carcinogenicity of these compounds, the metab-olites arising from epoxidation of the so-called bay region (Section 19.3) are respon-sible. Human exposure to PAHs can be attributable to occupational, environmental,and dietary sources. Around 70 different such compounds have been identified infoodstuffs; the most abundant are B[

a

]P and B[

a

]A (see Formula 19.5) present inthe greatest amounts in cooked or smoked meat products (Smith et al., 2001).

In food, PAHs are produced mostly during heating, especially open-flame heat-ing, such as grilling of meat. Under such conditions, fat from meat drips onto a hotsurface, such as hot coals during grilling, and is incinerated. The smoke from thefat pyrolysis containing PAHs is adsorbed by the meat. The levels of these com-pounds that can potentially be produced are relatively large: the surface of a two-pound, well-done steak was reported to contain an amount of benzo[

a

]pyrene equiv-alent to that found in the smoke from 600 cigarettes (Pariza, 1982). In the case ofsmoked meat and fish, smoke used during processing is also a source of carcinogenicPAHs (Sikorski, 1988). In addition, a number of food products contain measurableamounts of these hydrocarbons resulting from environmental pollution, such as fishcaught in heavily industrialized regions.

The concentration of PAHs detected in foods are in a range from several toseveral hundred ng per 1 g of food product (Anonymous, 1993). In feeding studiesin which volunteers consumed heavily charbroiled beef, a dose-dependent formationof PAH-DNA adducts in white blood cells was observed. Their level increased after1 to 4 days following ingestion, and they were eliminated within about 7 days. It

FORMULA 19.5

benzo[a]pyrene benzo[a]antracene


468


was thus unequivocally demonstrated that foodborne PAHs are capable of inflictingdamage on human DNA (Schoket, 1999).

19.5.4.3 Acrylamide and Furan

Acrylamide (AA), until the year 2000, was regarded solely as a product of thechemical industry, and based on animal studies, classified as a probable humancarcinogen (IARC, 1994). Therefore, its detection in persons not occupationallyexposed to this compound came as a surprise (Bengmark, 1997). Further studiesrevealed that AA is formed during heat processing of foods with a high starch content,such as potatoes, bakery items and cereals, nuts, and coffee and cocoa. Potato chipsand French fries are a particularly abundant source of AA (Tareke et al., 2000;Delatour et al., 2004). The carcinogenic properties of AA result from its ability todamage DNA after metabolic activation to glycidamide (Section 19.3) (Besaratiniaand Pfeifer, 2004).

AA is formed as a result of a heat-induced reaction of amino acids, peptides,and proteins with carbonyl groups of reducing sugars, such as glucose and fructose,concurrently with formation of so-called Maillard browning products (Friedman,2005). However, the prolonged heat processing decreases AA concentration due tothermal instability of this compound.

Another genotoxic compound proposed by the European Food Safety Authorityas a food carcinogen is furan (EFSA, 2004). It occurs in a variety of foods, such ascoffee and canned and jarred foods, including baby foods containing meat andvarious vegetables. The variety of foodstuffs in which furan was detected suggeststhat there are probably multiple routes of its formation. It was shown that furanarises upon thermal processing of carbohydrates and ascorbic acid, as well as duringexposure of these substrates to ionizing radiation (Fan, 2005). From available datait appears that possible human exposures match the doses that produce carcinogeniceffects in animals. However, it is too early for reliable risk assessment (EFSA, 2004).

19.5.4.4 Effect of Commercial Processing and Cooking Techniques

The content of foodborne mutagens resulting from processing is relatively small butvery variable, and is estimated to amount to from 0.1 to 500 ng/g of a given foodproduct. A World Cancer Research Fund panel of experts evaluated the evidenceand indicated that consumption of grilled or barbecued meat, fried foods, and a diethigh in cured meats possibly increases the risk of certain human cancers. Theyconcluded, however, that “there is no convincing evidence that any method ofcooking modifies the risk of cancer” (WCRF/AICR, 1997). This statement has beenargued against by several researchers who demonstrate in their studies that theformation of food carcinogens depends strongly on the cooking technique used, andmust therefore reflect the differences in health hazard. This is of special concern inthe case of animal protein foods because their processing involves methods partic-ularly liable to generate carcinogenic HAAs and PAHs. Another cooking-associated



469

exposure to PAHs and HAAs involves fumes, produced particularly abundantlyduring so-called stir-frying. Lung cancer is the most common cause of cancer deathamong women in Taiwan, though most of them are nonsmokers. There are recentreports confirming an association between PAH-DNA adduct levels in lung tissueand lung cancer incidence in Chinese women, many of whom reported that they stir-fried meat daily (Yang et al., 2000).

The temperature applied during processing has a decisive influence on the kindof HAAs formed, while the amount depends on cooking time and method, as wellas the type of food (Bartoszek, 2001; Cross and Sinha, 2006). Their content,however, can be effectively reduced. For instance, mutagens of the HAA type werenot detected in beef either processed in a microwave oven or stir-fried for threeminutes on high heat (Miller, 1985). It has also been established that mutagenicityof cooked meat decreases (mostly fried hamburgers were analyzed) after microwavepretreatment causing the leakage of juices, thereby diminishing the content of sugarsand creatinine—precursors of some HAAs. Addition of onion and some vitaminsalso effectively reduced mutagenicity of cooked hamburgers (Kato et al., 1998;Kato et al., 2000).

Cured meat and fish are the main source of nitrite and the greatest contributorsof preformed carcinogenic N-nitrosamines in the human diet. Another traditionalway of preserving protein foods—salting—also modifies cancer risk. Epidemiolog-ical studies showed that stomach cancer rates are highest in those parts of the worldwhere diets are traditionally very salty, for example, in Japan, China, or Chile. Saltis used extensively as a preservative and flavor enhancer throughout the world, butit was demonstrated to increase stomach cancer risk in a dose-dependent manner.This carcinogenicity enhancement is probably due to damage to the mucosal layerfacilitating

Helicobacter

pylori

infection (WCRF/AICR, 1997).In addition, commercial foods may contain traces of chemicals used in pack-

aging, and migration from food-contact materials can occur during their process-ing, storage, and preparation. These chemicals include monomers of polymericmaterials used in packaging, such as vinyl chloride and acrylamide, classified bythe International Agency for Cancer Research (1987) in group 2A (probable humancarcinogens). Cadmium also belongs to this group, which along with lead, arsenic,and other carcinogenic heavy metals, may contaminate foods, especially organmeats including liver and kidney, in which these metals tend to concentrate (Rojaset al., 1999).

Cooked meals are characteristic of most civilizations, and preparation and enjoy-ment of cooked food is intrinsic to social and family life. Meat and fish may becooked using water, fat, more or less fierce heat, direct flame, and in other ways.Curing and smoking have been used as a means of preserving meat and fish forthousands of years. Modern food technologies employ basically the same methods,but on a larger scale, to provide easy-to-prepare and time-saving convenience foodsdevoid of microbial contamination with increased shelf life. Relatively recently, ithas been realized that all the above benefits must be weighed against the possibilityof formation of a variety of carcinogenic compounds as a result of food processing.


470


19.5.5 M

UTAGENS

IN

T

EA

, C

OFFEE

,

AND

ALCOHOLIC BEVERAGES

Coffee brewed from roasted beans and that prepared from instant powder, includingthe caffeine-free type, all display mutagenic activity. Apart from natural mutagens,such as caffeic acid and its precursors chlorogenic and neochlorogenic acids, thesedrinks contain mutagenic products of pyrolysis—methylglyoxal and the less activeglyoxal and diacetyl (Ames et al., 1986).

These pyrolysis products were also found in roasted tea and brandy-type alco-holic beverages (Sugimura and Sato, 1983). In addition, as a result of ethanolmetabolism, mutagenic acetaldehyde is formed, while in coffee and tea, caffeine ispresent, which is an inhibitor of DNA repair synthesis and may also contribute tocancer risk.

These observations, made during studies carried out with the aid of microorgan-isms and experimental animals, suggested that tea and coffee might pose a serioushealth hazard, especially because both these drinks are consumed in substantialamounts almost all over the world. Epidemiological studies, whose results becameavailable ten years later, showed how misleading the extrapolation of data betweenspecies could be. Not only had no convincing evidence been found that dailyconsumption of tea or coffee increased cancer risk, it turned out that regular greentea intake decreased it owing to the presence of numerous phytochemicals exhibitinganticarcinogenic properties, which will be discussed later in this chapter. Caffeine,previously regarded as a harmful compound, has become an important (because ofsubstantial intake) factor in carcinogenesis prevention, mainly because it helps tocombat obesity constituting a well-documented risk factor of many widespreaddiseases, including heart ailments and cancer (WCRF/AICR, 1997; El-Bayoumy etal., 1997). Because of its wide spectrum of health benefits, some nutritionists suggestthat coffee should be treated as a functional food (Dorea and da Costa, 2005).

The opposite must be said in the case of alcoholic beverages. Experimentalresults did not indicate that they might play a role in cancer risk. This notion wassomehow supported by the epidemiological studies carried out in France, which ledto the discovery of the so-called French paradox. Contrary to common belief, despitehigh intakes of alcohol, the frequency of heart failures and possibly also tumorincidence are lower in the French population as compared to those of other countries.Currently, it is postulated that antioxidant substances present in colored alcoholicbeverages and particularly abundant in red wine (Figure 19.2) offer this protection.

The studies carried out in France are, however, the only ones that failed todemonstrate alcohol as a cancer risk factor. The data gathered in other regions

FORMULA 19.6

glyoxal

CH

CH

O

O

CH

C

O

O CH3

C

C

O

OCH3

CH3

methylglyoxal diacetyl


Mutagenic, Carcinogenic, and Chemopreventive Compounds in Foods 471

indicate alcohol as an important cause of carcinogenesis. The risk of cancer devel-opment increases with the amount of alcohol consumed, and becomes particularlyhigh when accompanied by cigarette smoking (WCRF/AICR, 1997; Doll, 1999).

19.5.6 OTHER RISK FACTORS

A number of epidemiological studies indicate that a high consumption of fat con-tributes to the development of breast and large intestine cancers in humans (Ames,1986). Carcinogenic effects are ascribed also to high-calorie and protein-rich diets.Animal studies suggest that all the mentioned risk factors come into play afterinitiation of tumorigenesis, while their mode of action relies on the increased pro-duction of oxygen radicals. Reactive oxygen species are generated in the organismas a result of normal metabolism. A diet rich in nutrients increases the intensity ofmetabolic processes, and hence oxygen radical production. These radicals are impli-cated in the induction of endogenous oxidative damage of macromolecules, includingthe formation of so-called oxygen DNA adducts (examples of such adducts resultingfrom hydroxylation of nucleobases are given below) and protein carbonyl derivatives(Youngman et al., 1992; Chevion et al., 2000). This type of lesion is believed toplay a significant role in the process of aging and the variety of degenerative age-related disorders including cancer.

Animal studies showed that calorie and protein restrictions markedly inhibit bothcarcinogenesis and accumulation of endogenous oxidative damage (Youngman etal., 1992; Rogers et al., 1993; Burcham, 1999). Convincing support is also lent byreports demonstrating that a diet containing ingredients with antioxidant propertiesconsiderably inhibits cancer development (WCRF/AICR, 1997; Thomas, 2000).

Moreover, although fats do not display mutagenic activity per se, some of theirconstituents, such as cholesterol and unsaturated fatty acids, are easily oxidizedduring thermal processing giving rise to reactive molecules, which in turn may trigger

FIGURE 19.2 Antioxidative properties of selected alcoholic beverages. The high antioxida-tive activity of beers may, to a considerable extent, result from the addition of antioxidants,vitamin C in particular. (Based on Bartosz, G., Janaszewska, A., Ertel, D., and Bartosz, M.,Biochem. Mol. Biol. Int., 46, 519, 1998.)

0 1 2 3 4 5 6 7

Antioxidative activity[arbitrary units]

light beer

dark beer

white wine

red wine

vodka

brandy

cherry



a chain reaction of lipid peroxidation leading to the formation of mutagens, promot-ers, and carcinogens. These include radicals, fatty acid epoxides and peroxides,aldehydes, and others (Ames, 1986). Aldehydes constituting the final stage of lipidperoxidation are particularly dangerous because of their relatively high stability,genotoxic potential, and the ease of absorption from diet. Therefore, in humans thetoxicity of oxidized polyunsaturated fatty acids appears to come mainly from alde-hydes (De Bont and van Larebeke, 2004).

Another important mechanism by which fat modulates carcinogenesis—someresearchers claim the most important—involves its interference with synthesis ofprostaglandins and leukotrienes as well as the development of so-called insulinresistance, which in turn stimulates proliferation of cells (colonic epithelium cellsin particular) (Woutersen et al., 1999; Bruce et al., 2000).

Another class of foodborne substances that have been postulated to influencethe frequency of cancer development, and that are drawing increasing attentionamong nutritionists are environmental pollutants. The most important are heavymetals and xenobiotics such as derivatives of dioxin, dibenzofuran, chlorinatedbiphenyls, and residues of pesticides (Biziuk and Bartoszek, 2006).

FORMULA 19.7

FORMULA 19.8

N

N

N

N

NH2

sugar

OH

HO

adenosine

N

N

N

N

NH2

sugar

H2N

OH

HO

guanosine

N

NH

O

O

CH3

sugar

OH

OH

thymidine

N

N

NH2

O

sugar

cytosine

OH

OH

OO

HH

CH2

O

H

OH

O

H

H11C5

malondialdehyde acrolein trans-4-hydroxynonenal

CH3 O

H

crotonaldehyde



These compounds display several activities regarded as important for cancerrisk. Dioxins and biphenyls were shown to form DNA adducts. Some are able toinduce oxidative stress in the organism. They are also so-called xenoestrogens.Xenoestrogens penetrate organisms through food, and they mimic or change theactivity of estrogens produced endogenously. To these compounds, whose abilityto promote the development of estrogen-dependent cancers (e.g., breast cancer),has been documented, belong among others, polychlorinated biphenyls (PCBs),formed during drinkable water chlorination, pesticide residues (DDT in particular),and some components of plastics used for food packaging. In the case of DDT andcertain PCBs, their association with breast cancer incidence was evidenced basedon human-derived biological material. These substances are extremely stable andpersist in the environment for many years, even in countries where DDT was bannedlong ago. It is estimated that decreased exposure to xenoestrogens would decreasethe frequency of breast cancer by 20%, that is, by 36,000 cases in the United Statesalone (Davis et al., 1993).

19.6 CHEMOPREVENTIVE FOOD COMPONENTS

Laboratory studies carried out over the past 20 years demonstrated that food, oneof the major components of the human environment, contains numerous mutagensand carcinogens. As described earlier in this chapter, they may be naturally occurring,but most of them are of anthropogenic origin and are found in food mainly as aresult of thermal processing of fat and protein-rich food products. These findingswere followed by epidemiological investigations, which confirmed the health riskassociated with the consumption of foods with high protein and calorie content.They revealed, however, that edible vegetables and fruits, apart from nutritive mac-roelements, contain numerous microelements and nonnutritive phytochemicals dis-playing different anticarcinogenic and other health-promoting biological activitieseffectively reducing human cancer risk (for the most extensive review, seeWCRF/AICR, 1997). The latter until recently have been regarded as unimportant tohuman health.

In the 1990s, the results of numerous investigations carried out in differentpopulations were published demonstrating that high vegetable and fruit content in

FORMULA 19.9

O

O

O

Cl

Cl

Cl

Cl Cl

dibenzodioxin dibenzofuran biphenyl DDT



the diet was associated with decreased cancer incidence. As a result of research onanticarcinogenic food components, a number of substances displaying such chemo-preventive properties have been characterized: lycopene found in tomatoes (Giovan-nucci, 1999), epigallocatechins in tea (Fujiki et al., 2000), sulforaphane in broccoli(Zhang et al., 1994), resveratrol in grapes (Jang et al., 1997), to name only a few ofthe most extensively investigated. The chemopreventive potential exhibited by plantfoods has now become one of the major and most promising fields of cancer researchbecause it may help to diminish the global cancer burden simply by implementingspecific dietary recommendations (Schatzkin, 1997; Wolf, 2001). In addition, phy-tochemicals isolated from edible plants are being tested with the aim of developingdietary supplements that could protect humans against cancer, as well as become ameans of cancer chemotherapy enhancement.

19.6.1 ANTICARCINOGENIC FOOD COMPONENTS

A number of natural and synthetic compounds are able to prevent cancer inductionor development when administered to animals before or concomitantly with carcin-ogens. These substances include vitamins, microelements, compounds of plant ori-gin, medicines, and others (Table 19.2). Although the studies on the modes of actionof cancer preventive agents are still underway in many laboratories and bring newdiscoveries each day, it was realized even before they were undertaken that anyfactor capable of counteracting the production of carcinogenic metabolites, inhibitingthe initiation or promotion of tumorigenesis, or inhibiting metastasis by malignantcells, may be considered an anticarcinogen.

Anticarcinogens are divided into three groups depending on the stage of carcino-genesis on which they act (Ames, 1986; Anonymous, 1993; Caragay, 1992). The firstof these groups includes blocking agents protecting cells at the stage of initiation ofneoplastic transformation. The second group—suppressing agents—are important dur-ing cancer promotion and uncontrolled growth of initiated cells, while factors makingcells more resistant to neoplastic transformation constitute the third group.

Blocking agents protect cells against substances that could initiate changesleading to malignancy. There are three major mechanisms of their activity. Firstly,they prevent the formation of carcinogens from precursors. For instance, vitamin Cinhibits, via an unknown mechanism, the formation of carcinogenic nitrosaminesfrom amines and nitrites present in food (Caragay, 1992). It has also been foundthat lactic acid bacteria from both fermented dairy (Gilliland, 1990) and nondairy(Thyagaraja and Hosono, 1993) foods display antimutagenic activity owing to theirability to bind mutagens. In binding, peptidoglycan present in the bacterial cell wallis involved, and this property is not abolished after sterilization. Similar physico-chemical sequestering of mutagenic and carcinogenic aromatic substances is dis-played by chlorophyllin, the sodium and copper salt of chlorophyll (Ardelt et al.,2001). Agents that protect cells against DNA damage belong to the second groupof blocking agents. These mechanisms are the best recognized. They involve thereduction of synthesis or inhibition of enzymes responsible for the metabolic acti-vation of carcinogens (phase I enzymes) and induction of enzymes taking part inthe detoxification of harmful substances (phase II enzymes). The ability to modulate



TAB

LE 1

9.2

Ant

icar

cino

geni

c Fo

odbo

rne

Subs

tanc

es: T

heir

Occ

urre

nce

and

Maj

or C

hem

opre

vent

ive

Act

ivit

y

Type

of

prev

entiv

e fa

ctor

Subs

tanc

eSo

urce

Che

mop

reve

ntiv

e ac

tivit

y

Blo

ckin

g ag

ents

vita

min

Cvi

tam

in E

caro

tene

sly

cope

neep

igal

loca

tech

ins

chlo

roph

yllin

pept

ydog

lyca

ngl

utat

hion

eis

othi

ocya

nate

s

citr

us f

ruit

plan

t oi

lsca

rrot

(an

d ot

her

oran

ge v

eget

able

s)to

mat

oes

tea

gree

n ve

geta

bles

cell

wal

l of

lac

tic b

acte

ria

garl

icbr

occo

li

antio

xida

ntan

tioxi

dant

antio

xida

ntan

tioxi

dant

antio

xida

ntar

omat

ic c

arci

noge

n se

ques

teri

ngca

rcin

ogen

seq

uest

erin

gch

emic

al b

indi

ng o

f el

ectr

ophi

les

deto

xify

ing

enzy

me

indu

ctio

nSu

ppre

ssin

g ag

ents

geni

stei

n, d

aidz

ein

geni

stei

nre

tinoi

dsis

othi

ocya

nate

s

soy,

sor

goso

yor

ange

-col

ored

veg

etab

les

cruc

ifer

ous

vege

tabl

es

antie

stro

geni

c ac

tivity

inhi

bitio

n of

ang

ioge

nesi

sst

imul

atio

n of

cel

l di

ffer

entia

tion

inhi

bitio

n of

onc

ogen

e ac

tivat

ion

Fact

ors

mak

ing

cells

mor

e re

sist

ant

tone

opla

stic

tra

nsfo

rmat

ion

isofl

avon

esdi

ally

l su

lfide

poly

enoi

c n-

3 fa

tty a

cids

vita

min

D +

Ca

+ P

soy

garl

icfis

h oi

lre

stri

cted

-cal

orie

die

t co

ntai

ning

inc

reas

ed l

evel

of

vita

min

D +

Ca

+ P

stim

ulat

ion

of c

ell

mat

urat

ion

anti-

Hel

icob

acte

r py

lori

act

ivity

mod

ulat

ion

of s

igna

l tr

ansd

uctio

nin

hibi

tion

of c

ell

prol

ifer

atio

n



the activity of cytochrome P450 isoenzymes, often implicated in carcinogen acti-vation, is displayed by numerous compounds, such as phenols found in edibleplants. The detoxifying enzymes, especially glutathione-S-transferases, are effec-tively induced by isothiocyanians present in cruciferous vegetables, such as broc-coli. The compounds capable of trapping DNA-damaging species belong to thethird group of blocking agents. The removal of toxic metabolites is usually accom-plished by nucleophilic substances, primarily glutathione and other sulfur-contain-ing compounds abundantly found in garlic and onion, which can bind electrophilicDNA reactive intermediates. Vitamins C and E, which trap oxygen radicals in lipidmembranes, as well as beta-carotene and other polypropenes, present in all chlo-rophyll-containing food products, particularly effective in neutralization of singletoxygen, protect DNA against oxidative damage. Similar roles are played by com-pounds containing selenium. Selenium is an essential component of the active siteof glutathione peroxidase, the enzyme responsible for destroying hydrogen peroxideand other peroxides generated during lipid peroxidation. Also polyphenols, majorphytochemicals present in all kinds of foods of plant origin, display antioxidativeproperties.

Suppressing agents, constituting the second group of anticarcinogenic factors,influence the process of transformation of initiated (procancerous) cells into trulymalignant cells. Numerous nonnutritive phytochemicals display the ability to slowdown or inhibit cancerous growth. Several protective mechanisms can be distin-guished (Wattenberg, 1997). They involve stimulation of cell differentiation (retinol),inhibition of oncogene activation (isothiocyanians), selective inhibition of prolifer-ation of tumor cells and antiangiogenic activity (genistein present in soy) disablingthe growth of new blood cells necessary to supply the neoplasm with nutrients andoxygen. Generally, suppressing agent mechanisms are poorly understood, as are theprocesses preventing cancer development at the later stages of carcinogenesis.

Agents belonging to the third group render cells more resistant to neoplastictransformation. These mechanisms are least known. They include stimulation of cellmaturation, an activity believed to be responsible for reducing breast cancer growthby soy isoflavones, and inhibition of cell division in target cells. Proliferationincreases the probability of the conversion of promutagenic DNA damage intomutation. Hence, reduction of its rate protects cells (in a way) against neoplastictransformation. It has been demonstrated that dietary enrichment in calcium, phos-phate, and vitamin D slows down the rate of cell division (WCRF/AICR, 1997;Wattenberg, 1997). Some garlic components can be included in this group of anti-carcinogenic agents because of their antimicrobial activity against Helicobacterpylori, a risk factor in the case of gastric cancers. Garlic components inhibit thegrowth of these bacteria and thereby prevent damage to epithelium, which makesthis tissue more resistant to the harmful effects of carcinogens (WCRF/AICR, 1997).

Vegetables and fruits are the major source of dietary anticarcinogens that canprotect human organisms against neoplastic diseases by different mechanisms, atvarious stages of carcinogenesis. Hence, a diet rich in plant-derived foods appearsto be a realistic nonpharmacologic approach against cancer. Apart from numerousanticarcinogenic substances, it also provides meals of low calorie and proteincontent. All these factors reduce cancer risk in humans. Therefore, the food and



pharmaceutical industries share interest in edible plants as a means of cancer chemo-prevention. In the case of the food industry, prevention will probably rely on dietaryrecommendations ensuring a high intake of protective phytochemicals, as well asenrichment of foods with anticarcinogenic vitamins and minerals. The pharmaceu-tical industry has begun to develop preparations, based on edible plants, exhibitingactivities desirable from a cancer prophylactic perspective.

19.6.2 CANCER CHEMOPREVENTION

Cancer chemoprevention can be defined as the prevention of neoplastic diseases byproviding people with one or more chemical substances in a special preparation oras naturally occurring dietary components. Cancer development is a slow multistageprocess that takes about 20 years on average. The number of new cancer casesestimated around the world for only 25 different cancers in 1990 amounted to 8.1million (Parkin, 1998). If this number is multiplied by 20 years of latent development,it may be expected that over 160 million people at the moment, are in one of thestages of neoplastic transformation, which is life threatening. These people are thetarget population of cancer prophylaxis, and hence chemoprevention.

Anticarcinogenic compounds found in edible foods display many advantagesfrom a chemoprevention point of view. Any substance consumed as a chemopreven-tive agent is supposed to be ingested by healthy people for a long time, and thereforeit must be devoid of toxicity. Numerous components of fruits and vegetables fulfillthis condition. Another desirable property of edible plants is the fact that theyrepresent a well-known element of human life and thereby facilitate the decision ofadopting health-promoting activities.

For about ten years, very extensive studies have been carried out on numerouscompounds, both natural and their artificial derivatives, with potential applicabilityin cancer chemoprevention. Here are four examples of promising substances isolatedfrom edible plants.

Sulforaphane is one isothiocyanian produced by vegetables from the cruciferousfamily, which gives them a characteristic taste. Broccoli is a particularly abundantsource of sulforaphane. It is capable of inducing liver II phase enzymes responsiblefor detoxification of mutagens and carcinogens (Zhang et al., 1994). Another prom-ising compound, epigallocatechin gallate, was isolated from green tea. This com-pound, which is a very potent antioxidant, constitutes about 50% of the dry weightof green tea extract (Fujiki et al., 2000). It is present in black tea extracts as well,though in smaller amounts. Antioxidative properties are also displayed by lycopene,one of the major carotenoids present in tomatoes, processed tomatoes in particular(Giovannucci, 1999). Another chemopreventive compound, resveratrol, is a phy-toalexin and was isolated from grapes. Resveratrol was demonstrated to activatedifferent mechanisms preventing cancer development. Studies in animals showedthat it induced II phase enzymes, scavenged oxygen radicals, and stimulated celldifferentiation; thus it inhibited carcinogenesis at various stages of neoplastic trans-formation. The health-promoting properties of red wine are also ascribed to resver-atrol (Jang et al., 1997).



Moreover, it has been postulated recently that dietary supplementation with foodantioxidants may provide a safe and effective means of enhancing the body’sresponse to cancer chemotherapy (Conklin, 2000). Much more research is neededto validate this claim, however; the stimulation of formation of oxygen radicals byantitumor drugs is a known cause of such side effects of chemotherapy like cardio-or nephrotoxicity. The approved chemoprotectants used clinically to date are notneutral to the organism either. In contrast, certain antioxidative food components,in doses that are without adverse effects, could improve the quality of life of patientsby ameliorating chemotherapy-induced side effects, and also enhance activity ofantitumor drugs by different mechanisms (e.g., inhibition of topoisomerase II).

The discovery of anticarcinogenic properties of many plantborne compoundspresent in foods is undoubtedly one of the most important developments that allowus to hope that the cancer death toll can be diminished. The fact that these substancesare found in foods that are liked and widely appreciated should facilitate the utili-zation of their precious chemopreventive properties.

19.7 FINAL COMMENT

Although cancer has always plagued humankind, it is primarily in modern timesthat these diseases have become epidemic, particularly in developed countries. Onein three individuals in the Western world will develop cancer in their lifetime, and

FORMULA 19.10

epigallocatechingallate

O

H

HOH

OHO

O

OH

OHHO

OH OH

OH CH3

SNCS

O

sulforaphane

OH

OH

OH

resveratrol

CH3

CH3

CH3

CH3 CH3

CH3 CH3

CH3 CH3

CH3

lycopene



one in five will die from cancer (Futreal et al., 2001). It is no longer a matter ofdebate that dietary factors influence cancer risk. What remains to be resolved is howdietary factors might interact to affect cancer risk and what preventive steps can betaken to minimize it. Figure 19.3 clearly shows that many foodborne cancer riskfactors are readily avoidable, and when the appropriate dietary recommendations arefollowed, food may play a protective role.

Evidence-based dietary guidelines aimed at curbing cancer risk have been for-mulated by numerous organizations and generally propose that individuals shouldreduce animal fat intake, include a variety of vegetables and fruits in the daily diet,consume alcohol in moderation, maintain a healthy weight and minimize consump-tion of processed (cured, smoked, or heated) meat, red meat in particular (Greenwaldet al., 2001). The U.S. Department of Health and Human Services and the U.S.Department of Agriculture have jointly published the Dietary Guidelines for Amer-icans every five years since 1980. The most recent revision (2005) promotes a healthylifestyle by putting more emphasis on weight management and physical activity.Also in contrast to previous versions, not only is reduction of fat intake advocated,but the kind of fats are stressed, and hence more specific recommendations aboutcertain foods as sources of fats are given (Weaver and Schneeman, 2005).

FIGURE 19.3 The influence of some food components and dietary preferences on the riskof the development of the most frequent human cancers. (Based on World Cancer ResearchFund and American Institute for Cancer Research (WCRF/AICR), Food, Nutrition and thePrevention of Cancer: A Global Perspective, AICR, Washington, DC, 1997.)

Lung cancer

Pancreas cancer

Colorectal cancer

Breast cancer

Prostate cancer

Kidney cancer

Veg

etab

les

Frui

ts

Car

oten

oids

Vita

min

C

Fibe

r

Alc

ohol

Mea

t

Hea

tpr

oces

sing

Hig

h ca

lori

e co

nten

t

Obe

sity

Decreased risk

convincing probable possible



Despite the institutional efforts to recommend proper dietary habits, usual eatingpatterns in the United States and European countries suggest that our diets are ofteninadequate in terms of meeting these recommendations for ensuring optimal health.People tend rather to use dietary supplements, which may be essential for vulnerablegroups, rather than shifting their preferences toward a healthier diet composition(ADA, 2005). However, there seem to be signs of change. There is growing awarenessof nutritionists, food scientists, the food industry, and consumers with respect to therelationship between diet and health. Hopefully, this awareness will result in con-sumer access to products that will help them meet dietary guidelines.

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20 The Role of Food Components in Children’s Nutrition

Grażyna Sikorska-Wiśniewskaand Małgorzata Szumera

CONTENTS

20.1 Role of Nutrition in Children’s Development .......................................... 48820.1.1 Food Pyramid Serving Recommendations ................................. 48820.1.2 Proper Nutrition in Childhood Can Prevent Chronic

Diseases in Adults ....................................................................... 48920.1.3 The Influence of Nutrition on Cognitive Development

in Childhood................................................................................ 48920.2 Lipids in Children’s Nutrition ................................................................... 490

20.2.1 Role of Lipids in Children’s Nutrition ....................................... 49020.2.2 Recommended Dietary Intake of Lipids..................................... 49220.2.3 Undesirable Dietary Fat Effects in Children .............................. 493

20.3 Saccharides ................................................................................................ 49320.3.1 The Role of Saccharides in Children’s Nutrition....................... 49320.3.2 The Glycemic Index.................................................................... 49420.3.3 Recommended Dietary Intake of Saccharides in Children

and Adolescence.......................................................................... 49520.3.4 Inappropriate Saccharide Intake ................................................. 497

20.4 Proteins ...................................................................................................... 49820.4.1 The Role of Proteins in Children’s Nutrition............................. 49820.4.2 Recommended Dietary Allowances of Proteins in Infancy

and Adolescence.......................................................................... 49920.4.3 Inappropriate Protein Intake ....................................................... 501

20.5 Mineral Components in Children’s Nutrition ........................................... 50220.5.1 The Role of Macro and Trace Elements in Children’s

Nutrition ...................................................................................... 50220.5.2 Calcium ....................................................................................... 50220.5.3 Magnesium .................................................................................. 50320.5.4 Zinc.............................................................................................. 50420.5.5 Iron .............................................................................................. 504



20.6 Vitamins in Children’s Nutrition............................................................... 50520.6.1 Introduction ................................................................................. 50520.6.2 Vitamin C .................................................................................... 50520.6.3 Vitamin B-Complex .................................................................... 50520.6.4 Vitamin A .................................................................................... 50720.6.5 Vitamin D .................................................................................... 50720.6.6 Vitamin E .................................................................................... 50820.6.7 Vitamin K .................................................................................... 508

20.7 Feeding Low-Weight Preterm Infants—A Challengefor Neonatologists...................................................................................... 509

20.8 Vegan Diet—Is It Really Adequate for Children and Adolescents? ........ 510References.............................................................................................................. 512

20.1 ROLE OF NUTRITION IN CHILDREN’S DEVELOPMENT

20.1.1 FOOD PYRAMID SERVING RECOMMENDATIONS

Nutritional recommendations for children are based on daily allowances of nutrientsestablished by the Food and Nutrition Board of the U.S. National Academy ofSciences; the latest update was published in 2005. Childhood is a period of growingup, which is why a considerable amount of nutrition from a well-balanced diet isrequired. A balanced diet contains a combination of several different food types—grains and pulses, fresh fruits and vegetables, meat, dairy products, and fats andoils. Energy from the diet should be supplied in the following approximate propor-tions: 30% proteins, 20% fats, and 50% saccharides. About 60% of proteins con-sumed by children should be of animal origin. Vegetable oils should supply 20%of total daily energy.

A balanced diet is low in fat and refined saccharides, and includes healthysaccharides and a moderate amount of protein.

The food guide pyramid consists of the following six food groups, starting at thebase: grain products such as bread, cereal, rice, and pasta (6 to 11 servings per day);vegetables (3 to 5 servings) and fruits (2 to 4 servings); dairy products and other protein-rich commodities (meat, beans, fish: 2 to 3 servings a day); and sparingly fats, oils, andsweets (American Academy of Pediatrics [AAP] 1998). Supplementation with vitaminsand mineral components is vital for proper development, especially in adolescence.Physical activity like cycling, gym, skating, ball games, or dancing should be includedfor at least 30 to 60 minutes per day, three to five times a week, as a complement tothe food guide pyramid to emphasize the importance of exercise in nutrition.

Proper nutrition in childhood and adolescence results in appropriate weightand height, as compared to standard growth charts. These measurements areimportant tools for monitoring a child’s progress, especially in the first years oflife. Having the right dietary habits and making the right lifestyle choices earlyin life will help young people develop health-promoting behaviors they can followthroughout their lives.


The Role of Food Components in Children’s Nutrition 489

20.1.2 PROPER NUTRITION IN CHILDHOOD CAN PREVENT CHRONIC DISEASES IN ADULTS

Adolescence is a time of building muscles and bones. About 45% of the adult skeletalmass is formed during adolescence, up to the third decade when the bones reach apeak bone mass (PBM). After this time, a consistent loss of 1% of bone mass isobserved every year.

All the Ca for skeletal growth must be derived from the diet. The largest gainsin bone mass are made in early adolescence, between the ages of 10 and 14 in girlsand 12 and 16 years in boys. The achievement of PBM during adolescence is crucialto reduce the risk of osteoporosis in later years. The efficiency of Ca absorption isaround 30%, so it is important to supply an adequate Ca intake in the diet by eatingdairy products like milk, yogurt, and cheese. The intake of Ca, vitamin D, andphosphorus is equal in importance to physical exercise, which promotes an incor-poration of Ca into the bones.

Physical activity and a well-balanced diet prevent obesity in children and ado-lescents. The reason for obesity is multifactorial: socioeconomic, biochemical,genetic, and psychological factors all closely interact, but the nutritional aspect seemsto be the most important. Fast foods and excessive consumption of saccharides resultin excessive weight in children and adolescents. Physical inactivity not only has aprime role in leading to obesity, but also contributes to the development of chronicdiseases in adults, such as heart disease, hypertension, and diabetes (Freedman etal. 1999). It appeared that breast-fed infants had lower blood pressure during ado-lescence (Fewtrell et al. 2002).

20.1.3 THE INFLUENCE OF NUTRITION ON COGNITIVE DEVELOPMENT IN CHILDHOOD

Cognition represents a complex set of abilities such as memory, reasoning, attention,psychomotor coordination, and behavior. Nutrition could affect this in infants, chil-dren, and adolescents by influencing the development of the hippocampus, myeli-nation of neurons, and operation of neurotransmitters (Bryan et al. 2004).

Protein-energy malnutrition (PEM) in early life has lasting effects on intelligencequotient (IQ) up to adolescence, taking into consideration that the first two years oflife are critical for brain growth and development. Certain brain areas, such as thefrontal lobes, develop throughout childhood (Mendez and Adair 1999). Key nutrientsfor cognitive development include glucose, thiamine, iodine, Fe, Zn, folate, vitaminB12, and n-3 polyunsaturated fatty acids (PUFA) (Bryan et al. 2004).

A rise in blood glucose level improves results in reaction time tasks, and allowsfaster information processing, better word recall, and improvement on cognitiveconflict tasks. The brain appears to be sensitive to variable levels of glucose, espe-cially hypoglycemia, which is especially important in children who omit breakfast.While the effects are inconsistent in well-nourished children, omission of breakfastdecreases mental performance in malnourished children. Children may be moresusceptible than adults to various levels of glucose because of their greater brainmetabolic demands (Pollit et al. 1981). Therefore, low-glycemic-index foods that



minimize glycemia fluctuations could improve cognition (Benton et al. 2003). Theexperimental evidence does not confirm the hypothesis that sucrose or food additiveintake (artificial sweeteners or preservatives) causes behavioral problems, especiallyattention deficit hyperactivity disorder (ADHD) (Bellisle 2004).

A diet composed of fast food can provoke thiamin deficiency and behavioralproblems. Thiamin treatment reverses aggressiveness in thiamin-deficient adoles-cents (Benton et al. 1997). An iodine deficiency induces hypothyroidism and has asignificant effect on cognitive performance including mental disability.

The cortex, hippocampus, and striatum are very sensitive to Fe deficiency, as itaffects the proper myelination of neurons and neurotransmitter function. Fe influ-ences myelination of frontal lobes throughout childhood. There is reasonable evi-dence for the beneficial effects of Fe supplementation on the cognitive performanceof older children. Zn plays a crucial role in DNA and protein synthesis and mayinfluence cognitive development, especially attention, activity, neuropsychologicalbehavior, and short-term memory (Bryan et al. 2004).

Folate, together with vitamin B12 and B6, share a metabolic pathway in methy-lation and synthesis of methionine in the central nervous system, so depletion ofthem may have implications for cognitive development, especially with respect tomemory performance and neural tube defects (Fleming 2001).

It has been proved that both n-3 and n-6 PUFAs influence memory, visual acuity,visual recognition, and mental development. Moreover, they may influence theoccurrence of ADHD, dyslexia, dyspraxia, and autistic behaviors.

Both undernutrition and micronutrient deficits are responsible for cognitive andbehavioral deficits in malnourished children (Wachs 2000). Therefore, nutrientcomposition and meal patterns can exert beneficial effects, mainly on the correctionof poor nutritional status. Even intelligence scores can be improved by micronutrientsupplementation in children and adolescents with very poor dietary status. It remainscontroversial whether additional benefits can be gained from acute dietary manip-ulations. Overall, the literature suggests that good and regular dietary habits arethe best way to ensure optimal mental and behavioral performance at all times(Bellisle 2004).

20.2 LIPIDS IN CHILDREN’S NUTRITION

20.2.1 ROLE OF LIPIDS IN CHILDREN’S NUTRITION

Fat, being the main source of concentrated energy in foods, is necessary in the dietof infants and young children due to their great energy needs. Lipids deliver abouthalf the energy in human milk and in most infant formulas. Fats also aid in theabsorption of fat-soluble vitamins and carotenoids. Lipids improve the taste of food,determine its texture and aroma, and play an important role in regulation of themotor activity of the gastrointestinal tract.

Approximately 98% of natural fats are triacylglycerols (TAG); the rest includefree fatty acids, monoacylglycerols, diacylglycerols, cholesterol, and phospholipids.TAGs serve as the main form of fat storage in adipose tissue, thus playing an



important role in thermoregulation. The stored fat also acts as a reserve of metabolicfuel for the body. Cholesterol is used as the precursor of steroids and in bile acidsynthesis.

Linoleic acid and α-linolenic acid, which cannot be produced by humans andtherefore must be delivered with food are important precursors in further metabolicchanges leading to formation of long-chain PUFAs (LC-PUFA)—arachidonic acid(AA) and docosahexaenoic acid (DHA)—which have high biological activities.LC-PUFAs are the precursors of prostaglandins, prostacyclins, and thromboxanesknown as eicosanoids. Different eicosanoids derived from LC-PUFAs play a cru-cial role in inflammatory and immune reactions, thrombocyte aggregation, andregulation of blood pressure. They may also have different effects on infant growth(Koletzko 2001). LC-PUFAs have received a good deal of attention as crucialmembrane constituents of the nervous system and retina (Lanting and Boersma1996). DHA is considered particularly important for brain function with respectto membrane fluidity and thickness and thus affects cell signaling (Uauy et al.2001). PUFAs are beneficial in the development of visual acuity and the cognitiveprocess in infants; some evidence also suggests that n-3 LC-PUFA may diminishthe symptoms of neurological disorders in older children (Bryan et al. 2004).Zhang et al. (2005) recently observed in schoolchildren that intake of PUFAs incontrast to cholesterol may be beneficial in terms of performance of the memoryand possibly reading ability. Lack of n-6 FA is manifested by growth retardation,desquamation and thickening of the skin, decreased skin pigmentation, muscularcontraction disorders, and increased susceptibility to infections. n-3 LC-PUFAdeficiency is the cause of brain dysfunction, manifested as learning and sleepdisorders, paresthesias, and abnormal visual function (Uauy et al. 2001). The extentof insufficient LC-PUFA supply in children’s food is unknown because the clinicalmanifestations occur only in extreme deficiency (Uauy et al. 2003).

Dietary lipids affect cholesterol metabolism and may play a crucial role indevelopment of cardiovascular diseases in later life. High intake of food lipidsmay contribute to obesity, atherosclerosis, and some types of cancer. Therefore,the main rationale for restricting the amount of fat during childhood is to preventthese disorders later in life. However, the results of some studies show that thereis no evidence that reducing fat intake during childhood protects against athero-sclerosis later in life. Moreover, young children who eat a fat-restricted diet appearto grow normally, but they are prone to consume insufficient amounts of manynutrients, especially Ca, Zn, Mg, phosphorus, vitamin E, vitamin B12, thiamin,niacin, and riboflavin (Olson 2000). Niinikoski et al. (1997) have proven thatmoderate restriction of fat intake to 25 to 30% of total energy is compatible withnormal growth.

The prevalence of obesity in children is increasing despite general awareness ofhealthy nutrition; it is considered now that this is due mainly to excessive saccharideintake and insufficient physical activity, rather than excessive fat consumption. Med-ical problems in obese children are common and concern mainly cardiovasculardiseases, endocrine system disturbances and mental disorders.



20.2.2 RECOMMENDED DIETARY INTAKE OF LIPIDS

The requirements for fat intake may be presented in different ways. Usually therecommended lipid intake is expressed as a percentage of energy intake, and is givenfor total fat, n-6 LC-PUFA, n-3 LC-PUFA, cholesterol, and saturated and trans fattyacids. Generally, the total fat intake is recommended as 30 to 35% of the total energyintake (Prentice et al. 2004).

High intake of fat in the first six months of life is important for providing anadequate supply of energy for the rapidly growing infant. The recommended fatintake in this period is based on Adequate Intake (AI), which reflects mean lipidsupplies in breast-fed infants (Food and Nutrition Board 2005). Assuming dailyhuman milk consumption of about 0.78 dm3/day in infants who are exclusively breastfed, and a mean content of 40 g fat per dm3, the AI for lipids is 31 g/day. As themean energy content of mature human milk is 2717 kJ/dm3, the dietary fat deliversabout 50 to 55% of the total energy to breast-fed infants. The proportion of totalenergy provided as fat subsequently decreases with a wider variety of foods in theinfant’s diet during the next six months. The AI for older infants is based on themean consumption of fat from human milk and complementary foods in 7- to 12-month-old children, and is calculated as 30 g/day of fat. It means that in this periodabout 40% of energy in the diet is delivered through fat.

According to the guidelines of the European Society for Pediatric Gastroenter-ology, Hepatology, and Nutrition (ESPGAN) Committee on Nutrition (Com. Direc-tive 1996), the total fat content in starting formulas should range from 4.4 to 6.6g/418 kJ and 3.3 to 6.5 g/418 kJ in follow-up formulas. The fat is usually a blendof several vegetable oils, predominantly soybean, safflower, sunflower, coconut, andpalm oil. These oils do not contain cholesterol, and therefore breast-fed infants upto four months of age receive more cholesterol than formula-fed babies due to itshigh concentration in human milk (30 mg/100 cm3). Cholesterol is not routinelyadded to infant formulas because the beneficial effects of this supplementation havenot been established.

Due to the very important role of PUFAs in infant diets, there is much interestnot only in the lipid amount, but also its composition, in infant formulas. Regardinghuman milk as a standard, the ESPGAN Committee has recommended inclusion ofLA and ALA in infant formulas. LA should supply 4.5 to 10.8% of total energy.Due to the competitive antagonism of the n-3 and n-6 acids, ratios of concentrationsof LA to ALA have been set at 5:1 to 15:1 (Aggett et al. 1991).

Premature infants have limited essential fatty acid stores and insufficient activityof elongase and saturase enzymes indispensable for the production of PUFAs. Thesepreterm babies have especially high requirements for LC-PUFAs because of rapiduse of these acids in the fast growing tissues, especially the brain. Therefore thesupplementation of infant formulas with LC-PUFA compared to human milk lipids(1% for n-6 LC-PUFA and 0,5% for n-3 LC-PUFA) is considered to improve thenutrient supply and to have beneficial effects on the early growth and developmentof formula-fed babies. At present there is no requirement for supplementation offormulas with LC-PUFAs, but it is recommended in preterm infants. Investigationsregarding the development of cognitive and motor functions, as well as growth rate,



in children up to 18 months have not shown any need to supplement the milkformulations with LC-PUFAs for healthy, full-term infants (Lucas et al. 1999).

For children after infancy the recommended daily fat intake is set at 3.1 to 3.3g per kg of body weight, which represents approximately 32% of the total energyrequirement. During the next years of life, fat consumption is gradually restrictedto 30% of the daily energy intake (Table 20.1).

20.2.3 UNDESIRABLE DIETARY FAT EFFECTS IN CHILDHOOD

Trans fatty acids, although unsaturated, mimic saturated fatty acids in biologicalactivity. They cannot be used to produce useful mediators due to their chemicalstructure, and may contribute to decreased LC-PUFA synthesis by impairment ofdesaturation and elongation of essential fatty acids during the perinatal period andchildhood. Large amounts of trans fatty acids incorporated into the cells create therisk of malformation of the cell membranes and other cellular structures. Trans fats,along with saturated ones, increase the concentration of low-density lipoprotein cho-lesterol (LDL) and lower the concentration of high-density lipoprotein cholesterol(HDL), thereby increasing the risk of heart disease in the elderly. Some studies havealso shown that high intake of trans fatty acids may be linked to a greater risk of type2 diabetes (Salmeron et al. 2001). Trans fats also have a detrimental effect on thenervous system; they are incorporated into the brain cell membranes and myelin, thusaltering the ability to transfer electric signals. The European Union has limited thecontents of trans fatty acids to 4% of total fat in foods for infants and young children(Aggett et al., 1991). In 2004, the U.S. Food and Drug Administration (FDA) FoodAdvisory Committee voted to recommend that trans fatty acid intake levels be reducedto less than 1% of energy. Most countries set the upper limit for the consumption ofsaturated fatty acids at 10% of energy intake (Prentice et al. 2004).

20.3 SACCHARIDES

20.3.1 THE ROLE OF SACCHARIDES IN CHILDREN’S NUTRITION

Saccharides, in the form of glucose, provide readily available energy to each cell ofthe body, particularly to the brain. Glucose is the brain’s only energy source. Thatis why glucose in the bloodstream has to be maintained at a certain constant level.

TABLE 20.1Dietary Reference Intake of Lipids

Range (percentage of energy)

Children1–3 years

Children4–8 years

Children9–13 years

Total fat 30–40 25–35 25–35n-6 PUFAS 5–10 5–10 5–10n-3 PUFAS 0.6–1.2 0.6–1.2 0.6–1.2



Several hormones, including insulin, regulate the flow of glucose to and from theblood to keep it at a constant level. In a low-saccharide diet, proteins are metabolizedto glucose in order to maintain its level. This means that these proteins are lessavailable for growth. Thus, saccharides in the diet have a protein-sparing effect. Adiet containing an optimum level of saccharides may help prevent body fat accumu-lation. Glucose can be converted to glycogen, which is stored in the liver and muscles,until the organism requires energy or until it is converted to some of the amino acidsused in the synthesis of proteins (Food and Agriculture Organization/World HealthOrganization [FAO/WHO] 1998).

Monosaccharides are absorbed directly by the small intestine into the bloodstreamwithout energy involvement. Disaccharides are broken down by the digestive enzymesand absorbed as monosaccharides. Lactose is the main sugar in both human milk andinfant formulas. Oligosaccharides are also broken down to monosaccharides prior toabsorption into the bloodstream. Some short-chain polysaccharides are resistant todigestion by the enzymes in the gut but are metabolized by colon microflora, thuspromoting the growth of Bifidobacteria and Lactobacilli in the colon (see Chapter 15).

Starch is readily digested. Nonstarch polysaccharides are the main componentsof dietary fiber. They include cellulose, hemicelluloses, pectin, and gums. Thevarious components of dietary fiber have different physical structures and properties.Dietary fiber helps to keep the bowel functioning correctly as fibers are not digestedin the small intestine, thus increasing the physical bulk in the bowel, stimulatingintestinal transit and protecting against constipation, irritable bowel syndrome, anddiverticular disease. Healthy polysaccharides are present in whole-grain cereals,brown rice, oatmeal, whole-grain breads, cereals, fruits, and vegetables. They aremetabolized to glucose slowly in the organism, thus the level of glucose is easy tocontrol. Healthy polysaccharides work quickly to aid satiety, therefore childrenconsuming diets high in healthy saccharides are less likely to overeat.

Very few dietary polysaccharides are converted to body fat mainly because thisprocess is very inefficient for the organism (Hellerstein et al. 1991). In addition tofiber, whole grains contain more essential fatty acids, vitamin E, Mg, niacin, thiamin,riboflavin, phosphorus, Fe, and Zn than their processed equivalents. Some whole-grain foods are folic acid fortified.

Polyols like isomalt, sorbitol, and maltitol are sweet, but cannot be used forinfant feeding due to their laxative effect.

20.3.2 THE GLYCEMIC INDEX

When a saccharide food is eaten, the glucose level in the blood rises depending ona metabolism rate, and subsequently decreases what is known as the glycemicresponse. The impact of different saccharide foods on the glycemic response of thebody is compared to a standard food like white bread or glucose and is known asthe glycemic index (GI).

There are a few factors influencing the glycemic response, such as the type ofsaccharide, the methods used for preparing food, the addition of other nutrients likefat or protein, and the individual metabolism of the organism.

It was believed that complex saccharide foods, such as bread, rice, and potatoeswere digested slowly, causing a gradual increase in blood sugar levels. It appeared



that many starchy foods, like rice, broke down quickly during digestion, and thishad the highest GI. They raise blood sugar levels higher and more quickly than foodswith a low GI. However, foods such as beans break down more slowly and have alow GI. Foods containing sucrose actually show a quite low-to-moderate bloodglucose response—lower than foods like rice. Foods with a low GI of less than 55(glucose as a standard 100) include noodles and pasta, lentil, apples and apple juice,oranges and orange juice, pears, grapes, low-fat yogurt, baked beans, and chocolate.Foods with an intermediate GI factor (55 to 70) include basmati rice, banana, softdrinks, sweet corn, pineapple, and white sugar. Foods with a high GI (greater than70) include bread (white or whole meal), baked and mashed potatoes, cornflakes,French fries, honey, and white rice (Foster-Powell et al. 2002).

A higher intake of low- rather than high-GI foods results in slower digestion ofsaccharides and slower absorption of sugar into the bloodstream. This in turn mayhelp to regulate blood-sugar levels and insulin concentrations, although long-termstudies on overall health benefits are not yet available. A diet that is composed largelyof saccharide-rich, low-GI foods also tends to be low in fat, which may benefitweight control. Hence it may have implications for diabetes, hypertension, andobesity. Food products having a high GI have an amount of carbohydrates in oneserving (glycemic load [GL]) of 20 or more. Foods with a medium GI have a GLranging from 11 to 19, while those with a low GI have a GL of 10 or less. Somefoods (i.e., carrots) have a high GI but low GL (Table 20.2).

20.3.3 RECOMMENDED DIETARY INTAKE OF SACCHARIDES

IN CHILDHOOD AND ADOLESCENCE

The recommended dietary intake of saccharides was defined in a few Europeancountries with a variation in reference intakes due to the methodology used, and isexpressed as a percentage of energy intake or as grams/day. A healthy balanced dietcontains 45 to 65% energy from saccharides (saccharose no more than 10%) and0.5 g/kg/d or 7 to 25 g (depending on age) of dietary fiber per day for children over2 years of age (Prentice et al. 2004).

TABLE 20.2Glycemic Index and Glycemic Load of Certain Foods

Food Glycemic index Glycemic load

Apple 40 6Baked potato 85 26Brown rice 50 16Carrot 92 5Corn flakes 92 24Orange juice 50 13Plain bagel 72 25Potato chips 54 11Pound cake 54 15Sucrose 58 6



Breast-fed infants receive less saccharides than infants fed with infant formulas(Alexy et al. 1999). Human milk contains lactose as a main disaccharide, but alsoa complex mixture of oligosaccharides in minute amounts (1.85 g/100 cm3) with amaximum level in the first week of lactation. The oligosaccharides may serve assubstrates for colonic fermentation with consecutive production of short-chain fattyacids, which are nutritive for colonocytes. Whether the addition of short-chaincarbohydrates to infant formulas and follow-up formulas would bring beneficialeffects is under investigation (European Commission—Scientific Committee onFood, September 2001).

Infant formulas contain 1.6 to 3.3 g/100 kJ saccharides (40 to 47%), includinglactose of at least 0.8 g/100 kJ, and saccharose up to 20%. Minute amounts of othersugars like maltose, glucose, fructose, dextrins, and starch are allowed, especiallyfor infants older than 6 months, having active pancreatic amylase (Table 20.3).

Up to 4 to 6 months of age, infants should be given human milk or infant formulasonly. Despite introducing other products, human milk or infant formula feedingshould be continued until 12 months of age. After 4 to 6 months of age, fruit juicesand vegetable soups are recommended, including sugars and fiber decomposed inthe cooking and smashing process. Fruit juices should be limited to 120 to 180 cm3

daily. Fruits and cereals with modified starch and fibers are introduced at 7 to 8months of age. In the 10th month of life, white bread, smashed vegetables containinggluten, and less modified starch enrich the menu. The introduction of natural cow’smilk should be delayed until 1 year of age; cow’s milk given to infants during thesecond year of life should not be defatted (FAO/WHO 1998, Briefel et al. 2004).

Children over 2 years of age should consume a minimum amount of fiber, ingrams equal to their age in years plus 5 to 10 g/day (Prentice 2004). In the absenceof a strictly defined daily requirement, a wide range of saccharide-containing foodsshould be eaten, so that the diet is sufficient in essential nutrients and dietary fiber.On average, most 2- to 3-year-old children need 154 to 182 g of grains per day;school-age children, about 170 to 227 g; and active teens may need as many as 255or 284 g. At least half of those servings should come from whole grains. The otherhalf can come from the more common enriched grains, such as enriched white flour(FAO/WHO 1998) (Table 20.4).

TABLE 20.3Carbohydrate Contents in Infant Formulas (g/100 cm3)

Products Saccharose Lactose Starch Other

Human milk 0 7.0 0 0Neonatal formula 0 5.3 0 2.6Starting formula 0 (max 30%) 6.5 (min 3.5) 0.1 (max 20%) 1.1Follow-up formula 0 (max 20%) 6.4 (min 1.8) 0.6 (max 20%) 0.7 (max 40%)

Fiber not present in all products

Source: Dir. European Commission (EC) 91/321-1991, http://www.meadjohnson.com/products/hcp-infant/prosobee.htlm).



There is no added sugar in 100% fruit juice, but the energy from the naturalsugars found in the juice add up to the daily energy intake. In order to avoid obesityin children, the American Academy of Pediatrics (AAP) recommends limiting juiceintake to 120 to 180 cm3 for children less than 7 years of age, and no more than 240to 350 cm3 for older children and teens (AAP 1998).

20.3.4 INAPPROPRIATE SACCHARIDE INTAKE

Childhood and adolescence are of equal importance, and in prevention, even moreimportant than maturity in the treatment and prophylaxis of obesity, hypertension,diabetes, and cardiovascular diseases.

The huge amount of refined, unhealthy carbohydrates in candy, soda, white rice,white flour, breads, pastries, cookies, cake, frozen desserts, and some fruit juiceseaten by children has led to a dramatic rise of obesity in recent years. Each 355-cm3 serving of a carbonated, sweetened soft drink contains the equivalent of 10teaspoons of sugar and 0.6 kJ. Sweetened drinks are the largest source of addedsugar in the daily diets of U.S. children. Consuming 355 cm3 of a sweetened softdrink daily increases a child’s risk of obesity by 60% (FAO/WHO 1998).

TABLE 20.4The Recommended Dietary Intake of Saccharides in Girls and Boys as Defined by the Mayo Clinic

Boys and girls, ages 2 to 4

Energy (kJ) 4250–5950, depending on age and activity levelDigestible saccharides 45% to 65% of daily energy intake (at least 130 g)Fiber 19 g a day


Energy (kJ) 5100–8500, depending on age and activity levelDigestible saccharides 45% to 65% of daily energy intake (at least 130 g)Fiber 25 g a day


Energy 6800 to 11050, depending on age and activity levelDigestible saccharides 45% to 65% of daily energy intake (at least 130 g)Fiber 26 g a day


Energy (kJ) 7650 to 13600, depending on age and activity levelDigestible saccharides 45% to 65% of daily energy intake (at least 130 g)Fiber 26 to 38 g a day

Source: Adapted from Mayo Foundation for Medical Education and Research(MFMER) July 20, 2005.



The contribution of different foods to the average daily nonmilk extrinsicsugar (NMES), institutes about 17% of food energy consumed by British school-children. The main sources of NMES among children are carbonated soft drinksand chocolate confectionery, with the portion provided by carbonated soft drinksincreasing with age, which is in reciprocal relationship with obesity (Hendersonet al. 2003).

It is thought that high insulin levels are one of many factors in the developmentof heart disease and hypertension. The consumption of a diet rich in low-GI foodswill help to hold down insulin levels.

Eating too many sugary foods can also lead to tooth decay, although the opinionis controversial in today’s fluoride- and oral hygiene-aware populations. Foodscontaining sugars or starch produce acids leading to demineralization of toothenamel. Saliva provides a natural mineralization, which rebuilds enamel. The morefrequently sugars are consumed, the longer the time during which the tooth isexposed to the low pH levels at which demineralization occurs. At alkaline pH levels,the rate of demineralization is much lower. When foods containing sugars areconsumed too frequently, this natural repair process is overwhelmed and the risk oftooth decay is increased.

With regard to dental health, research from recent years allows a more rationalapproach to the role of saccharides in dental caries. It is now recommended thatprograms to prevent dental caries focus on fluoridation, adequate oral hygiene, anda varied diet, and not on the control of sugar intake alone (FAO/WHO 1998).

20.4 PROTEINS

20.4.1 THE ROLE OF PROTEINS IN CHILDREN’S NUTRITION

Proteins in the diet provide amino acids for forming the body’s proteins, includingthe structural proteins for building and repairing tissues, enzymes, hormones, andantibodies for carrying out metabolic processes. A constant supply of proteins isessential in childhood in order to support growth.

The body cannot store amino acids, so it is constantly breaking down andsynthesizing proteins. This protein turnover must be constantly fueled by the diet.Adequate energy is also critical, as the lack of it causes proteins to be used as asubstrate for energy at a rate of 17 kJ per 1 g, rather than for synthesizing tissue. Itoccurs when the preferred fat and carbohydrate supply runs low.

The proteins present in human milk are casein and whey proteins, such as α-lactalbumin, serum albumins, lactoferrin, lysozyme, and immunoglobulins. Humanmilk casein is much better assimilated than bovine milk casein. It has a positiveinfluence on bowel motor activity, assimilation of Ca, and stimulation of Bifidobac-terium growth. The main whey protein, α-lactalbumin, contains all essential aminoacids. Lactoferrin has a beneficial effect on Fe assimilation and bacteriostatic activity(Raiha 1994). Besides stimulation of the immune system, lactoferrin also preventsthe growth of pathogens, exerts antibacterial and antiviral properties, controls celland tissue damage caused by oxidation, and facilitates iron transport. It is presentin both breast and cow’s milk, with concentrations in human milk being 5 to 10



times higher than in bovine milk. Both lysozyme and immunoglobulin A accumu-lated on the intestinal epithelium play a positive role in the immunity of bowelsupregulating the inflammatory response. The activity of lipase, amylase, α-1-anti-tripsine, and hormones (GH, IGF-1, GM-CSF, TGF-β) is much higher in humanthan in bovine milk, which plays a role in nutrition. In human milk, a minute amountof β-lactoglobulin is found, which is predominant in bovine milk.

20.4.2 RECOMMENDED DIETARY ALLOWANCES OF PROTEINS

IN INFANCY AND ADOLESCENCE

Human milk is the best food for human infants as it covers the recommended dietaryallowances (RDA) in all aspects, providing energy and components that favor growthand immunological protection.

Human milk contains from 2.2 g protein/100 cm3 in the first days of lactation,to 1.0 g/100 cm3 in the 2nd and 3rd months (mean 1.2 g/100 cm3), whereas the realamount of absorption is 0.7 g/100 cm3. So, breast-fed children obtain 1.6 to 1 g/kg/din the 1st month, 1 g/kg/d in the 3rd and 0.9 g/kg/d up to the 6th month of life. Ifextra meals are introduced it is even higher (1.2 to 1.4 g/kg/d from the 4th to the6th month) (Raiha 1994).

The safe level of protein intake for infants in the first year of life was decreasedby IDECG (1994) to 70% of that recommended by FAO/WHO/UNU (1985) (seeTable 20.5). However, the real consumption of proteins in Europe and North Americais far higher than recommended by WHO/FAO/UNU, i.e., 40 g/day (3.5 g/kg/d) at2 years, 3 g/kg/d (60 g/d) at 3 years and 100 g/d at 13–15 years (Prentice et al. 2004).

In 1994 the International Dietary Energy Consultancy Group (IDECG) con-cluded that the requirement was approximately 90 to 100 mg N/kg/day at all ages,in comparison to 120 mg N/kg/day introduced earlier (FAO/WHO 1985). TheIDECG report recommends that the 50% increase in the protein allowance forgrowth in the 1985 report, should be reduced to 24 to 48% during the first year(Dewey et al. 1996).

TABLE 20.5Safe Levels of Protein Intake for Infants

Age (months) Safe protein level (g/kg/d)

1994 IDECG 1985

0–1 2.7 2.54–5 1.3 1.96–9 1.1 1.7

9–12 1.0 1.5

Source: FAO/WHO (Food and Agriculture Organization/World HealthOrganization), 1985, Energy and Protein Requirements, WHO Tech-nical Report Series 724, WHO, Geneva; Scrimshaw, N.S., Waterlow,J.C., and Schürch, B., Eds., 1996, Eur. J. Clin. Nutr., 50, S1197.



A healthy, balanced diet containing 8 to 15% of kJ intake from proteins isrecommended, which is quite different than human milk values—5.5% (Prentice etal. 2004). This means that the protein intake of some children not being breast-fedis 3 to 4 times above the requirement. Whether it has any significance for health isunder investigation (Lambert et al. 2004).

The amino acid score (AAS) values in different types of infant formulas, follow-up formulas, and special formulas vary from 0.71 to 0.88. This means that they differslightly from the European Economic Community (EEC) recommendations (EEC1991). The essential amino acid content in human milk and standard proteins arealso slightly different (see Table 20.6).

Human milk, selected infant formula, and follow-up formula, differ in theircontent of essential amino acids. According to EEC recommendations, protein con-tent in infant formulas based on cow’s milk should range from 0.45 to 0.7 g/100 kJ,and in infant formulas based on partly hydrolyzed proteins from 0.56 to 0.7 g/100kJ, whereas follow-up formulas include 0.5 to 1.0 g protein/100 kJ. In modifiedinfant formulas there is 1.5 to 2.5 g of protein/100 cm3, so the daily intake of proteinis 2 to 4 g/kg (Raiha 1994).

Infant formulas should be enriched with taurine (at least 5.3 mg/418 kJ) and L-carnitine (1.2 mg/418 kJ), both important in lipid metabolism. Taurine is vital forthe development of the retina.

The differences in the efficiency of utilization of various proteins must be takeninto consideration. Formula-fed infants may have higher dietary protein requirementsthan those who are breast-fed. Protein allowances must also be increased for catch-up growth following episodes of malnutrition or infection in children of all ages.An additional intake of at least 30% is required for this purpose (Dewey et al. 1996).

TABLE 20.6Amino Acid Content in Human Milk and Standard Proteins

Essential amino acids

Standard protein (g/100 g of protein)

Human milk Chicken egg FAO

Arginine 3.8 6.2 3.7Histidine 2.5 2.3 —Isoleucine 4.0 5.9 4.0Leucine 8.5 8.6 7.0Lysine 6.7 6.4 5.7Methionine 1.6 3.4 2.2Phenylalanine 3.4 5.6 2.8Threonine 4.4 4.8 4.0Tryptophan 1.7 1.5 1.0Valine 4.5 7.0 5.0

Source: FAO/WHO (Food and Agriculture Organization/World Health Organi-zation), 1973, Energy and Protein Requirements, Technical report series 522,WHO, Geneva.



Comparisons show slightly higher recommendations for protein intake in child-hood and adolescence in Canada. The estimated American average requirements forproteins are lower: the daily intake for boys 9 to 13 years of age is 0.77; 14 to 18years, 0.75 g/kg; and girls 9 to 18 years, 0.73 g/kg (Petrie et al. 2004) (see Table 20.7).

In cases of allergies to cow’s milk and carbohydrate metabolism disturbances,soybean formulas for infants are applied. Due to the low methionine content insoybean protein, methionine is added to improve the amino acid composition of theformula. According to the recommendations of the EEC, soybean-based formulasshould contain methionine in at least in the same amount as in human milk(1.6 g/100 g) (EEC 1991). Possible influences of soybean formulas on childrengrowth and health is still under investigation, although no differences in growth anddevelopment have been observed so far.

20.4.3 INAPPROPRIATE PROTEIN INTAKE

A diet with a high amount of proteins has too much energy, resulting in weight gain,and development of obesity in the future. Infants with a high protein intake have anincreased glomerular filtration rate and renal size. A high level of amino acids inthe diet may stimulate secretion of insulin and IGF-1. This results in increased growthrate, muscle mass, and adipose tissue in infancy. No data are available, however, toconfirm the above hypothesis. It is essential to eat a well-balanced diet, but there isno need to go overboard on protein (Wharton et al. 2000).

Protein energy malnutrition (PEM) describes disorders occurring mainly indeveloping countries. It affects young children as a result of both too little energyand not enough protein in the diet. Deficiencies of protein or of one or more ofthe essential amino acids leads to reduced growth or loss of muscle mass inchildren. The two extreme forms of PEM are marasmus and kwashiorkor. Maras-mus follows a chronic deficiency of both protein and energy, and is characterizedby muscle wasting and an absence of subcutaneous fat. The child becomes severelyunderweight, very weak, and lethargic. Kwashiorkor occurs in older children ona diet composed solely of starchy foods, and is characterized by a deficiency ofprotein quantity and quality, which leads to malnutrition and edema. A child with

TABLE 20.7Comparison of Protein Intake Recommendations in g/kg/day in Europe and Canada, Compared to FAO/WHO Guidelines

Age 2 years 3 years 5 years10 yearsgirls/boys

15 years girls/boys 18 years

Europe 1.13 1.09 1.02 1/0.99 0.87/0.92 0.75Canada 1.16 1.16 1.06 1.01 0.95/0.98 0.88FAO/WHO 1.15 1.1 1 1 0.9/0.95 0.75

Source: Adapted from Prentice, A., Branca, F., Decsi, T., Michaelsen, K.F., Fletcher, R.J., Guesry, P.,Manz, F., Vidailhet, M., Pannemans, D., and Samartin, S., 2004, Brit. J. Nutr., 92, suppl. 2, S83–S146.



kwashiorkor is severely underweight, but this is often masked by edema causedby hypoalbuminemia. The hair is characteristically thin and discolored.

The differences in protein recommendations for any given population in differentcountries depends on geographical, environmental, genetic, and lifestyle factors, andshould be discussed at the regional level to optimize growth, development, and healthof children and adolescents.

Human milk represents standards of excellence impossible to imitate in allaspects in infant formulas. The functions of the human proteins present in humanmilk are of great benefit to infants and newborns due to their nutritive influence andimmunoenhancement. Children should be breast-fed whenever possible.

The problem of protein intake above the recommended daily requirements andthe immediate or long-term metabolic effects on growth, body composition, andadiposity cannot be ignored.

20.5 MINERAL COMPONENTS IN CHILDREN’S NUTRITION

20.5.1 THE ROLE OF MACRO AND TRACE ELEMENTS IN CHILDREN’S NUTRITION

Mineral components play an important role in the human organism. They are theingredients of soft tissues, bones, and body fluids, taking part in central nervoussystem and muscle activity and maintaining the acid–base equilibrium. Many activeforms of enzymes and hormones contain trace elements in order to possess biologicalactivity, and therefore microelements are sometimes called activators or biologicallyactive substances (see Chapter 4).

The depletion of mineral components may lead to clinical symptoms, which canbe prevented by supplementation of the missing substance. Physical growth andmental development in children may be compromised due to subclinical or apparentdeficiencies of macro- and micronutrients. Not all mineral components are consid-ered indispensable, and some of them may be even toxic.

20.5.2 CALCIUM

About 99% of total body Ca serves as a building material of bones and tooth enamel.The rest of this macroelement is present in blood, muscles, and other tissues, andplays an important role in the coagulation process as well as in muscle contractionand permeability of cellular membranes. It regulates heart rhythm, blood pressure,and absorption of vitamin B12.

Recent studies and dietary recommendations have emphasized the importanceof suitable Ca intake, especially in children undergoing rapid growth and mineral-ization of the skeleton during pubertal development. Epidemiological data obtainedin 2004 show great variations in Ca intake in children and adolescents in Europeancountries (Lambert at al. 2004).



Appropriate Ca consumption in children and during the maturing period isnecessary in calcification of the skeleton. Increasing peak bone mass may minimizethe risk of osteoporosis later in life (Miller and Weaver 1994). Correct concentrationof Ca also reduces the risk of heart disease, stroke, intestinal malignancies, andkidney calculus. A very low Ca intake can contribute to the development of ricketsin infants, especially those who receive a restrictive diet. Recent data also suggestthe possibility of an increased risk of bone fractures in children with low bone massdue to lack of Ca intake. Wyshak and Frisch (1994) report a positive relationshipbetween high cola consumption and increased frequency of bone fracture in children,yet it is uncertain whether it depends on the excessive phosphorus content in colaor a lack of dairy products, which are replaced by various beverages. In childrenwith chronic diseases that require steroid therapy, the risk of decreased calcificationof bones is much higher than in healthy children, although the benefits of increasingCa consumption in those patients remains unclear (Abrams 1995). Usually in medicalpractice these children are supplemented with additional doses of vitamin D in orderto increase Ca absorption from the intestine.

The main source of Ca in infant diets is human milk, or if a child is not breast-fed, infant formula. The data show that bioavailability of Ca from human milk isgreater than from cow’s milk. Relatively higher Ca concentrations are reached incasein hydrolysates and soy formulas. There is no research data justifying the useof high doses of Ca in the diets of full-term infants (AAP 1999). After infancy, thedevelopment of eating patterns that ensure adequate Ca intake is of great value.Therefore, children should be given a sufficient amount of milk and dairy products,as well as vegetables, because of their high Ca bioavailability.

Decreased Ca absorption may be due to lactose intolerance. In these cases,patients can drink only a small amount of fresh milk in order to avoid colic painand diarrhea, so that the main source of Ca in their diets are solid dairy productssuch as cheeses and yogurt. Consuming lactose-free preparations increases the riskof inadequate Ca intake because lactose facilitates its absorption.

20.5.3 MAGNESIUM

Mg requirements of infants, children, and adolescents are greater than that of adults,except pregnant and lactating women, those under stress, and those in a convalescentperiod. Infant requirements depend on the amount of maternal supply during preg-nancy, as well as the condition of the baby (premature infant, small for gestationalage, or born after a complicated gestation or delivery). The status of the digestivesystem and kidneys affects Mg status. Children with Crohn’s disease, celiac disease,and other chronic malabsorptive problems tend to present decreased Mg bloodconcentrations. Some medicines used in pediatric patients (antibiotics, diuretics,antineoplastic and immunosuppressive drugs) may also contribute to its deficiency,and supplementation should be instituted.

Signs of Mg deficiency in children include fatigue, weakness, memory andconcentration disorders, lack of coordination, excessive drowsiness, hair loss, syn-cope, stammering, nycturia, numbness, and seizures. Severe Mg deficiency can result



in decreased Ca and K concentrations in blood. Adequate supplementation is nec-essary to prevent blood hypertension and other circulatory diseases later in life.

Chocolate is the most desirable Mg source for children; an average chocolatebar of 100 g provides about 20% of the adult daily requirement.

20.5.4 ZINC

Zn is essential for childhood growth and development. A too-low supply of Zn maycontribute to delayed mental and physical development and reduced appetite (Sal-gueiro et al. 2002). Impaired cellular immunity and wound healing as well as skinlesions, which are easily infected by bacteria, may also be observed in patients withZn deficiencies. Due to its role in acute-phase inflammatory response and reducingantimicrobial resistance, Zn may promote recovery from severe infectious diseasesin the young. During the first year of life, the requirement for this element is relativelyhigh. Although human milk is a rich source of Zn with high bioavailability, theconcentration decreases with the duration of lactation and sometimes the infant’sneeds are not fully satisfied (Krebs and Westcott 2002).

20.5.5 IRON

Studies indicate that children with recognized hypochromic anemia in early child-hood are at risk for poor cognitive and motor development. There is also evidenceof behavior problems and minor neurological dysfunctions in such children(Grantham-McGregor and Ani 2001). Fe storage in newborns is usually sufficientfor the first 6 to 8 weeks in premature babies and for 12 weeks in full-term infants.Fe absorption from formulas or cow’s milk is 4 times lower than from human milk,mainly due to the higher content of casein and interactions with other componentsof cow’s milk. Cow’s milk also decreases the absorption of Fe from other dietarysources and may cause the presence of occult blood in the gastrointestinal tract,particularly in infants with an allergy to cow’s protein. Therefore, the exclusion ofcow’s milk in allergic patients in the first year of life is essential in preventinghypochromic anemia. All infants who are not breast-fed should receive Fe-fortifiedformulas appropriate for their age. Supplementation of formulas with Fe hasdecreased the frequency of anemia in formula-fed infants. Although human milk isan ideal source of nutrients for most infants, exclusive breastfeeding after six monthsof life puts infants at risk for Fe deficiency due to the reduced content of this element.In order to prevent hypochromic anemia, the AAP recommends daily Fe supplemen-tation in a dose of 1 mg/kg in infants older than 6 months. Gradual introduction ofsolid foods in the second 6 months of life should complement the breast-milk diet.

Not only infants are at high risk of developing Fe deficiency anemia; childrenat a low socioeconomic level, immigrants from developing countries, adolescents(particularly girls with excessive menstruation), patients with malabsorption syn-dromes, and those with recurrent bleeding, for example from the nose, also requireFe supplementation.



20.6 VITAMINS IN CHILDREN’S NUTRITION

20.6.1 INTRODUCTION

Vitamins make up a group of organic substances that are essential in small quantitiesfor normal metabolism in children and adults. They play an important role in proteinmetabolism and are necessary in almost all biochemical processes. They must bedelivered with food, and usually a healthy, varied diet can satisfy the body’s demandsfor them. Fat-soluble vitamins (A, D, E, K) can be stored in the liver and thereforethey are not needed every day in the diet; their overdosage may be toxic. Water-soluble vitamins include the B-complex group and vitamin C. They are not stored inthe body because of elimination in urine, so that intake must be assured every day.No symptoms of their excessive intake are known, except for folic acid and nicotinicacid. Water-soluble vitamins are easily destroyed during food preparation and storage.

20.6.2 VITAMIN C

Vitamin C is regarded as the most important water-soluble antioxidant in humans;it works with vitamin E as a free-radical scavenger, and thus plays a role in preventingneoplastic diseases and may assist in conventional chemotherapy of certain tumors(Goldenberg 2003). It is also vital for good functioning of the immune system, andtherefore in children with viral or bactericidal infections it is always used to supportantiinfectious therapy. It strengthens blood vessel walls, and it may be useful inpreventing hemorrhagic processes caused by increased permeability or fragility ofcapillaries. Vitamin C aids in wound healing and bone and tooth formation; it is alsonecessary for absorption of Fe from the intestines and for bile acid and steroidsynthesis. Insufficiencies may result in easy bruising, hemorrhagic diathesis, poorwound healing, and recurrent infections of the respiratory tract. Scurvy is the onlydisease known to be well treated with high doses of vitamin C.

20.6.3 VITAMIN B-COMPLEX

All vitamins from this group take part in the metabolism of carbohydrates, fats, andproteins. They are essential in maintaining the appropriate tone of digestive systemmuscles, as well as the good condition of nerves, skin, mucous membranes, hair,nails, and liver.

A long-lasting thiamine deficiency in infants may lead to irreversible brain dam-age, cardiac disorders, and failure to thrive. The classic thiamine deficiency in humansis beri-beri, characterized by anorexia with weight loss and neuromuscular abnormal-ities such as paresthesia, muscle weakness, and a tingling or burning sensation in thehands and feet. The disease is still common in Southeast Asia, where polished riceis a dietary staple, while in developed countries it is now rare due to the high amountsof thiamine in enriched cereal products. Fattal-Valevski et al. (2005) have reportedrecently on several infants in Israel in whom severe neurological and cardiac symp-toms were recognized, probably due to insufficient thiamin intake from soy-based



formulas. Riboflavin is delivered mainly through dairy products, meat, fish, and darkgreen vegetables. Poor riboflavin status interferes with Fe handling and may contributeto hypochromic anemia; deficiency is also a risk factor for cancer and cardiovasculardisease. Adequate riboflavin intake is important to assure appropriate developmentof the gastrointestinal tract as well as normal night vision (Powers 2003). School-children along with lactating women and their infants are at risk of low riboflavinintake, usually due to insufficient consumption of milk products.

Cobalamin, which is found only in animal foods and milk products, is essentialfor the production of red blood cells and genetic material in humans. It is also calledthe antistress vitamin because it enhances immune functions and improves the body’sability to cope with stressful conditions. Coppen and Bolander-Gouaille (2005)suggest that supplementation of both vitamin B12 and folic acid may improve treat-ment outcomes in depressive patients, yet there are no data for that use in children.Risk groups for cobalamin deficiency include vegetarian children and those withgastrointestinal diseases, especially with certain intestinal infections, such as tape-worm and, possibly, Helicobacter pylori. The lack of vitamin B12 is frequent ininfants of mothers with pernicious anemia caused by a lack of intrinsic factor, asubstance that allows absorption of this vitamin from the intestine. Clinical presen-tation of insufficient cobalamine intake in children may lead to degeneration ofperipheral nerves and other severe neurological symptoms, macrocytic anemia (acondition characterized by production of larger red blood cells with decreased abilityto carry oxygen), and cognitive impairment.

Folic acid has a similar positive influence on the production of red blood cells inbone marrow as cobalamine; therefore a deficiency may be presented in blood mor-phology in a similar way. Yet now the most widely recognized beneficial role of folicacid concerns pregnant and preconceptive women, because if taken before conceptionand during the first weeks of pregnancy, it reduces the risk of neural tube defects inneonates. Augmented intake of folic acid has also been discussed with respect towhether it may prevent cancer and cardiovascular diseases in adults (Staff et al. 2005).

Pellagra is a classic syndrome resulting from extreme niacin deficiency. It ischaracterized by digestive disturbances, weight loss, dermatitis in sun-exposed areas,glossitis, and abnormal mental functioning. It is rarely reported today in industrial-ized countries, although it may still affect people in China, India, and Africa or inregions where maize is a dietary staple. Maize contains niacin, but in a form disablingits absorption from the intestine. Secondary pellagra may also occur in children withprolonged diarrhea or liver cirrhosis. Due to the beneficial effect of niacin in reducingTAG and LDL while increasing HDL levels, it is useful in treatment of a wide varietyof lipid disorders (McKenney 2004).

Biotin deficiency in children has been documented during prolonged parenteralnutrition without biotin supplementation as well as in malabsorption syndromes orchronic diarrhea due to short bowel syndrome (Sikorska-Wiśniewska et al. 2004).There are also data suggesting that humans consuming high amounts of raw eggwhite may develop symptoms of vitamin H deficiency (Sweetman et al. 1981), whichis caused by the presence of avidin—a protein-blocking biotin absorption from theintestine. The clinical findings in children with biotin deficiency include alopecia,



dry and scaly skin, fatigue, nausea, ataxia, and developmental delay. Appropriatesupplementation of biotin resolves the clinical symptoms.

20.6.4 VITAMIN A

Vitamin A, and its precursor beta-carotene, are considered as natural antioxidants,so they may have a protective response against cardiovascular and malignant diseasesin the elderly. Some data show the synergistic action of beta-carotene, vitamin C,and vitamin E in protection of lipids in cell membranes. Vitamin A also plays animportant role in tissue regeneration, bone growth, tooth development, and repro-duction, as well as in enhancing immune functions in growing children. There arealso data on the beneficial role of carotenoids in patients with cancer, although someof them indicate increased incidence of lung cancer in adult smokers (Wolf 2002).Insufficient vitamin A supply mainly affects children from poor countries, in whichvitamin A deficiency is a major public health problem. Such depletion does not occurin Europe, although it also may be seen in malnourished children and those withsevere liver dysfunction, celiac sprue, or cystic fibrosis. Clinical symptoms of a milddeficiency of beta-carotene include night blindness and diarrhea, particularly com-bined with intestinal infections. Severe depletion may cause keratinization of theskin and blindness in children. Excessive vitamin A consumption in children maylead to chronic or acute toxicity. Acute toxicity manifests itself as increased intrac-ranial pressure, which may mimic a cerebral tumor. Chronic toxicity develops withina few weeks and may appear as hair loss, growth retardation, enlargement of theliver and spleen, and generalized weakness and arthralgias.

20.6.5 VITAMIN D

Although classified as a vitamin, vitamin D (cholecalcyferol) should rather be con-sidered as a prohormone, due to its high production in humans through the conversionof skin 7-dehydrocholesterol to vitamin D upon exposure to ultraviolet-B radiationfrom sunlight. The active metabolite, 1,25(OH)2D, plays a critical role in the body’shomeostasis of Ca and P. It increases Ca absorption in the intestine and kidney, andtriggers osteoclastic activity in the bones. A proper supply of vitamin D is especiallyessential in infants and small children, and protects them from failure of bonemineralization. A deficiency of vitamin D in this group of patients may lead torickets, the consequences of which may persist until the end of life. In adults, vitaminD deficiency causes osteomalacia, a condition in which bone mineral componentsare progressively lost. It may also lead to muscle weakness and pain.

Exclusively breast-fed infants who do not receive sufficient supplementation ofvitamin D are at high risk of deficiency, particularly when they are not exposed tosunshine. Such sun avoidance in infants less than 6 months old is recommended by theAmerican Academy of Pediatrics (Pettifor 2005). The risk of vitamin D deficiency alsoconcerns people with dark skin who produce lesser amounts of cholecalcyferol, andchildren and adults with fat malabsorption syndrome and inflammatory bowel diseases.

As the dietary content of vitamin D is generally insufficient to prevent a defi-ciency, infants and small children should receive fortified foods, which is particularly



important for those who are exclusively breast-fed. Formula-fed infants do notrequire an additional supply of vitamin D due to fortification of the milk.

Vitamin D toxicity may affect children who are treated with high doses of thevitamin without controlling serum Ca levels. In the past it was more frequent thanit is now because very high doses of vitamin D were administered in infants withrickets. Anorexia, nausea, and vomiting, followed by polydipsia, polyuria, irritability,muscular weakness, and impaired renal functioning are the first symptoms of chole-calcyferol overdosage.

20.6.6 VITAMIN E

Alpha-tocopherol is the most active form of vitamin E in humans. It acts as a powerfulantioxidant, protecting cell membrane lipids from destruction by free radicals, aswell as lipids in LDLs from oxidation. Oxidized LDLs are considered as importantfactors of cardiovascular diseases. Vitamin E has also been shown to decrease theaggregation of platelets and to enhance vasodilatation. Vitamin E deficiency is rarelyobserved in humans; it may appear in individuals with severe malnutrition and fatabsorption disorders. Children with cystic fibrosis and chronic cholestatic liverdiseases (who have an impaired ability to absorb fat, and therefore also fat-solublevitamins), may develop clinical symptoms of vitamin E deficiency. Supplementationis an important part of routine therapy in these children. A lack of vitamin E resultsmainly in neurological disorders: impaired balance and coordination as well assensory nerve injury, muscle weakness, and damage to the retina. In extremely severecases, vitamin E deficiency may cause an inability to walk.

Healthy individuals who eat a balanced diet rarely need supplements. VitaminE supplementation is reported to be beneficial in patients with chronic inflammatory,cardiovascular, and neoplastic diseases, as well as in individuals with impairedcognitive function. It is also established that vitamin E supplementation in preterminfants with very low birth weight decreases the risk of conditions characteristic ofthis special group of patients: intracranial hemorrhage, severe retinopathy, and blind-ness (Brion et al. 2003). Vitamin E overdosage may lead to increase tendenciestoward bleeding, tiredness, and impaired immune functioning. In preterm infants itincreases the risk of sepsis (Brion et al. 2003).

20.6.7 VITAMIN K

Vitamin K is delivered to the human organism through diet (vitamin K1, phytonadi-one) as well as being produced by bacteria in the intestines (vitamin K2,menaquinone). It plays a crucial role in normal blood clotting—controlling the for-mation of coagulation factors II, VII, IX, and X in the liver. Vitamin K is also involvedin bone metabolism, regulating its formation and repair functions and subsequentlyin the prevention of osteoporotic fractures (Ryan-Harshman and Aldoori 2004).

Vitamin K deficiency mainly affects newborns in the first days of life because lipidtransport through the placenta is relatively poor, and because their intestines are sterile,so the synthesis of menaquinone is impossible. Exclusively breast-fed babies are atmore risk of vitamin K deficiency due to the very small amount of vitamin K in human



milk. The clinical picture of vitamin K deficiency (called hemorrhagic disease) of thenewborn occurs usually in the first week of life and is manifested by umbilical,cutaneous, and mucosal bleeding, and in the most serious cases by intracranial hem-orrhage. Later forms of hemorrhagic disease affect infants aged 2 to 6 months, espe-cially those with malabsorption syndromes, liver diseases, and those breast-fed babieswhose mothers are taking antibiotics, anticoagulants, or anticonvulsants. In olderchildren, vitamin K deficiency may also be caused by marginal dietary intake, pro-longed parenteral nutrition, or broad-spectrum antibiotic therapy. Easy bruising andbleeding from nasal mucosa and in the gastrointestinal tract and genitourinary systemin that group of patients may suggest vitamin K deficiency.

According to the recommendations of American Academy of Pediatrics (1993),all breast-fed infants should receive 1 mg of vitamin K intramuscularly in the firstday of life.

The only toxic form of vitamin K is menadione, a synthetic analog of vitamin K.It can cause hemolytic anemia, jaundice, and severe neurological problems. Therefore,this form of vitamin K may be used only in proper doses in medical treatment.

20.7 FEEDING LOW-WEIGHT PRETERM INFANTS—A CHALLENGE FOR NEONATOLOGISTS

Over the past two decades, attention has been directed toward improving the nutritionof immature preterm infants. In premature infants, gastroesophageal reflux occursdue to lower esophageal sphincter tone and immature neural regulation, which iswhy overall duodenoanal transit is prolonged (Berseth 2001). Insufficient motorfunction of the gut seems to be the major problem in extremely low-weight children.New techniques for feeding of low-weight infants (those with no sucking reflex)have been established, such as central line total parenteral nutrition (TPN), enteralfeedings, such as bolus feeds or continuous infusions by orogastric and transpylorictubes. Enteral feeding, especially fortified breast milk or preterm formulas, even inminute amounts, is the method of choice as it triggers maturation of the motorfunction of the gut and hormone release. On the contrary, an increased volume offeeding (more than 25 cm3/kg/d) may provoke necrotizing enterocolitis (NEC) inlow-weight preterm children. Human milk has many benefits for preterm infants: itis better tolerated and the risk of NEC is lower (Berseth 2001, Fewtrell 2002).

Human milk may not meet the requirements of preterm infants. The recom-mended intake of energy for preterm infants is 460 to 502 kJ; proteins, 3.6 to 3.8g/kg/d; and carbohydrates, 3.8 to 11.4 g/kg/d due to their increased growth rate.Human milk should be fortified with phosphate and calcium to prevent the devel-opment of metabolic bone disease resulting in reduced height and low peak bonemass. Fortified breast milk is viewed as adequate for children, but it is known thatbreast-fed infants grow more slowly and have lower bone mass than formula-fedinfants (Fewtrell 2003).

A higher energy intake (up to 480 to 520 kJ/kg/d (400 to 440 parenterally) andprotein (up to 4 g/kg/d) is advised on an individual basis in cases of insufficientgrowth (Denne 2001).



The nutritional components of fortified milk and preterm infant formulas differfrom milk for mature infants; they have more energy because of the addition ofproteins, carbohydrates, and fat (Table 20.8).

Choosing the proper type of feeding—fortified breast milk or special formulas—depends on infant maturity and daily weight gain. Infant maturity involves adequatemucosal and motor function of the gastrointestinal tract. Most enzymes are presentby the second trimester; however, lactase does not appear until the 34th week.

The addition of LC-PUFAs to preterm infant formulas is still being debated.Preterm infants fed on formulas have lower LCPUFA in the phospholipids and redcells. ESPGAN recommends enriching low-birth-weight infant formulas withdocosahexaenoic acid and arachidonic acid at least to the levels found in humanmilk due to a reduced ability to synthesize LC-PUFAs (ESPGAN 1991). However,the addition of n-3 fatty acids may be associated with growth delay lasting longerthan supplementation. Its significance for future development is controversial (Few-trell 2002).

Assuming the use of special nutrient-enriched postdischarge formulas enrichedwith proteins, vitamins, phosphorus, Ca, Zn, trace elements, and a modest increasein energy are essential for feeding low-weight preterm infants up to 9 months. Breast-fed preterm infants may also benefit from nutritional supplementation (Fewtrell 2003,AAP 1998).

20.8 VEGAN DIET—IS IT REALLY ADEQUATEFOR CHILDREN AND ADOLESCENTS?

A vegan diet includes only plant foods—grains, vegetables, fruits, nuts, seeds, andvegetable fats. The nutritional content of vegan food is usually sufficient, with theexception of Ca, Fe, iodine, and vitamin B12, and may result in decreased bonemass, anemia, hypothyroidism, and pernicious anemia. However, vegan diets have

TABLE 20.8Nutritional Content of Milks and Formulas for Preterm Infants (in 100 cm3)

Mature breast milk

Fortifiedbreastmilk

Preterm infant formula

(Cow and Gate Nutricia)

U.K.post-discharge

formula (PremCare)

Term formula

Energy (kJ) 1207 1470 1384 1240 1157Protein (g) 1.3 2.5 2.4 1.85 1.4Carbohydrates (g) 7 9.7 7.9 7.2 7.5Ca (mg) 35 112 108 70 53Na (mg) 15 37 41 221 19LCPUFA + + + + +

Source: After Berseth, C.L., 2001, Semin. Neonatol., 6, 417–424; Fewtrell, M.S., 2003, Semin. Neonatol.,8, 169–170; and Tormo, R., Potau, N., and Infante, D., 1998, Early Human Development, 53, Suppl,165–172.



a lower level of total fat, saturated fat, and cholesterol, which reduces the risk ofadult diseases.

The recommended intake of amino acids for vegan children is higher than thatfor nonvegans, but some essential amino acids may be lacking (Table 20.9). More-over, plant food protein is 85% digestible. For example, grain protein has a lowlysine but high methionine level, while white bean protein has limited methioninebut more than enough lysine. When both are eaten together, the essential aminoacids are complete. Soy is very rich in protein. Soy milk contains almost twice asmuch protein per calorie as cow’s milk, and about five times as much as human milk(Young and Pellett 1994).

Ca intake of vegan children is 39 to 48% of current recommendations, which maylower bone mass and increase the risk of fractures (Sanders 1992). Ca absorption fromvegetables (broccoli, turnip greens, spinach, dried figs) is higher than from milk. SomeCa-fortified foods, such as orange juice, soymilk, and apple juice may be advisablefor vegan children. Soy isoflavones stimulate bone growth (Ishida et al. 1998).

The low iodine levels in many plant foods reflects the low iodine levels in thesoil. The recommended level of 150 micrograms per day is usually unmet in veganchildren, which is why iodized salt and iodine-rich seaweeds should be included inthe vegan diet.

Vitamin B12 cannot be found in plants, so a vegan diet should contain somevitamin B12 -fortified foods, such as breakfast cereals, yeasts, and fortified soymilkto prevent pernicious anemia.

Mean Fe intake in vegans is above recommendations due to its high content inplants, but the nonheme form of Fe is less easily absorbed (Hunt and Roughead1999). Vitamin C is necessary for the absorption of Fe, and its level is usually highin a vegetarian diet. The rates of anemia are not higher among vegetarian childrenthan their “all food” peers.

Vegetarian children consume less fat than omnivore children. The n-3 fatty acidsmust be taken into consideration for younger children because growing brain and eyesneed more of these nutritious fats. Short-chain n-3 fatty acids can be found in soybeans,

TABLE 20.9Protein Recommendations for Vegan Compared with Nonvegan Children (boys/girls)

Age (years)Suggested range for

proteins (g/kg)Recommended protein intake for vegans (g/d)

Recommended protein intake for nonvegans

(g/day)

1–2 1.6–1.7 18–19 132–3 1.4–1.6 18–21 164–6 1.3–1.4 26–28 24

7–10 1.1–1.2 31–34 2811–14 1.1–1.2 50–54/51–55 45/4615–18 1.0–1.1/0.9–1.0 66–73/50–55 59/44

Source: Adapted from Messina, V. and Mangels, A.R., 2001, J. Am. Diet. Assoc., 101, 6, 661–669.



walnuts, avocado, green leafy vegetables, and vegetable oils. Vegan children have highintake of linoleic compared to linolenic acid, so it is crucial to enrich the diet with n-3s to enhance conversion of linolenic acid to DHA (Messina and Mangels 2001) (seeTable 20.9).

Regular exposure to sunlight is enough for vitamin D production, but sometimesvitamin D–fortified foods are suggested. Rickets may occur in macrobiotic childrensuffering from a lack of sunlight (Dagnelie et al. 1990).

The bioavailability of Zn found in whole grains, wheat germ, nuts, and fortifiedcereals is reduced by the presence of phytate. Attention should be focused onproviding children with Zn-rich foods like nuts and legumes.

The American Health Foundation recommends a higher fiber intake for veganchildren than for nonvegan (age plus 5 to 10 g per day) (Wiliams and Bollella 1995).Usually fiber intake in vegan children exceeds suggested amounts. Its negativeinfluence on health is not known, but in some cases, a low-fiber diet may be beneficial(refined grains, fruit and vegetable juices).

Special attention must be focused on teenagers to ensure a proper nutritionalintake, in light of their disordered eating behaviors. According to the AmericanDietetic Association and the American Academy of Pediatrics, a properly plannedvegan diet can support normal growth and development in children if it is enrichedwith fortified food (American Dietetic Association 1997, American Academy ofPediatrics 1998).

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517

Index

A

Acesulfame K, 365Acetaldehyde, 296, 299, 300,312, 343, 470Acetylation, 115, 288, 460Acetyl-3-hydroxyfuran, 109Acid casein, 150Acid,

acetic, 8, 105, 114, 169, 299, 300, 312, 316acetylneuraminic, 133acrylic, 124, 352adrenic, 196alginic, 81, 123aminobutyric, 428aminolevulinic, 87apocarotenic, 251arachidic, 184arachidonic, 431, 443, 491, 510ascorbic, 5, 10, 24, 65, 70, 81, 91aspartic, 131, 200, 428behenic, 194benzenecarboxylic, 82benzoic, 81, 360, 362, 363betalamic, 267brassidic, 185butyric, 299, 312,313, 326, 393caffeic, 470capric, 184caproic, 184caprylic, 184carboxylic, 103, 273, 344carminic, 268cerotic, 184cetoleic, 185chloroacetic, 115chlorogenic, 265cis-parinaric, 134citric, 26, 139, 147, 362, 429dehydroascorbic, 144, 168, 348, 353dihomo-γ-linolenic, 186docosahexaenoic, 341, 437, 442, 491eicosapentaenoic, 443elaidic, 185, 192erucic, 185, 194erythronic, 107, 108ethylenediaminetetraacetic, 70, 91ferulic, 315

folic, 386, 431, 432, 437, 446–448, 494, 505, 506, 513, 515

formic, 166gadoleic, 185galactaric, 109galacturonic, 109gallic, 362, 363gluconic, 104glutamic, 75, 80, 82, 131, 344, 349, 428glyceric, 107glyoxalic, 107, 108gondoic, 185hexanoic, 307hialuronic, 96hydroxy, 353hydroxycarboxylic, 366hydroxytricarballylic, 80ioleocapric, 185lacceric, 184lactic, 117, 164, 169, 352lauric, 184, 343lauroleic, 185lignoceric, 184linderic, 185linoleic, 19, 138, 303, 306, 317, 319, 321, 323,

328, 491linolenic, 138, 317, 449, 512

α-linolenic, 19, 138, 186, 442, 444, 491γ-linolenic, 186

malic, 26, 121margaric, 184mead, 186melissic, 184montanic, 184mucic, 109myristic, 184nervonic, 185nitrous, 170, 361nordihydroguaiaretic, 71

α-linolenic, 138, 442, 444, 491obtusilic, 185oleic, 182, 185, 187, 196, 202oleomyristic, 185oleopalmitic,185oxalic, 26palmitic, 184. 201, 202pelargonic, 184



petroselinic, 185phosphoric, 99, 119, 123, 133, 169physeteric, 185propionic, 299pyroligenious, 312ricinoleic, 314salicylic, 362sorbic, 287, 358, 360–362, 372, 373, 388stearic, 184, 187, 202stearidonic, 186sulfobenzoic, 365tartaric, 26thiobarbituric, 71thiolactic, 312timnodic, 196trans fatty acids, 4, 492, 493tzuzuic, 185uric, 11, 69uronic, 115vaccenic, 185, 187, 188valeric, 184

Acidulants, 314, 357, 359, 367, 368, 372Acrylamide, 13, 14, 352, 353, 482–484Actin, 17, 164, 165, 172, 225, 345Actomyosin, 143, 355Acylation, 102, 103, 130, 163, 169, 170Additives, 1, 5, 6, 12, 14, 21, 26, 50, 67, 68,

75–77, 80, 82, 83, 88–92, 116, 121, 123, 138, 141, 167, 258, 272, 273, 276, 286, 287, 291, 331, 333, 227, 344, 351, 355, 357, 359, 361, 363–365, 367, 369, 371–373, 375, 377, 378, 381, 384, 386, 388, 390, 430, 452, 459

Adenine, 70, 257, 459Adenosine diphosphate, 11Adipates, 116Adsorption isotherm, 46Aflatoxins, 462Agar, 95, 232, 365, 397Alanine, 131, 280, 350Albumin, 70, 125, 132, 142, 225, 232, 233, 279,

282, 285, 289, 293, 335Alcalase, 150Aldehydes, 71, 94, 115, 116, 297, 299, 300, 303,

305–308, 312, 313, 319, 336, 339, 342, 349, 351, 364, 365, 372

Aldoses, 94, 102, 109Aldosylamines, 102Aldosylamino acids, 102Aleurone layer, 22, 117Alginates, 5, 72, 95, 114, 122, 257, 346, 347, 365Alkaloids, 2, 263, 341, 430, 462Alkylation, 130, 168Alkylpyrazines, 108

Allene oxide, 304Allergen cross-reactions, 275, 289Allergenicity, 275, 281, 285, 287, 289, 292, 294,

388Allergens, 12, 14, 154, 277–294Alliin, 302Allspice, 296Allyl disulfide, 312Allylthiocyanate, 303Aluminum, 61, 75–79, 82, 88, 89, 268Amino acid sequences, 280Amino acids, 2. 4–8, 12, 39, 63, 75, 93, 102, 121,

122, 125, 127, 130, 131, 138, 161, 270, 271, 280, 296, 301, 305, 308, 313, 328, 341, 342, 363, 365, 427–429, 432, 465, 466, 468, 494, 498, 500, 501, 511, 514

essential, 429, 498, 500, 501, 511 flavor from, 8, 122, 305

Ammonia, 10, 44, 102, 121, 268, 270, 305, 313, 400

Ammonolysis, 103Amylases, 22, 298Amylopectin, 96, 99, 100, 111, 113, 116, 118,

119, 122, 124, 226, 237, 247Amylose, 94, 96, 98–100, 111, 113, 116, 118,

119, 124, 125, 226, 237, 239, 242, 337, 347

Anhydrosugars, 105Anise, 290, 296Anaphylactic reactions, 287, 291Annatto, 251Antithixotropy, 217Anthocyanins, 8, 245, 246, 260–265, 274, 341,

446Anthraquinone, 268Antiadhesive activity, 391, 405, 406Antibiotic, 291,294, 392, 395, 406, 414, 509Antibleaching agent, 80Antibodies, 276, 278, 280, 288, 291, 292, 417,

419, 420, 498Antibrowning agent, 83Anticlotting agents, 359Antiestrogenic activity, 475Antifoaming agents, 359Antifreeze proteins, 5, 329, 333, 353Antigen, 278, 281, 282, 291, 292, 419Antigen-presenting cell, 278Antimutagenicity, 484Antioxidants, 3, 14, 26, 67, 70, 72, 109, 139, 153,

159, 172, 180, 252, 264, 267, 271, 274, 287,306, 311, 323, 339, 342, 346, 351, 354, 357–359, 361–363, 372, 386, 430, 439, 445, 448, 465, 471, 478, 480, 484, 507


Index 519

Antiviral properties, 151, 498Apocarotenoids, 246, 252Appotransferrin, 70Aquaporins, 44, 59Arabinofuranosyl, 304Arabinogalactan, 96, 338Arabinose, 95, 241, 261Arginine, 131, 342, 447, 464, 482, 500Aroma, 123, 287, 298, 300, 303, 330, 490Arsenic, 3, 51, 55, 61, 75, 79, 84, 88, 89, 91, 469Arsenobetaine, 84, 89Arsenocholine, 84, 89Arthus reactions, 277Arylhydrazones, 102Ascorbate, 70, 80, 82, 171, 259, 339, 343, 346,

350, 361, 385, 465Asparagine, 131, 335, 352Aspartame, 105, 123, 126, 286, 287, 364, 365,

371–373, 429Aspartic protease inhibitor, 279Atherosclerosis, 267, 439, 440, 445, 447–450,

491Atopic reactions, 277ATP, 11, 43, 68, 257, 333, 345, 429, 441

B

B vitamins, 21, 430, 448Baking, 110, 122, 237, 273Baking powder, 79Barium, 51, 55Batochromic shifts, 263Benzaldehyde, 312, 313, 315, 316, 320Benzoate, 80–82, 287, 371Betalains, 8, 245, 246, 265, 267, 274, 341BHA, 362, 363, 372BHT, 180, 363Bile acids, 396, 445Biochemical oxygen demand, 56Biohydrogenation, 187, 188, 192, 193Bisulfite, 56, 341, 342, 360Bixin, 246, 249 251Bleaching, 78, 83, 251, 340Blocking agents, 78, 83, 251, 340Bloom, 165, 349Borneol, 300Boron, 66, 189Botulism, 49, 381Bound water, 38, 42, 43, 45Bowman-Birk trypsin inhibitor, 132Bromine, 192Browning of food, 102Buffering agents, 67Bulk-phase water, 39, 98, 299, 301

Butterfat, 313, 323Butyrolactone, 109

C

Cadmium, 51, 53, 61, 63–65, 74, 75, 85, 86, 88, 89, 469

Caffeine, 430, 470Calcium, 10, 17, 19, 55, 62, 65–68, 77, 79–88,

90, 122, 136, 137, 145, 150, 164, 172, 175, 201, 281, 502, 509, 512, 514–516

Calpains, 164, 165, 333Cancer, 62, 68, 89. 138, 193, 207, 253, 255, 258,

264, 371, 383, 384, 389, 391, 393, 395, 396, 405, 407–499, 414, 415, 418, 420, 450–456, 459–462, 464–474, 476–485, 491, 506, 507

Canthaxanthin, 246, 248, 251–253, 340Capsanthin, 248, 250, 251, 340Capsorubin, 250, 251Caramel, 95, 108, 121, 122, 245, 270, 305, 429Caraway, 290, 296Carbohydrates, 67, 93, 94, 100, 113, 117, 119,

122, 124, 125, 128, 257, 271, 281, 289, 307, 313, 369, 371, 373, 407, 408, 411, 429, 495, 514

Carbon dioxide, 51–53, 56, 69, 324Carbonate, 80, 81, 107Carbonyl compounds, 168, 169, 175, 307, 319,

322, 331, 343, 360Carboxylic groups, 114Carboxymethyl cellulose, 115, 123, 145Carcinogens, 188, 361, 379, 390, 393, 415,

451–456, 459–462, 464–466, 468, 469, 472–474, 476, 477, 480, 483–485

Cardioprotective effects, 446Cardiovascular diseases, 446, 491, 497, 506, 508Carotene, 178, 246, 248–255, 436, 442, 444, 476,

507Carotenoids, 5, 8, 19, 178, 203, 245–257, 274,

298, 329, 338–341, 351, 354, 490, 507

Carrageenans, 72, 96, 115, 122, 123, 225, 335,, 365

Caryophyllene, 312Caseins, 19, 136, 149, 164, 167, 170, 278, 332Castor oil, 314Catalase, 70, 159Catechins, 265, 446Cathepsin D inhibitor, 279Cathepsins, 164, 165, 345Cationic dyes, 273



Celiac sprue, 65, 152, 507Cellobiohydrolase, 118Cellobiose, 94, 118Cellulose, 21, 24, 26, 45, 65, 94, 96, 98, 100, 105,

111, 114, 115, 118, 122–124, 145, 179, 225, 227, 338, 344, 365, 494

acetate, 115Ceruloplasmin, 70, 71Chelating agents, 70, 71Chinese restaurant syndrome, 287Chinons, 26Chirality, 93, 100Chitin, 94, 95, 99, 109, 114Chitosan, 94, 95, 109, 125

19, 37, 55, 66, 71, 72, 80, 81, 86, 90,107, 124, 153, 189, 469,

Chlorination, 56, 105, 364, 473Chlorine, 102Chlorogenoquinone, 265Chlorophyll, 158, 245, 249, 255–258, 274, 340,

351, 474, 476Cholemyoglobin, 259Cholesterol, 21, 59, 68, 138, 178, 181, 192, 264,

385, 396, 490–493, 511, 514 Choline, 6, 21Chromium, 10, 51, 55, 62, 66–68Chromoproteins, 156Cinerarin, 263Cinnamaldehyde, 310Citral, 312, 318Citrate, 71, 80, 153Citronellol, 300, 304, 312CLA, 188, 189, 192, 193, 203Clarifying agents, 357, 359, 367Clathrate hydrates, 40Coacervation, 124Coalescence, 146, 147, 335, 366, 367Cobalt, 66, 68, 354Cochineal, 268, 272Cocoa butter, 203, 249Cohesion, 34, 348Collagen, 17, 20, 132, 139, 145, 154, 155, 168,

225, 233, 234Colorants, 1, 5, 26, 93, 102, 121, 123, 245–249,

251–253, 257, 259, 261, 263–274, 357, 359, 363

Compartmentalized water, 42Complexing agents, 116, 359Conformation, 4, 16, 39, 43, 94, 98, 129, 130,

133–137, 146, 153, 158, 169, 224, 227, 247, 285, 287, 332, 351, 365,

Coniferaldehyde, 315Conjugated linoleic acid, 138, 177, 187, 188, 207Contaminants, 6, 9, 12, 63, 73, 83, 92, 100, 284,

372, 376, 389, 482

Cooking, 3, 1, 65, 73, 74, 91, 96, 110, 141–143, 148, 152, 154, 165, 287, 295, 298, 306–308, 313, 325, 333,339, 340, 350, 368, 377, 385–387, 447, 460, 461, 465, 468, 469, 481, 484, 485, 496

Copigmentation, 263, 264, 274Copper, 10, 51, 62, 64–66, 69–71, 74, 85, 88. 91,

257, 354, 355, 427, 434, 474Coprecipitation, 124, 150Cornstarch, 119, 123, 127Costamers, 17Cow’s milk allergens, 275, 278Creaminess, 336, 368Creaming, 145, 146, 349, 367Creatine, 447, 466Creatinine, 466, 469Creep recovery, 209, 218, 220, 221, 240Crocetin, 251Crocin, 251Cross-linking, 72, 116, 123, 132, 139, 141, 143,

153–155, 157, 158, 162, 163, 167–170, 233, 287, 329, 330, 334, 335, 337, 338, 344–351, 353, 347

of proteins, 163, 167Crude protein, 2, 3, 16, 23, 346Crustacea allergens, 275, 281Cryoprotectants, 148, 149, 172, 337Cryptoxanthin, 249–251, 253Crystallization, 25, 96Curcumin, 245, 268, 269CVD, 439–442, 444–446, 448Cyanide, 51, 385Cyaidin, 261, 264Cyclamates, 105, 123Cyclization, 108, 109, 246Cyclodextrins, 118, 124, 127Cycloglucans, 118Cyclooxygenase, 443Cysteine, 64, 70, 75, 131, 159, 165, 279, 284, 285,

302, 305, 320–322, 336, 342, 351, 446

protease inhibitor, 279Cytochrome oxidase, 65, 69, 339Cytochrome P450 complex, 456Cytokines, 419, 443Cytoskeletal proteins, 17, 28, 164, 165, 345Cytotoxic cells, 419

D

Daidzein, 446, 475Deamination, 168, 395Deborah number, 210, 211, 224


Index 521

Decadienal, 302, 307, 308, 312, 328Decalactone, 312, 314Decalcification, 89Decanal, 297, 300Decarboxylation, 4, 432Deformation, 209–212, 217–220, 222, 223, 226.

238, 240, 241, 440Dehydration, 47, 48, 59, 105, 271Dehydroalanine, 157, 158, 350Delphinidin, 261, 264Demethylation, 85Denaturation of proteins, 8, 137, 141, 231, 287, 330Deoxyalliin, 302, 307, 328Deoxysugars, 371Desaturase, 196, 443Desmin, 17, 165, 346Desorption isotherm, 46Desoxysaccharides, 105Detergents, 56, 105, 116, 136, 277Detoxification, 75, 85, 455, 458, 474, 477Dextran, 96, 117, 404, 410Dextrins, 114, 118, 122, 124, 496Dextrose, 270, 311, 369DHA, 186, 192, 196, 198, 431, 442, 444, 491, 512Diacetyl, 271, 312, 316, 348, 470Diacylglycerols, 5, 179, 490Dialdehydes, 107Diallyl sulfide, 138, 475Diazonium, 361Dichlorobenzene, 55Dichloroethane, 55Dietary fat, 368, 449, 467, 482, 485, 487, 492,

493, 515, 516Dietary fiber, 5, 23, 63, 96, 126, 385, 439, 445,

449, 494–496Diethylacetal, 312Diglycosides, 261, 262, 304Dihydrogen phosphate, 80–82Dimagnesium phosphate, 81Dimethyl sulfoxide, 115Dimethylsulfide, 312Dioxin, 377, 472Diphenyl oxide, 312Disaccharides, 94, 95, 109, 115, 117, 271, 494DNA, 68, 79, 296, 314, 317, 324, 379, 390,

395–397, 404, 407, 417, 425, 432, 433, 446, 451, 456–459, 467–471, 473, 474, 476, 462–484, 490

Dough, 22, 78, 80, 81,, 89, 116, 138, 152, 154, 155, 167, 211, 212, 218, 221, 237, 298, 321, 328, 336, 338, 347, 348, 355, 360

DPA, 186, 196, 198Drip formation 8, 74, 141, 155, 172Dyes, 246, 272, 273, 286, 360, 364, 365

E

Eicosanoids, 443, 491Egg allergens, 275, 280, 281Elastic compliance, 220, 221Elastic solid, 209–211, 219, 238, 240Elastin, 65, 240Electrostatic interactions, 38, 41, 143, 146, 169Elongase, 196, 492Emulsification, 147, 311, 320Emulsifying agents, 5, 75, 79, 89, 197, 359Emulsions, 5, 145, 146, 180, 184, 213, 215, 216,

235, 236, 319, 330, 336, 344, 348, 366, 367

Enantiomers, 117Encapsulation, 93, 123, 308Endiols, 109Endoglucanase, 118Endomysium, 17, 19Endopeptidases, 132, 161, 166Endosperm, 21–23, 73, 331, 348Endrin, 55Enolization, 109Enthalpy, 38, 39, 132, 125, 136, 151, 174Entropy, 38–40, 133–135, 139, 140, 332Enzyme inhibitors, 4, 7, 330, 342, 345, 385Enzymes, 3, 17, 22, 25, 63, 67–69, 121, 152, 163,

164, 196, 285, 314, 318, 320, 334, 344, 345, 359, 368, 429, 432, 498

EPA, 186, 195, 196, 198, 442–444Epigallocatechin, 477, 478Epimysium, 19Epithelium, 65, 75, 152, 296, 415–420, 472, 475,

499Epitope, 284, 290, 292–294, 516Epoxides, 105, 252, 336, 472Erythrocytes, 44, 87Erythrose, 94Erythrosine, 158, 273, 351Erythrulose, 94Essential oils, 296, 297, 300, 310–312, 320, 323,

327Esterases, 315, 319Esterification, 99, 102, 115, 116, 132, 182, 315Esters, 3, 8, 39, 79, 116, 161, 162, 178, 180–182,

251, 297, 300, 312, 315, ,316, 323, 327, 362, 369, 431

Estrogens, 473Ethanol, 114, 120, 131, 161, 179, 181, 252, 257,

269, 273, 296, 299, 326, 343, 431, 435, 436, 470

Etherification, 105, 115, 116Ethers, 115, 116, 312, 365Ethyl acetate, 300, 312Ethyl butyrate, 312, 313, 325



Ethyl hexanoate, 312, 313Ethyl phenyl acetate, 312Eugenol, 310, 312, 315Evening primrose oil, 203–205Extrusion cooking, 154, 308, 325

F

Fats, 4, 8, 15, 25, 26, 430, 431, 448, 471, 479, 488, 490, 493, 505, 510, 511

oxidation of, 363rancid odor of, 307substitutes of, 116, 357, 359, 368

Fatty acid composition, 3, 12, 177, 181, 193–195, 206, 343

Fatty acid methyl esters, 182Fatty acids, 4, 6–9, 12, 19, 22, 25, 26, 39, 63, 98,

103, 138, 167, 177, 178, 182, 184–186, 194, 197, 199, 252, 296, 298, 301, 303, 306, 307, 311, 314, 316, 317, 321, 322, 330, 336, 343, 368, 369, 385, 392, 393, 407, 408, 431, 442–444, 446–450, 471, 472, 475, 589, 510, 511, 514, 516

long chain, 8, 63medium chain, 8, 63polyenoic, 12, 177, 193, 343short-chain, 393, 407, 408, 496trans, 4, 177, 184, 185, 492, 493unsaturated, 63, 431, 442, 4718, 10, 24, 57, 67, 80, 147, 152, 160, 165, 288,

297, 298, 314, 315, 325, 327, 368, 369, 371, 387, 395, 397, 404, 407–410, 421, 423, 429, 496

Ferritin, 70, 86, 514Ferroxidases, 71Fish allergens, 275, 281Folch procedure for isolation of lipids, 180Fiber, 5, 6, 10, 12, 17, 18, 45, 64, 65, 96, 126,

138, 139, 212, 320, 336, 345, 370, 385, 439, 442, 445, 446, 449, 479, 494–497, 512, 514, 516

dietary, 5, 23, 63, 96, 126, 439, 445, 449, 494–496

muscle, 17–19, 345Film formation, 129, 141, 146, 334Firming agents, 79Flavonoids, 26, 70, 260,263, 264, 430, 462Flavor, 1, 4, 6–8, 11, 50, 67, 70–72, 80–82, 91,

93, 96, 120, 122, 123, 152, 153, 156, 160, 164–166, 192, 253, 270, 271, 295–330, 335, 339, 241–344, 353, 357–359, 361, 362, 364, 365, 367–369, 371, 372, 379, 382, 469

in cheese, 323compounding of, 296, 312green-grassy notes in, 295, 304, 326intensity of, 299of meat, 71, 313, 320, 325, 326, 339, 361process, 296, 305, 313, 314

Flocculation, 54, 57, 145, 367Fluoride, 50, 51, 53, 55, 62, 77, 88, 91, 498Fluorine, 66, 67Foam, 8, 95, 96, 116, 138, 147, 148, 150, 239,

240, 359, 367Folate, 138, 385, 386, 388, 432, 449, 455, 489Food allergens, 14, 275–294Food additives, 5, 67, 75, 80, 82, 83, 86, 88,

90–92, 141, 258, 275, 276, 286, 387, 357–373, 384, 388, 390, 452

Formaldehyde, 10, 114, 167, 168, 175, 330, 334, 346

Free energy change, 30, 135Free water, 36, 37, 45Freshness, 7, 9, 11, 160, 257, 326, 429Fructan prebiotics, 398, 399Fructans, 109, 127, 391, 395, 396, 399, 408Fructo-oligosaccharides, 370, 371, 394, 406Fructofuranosidase, 279, 286, 398Fructofuranosyl, 364, 370, 398Fructopyranose, 105Fructose, 6, 24–26,, 95, 101, 107, 109, 117, 118,

120, 121, 341, 370, 496 Fructosyltransferase, 370Frying, 4, 110, 122, 148, 307, 327, 328, 368, 369,

447, 465, 466, 469, 485Fucose, 95Furan derivatives, 108, 109Furcellaran, 96, 115, 122, 123, 347Furfural, 271, 309Furosine, 350

G

Galactan, 96Galactaric acid monolactone, 109Galacto-oligosaccharides, 391, 394, 399, 409Galactopyranose, 105Galactose, 95, 107, 109, 133, 261Galactosidase, 370, 399, 409, 416Galactosucrose, 105Galacturonans, 365Garlic, 138, 296, 301, 302, 307, 310, 319, 322,

328, 352, 435, 475, 476Gelatin, 126, 142, 143, 145, 154, 163, 173, 232,

267, 367Gelatinization, 22, 111, 154, 155, 337, 347, 371


Index 523

Gelation, 72, 90, 100, 119, 142–144, 149, 150, 163, 164, 173, 175, 231, 233, 237, 241, 242

Gellan, 232, 233, 242, 365Gelling agents, 5, 123, 150Gels, 5, 72, 99, 100, 114–116, 124, 125, 127,

141–144, 148, 151, 154, 160, 163, 165, 172, 173, 175, 220, 233=235, 242, 243, 185, 329, 330, 337, 338, 344, 346, 348, 398

Genistein, 446, 475, 476Gentio-oligosaccharides, 391, 394, 402–404, 410Geosmin, 216, 325Geraniol, 301, 304, 305, 308, 312Glazing agents, 359GLC, 177, 203, 206Globulins, 139glucans, 138, 365Gluconate, magnesium, 80Glucono-δ-lactone, 102Glucopyranosides, 304Glucose, 10, 24–26, 44, 66, 68, 94–96, 98, 99,

101, 102, 105, 107, 109, 114, 115, 117, 118, 120, 121, 125, 126, 226, 233,m267, 270, 281, 305, 328, 341, 352, 369, 385, 399, 403, 404, 407, 440, 441, 448, 468, 489, 493, 494–496

isomerase, 102, 118syrup, 114, 120

Glucosidases, 265Glucosinolanes, 7Glucosylamine, 95Glucuronyltransferases, 456Glutamate, 80, 82, 286, 287, 365, 428, 431Glutamine, 131, 162, 348Glutathione, 159, 434, 447, 456, 459, 475, 476Glutelins, 139, 152Gluten, 5, 9, 12, 22, 144, 145, 155, 170, 175, 218,

221, 232–235, 237, 242, 285, 332, 335, 336, 343, 348, 354, 496

Glycans, 99Glycemic index, 429, 431, 445, 487, 489, 494,

405, 514, 516Glycemic load, 495, 514Glyceroaldehyde, 94Glycerol, 49, 178, 179, 187, 199, 312, 364, 366Glycine, 131, 280, 284, 323, 373, 428Glycinin, 136, 163,279, 292Glycogen, 4, 8, 96, 100, 120, 432, 494Glycolipids, 179Glycomacropeptide, 133, 152, 164, 411Glycoproteins, 94, 99, 131, 133, 276, 278, 333,

335, 340Glycosidase, 305

Glycosides, 94, 95, 150, 178,260, 262, 264, 265, 303–305, 321, 322, 325, 343

Glycosylation, 132, 150, 172, 261, 263, 406Glycosylsucrose, 370Glycosyltransferases, 132Grilling, 457Grinding, 149, 152, 285Grits, 152Guar gum, 225, 227, 335, 445Gum Arabic, 335, 338Gums, 94–96, 114, 122, 127, 311, 337, 355, 365,

494Gut microflora, 391–393, 424

H

Halide, 105Haloacetates, 168Haloamides, 168Halogenation, 105, 115HDL, 192, 441, 442, 448, 493, 506Heat pasteurization, 7Heavy metals, 7, 12, 158, 452, 469, 472Helical complexes, 98, 99Heme, 17, 70, 87, 245, 259, 260, 361, 434Hemiacetals, 94, 312Hemicellulose, 65Hemichrome, 259Hemiketals, 94Hemoglobin, 62, 67, 69, 142, 259, 260, 334, 338,

480, 482Heparin, 96, 115, 123Herbs, 77, 295–197, 310, 311Heteroaromatic compounds, 102Heterocyclic aromatic amines, 7, 352. 457–459, 465Heterocyclic compounds, 299, 300, 305, 308, 312Hexametaphosphate, 71Hexanal, 296, 301, 304, 306–308, 312, 316, 317Hexane, 181, 182, 189, 203–205, 252Hexanol, 304, 312, 316, 317Hexosanes, 113Hexoses, 94, 95, 108, 117, 271, 371hexuloses, 94, 95Hilum, 100Histidine, 64, 131, 169, 175, 460, 500HLB, 146, 366, 367Homocystine, 439, 446–448Hookean elastic solid, 210Hotrienol, 305HPLC, 13, 177, 189, 190, 202–205, 274, 372Humectants, 6, 49, 121Hydration, 37–39, 42, 137, 140, 142, 153, 173,

175, 236, 237, 262, 334, 337water, 42



Hydrazine, 102, 462Hydrazo compounds, 360Hydrocarbons, 3, 12–14, 37, 39, 56, 98, 178, 246,

252, 258, 297, 308, 312, 451, 459, 467, 484

aromatic, 12, 14, 258, 457, 459, 467, 484Hydrocolloids, 72, 90, 93, 122, 241–243, 337,

338, 343, 355, 372Hydrogen bonds, 5, 29, 31–41, 43, 45, 94, 107,

110 111, 116, 135, 143, 153, 233, 332–335, 337, 341, 346, 348, 353, 364, 457

Hydrogen peroxide, 79, 307, 476Hydrogen sulfide, 56, 299, 305, 313Hydrogen sulfite, 82Hydrogenation, 8, 102, 185–188, 192, 103, 251Hydrolases, 19, 326, 343, 344, 456Hydrolysates, 133, 148, 151, 160–162, 165–167,

173, 287, 289, 313, 350, 503Hydrolysis, 7, 19, 22, 50, 114, 115, 117, 150, 151,

154, 161, 162, 165, 166, 172, 179, 200–202, 206, 257, 267, 278, 280, 281, 288, 289, 304, 305, 309, 315, 325, 332, 333, 340, 341, 343, 346, 348, 349, 370, 371, 398, 402, 403, 409

acid, 114, 151, 206, 266, 280, 304, 309enzymatic, 15o, 172, 201, 202, 325, 398, 402,

403of protein, 288, 349

Hydroperoxide, 312, 317lyase, 303, 304, 316, 317, 319, 321, 322, 324,

325, 327Hydrophopicity, 5, 129, 131, 133, 134, 136, 139,

144, 146, 147, 161, 169, 170, 172, 338

surface, 129, 133, 136, 139, 144, 146, 147, 172, 338

Hydroxy-2-methylpyran-4-one, 109Hydroxylamine, 102, 459Hydroxylases, 132Hydroxylation, 87, 132, 432, 457, 471Hydroxymethylfuran-2-aldehyde, 108, 109, 113Hydroxyproline, 132Hyperchromic effect, 263Hypoxanthine, 9, 11, 69

I

Imidazoles, 305Imine groups, 39Immunogenicity, 292, 420Immunoglobulins, 154, 291, 498Immunoreactivity, 284, 288, 292

Indigoid dyes, 273Inositol, 117, 179Insulin, 67, 120, 123, 428–430, 441, 445, 446,

448. 472, 501 Interesterification, 368Intrinsic fluorescence, 133, 134Inulin, 96, 115, 369–371, 394, 396, 398, 399,

405–409, 424Invert sugar, 107, 270, 343Iodide, 62Iodine, 66, 67, 69, 489, 490, 510, 511Ionic strength, 136, 139, 143, 173, 239Ionization constant, 73Ionone, 252, 253, 297, 308, 312Iron, 22, 52, 55, 62–73, 76, 82, 85–88, 91, 150,

259, 351, 361, 387, 427, 434, 461, 487, 498, 504, 514

Isoamyl acetate, 316, 322Isoascorbate, 361Isoelectric point, 122, 131, 365Isoeugenol, 315, 326Isoflavones, 138, 475, 476, 511, 514Isoleucine, 131, 500Isomalto-oligosaccharides, 391, 394, 401, 409Isothiocyanates, 303, 310, 320, 475, 485

K

Kamaboko, 9, 10, 148, 154, 163, 325Keratin, 20, 21Ketones, 71, 94, 102,115, 253, 300, 306, 308, 312,

336, 342Ketoses, 94, 109Ketosylamines, 102Ketosylamino acids, 102Kynurenine, 159

L

Lactalbumin, 150–151, 172, 278–280, 293, 411, 498

Lactase, 409, 510Lactoferricins, 151Lactoferrin, 150–151, 290–291, 498Lactoglobulin, 144, 150, 154, 233, 235, 278–280,

371, 499Lactones, 102, 107, 300, 312, 319Lactoperoxidase, 150–151, 290, 482Lactose, 10, 19, 65, 94–95, 101, 105, 109, 120,

153, 270–271, 288, 309, 370, 399–400, 407, 409

Lactosucrose, 370


Index 525

Lactulose, 109, 113, 354, 394–396, 400–401, 406–407, 409

Lactylate, 366Lakes, 264, 268, 273Lanthionine, 7LC-PUFA, 182, 187, 196, 491–493 LDL, 192, 440–442, 444–446, 450, 493, 506Lead, VI, 7, 9, 51, 55–56, 61, 63–65, 67, 75–76,

86, 88, 90, 105, 114, 135–137, 142, 154–158, 163–165, 170–171, 182, 259, 287, 311, 334, 337–338, 340, 343, 345–351, 359, 379–380, 396, 431, 435–436, 448, 453, 465, 469, 498, 502, 505–508

Leavening agents, 78, 89, 359Lecithins, 320, 348Legumin, 163, 172, 282, 294Lemons, 26Leucine, 131, 324, 500Leukotrienes, 443, 472Levan, 117Lignin, 315Lime, 80, 367, 385Limonene, 297, 308, 312, 318Linalool, 297, 301, 304–305, 308, 316Linalyl acetate, 312Lindane, 55Linear viscoelastic properties, 209, 229Linseed oil, 317, 324Lipases, 22, 179, 315–316, 322–323, 369Lipids, 177–207

acidolysis of, 199, 200alcoholysis of, 199, 200autoxidation of, 71, 168complex, 177–179composition of, 3, 512–513emulsification of, 147esterification of, 182, 315interesterification of, 368metabolism of, 68, 188, 207, 370, 396, 440,

448, 449,500oxidation of, 8, 49, 67, 70, 71, 91, 156,160,

168, 175, 252, 265, 295, 306–308, 310, 339, 343, 346, 349, 350, 351, 354, 372

peroxides, 158, 253, 339rancid odor from, 307structure of, 43

Lipolysis, 177, 200Lipoxygenase, 70, 152, 167, 252, 296, 298,

303–304, 306, 314, 316–317, 319–327, 340, 443

Lithium, 433Lobry de Bruyn-van Ekenstein rearrangement,

109

Long-chain alcohols, 178Long-chain fatty acids, 8, 63Long-chain polyenoic fatty acids 177, 193Low-density lipoprotein, 192, 264, 493Lutein, 246–247, 249–251, 253–255Lycopene, 246–250, 253–255, 385, 474–475,

477–478, 482Lysine, 7, 49, 131, 152, 163, 271, 309, 342,

348–351, 354, 386, 500, 511Lysinoalanine, 7, 158, 355Lysozyme, 136, 143, 151, 174–175, 280, 498–499

M

macroelements, 61–62, 66, 68, 74, 473Magnesium, 55, 62–63, 66–68, 73–74, 77, 80–81,

121, 175, 255, 430, 446, 450, 487, 503

Maillard reaction, 8, 102, 114, 136, 156, 270–271, 295, 298, 305–309, 313, 320, 322–325, 336, 350, 360–361

Malondialdehyde, 168, 472Maltol, 109, 112, 299Maltose, 24, 94–95, 97, 105, 107, 114, 118,

120–121, 401, 404, 410, 496Malvidin, 261Manganese, 51–52, 55–56, 66, 69, 73Mannans, 96Mannitol, 96, 101, 105, 120, 346Mannopyranose, 105Mannose, 95, 101, 109, 133Maple syrup, 120, 271Margarine, 25, 179, 215, 251, 335, 349, 366, 447Melanoidins, 245, 246, 270,271, 274, 341Mercaptide bonds, 75Mercury, 3, 51, 55, 61, 64, 75, 84–85, 88, 90Metabisulfite, 82–83, 360Metallothioneins, 75Methane, 58Methanethiol, 299, 305Methanol, 13, 105, 114, 180, 181–182, 189, 203,

256–258, 465methionine, 131, 152, 283, 285, 342, 435, 446,

455, 490, 500–501, 511Methoxychlor, 55Methyl cinnamate, 312–313Methyl dihydrojasmonate, 312Methylation, 85, 99, 105, 115, 132, 260, 403,

454–455, 483, 490Methyldehydroalanine, 157Methylglyoxal, 470Methylmercury, 84–85, 88, 90Metmyoglobin, 136, 156, 259–260, 330, 338, 360



Micelles, 40, 99, 122, 136–137, 142–143, 164, 169, 172, 332, 334–336, 366

Microbial gums, 365Microelements, 27, 62, 64, 66, 68, 433, 473–474,

502Microencapsulation, 116, 124, 126Microfibrils, 100, 144, 345Milling, 22, 73–74, 152, 232, 331, 386Minerals, 1–3, 19, 21–22, 26–27, 52, 54, 61–68,

73–74, 86, 88, 91, 121, 155, 375, 387–388, 417, 477

Mitochondria, 70, 87, 163Models

flickering clusters, 35–37Stillinger, 35, 36, 59Wiggins, 36, 59

Modori, 143, 165Moisteners, 359Molybdenum, 66, 69Monoacylglycerols, 19, 155, 335, 491Monobasic potassium phosphate, 81Monocalcium benzoate, 80Monocalcium phosphate, 79–80Monokines, 419Monopotassium dihydrogen ortophosphate, 81Monosaccharides, 26, 94–95, 107, 113–114, 117,

261, 442, 494Monosodium L-glutamate, 82Monoterpenes, 297, 304–305, 310, 327Mood food, 427–437MSG, 68, 82, 287Multiphase food materials, 209, 235, 236Mucin, 21MUFA, 444Muscles, 4, 8, 67, 71, 86, 91, 96, 131, 156,

164–165, 167–168, 281, 333, 344, 447, 489, 494, 502, 505

Mutagens, 451–453, 456, 460–462, 465–466, 468, 470, 472–474, 477, 480, 483–485

Mutarotation, 101Mycoprotein, 129, 153, 175Myofibrillar proteins, 17, 141, 148, 164–165, 167,

346Myoglobin, 67, 69, 156, 259–260, 330, 339, 360Myosin, 17, 142, 144, 153, 163–165, 172, 174,

225, 330, 333, 335, 345–346Myrosinase, 7

N

NAD, 117NADH, 70, 150, 339NADPH, 70, 150, 173, 257, 460, 480

Naphtol, 98Naringenin, 459Nectarine, 250, 305Neohesperidin dihydrochalcone, 105Nerol, 304–305Neurotransmitters, 427–429, 434, 489Newtonian viscous liquid, 210Niacin, 350, 355, 385, 491, 494, 506, 514Niacytin, 385Nickel, 51, 66, 102, 185, 275–276Nitrate, 51, 55, 83, 187, 339, 361, 388, 464Nitric oxide, 351, 361, 464Nitrite, 51, 83, 156, 168, 259, 339, 357, 360–361,

372–373, 464, 469, 481–482Nitrogen, 2, 11, 14, 32, 56, 64, 116, 180, 182,

259, 299, 303, 305, 312, 419, 459, 480

Nitrogen to protein (N:P) conversion factor, 2Nitrosamines, 388, 451, 457, 462–465, 469, 474,

482Nitrosation, 130, 170–171, 351, 464Nitrosomyoglobin, 259, 361Nitrosylhemochromogen, 156, 174Nonadienal, 307Nondigestible oligosaccharides, 369, 399Nonheme iron, 70–71, 91, 434, 514Non-Newtonian liquids, 209, 213Norbixin, 251Nucleases, 315Nucleotides, 4, 102, 122, 314, 346, 365, 454Nut allergens, 275, 282Nutraceuticals, 6, 14, 439, 447

O

Ochratoxin A, 462–463Ocimene, 312Octanol, 299, 304Octen-3-ol, 303–304, 316, 319, 328Odor, 57–58, 253, 287–288, 295, 298–300,

302–303, 305, 307, 316–317, 323, 327, 330

Oil, 8–9, 12–13, 19, 25, 44, 144–147, 172, 178–179, 184–187, 189–190, 192, 194, 197, 199, 201, 203–206, 213, 251, 258, 273, 297–298, 301–302, 306–308, 310–311, 314, 316–319, 321–324, 327–328, 335–336, 338, 348–349, 366–367, 386, 444, 450, 466, 475, 485, 492

Oleoresin, 251, 269, 300, 311Olestra, 369, 372Oligosaccharides, 6, 93–94, 101–103, 107, 114,

117–118, 122–123, 335, 337, 346,


Index 527

353, 369–371, 373, 391, 394, 396, 398–399, 401–411, 494, 496

Olive oil, 8, 12, 178Oncogene activation, 475–476Opioid activity, 151Opsonization, 419Organoarsenic compounds, 84, 89Ornithine, 157Ortophosphate, 71, 81–83Osazones, 102Osuloses, 109Ovalbumin, 126, 142–144, 147, 172, 175, 225,

280, 335Ovomucoid, 143, 147, 280, 335Ovotransferrin, 147, 280Oxalates, 63Oxazoles, 305Oxidation, 3, 4, 7, 8, 19, 49, 56, 61. 67, 70, 71,

91, 102, 107, 109, 115, 1116, 118, 122, 130 139, 140, 150, 153, 156, 158–160, 167, 168, 175, 180, 193, 246, 252, 259, 260, 264, 265, 271, 295, 297, 300, 304,–308, 327, 328, 330, 331, 338–343, 346, 349, 351, 352, 354, 360, 362, 363, 372, 383, 385, 388, 429, 440, 443, 444, 446, 447, 481, 482, 485, 498, 508

of carotenoids, 340of fatty acids, 167, 306, 443

of heme, 259of lipids, 3, 4, 8, 19, 49, 70, 71, 91, 156, 158,

159, 160, 167, 168, 180, 193, 252, 264, 271, 295, 297, 300, 305–308, 327, 328, 330, 331, 339, 343, 346, 349, 352, 354, 363, 372, 385, 388, 443, 444, 446, 508

Oxide, 79, 81, 115, 158–159, 167, 302, 304, 312, 330, 346, 351, 361, 464

Oxidizing agents, 158, 273, 339, 348, 351Oxidoreductases, 19Oximes, 102Oxygen, 3–4, 6, 19, 31–32, 39, 52, 56, 58, 64, 67,

69–71, 94, 107, 118, 123, 144–145, 158, 175, 180, 246, 248–249, 252–253, 259, 262, 265, 274, 277, 296, 308, 330, 334, 338, 341, 358, 363, 419, 425, 434, 454–455, 459, 471, 476–478, 506

P

Partially hydrogenated fats, 188, 192Parvalbumin, 279, 281P-cymene, 308

Pancreatic lipase, 179, 200–202Papain, 167. 284, 285Paramyosin, 131Paratope, 292Parenchyma, 23, 27Peanut allergens, 284Pectic oligosaccharides, 391, 403, 406, 410, 411Pectins, 5, 65, 95–96, 114–115, 122–123, 125,

127, 220, 225, 335, 337, 347, 365, 367, 403, 410–411

Pelargonidin, 261Pentosanes, 113Pentoses, 94–95, 108, 271, 371Pentuloses, 94Peonidin, 261Peppermint, 297, 322Peptides, 2, 8, 131, 136, 138, 150–152, 160–165,

170, 255, 277–278, 280, 289, 292, 305, 320–322, 367, 428, 468

biologically active, 150, 151Peptones, 165Peptydoglycan, 475Perimysium, 19Peroxidases, 159, 265, 456Peroxidation, 70, 92, 203, 205, 252, 264, 267Peroxide value, 9Peroxides, 158, 203, 252–253, 303, 316, 319, 321,

336, 339, 343, 472, 476Peroxyl radicals, 252, 264Pesticides, 56, 73, 89, 378, 380, 383–384, 388Petunidin, 261Pheophytin, 257–258, 340, 344Phagocytic activity, 419Phellandrene, 312Phenolases, 265Phenolics, 246, 252, 261, 274, 331, 450, 462Phenoloxidase, 262, 342, 354Phenols, 158, 274, 351, 360, 363, 430, 436–437,

445–446, 450, 476Phenylalanine, 131, 162, 315, 324, 342, 365, 371,

500Pheophorbide, 257–258Phloem, 27Phosphatases, 167Phosphate, 11, 20, 39, 43, 65, 70–71, 77–83, 89,

94, 116–117, 123, 126–127, 130, 136–137, 141, 150, 153, 167, 170–172, 179, 257, 270, 281, 305, 333, 346, 365, 388, 432, 476, 509

Phosphatidylcholine, 336Phospholipids, 5, 19, 22, 40, 70, 99, 179, 181,

255, 285, 307, 324, 335–337, 346, 353, 431, 490, 510

Phosphorus, 19, 65–68, 77, 81, 88, 99, 170, 430, 489, 491, 494, 503, 510, 516



Phosphorylation, 133, 167, 170, 175Phosvitin, 130, 281, 334Photooxidation, 249Phytates, 64, 434Phytin, 117, 257–258, 340, 344Phytoalexin, 470Phytoncides, 26Phytosterols, 138, 178Pica, 86Pigments, 3–4, 8–9, 17, 20–21, 25, 40, 70, 121,

148, 158, 167, 245–246, 249, 251–254, 256–260, 262–270, 272–274, 306, 311, 329, 331, 338–342, 372

Pinene, 312Plant gums, 95, 114, 122Plasma, 77, 85, 88, 111, 126, 143, 152, 172, 323,

381, 419, 434, 445–447, 450Plasmin, 70–71, 164, 371Plasmolysis, 120Plasteins, 160–162Plastic behavior, 209, 215, 236Plasticizers, 114, 142, 145, 239, 347Pollen, 121, 276, 277, 282, 285, 286, 290, 293Polychlorinated biphenyls, 473Polycyclic aromatic hydrocarbons, 12, 14, 258,

451, 459, 467, 484Polyhydroxyketones, 94Polymerization, 93, 111, 113–115, 117, 124–125,

144, 148, 155, 160, 163, 165, 173, 265, 300, 341–342, 346, 369–370, 398

Polyols, 102, 304, 368, 494Polyphenol oxidase, 167, 341, 360Polyphenolic compounds, 70Polyphenols, 158, 224, 430, 436, 437, 445, 446,

450, 476Polyphosphates, 116, 141, 153, 167, 171–172,

346Polypropenes, 476Polyrybosome, 132Polysaccharides, 4–5, 26, 72, 90, 93–95, 99–100,

103, 105, 107, 109–111, 113–119, 122–123, 125–126, 141–142, 150, 225, 227, 229, 231–233, 236, 242, 335–337, 342, 346–347, 365–366, 369, 405, 410, 442, 494

Porphyrins, 64, 246Potassium, 27, 55, 62, 67, 68, 73, 74, 80–83, 182,

372, 446, 450Prebiotics, 6, 126, 357, 359, 369, 391–411, 424,

426Preservatives, 5, 26, 286–287, 357–360, 362–363,

367, 371, 383, 386, 461, 465, 490Pressurization, 153–154, 173

Prions, 379Probiotics, V, XII, 6, 369, 393, 396, 400, 402,

404–407, 413–415, 417–419, 421–426

Processing of food, 62, 340, 376, 390, 468, 483Procollagen, 132Profilin, 279, 286, 294Prolamines, 130, 139, 163Proline, 131–132, 334Prooxidants, 71–72, 158, 252, 351Propanol, 139, 148, 203–205, 299Propionaldehyde, 299Propyl gallate, 71Propylene glycol, 312–313, 366Propylene oxide, 115Prostacyclins, 443, 491Prostaglandins, 276, 276, 443, 472, 491Proteases, 22, 73, 163–165, 285, 298, 315Protein bodies, 3, 332Protein-phospholipid membranes, 4Protease inhibitors, 138, 143, 285Protein-energy malnutrition, 489Proteins, 130–175

acylation of, 130, 163, 169, 170alkylation of, 130, 168amino acid composition of, 2, 14, 129, 130,

282, 290, 501chemical modification of, 168conformation of, 16, 129, 130, 133–137, 146,

153, 158, 169, 165, 332, 357 denaturation of, 8, 129, 136, 137, 139, 141,

142, 144, 145, 150–154, 156, 174, 231, 259, 280, 284, 287, 330, 333, 346, 355

gelation of, 142–144, 146, 149, 150, 163, 164, 173, 233, 241

hydrophobicity of, 129, 131, 133, 134, 136, 139, 144, 146, 147, 161, 168–170, 172, 338

oxidation of, 158, 160solubility of, 130, 137–141, 146, 150,

153–155, 160, 170–172, 334, 335Proteolysis, 154, 160, 163–167, 175, 278, 284,

333, 345, 355, 395Protopectin, 95–96Psicose, 109, 113PUFA, 182, 187, 188, 192, 193, 196–198, 298,

443, 444, 489, 491, 492Pullulan, 117–118Purine, 70, 274Pyrazine, 102, 122, 300–301, 305–306, 308, 312,

328Pyridine, 122, 300, 302, 305–306, 312, 466, 483Pyridoxal, 386, 388, 432Pyridoxine, 386, 432


Index 529

Pyrrole, 259Pyrrolizidine alkaloids, 462

Q

Quaternary structure, 130, 136, 142Quercitin, 446Quinoline, 466, 480, 484Quinones, 158, 246, 265Quinoxaline, 466, 483–484

R

Racemization, 158Radicals, 70, 75, 122, 125, 153, 158–160,

252–253, 264, 272, 277, 331, 339, 343, 352, 363, 372, 445, 447, 454, 471–472, 476–478, 484, 508

Radium, 55Raffinose, 105, 369, 402Rancidity, 70–71, 90–91, 307, 343, 363Rapeseed oil, 194, 197, 200Recommended dietary intake, 487, 492, 495, 497RDA, 66–67, 385, 499Reactive oxygen species, 158, 253, 454–455, 459,

471Reductants, 70, 363Reduction, 52, 56, 59, 62, 70, 72–73, 87, 94–96,

98, 101–102, 107, 114–115, 117, 119, 156, 254, 260, 270, 296, 303, 319–320, 339, 360–361, 364, 366, 370, 383, 391, 395–396, 401, 411, 418, 412, 435, 464, 474, 476, 479

Reductones, 109, 271Relative humidity, 46, 48, 339Relative sweetness, 105, 120Relaxation time, 210, 219, 220, 228, 234, 240Rennet casein, 150Resistant starch, 112, 369, 371, 385Resveratrol, 445, 474, 477–478, 483Retardation test, 220, 231, 232, 240Reticuloendothelial system, 419Retinal, 254Retinoids, 253, 475Retinol, 253–254, 280, 476Retrogradation, 5, 116, 123, 347Reverse osmosis, 57Rhamnopyranosyl, 304Rhamnose, 95, 261Rhamnosidase, 305Rheological models, 214Rheological properties, XI, 6–8, 17, 61, 72, 114,

143–144, 147, 153–154, 163,

209–211, 213, 215–217, 219, 221, 223–225, 227, 229, 231, 233–237, 239, 241–243, 338, 344, 348–349

Rheopexy, 217Rhizomes, 296Riboflavin, 96, 158, 245–246, 269–270, 351, 386,

389, 491, 494, 506, 515RNA, 68, 433Roasting, 122, 152, 353Rose, 179, 297, 312Rose oxide, 312Rosemary, 296, 352Rotational mobility, 43Rutinosides, 304

S

Saccharides, V, XI, 1–2, 4–6, 8–10, 16, 23–26, 39, 41, 93–97, 99, 101–103, 105–109, 111, 113, 115, 117–123, 125–128, 133, 138, 145, 148, 152–153, 155–156, 160, 163, 168, 281, 333, 335, 337, 343, 347, 349–350, 354–355, 392, 394, 396–397, 428, 487–489, 493–498

Saccharin, 105, 123, 364–365Saccharose, 2, 9, 24, 429, 495–496Safflower oil, 197Saffron, 252, 296Salting, V, 4, 48, 140–141, 165, 340, 469Saponins, 95, 138, 462Sarcolemma, 17, 19Sarcoplasm, 17, 164–165, 333, 345–346Sarcoplasmic proteins, 17, 143, 148, 173Saturated fatty acids, 184, 442, 493Schardinger dextrins, 118Schiff bases, 102Scutellum, 22–23Selenium, 51, 55, 62, 65–66, 69, 85, 90, 255, 427,

434–437, 447, 476Selenoglutathione peroxidase, 69Selenomethionine, 62, 435Semicarbazide, 102Sensitizers, 158, 351Sequestrants, 67, 73, 363Serine, 131, 164, 433Serotonin, 87, 428–429, 432, 434Setting, 142, 147, 155, 163, 174, 237, 379Shallot, 301Shear, 117, 142, 144, 209–220, 223–224,

226–229, 235–236, 238, 240, 242, 344, 366

modulus, 142, 219, 238power law, 215



rate, 212–218, 224, 226–229, 235–236strain, 210, 213stress, 213, 216, 220thickening, 213, 215, 217thinning, 213–216, 227–228, 242

Shelf life, 6–8, 29–30, 43, 46, 48, 50, 67, 72, 267, 357–358, 366, 453, 469

Short-chain fatty acids, 393, 407–408, 496Shortenings, 349Silage, 165–166Silver, 55, 189–190, 340Singlet oxygen quenchers, 252Sitosterol, 447SOD, 70Sodium,

alginate, 82aluminum phosphate, 78–79, 82ascorbate, 82, 171benzoate, 82, 371caseinate, 367chloride, 37, 71–72, 90, 153, 346chlorophyllin, 81citrate, 71dihydrogen phosphate, 82dodecylsulfate,glutamate, 82, 286–287, 365hypochlorite, 115nitrate, 83oxalate, 71phosphate, 83trimetaphosphate, 170trialuminum tetradecahydrogen,

Sorbitol, 96, 101, 103, 105, 120–121, 346, 369, 464

Sorbose, 95Sorghum, 21, 332Sorption isotherm, 46–47, 49Soy allergens, 284Soybean oil, 187, 189–190, 197, 213, 298, 302,

307, 316, 322, 338Soybean oligosaccharides, 391, 394, 402, 409Speciation analysis, 63, 76Specific rotation, 100Spices, 8, 62, 77, 89, 286–287, 295–297,

310–312, 321, 325, 352, 355Spinacine, 169Squalene, 3, 178Stabilizers, 78–79, 89, 150, 153, 216, 336, 357,

365–366Stachyose, 105, 369, 402Staling, 5, 95, 116, 338, 383Starch,

anionic, 105, 123cationic, 105degradation of, 118

depolymerization of, 114, 125dextrinization of, 114esterification of 105, 115, 116etherification of, 105, 115, 116gelatinization of, 22, 111, 337, 347, 371granules, 22, 24, 99–100, 111, 113, 116, 119,

126, 155, 237, 332, 337, 348retrogradation of, 4, 116, 347sulfates, 116

Steric exclusion, 142Sterigmatocystin, 462Sterols, 3, 19, 40, 178, 396Stiffening agents, 359Stilbene, 315Stokes-Einstein relation, 41Storage modulus, 222, 234, 240Strain, 36, 143, 187, 209–213, 215, 217–219,

221–222, 229, 238–240, 288, 323, 331, 400, 409, 413, 415–417, 419–423, 425–426, 449, 460

Strecker degradation, 313, 328, 335Stress, 143, 151, 209–210, 212–224, 228, 234,

236, 238–240, 253, 277, 288, 296, 331, 333, 348, 414, 436, 447, 454, 473, 503, 515

Styrene, 124Sucralose, 364–365Sucrose, 25–26, 58, 94–95, 97, 103, 105, 107,

117–120, 122–123, 126, 233, 343, 346, 364–365, 368–370, 395, 398, 404, 408–409, 436, 490, 495

Sugar, 2, 6, 9, 44, 94–94, 102, 105, 107–108, 116–117, 120–122, 127, 147, 214, 270, 273, 299, 305–306, 313, 315, 343, 353, 365, 367, 370–371, 394, 429–430, 432, 442, 457, 459, 472, 483, 494–495, 497–498

Sulfate, 77–79, 81, 89, 105, 115, 123, 180, 266, 281, 365, 393, 405, 411

Sulfides, 301–302Sulfur, 7–8, 52, 64, 67, 82–83, 85, 131, 156–157,

159, 262, 265, 297, 299, 301–305, 308, 310, 312, 319, 321, 324, 360, 435, 476

Sulfmyoglobin, 259, 338Sulfocatechols, 360Sulforaphane,474, 477–478, 485Sulfotransferases, 456Sulfur, 7, 8, 52,64, 67, 85, 131, 156, 157, 297,

299, 301–305, 308, 310, 312, 319, 324, 360, 435, 476

dioxide, 82–83, 262, 265, 360Sunflower oil, 205–206Superoxide dismutase, 69–70, 90Suppressing agents, 474–476


Index 531

Surface active agents, 103, 197, 252, 366Surface tension, 34, 140, 145Surimi, 10, 148–149, 163, 172–175, 293, 346, 354Sweeteners, 5, 52, 114, 119–121, 123, 127,

286–287, 357–359, 363–363, 372, 490

Sweetness, 105, 119–120, 127, 364, 372Synergist, 82Syrup, 107, 114, 120, 233, 270, 271, 401

glucose, 114, 120maltotetraose, 120maple, 120, 271

T

TAGs, 178, 179, 188, 197, 199–206, 490Tannins, 26Targeted prebiotics, 391, 404, 405Tensile modulus, 238

strength, 4, 145, 335, 337, 345Terpenes, 26, 311. 331, 343Terpenyl acetate, 308Terpineol, 301, 305, 309Tertiary butylhydroxyquinone, 363Tertiary structure, 99, 130, 136, 139, 284, 363Tetracycline, 63Tetradecatrienone, 306Tetrahydrofurane, 252Tetrapyrrole pigments, 340Tetraterpene, 246Texture, 5, 8, 21, 27, 79, 93, 99, 122, 123, 138,

141, 142, 144, 147, 149, 152, 154, 160, 165, 167, 172, 173, 192, 241, 329, 330, 335, 336, 344, 346–349, 358, 368, 490

Thaumatin, 279, 364, 365, 371Thermal stability, 107, 132, 135, 142, 144, 145,

150, 153, 154, 173Thermolysis, 111, 114, 125, 127Thiamine, 7, 22, 305, 319, 321, 350, 385, 388,

432, 489Thiazoles, 300, 305, 306, 312Thickening agents, 357, 365Thiolanes, 300Thiols, 5, 70, 301Thiophenes, 300, 305Thiosemicarbazide, 102Tixotropic flow,Threonine, 131, 433, 500Threose, 348Threshold of flavor,Thrombosis, 443Thromboxanes, 443, 491Thymol, 310

Thyroid, 69Thyroxine, 67Tin, 65, 387Tocopherol, 267, 339, 351, 465, 508Toxaphene, 55Toxins, 3, 7, 56, 383, 393, 403, 411, 415, 419,

461–462, 464Transesterification, 103, 199–200, 257, 315, 368Transglycosylation, 401Transgalactosylation, 370Transglutaminase, 130, 142, 162, 172–174, 233,

334Transpeptidation, 161Trehalose, 94, 369Triacylglycerols, 3–4, 19, 22, 26, 63, 134,

177–178, 181, 201–203, 349, 368, 396, 440–442, 490

TAG, 180–181, 199–203, 205, 490, 506Triarylmethane, 273Trichloroethane, 55Trihalomethanes, 55Trimethylamine, 9, 11, 167, 312, 330, 346Tripolimetaphosphate,Trisaccharides, 24, 261, 399Trithianes, 305Trithiolanes, 305Trolox equivalent of antioxidanr capacity,Tropocollagen, 132, 139Troponin, 17Tryptophan, 87, 131, 173, 342, 351, 354,

428–429, 432, 437, 500Turbulent flow,Turmeric, 245, 268–269, 272, 296Tyrosine, 131, 271, 342, 407, 428

U

Ubiquinone, 5Undecalactone, 300, 312–313Unsaturated fatty acids, 19, 22, 25, 306–307, 322,

431, 442, 471Urea, 2, 10, 136

V

Valine, 131, 280, 500Van der Waals interactions, 32, 332–334Vanadium, 66Vanillin, 287, 311–312, 315–316, 319, 323,

326–327, 371Vicinal water, 39, 42–42, 46Vinculin, 17–18Vinyl chloride, 55, 124, 469



Violaxanthin, 246, 258–260Viscoelasticity, 209, 211, 217, 220, 223, 233Viscosity, 21, 41–42, 115, 120, 126, 137–138,

145–147, 154–155, 163, 167, 209, 212–217, 220, 223–229, 234–236, 240–243, 271, 309, 334–338, 343, 347, 366–367

Viscous liquid, 209, 210, 214, 240Vitamin A, 20, 69, 252, 254, 274, 340, 351,

386–387, 389, 488, 507B group, D, 65, 87, 386, 433, 475–476, 488–489, 503,

507–508, 512, 515E, 21–22, 178, 255, 339, 354, 386, 442, 444,

475, 488, 491, 494, 505, 507–508, 513

K, 386, 488, 508–509, 512, 515Vitamins, V, 1–7, 12, 17, 19–22, 24–26, 40,

73–74, 264, 274, 375, 385–388, 393, 415, 417, 427, 430–432, 436, 444, 446–448, 469, 474, 476–477, 488, 490, 505, 508, 510

Vulgaxanthin, 266

W

Warmed-over flavor, 70–71, 307, 325Wastewater treatment, 29, 57Water,

activity, 5, 29, 46–50, 59–60, 122, 271, 331, 352

drinkable, 53–54, 473in food, 29, 44pollution, 29, 54, 56thermal properties of, 29, 36–37treatment, 54, 57

types of, 42, 54Water-holding capacity, 17, 22, 28, 129, 141, 143,

332, 333Water-in-oil emulsions, 367Wax esters, 3WHC, 141, 150, 170, 172Wheat allergens, 275, 285Whipped cream, 147, 216, 335, 344Whiteners, 367Wood molasses, 113

X

Xnthan gum,xanthene, 273Xanthophylls, 8, 246, 249, 251, 253, 257Xantine, 69Xenoestrogens, 473, 481Xylans, 95, 114Xylem, 27Xylitol, 95–96, 105, 114, 120Xylo-oligosaccharides, 391, 394, 396, 402Xylose, 95, 113–114, 305–306, 319, 341

Y

Yield stress, 214–216, 234, 236, 240

Z

Z disk, 346Zeaxanthin, 249, 251, 253–254Zinc 62–66, 69, 74–75, 85, 88, 107, 387, 427,

433–434, 437, 487, 504, 514–515


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