food micro bio loy

8/22/2019 Food Micro Bio Loy

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S.-O. Enfors: Food microbiology

FoodMicrob io logy

Sven-Olof Enfors

KTH - BiotechnologyStockholm 2008


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Content

Chapt 1 Introduction...................................................................................1

Chapt 2. The ecological basis of food spoilage ...........................................52.1 The microflora ........................................................................52.2 The physico-chemical properties.............................................82.3 Chemical reactions ................................................................ 15

Chapt 3. Spoilage of different types of food .............................................22

Chapt 4. Foodborne pathogens..................................................................384.1 Microbial food intoxications .................................................394.2 Foodborne microbial infections.............................................44

Chapt 5. Food preservation.......................................................................515.1 Heat sterilisation and pasteurisation ...................................... 515.2 Chemical preservatives..........................................................655.3 Classification of preserved food ............................................65

Chapt 6 Fermented foods .........................................................................736.1 Beer brewing.........................................................................746.2 Fermented milk products ......................................................816.3 Fermented meat products .....................................................886.4 Fermented vegetables........................................................... 89


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1


Chap 1 Introduction

Living organisms are usually classified as animals, plants, algae, protozoa,bacteria, archae or viruses. All viruses, archae, bacteria, and protozoa plus theunicellular algae and some fungi, so called micro-fungi, are collectively calledmicroorganisms. The microfungi can be further divided into yeast and molds, aclassification that is based on the cell morphology. Based on DNA analysis, thegroup previously called bacteria is further divided into eubacteria and archaeand today the word bacteria is usually used as synonym to eubacteria.

Most microorganisms that we encounter in the normal spoilage of food belongto the eubacteria, here called bacteria, yeasts and molds. When it comes tofoodborne diseases, also viruses, some protozoa and archae, i.e. the blue-greenalgae, are involved.

A full species name is composed of two parts: the genus name plus thespecification defining the species within that genus. sometimes these genera aregrouped into families. This is illustrated in Table 1.1. Note that the genus nameis spelt with leading capital letter, while the species name is spelled with lowercase letters: Eschericia coli, Penicillium chrysogenum. The family, genus, andspecies names should always be written with italic letters. It is common in foodmicrobiology literature that the full species name is not used since many specieswithin the same genus are discussed. Then, Bacillus sp. means one not definedBacillus species and Salmonella spp. means several not defined Salmonella

species.

Table 1.1. Examples of family names, genus names and species names

Family Genus Species

Enterobacteriacae Escherichia Escherichia coliSalmonella Salmonella typhimurium

Salmonella enterica

Bacillacae Bacillus Bacillus subtilisBacillus cereusBacillus anthracis

Clostridium Clostridium botulinumBergeys Manual of Determinative Bacteriology divides bacteria into 35 groups. Groups,families, and genera which are most relevant in food microbiology are listed in Table 2.1.

In bacterial classification, the cell morphology, the relation to oxygen, and theGram staining reaction are important parameters. Most commonmorphological types are rods, cocci (spheric cells), and vibrioforms (short bentrods). The Gram reaction gives information about the cell envelope. Gramnegative cells have an outer membrane outside the cell wall which prevents thestaining. Obligate aerobes require molecular oxygen for their energy

metabolism (aerobic respiration). Anaerobes have an alternative energymetabolism that does not need oxygen. It may either be anaerobic respiration


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Introduction 2


(with e.g. nitrate as electron acceptor) or fermentation. Oxygen is often toxicfor anaerobic cells. Facultative anaerobic cells use oxygen and aerobicmetabolism if oxygen is available but switch to anaerobic metabolism inabsence of oxygen. Microaerophilic cells require low concentrations ofoxygen, while normal air contact is inhibitory. Lactic acid bacteria (e.g.Lactobacillus and Lactococcus) have an obligately anaerobic metabolism butare still resistant to oxygen.

Table 2.1. Some of the bacterial groups (according to Bergeys Manual of DeterminativeBacteriology) which are commonly encountered in food microbiology.

Groupnr

Description Food related organisms

2 Gram-neg., aerobic, mobile, vibrio-

formed

Campylobacter

4 Gran-neg., aerobic rods or cocci Pseudomonas, Shewanella, Legionella

5 Gram-neg., facultatively anaerobicrods

FamilyEnterobacteriacae(e.g.Escherichia, Enterobacter,Salmonella, Shigella, Yersinia, Erwinia)

Vibrio

17 Gram-pos. cocci Staphylococcus, Streptococcus,Lactococcus, Enterococcus, Micrococcus,Leuconostoc

18 Gram-pos endospore formers

aerobic or facultatively anaerobic:

obligate anaerobes:

Bacillus

Clostridium

19 Gram-pos, non-sporulating rods Lactobacillus

Brochothrix

Listeria

There is a number of often used group names of microorganisms. Some foodrelated examples are:

Gram-negative psychrotrophic rods: This includes the generaPseudomonas,Achromobacter, Alcaligenes, Acinetobacter, andFlavobacterium.

Lactic acid bacteria (LAB) includes the food related genera Lactobacillus,Lactococcus, Pediococcus ochLeuconostoc.

Coliform bacteria is not synonymous toE. coli but includesEscherichia coliand Enterobacter.


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Introduction 3


A special problem with the microbial taxonomy is that the names often are datedependent due to repeated re-classification of species. One example is thelactic acid bacteria which previously were called Streptococcus lactis,Streptococcus cremoris a.o. These so called lactic streptococci are nowreferred to a new genus and galled Lactococcus lactis, Lactococcus cremorisetc. Other previous Streptococcus spp. wich are associated with the intestinesare now called Enterococcus, while yet another group of the previousStreptococcus genus remain as Streptococcus. When it comes to pathogenicorganisms a further classification problem is that only some strains of a certainspecies may be pathogenic while other strains are harmless. An example isEscherichia coli to which species the feared EHEC (enterohaemorrhagicE. coli)belong. In such cases immunological or DNA analyses are required for properclassification.

Streptococcus is a genus with species of very different impact for humans. Someof todays Lactococcus and Enterococcus were previously classified asStreptococcus. They were then referred to as the lactic group and the enteric

group of the streptococci, respectively. A classification of the old streptococciaccording to current nomenclature is:

1. Lactococci (Lactococcus lacits, L. cremoris a.o.). These organisms are oftenused for fermentation of food.

2. Enterococci (Enterococcus faecalis, E. faecium a.o.) are in most cases notpathogenic, but certain strains have been reported to cause serious infections.Such contradictions are due to the limitation in the current nomenclature which is

based on phenotypic properties. These organisms are common in the intestinalflora. The presence of enterococci in food is not considered to be a health riskper

se, but it is used as an indication of bad hygiene and that constitutes a risk, sinceother organisms of faecal origin like Salmonella may be present. For this reasonenterococci (together with the coliforms) are called indicator bacteria.

3. Hemolytic streptococci. There are two types of hemolytic streptococci, and

these organisms remain in the genus Streptococcus: -hemolytic and -hemolytic.

The -hemolytic streptococci are named the viridans group and they are commonon mucous membranes in the mouth and respiratory tract and on the teeth. The -hemolytic streptococci are named the pyogenes group and among them there are

serious pathogens involved in several diseases and wound infections. -hemolytic

organisms produce a greenish discolorisation zone around the colonies on bloodagar while -hemolytic cells produce a clear zone.

Lactic acid fermentation is to a large extent also employed for production of

food, namely some of the fermented foods: cheese, yoghurt, fermentedsausages, and fermented vegetables like sauerkraut, pickles, olives, and others.


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Introduction 4


However, this fermentation is also involved in food spoilage. Then the type oflactic acid fermentation may be important for the taste development. Somelactic acid bacteria mainly produce lactic acid which while others also produceother products.

The lactic acid bacteria are grouped according to their type of lactic acidfermentation. Homofermentative lactic acid bacteria produce mainly lacticacid from the sugar, and no CO2. To this category belong

all Streptococcusall Lactococcusall Pediococcussome Lactobacillus

Heterofermentative lactic acid bacteria produce, besides lactic acid, alsoacetic acid, ethanol, CO2 and formic acid. Some can also convert citric acid(in milk) to diacetyl. In this group are

all Leuconostocmost Lactobacillus


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5


Chapter 2 The ecological basis of food spoilage

2.1 The microflora

Food consists to a large extent of cells from plants or animals (meat, fish,fruits, vegetables) and biological material with this origin (milk, juice, fat,starch etc). When discussing the shelf life of food it must be done from anecological viewpoint. All biological material in Nature is degraded to simplemolecular components, eventually down to inorganic components. This iscalled mineralization and it is a integrated part of the carbon and nitrogencycles in Nature (Fig 2.1) which is a prerequisite for life on Earth. If the

process is interrupted all nutrients would eventually be bound in deadbiological material. The circumstance that we select some part of this

biological material for food purpose does not change the natural fate of thefood, namely microbial degradation. However, it means that our interest in along shelf-life of food is in conflict with the natural processes.

Fig 2.1. Microorganisms,especially bacteria and fungi,account for the mainrecirculation of carbon and

nitrogen to the atmospherefrom where it is adsorbed forgeneration of plants whichconstitute the original sourceof food.

The degradation of biological material is mainly catalysed by microorgansims,which together carry an enormously diversified metabolic capacity. This is

illustrated in fig 2.2 which summarises the main paths of the biological energymetabolism.

All energy is generated, with exception of photosynthesis, by oxidation(combustion) of reduced substances (energy sources). Higher organisms likeanimals and also some microorganisms make this by oxidation of reducedcarbon compounds, e.g. sugars. These compounds are oxidised in many stepsin which oxidised co-enzymes (e.g. NAD

+) constitute the oxidant, which then

becomes reduced (e.g. NADH). These co-enzymes must be re-oxidised andeventually molecular oxygen in the air is used as the ultimate oxidant for this in

the respiration. The reduced compound or energy source is called electrondonor and the ultimate oxidant (oxygen) is called electron acceptor in this

Animals

Dead organisms

Plants Organicmateria

CO2 + N2

Light

Archae

BacteriaFungi

AlgaeProtozoa


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2. The ecological basis of food spoilage 6


energy metabolism. The electron donor in this case ends up as carbon dioxidewhile the electron acceptor oxygen is reduced to water. This respiration processis also coupled to phosphorylation of ADP to ATP.

Fig 2.2. Summary of different types of energy metabolism. Common principle is that energyis derived by oxidation in several steps of a reduced compound (C, N, S, Fe, H2 a.o.) bymeans of co-enzymes, here represented by NAD+. Re-oxidation of the reduced co-enzymecan be achieved with respiration, in which molecular oxygen, nitrate or nitrite, and sulphateare common oxidants (electron acceptors). An alternative to respiration is fermentation, inwhich a partially oxidised carbon compound from the metabolic path (e.g. pyruvate) is used

as electron acceptor for re-oxidation of the co-enzyme and then becomes reduced, in thiscase to ethanol.

When oxygen is used as electron acceptor the process is called aerobicrespiration, while the use of alternative electron acceptors like nitrate, nitrite,sulphate etc. is called anaerobic respiration. Many facultatively anaerobic

bacteria use oxygen if it is available but can switch to anaerobic respiration(e.g. nitrate respiration) or fermentative metabolism in absence of molecularoxygen. Of these respiration types, it is mainly the aerobic respiration andnitrate respiration that take place in food.

Some microorganisms can use other reduced compounds than carboncompounds as energy source. Some examples are ammonia and nitrite whichare oxidised by nitrifying bacteria, and sulphide, ferrous iron, and hydrogengas. These reactions are very important in the environment but seem to playlittle role in the handling of food.

One alternative type of energy metabolism which is common inmicroorganisms growing in food is fermentation, in which a reducedintermediate is used as electron acceptor in the re-oxidation of reduced co-

enzymes. There is a number of different fermentative metabolic pathways,

Cred NH3 Fe2+S 2-

CO2 NO3-

SO42- Fe

3+

NADH

NAD+

ATP

ADP

S 2-N2H2O

O2NO3SO4-

-

H2

H2O

Ethanol

Pyruvate

NAD+

NADH

Fermentation Respiration

Electron donors (energy source)Re-oxidation of co-enzymes

Electron acceptors


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named according to the dominating products, like ethanol fermentation, lacticacid fermentation, mixed-acid fermentation etc. Some of these reactions aredetrimental for the food while others are utilised in processing of food. Themain fermentative pathways and their role in food microbiology are furtherdiscussed in the section on degradation of carbohydrates.

To increase the shelf-life of food means that the progress of the naturaldegradation path must be prevented or delayed. However, food spoilage is notexclusively a matter of microbial degradation. Other spoilage reactions aredehydration, oxidation of fat, and endogenous metabolism (over-maturation offruits and vegetables), but microbial metabolism is the most important type ofreaction that reduces the quality of food during storage.

The common microbial food spoilage usually does not make the food unsafe oreven reduce its nutritional value, but it makes the product unpalatable. Thenegative perception of food which is severely contaminated by microorganismsis an important defence mechanisms for us, since the risk associated witheating food increases considerably if it is spoilt by microbial metabolism. Thisis due to the risk that some organisms among the spoilage flora may be

pathogens.

It is impossible to give a simple and yet comprehensive description of themicrobial spoilage of food since this is a very diversified process. What is said

in this booklet must be seen as typical and common cases, to avoid the use ofvery large lists of microbial names. When, for instance, it is stated below thatthe activities ofPseudomonas spp. limits the shelf-life of refrigerated freshmeat and fish, it means that most investigations - but not all- show thatPseudomonas species dominate the spoilage flora but there are usually anumber of other species involved, usually in the group "psychrotrophic, Gram-negative rods". Another problem is that it is not always sure that thedominating microflora is responsible for the main spoilage reactions. Anexample is that it may require 10 times more Achromobacter cells than

Shewanella cells to make fresh fish unacceptable in taste. Another example isthe lactic acid bacteria of the homo-fermentative type which have a relativelylow impact on the spoilage due to the domination of lactic acid in the metabolic

products.

Most food raw materials have a primary flora of microorganisms whichorigins from the production environment. During the continuing processing ofthe raw material and additional contamination (or secondary) flora infects thefood. It may come from the air, especially from dust in the air, from processwater, process equipment, or from humans which handle the food. During the

subsequent storage of the product the different species develop differentlydepending on the environment. The primary plus initial contamination flora


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usually is in the order of 103

cells/cm2

of solid foodstuff if the quality is verygood (see table 2.1). Depending on the conditions for growth some of thesespecies will grow exponentially (see Fig 2.3) up to concentrations above 10

7/

cm2

(or per gram). The finally dominating microflora may origin from theprimary or the contamination microflora. When the number of cells exceed 107to 10

8cells/cm

2(or per gram) the product usually develops bad smell and the

microflora is then called the spoilage flora. It is the nutritional (formicroorganisms) properties of the food and the environment (temperature,water activity, pH etc.) that determine which species will dominate thespoilage flora, their metabolic products and how fast this spoilage process will

proceed. In the sections below the environmental parameters will be discussedand in Chapter 2.2 the most important chemical reactions of food spoilage are

presented.

Table 2.1 Typical size of different food microfloras at goodproduction hygiene

Product Microbial concentration

Internal tissues of healthy animals 0

Plant surfacesFish skinEgg shell

Primary flora 103 cells/ cm2

Milk Contamination flora 103

cells / ml

MeatFish fillet

Contamination flora 103 cells / cm2

Spoilage flora on most food types 107- 10

8cells / cm

2or gram

2.2 The physico-chemical properties

The possibility of the food to serve as a substrate for microbial growth dependson a number of physical and chemical properties:

- Temperature- Water activity (aw)- pH and buffer capacity- Oxygen concentration and transfer

- Mechanical barriers- Metabolisable energy sources- Metabolisable nitrogen sources- Chemical inhibitors

Temperature. The temperature influences of course the rate of growth, andthereby the shelf-life of the product. But it has also an impact on selection ofspecies in the microflora. This is probably the explanation why reduction of

temperature in the refrigeration range (0-8C) has such a dramatic influence onthe growth rate, as demonstrated by experimental data Fig 2.3. The organisms


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growing at 20C have an initial generation time of about 4.8 h, while thegeneration time at 0C is about 25 h, and represents psycrotrophic organisms.

Fig 2.3. Influence of temperature on the total bacterial count (colony forming units, cfu)

on fresh meat. The dotted line indicate the typical level of spoilage. Note that the growth

initially is exponential.

Microorganisms are usually classified in four groups according to theirrelationship to temperature. Fig 2.4 illustrates this. In general, the mesophiles

have the highest maximum growth rate and an optimum temperature in the

range of 30-40 C .

Fig 2.4. Schematic illustration of the temperature dependence of the growth rate of different

classes of microorganisms. There are no general and exact limits for the temperature ranges.

0

4C8C10C

20C

00

Time (days)15

10Log

3

5

7

9

1

0 10 20 30 40 50 60 C

Relativegrow

thrate

Psychro-philes

Psychrotrophes

Mesophiles

Thermo hiles


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The psychrophiles have the lowest maximum growth rate, but can grow quite

fast at refrigerator temperature. Thermophiles have an optimum above 40C

and some can grow even above 100C. The psychrotrophic organisms

constitute an important group in food microbiology. They grow well in the 20-35 C range like the mesophiles but they can also grow relatively fast at

refrigerator temperature.

The growth rate of microorganisms is expressed either with the generation time(tg, h) or with thespecific growth rate constant (, h

-1). The generation time is thetime needed to double the amount of cells. The specific growth rate expresses therate of cell formation per cell. The correlation between these parameters can bederived from a mass balance of the cell number:

dN

dt=

N

whereNis the number of cells, (h-1) is the specific growth rate and t(h) is time.Integration withN0 cells at t= 0 andNtcells at time t, gives:

ln(Nt)

ln(N0)= t

After one generation time, tg , the cell number becomes 2N0. Insertion of this inthe equation above gives:

ln(2N0)

ln(N0)= tg

from which the correlation between generation time and specific growth rate isobtained:

ln(2)

= t

g!

0.69

Water activity (aw). The water activity is one of the main parameters whichdetermine how fast and by which type of organisms the food is spoilt. Thewater activity of food can be determined as the water vapour pressure (pH2O) in

a closed vessel in which the product is enclosed in relation to the water vapourpressure of purewater (pH2O*):

For a water solution with low molecular weight compounds (e.g. salt or sugar)the water activity is approximately:

aw=pH2O

pH2O*

aw!

nw

nw+ n

S


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wherenw= number of moles waterns= number of moles of dissolved molecules

Some common food components that reduce the water activity are:- Ions (e.g. salts)- Dissolved molecules (e.g. sugars)- Hydrophilic colloids (e.g. starch)- Ice

The water activity is a measure of the availability of the water for themicroorganisms. It is not only the water concentration that determines thewater activity but also the capacity of the material to bind water. This isillustrated in Fig 2.5 which shows sorption isotherms for some materials with

different water binding capacity. Cellulose get a relatively high water activityand starch a lower water activity at the same water concentration.

Fig 2.5. Sorption isotherms for differentmaterials show that aw is not the same aswater concentration

Fig 2.6. Schematic view of how theaw influences the rate of enzymereactions and microbial growth.

Most biochemical reaction rates decline with declining water activity.However, the sensitivity to reduced water activity varies, as illustrated in Fig2.6. Among microorganisms, molds and yeasts are generally more resistant tolow water activity and many enzymes retain their activity at even lower wateractivity. But there are many exceptions to this rule. Three types ofmicroorganisms prefer reduced water activity. These are osmophilic (sugar

preferring) yeasts, xerophilic (drought preferring) fungi, and halophilic (saltpreferring) bacteria. These organisms not only grow faster than most otherorganisms at lower water activity, but they also prefer a reduced water activity.

See further in Table 2.2.

0 0.3 0.6 0.9

Water activity

Waterconcentration(%)

30

20

10

0

Fruit

Starch

Cellulose

Meat

Water activity

BacteriaFungi

LipolysisProteolysis

Lipidoxidation

0 0.2 0.4 0.6 0.8 1

Relativerate


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Table 2.2 Examples of typical minimum water activity for growth of somemicroorganisms and corresponding aw in some foods.

Organism Min aw Food examples Food aw

Milk, fish, meat 0.99

Pseudomonas 0.97E. coli 0.96 Sausage, 7% salt 0.96

Clostridium 0.95

Brochothrixthermosphacta

0.94

Bacillus 0.93 Ham, 12% salt 0.93

Lactobacillus 0.93Streptococcus

LactococcusMicrococcus

0.93

Salmonella 0.91 Jam, 50% socker 0.91

Hard cheese, breadHerring, 20% salt 0.87

Staphylococcus 0.86

Yeasts in general 0.85

Molds in general 0.80

Halophilic bacteria 0.75

Grains w.10% water 0.7

Xerophilic molds 0.65

Osmophilic yeasts 0.60 Dried fruits, 15% water 0.6None Dry milk, soups etc.

Dry bread< 0.5

Halophilic = salt preferring;xerophilic = drought preferring;osmophilic = preferring high osmotic pressure (of sugar).

The water activity of food has a large impact on the rate of spoilage but also onthe type of spoilage since it exerts a selection pressure on the microflora. Manyof the common food spoiling microorganisms are very sensitive to reducedwater activity and the growth rate of these declines rapidly when the wateractivity drops below the optimum, which is close to 1 forPseudomonas andEnterobacteriacae. Many conclusions can be drawn from Table 2.2.Pseudomonas, which dominate the spoilage of refrigerated fresh meat and fishdoes not create problems in sausages and salted herrings or if meat and fish isdried. Such products get a spoilage flora of more low-aw resistant organismslike lactic acid bacteria, molds and yeasts. The table also explains why moldsare the main problem during storage of cheese and bread, and why dried

products like flour, grains, dry milk are not attacked by microorganisms at all,provided they are stored in a dry environment so they do not absorb water. It isalso obvious that the toxin producing Staphylococcus, which are commonly

present on human hands, constitute a threat at "smrgsbord" and other buffets.

Note that the figures in Table 2.2 are collected from different sources. Theactual minimum aw for and organism depends on other parameters like pH,


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temperature, and nutritional conditions. Thus, such data are only approximateand indicative of relative sensitivities.

pH is another parameter with large impact for the shelf-life of food. The pHinfluences both the growth rate and the type of organisms that will dominateduring storage. Most food products have pH below 7 (Table 2.3) and mostfood spoiling bacteria require a relatively neutral pH (Table 2.4), with theexception lactic acid bacteria wich grow well down to a pH in the range 4-5.In Nature there are many examples of bacteria that can grow at very low andvery high pH values, but these organisms are not relevant in foodmicrobiology. Comparing these tables give one reason why fruits and manyvegetables mainly are degraded by molds and sometimes yeasts.

Table 2.3. Typical pH-values of common food productsShrimps 7Fish 6.7Corn 6-7Milk 6.5Melon 6.5Butter 6.2Meat 5.1-6.4Cheese 5.9Oysters 5-6

Cabbage 5.5Potatoes 5.5Tomatoes 4.2Orange juice 4Yoghurt 3.5Apples 3Lemon 2

Table 2.4. Generalised picture of pH ranges for microbial growth

pH range pH optimum

Most food spoilage bacteria 6 - 9 71Lactic acid bacteria 4-7Molds 2 - 11 51Yeasts 2.5 - 7 4-5

Oxygen availability and the diffusion rate of oxygen are important parameters

that influence the type of metabolism. The rate of growth may be slower inanaerobic than in aerobic environments but on the other hand is the anaerobicmetabolism associated with much more detrimental products for the shelf-life.An exception to this is the lactic acid bacteria which have anaerobicmetabolism but usually produce less ill-smelling compounds than most otheranaerobic organisms. Anaerobic conditions are a prerequisite for growth of thedangerous pathogen Clostridium botulinum, and therefore special precautionsmust be taken when storing some types of food under anaerobic conditions.

The mechanical structure may be important for the shelf-life of food. On

whole meat bacteria grow only on the contaminated surface, where they dwell


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on the exudate, i.e. the glucose and amino acid rich liquid which leaks fromdamaged cells and blood vessels. If the meat is minced this surface andexudate increase enormously which leads to much higher microbial activityand growth in the inner anaerobic parts of the minced meat. Fruits andvegetables are protected from microorganisms by the outer shell or skin and bythe gelatine-like pectins which cements adjoining plant cells together. Outsidethe skin/shell the water activity is low and there is a lack of nutrients forgrowth of the contaminating microflora. But if the product is mechanicallydamaged or if the organism can produce pectinases the nutrients becomeavailable and the spoilage rate increases. It is mainly molds that produce

pectinases, and this, together with the often low pH of these products, explainswhy this type of food often is spoilt by molds. Yeasts, which also grow well atlow pH, often come as a second infection after the initial mold attack. Erwinia

is one of few bacterial genera with pectinase producing species which attackplant material.

Antimicrobial substances. Many food raw materials, especially vegetables andother food with plant origin, contain antimicrobial compounds which hamperthe microbial growth. Some examples are listed in Table 2.5.

Many microorganisms produce antimicrobial substances (antibiotics) and infood there is often growth of lactic acid bacteria, some of which produceantibiotics (Table 2.6). Nisin is a polypeptide antibiotic naturally produced in

fresh (unpasteurised) milk by Lactococcus lactis which belong to the normalflora transmitted during milking. Other antibiotics, like acidocin B and reuterinare mainly produced in processed milk if it is inoculated with the producingorganism.

Table 2.5. Some examples of naturally occurring antimicrobial substances.

Food Inibitor

Horseradish Allyl isothiocyanateOnion and garlic Allicin and diallylthiosulphinic acidTomato Tomatin

Radish RaphaninLingonberry Bensoic acidOregano Eteric oils

Table 2.6. Antibiotic substances produced by lactic acid bacteria

Antibiotic Organism

Nisin (in milk) Lactococcus lactisSalvaricin Lactococcus. salvaricusAcidocin B (fermented milk) Lactobacillus acidophilusReuterin (fermented milk) Lactobacillus reuterii


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Some definitions of antimicrobial compounds

Antibiotics Microbial product with an antimicrobial (bactericide/

fungicide or bacteristatic/fungistatic) activity and whichhave low toxicity to humans. If the latter is not added tothe definition most mycotoxins would also be classified asantibiotics.

Probiotics Microbial cultures, mainly lactic acid bacteria, which areconsumed for stabilisation of the intestinal microflora ofhumans or animals. They are believed to act byestablishing on the intestinal mucouse membrane and

prevent, possibly by production of antibiotics, the growthof other disturbing organisms.

Prebiotics Components (oligosaccharides) in the food that are notdigested in the intestines but are assumed to promote the

beneficial microflora.

Bacteriocines Bacterial proteins or peptides with bactericidal effectmainly on related species and strains.

bactericide = bacteria killing; fungicide = fungi killing;bacteri/fungi-static = inhibiting growth of bacteria/fungi.

2. 3 The chemical reactions

The most important chemical reactions involving food components duringmicrobial spoilage of food are:

- Degradation of N- compounds- Degradation of fat- Degradation of carbohydrates

- Pectin hydrolysis

Degradation of nitrogen compoundsThe dominating and usually the first reaction is oxidative deamination ofamino acids:

amino acid + O2 NH3 + organic acid

This reaction is assumed to be the dominating spoilage reaction in refrigeratedfresh meat and fish. The amino acid is then used as energy source by splitting

off the amino group with an oxidative deaminase, which leaves the organicacid that enters the energy metabolism.

deaminase


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Proteolysis. One could expect that proteolysis should be a common spoilagereaction. However, most microorganisms do not secrete proteases and thosewho do, usually do not produce them until there is a lack of nitrogen source.In later stages of spoilage, however, proteases and peptidases may degrade the

protein:

Proteins peptides amino acids

Many peptides have strong taste, bitter or sweet, and this sometimescontributes to the spoilage. These reactions are also important for thedevelopment of characteristic tastes of many fermented products.

Putrification is a set of anaerobic reactions with amino acids which results in amixture of amines (e.g. cadaverine, putrescine, histamine), organic acids, andstrong-smelling sulfur compounds like mercaptans and hydrogen sulphide:

Many of these compounds have terrible odour. Cadaverine, putrescine, andhistamine are formed by decarboxylation of lysine, ornithine, and histidine,respectively (Fig 2.6) While cadaverine and putrescine in food probably haveno health impacts, only spoil the food due to the odour, histidine causesintoxication problem since it may induce a serious anaphylactic shock. This isoften associated with microbial activity in histidine rich fishes of mackereltype, e.g. tuna fish.

Putrification is typical for microbial degradation of meat and other protein richfoods at higher temperature (> 15C). Bacillus and Clostridium species may

then grow fast and rapidly make the food toxic, but under refrigerationconditions these organisms are usually not active and under these conditionsthe oxidative deamination spoils the food before the putrification becomesdominating.

proteinase peptidase

amino acidsAnaerobic

metabolism

Amines

Organic acids

S-compoundsIndol


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Fig 2.6. Histamine, cadaverine and other amines are formed by decarboxylation of aminoacids.

Reduction of trimethylamine oxide (TMAO). Marine animals may contain high

concentrations of trimethylamine oxide, which is believed to have a function in

protecting proteins from denaturation at low temperatures, high pressure and

high osmolarity. Certain microorganisms, like Pseudomonas and Shewanella,can utilise TMAO as electron acceptor in anaerobic respiration:

This results in formation oftrimetylamin (TMA) which gives a typical "fishy"

smelling. TMA can also be formed by enzymatic hydrolysis of lecithin.

Degradation of fatWhen fat is degraded it becomes rancid and this rancidification depends onmany different reactions which are not all well known in detail. One attempt ofclassification is shown in Fig 2.7. The hydrolytic rancidification results in freefatty acids (FFA) and glycerol. Our organoleptic tolerance of free fatty acidsdepend on the type of the fatty acids, especially the carbon chain length. Up to

15% FFA is said to be acceptable in beef, which has long fatty acids, whileonly up to 2% is acceptable in olive oil. If very short FFA are formed, e.g.

H3C - N = O

CH3

CH3

H3C - N

CH3

CH3

TMAO-

reductase

TMAO TMA


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butyric acid from butter, only traces of the acids can be accepted. Thehydrolysis can be spontaneous but then at a very low rate, while it may proceedfast if lipolytic enzymes from the foodstuff or from the contaminatingmicroflora are present.

Fig 2.7. Different types of rancidification reactions.

The oxidative rancidification requires presence of oxygen. Autooxidativerancidification is catalysed by metal ions and is accelerated by light. In this

process peroxide radicals (ROO*) are produced and they react with other fattyacids to form instable hydroperoxides (R-OOH) which later on decompose toaldehydes and ketones which give the rancid taste (Fig 2).

Fig 2.8. Autooxidation of a fatty acid (RH) results in aldehydes and ketones. Thechain reaction is initiated by a radical (R*) which is produced from the fatty acidunder catalysis of Fe2+ and other metal ions and light. The radical reacts withmolecular oxygen to form a peroxide radical (ROO*). Antioxidants in food are usedto scavenge the peroxide radical that otherwise continuous the chain reaction byreacting with another fatty acid to produce a new radical (R*) and a hydroperoxide

(R-OOH). The hydroperoxide is instable and decomposes to ketones or aldehydes.


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-oxidation is the common metabolic route for degradation of fatty acids andeach cycle results in generation of one acetyl-CoA and a new fatty acid with 2C shorter C-chain (Fig 2.9). Some microorganisms have a side reaction in thelast step of the -oxidation cycle, by which very aromatic methyl ketones areformed and may contribute to bad taste (rancidity) of the food.

Fig 2.9. Methyl ketones may be formed as by-products in the -oxidation of fatty acids.

Lipoxydaserare common enzymes in plant and animal tissues and they are alsoproduced by some molds. The enzyme oxidises unsaturated fatty acids withcis-cis 1-4 pentadien configuration to hydroperoxides which decomposespontaneously to ill-tasting aldehydes and ketones. This configuration is

present in linolic and linolenic acids in plants and in arachidonic acids inanimal tissues. To prevent this type of rancidification during storage somevegetables, e.g. frozen spinach and peas, are heat treated to inactivate the plantenzyme. However, these aldehydes and ketones are not always unwanted

products in food. They are also important ingredients in certain types ofcheeses (see Chapter 6).

Degradation of carbohydratesMicroorganisms growing on food mainly use various sugars as carbon- andenergy source. Under aerobic conditions the energy source is combusted tocarbon dioxide and water but under oxygen limiting or anaerobic conditionsmany species switch to fermentative metabolism which results in variousfermentation products (see Fig 2.10). The most common fermentative

pathways are listed in Table 2.7.


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Table 2.7. Common fermentation types

Fermentation type Products

Alcohol fermentation Ethanol, CO2

Homofermentative lactic acid fermentation Lactic acid

Heterofermentative lactic acid fermentation Lactic acid, Acetic acid, Ethanol,CO2

Propionic acid fermentation Propionic acid, Acetic acid, CO2

Butyric acid fermentation Butyric acid, Acetic acid, CO2, H2

Mixed-acid fermentation Lactic acid, Acetic acid, CO2, H2, Ethanol

2,3-butanediol fermentation CO2, Ethanol, Butanediol, Formic acid

Of these fermentation types, it is the butyric acid, mixed acid and butanediolfermentations which are most detrimental for the food taste. The mixed-acid

and butanediol fermentations are typical for organisms in theEnterobacteriacae family. Butyric acid fermentation is common amongsaccharolytic Clostridium. Lactic acids is mainly produced by lactic acid

bacteria but it proceeds also under aerobic conditions since these bacteria arerelatively indifferent towards oxygen although they always use thefermentative metabolism. A more detailed picture of the different fermentation

pathways from glucose via the common intermediate pyruvate is shown in Fig2.10.

Fig 2.10 Summary of the six main fermentative pathways. The main end products are

emphasised by frames. Sites of co-enzyme generation and ATP formation are indicated.

Glucose

Pyruvate Acetaldehyde EthanolLactate

FormateAcetylCoA +H2CO2Oxaloacetate

Succinate

AcetateAcet-

acetylCoA

Propionate

Acetoin

Butandiol

Butyrate Acetone

Butanol 2-propanol

AcetylCoA +

Propionic acidfermentation

Ethanol fermentationLactic acid fermentation

Mixed acid fermentation

Butyric acid fermentation

Butandiolfermentation

NAD+

NAD+

NAD+

NAD+

NAD+

NAD+

NAD+

NAD+

NAD+

NADH

ATP

ATP

ATP

ATP

ATP

Acetate Ethanol H2 CO2


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Pectin hydrolysisPectins are carbohydrate polymers mainly composed of partially methylated

poly--(1,4)-D-galacturonic acid. They are present in all fruits and vegetableswhere they function as a glue between the plant cells which gives mechanicalrigidity. During ripening of fruits and berries indigenous pectinases aresynthesised or activated and start hydrolysing the pectins which makes thestructure soft. Also mechanical damages on fruits and vegetables activate

pectinases and this opens for microbial attack. However, also somemicroorganisms produce and secrete pectinases. Many molds have thiscapacity and among bacteria plant pathogens in the genus Erwinia also

produce pectinases which serve as tools for the microbial invasion resulting insoft rot.

Slime productionMicrobial spoilage of meat and fish sometimes results in a slimy surface layer,composed of microbial polysaccharides. Such polysaccharide slime can alsoappear as a result of microbial growth on vegetables, wine and vinager. Aspecial case of slime formation is the so called ropiness of bread which iscaused by B. subtilis which may survive the baking as spores and thengerminate and grow if the water activity is high and the temperature kept toohigh after the baking. The slime formation on cold-stored fresh meat usuallycomes after the meat has become unacceptable due to smelling. Some speciesof lactic acid bacteria produce polysaccharides and this is sometimes utilised in

various fermented milk products to give a higher viscosity (yoghurt, Swedishlngmjlk). However, the viscosity of yoghurt is mainly caused by protein

precipitation due to low pH.


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22


Chapter 3. Spoilage of different types of food

From a microbiological viewpoint it is convenient to classify different typesof food according to the conditions they provide for microbial growth whichgives an indication of the food shelf-life. One such classification is shown inTable 3.1.

Table 3.1 Food categories with different protection against microbial spoilage.

Food properties Example Protection

Water-rich

Protein-rich

Relatively neutral pH

Meat

Fish

Milk

Cooked food

None

Water-rich

Protein-poorRelatively sour

FruitsVegetablesRoot-fruits

Low pH

InhibitorsMechanical structure

Water-poorGrains

Flour

Bread

Low aw

Fermented foodSee Chapter 6 Often low aw + low pH

Microbial competitors

Microbial inhibitors

Preserved foodSalted/dried

Pickled

Smoked

Sterilised

Pasteurised

Low aw

Low pH

Low pH, low aw, inhibitors

No microflora

Small initial microfloraOften in combination with

chemical preservatives

3.1 Water and protein rich foods

Fresh meat, fish and milk belong to this category. They have a water activityclose to 1, contain lots of energy sources and other nutrients for microbialgrowth, are relatively pH neutral and contain no or little microbial inhibitors.If not treated by preservation methods these food stuffs are spoilt by microbialactivity in a couple of days or shorter at room temperature. Therefore these

products are always stored at refrigerator temperatures to reduce the rate ofmicrobial growth.

At a first look one would expect that eggs should belong to this category, butfor obvious reasons Nature has build a sophisticated system which keeps the


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3. Spoilage of different types of food 23


egg protected from microbial attack for several weeks at room temperature.This is described in Fig 3.11.

MeatAt the moment of slaughter, the animal's breathing and the aerobic respirationcease abruptly but the cells in the body tissues continue their metabolism forseveral hours and these reactions are important for the later microbialdevelopment. During the post mortem metabolism glucose is metabolisedthrough the glycolysis, but due to lack of oxygen, lactic acid is produced fromthe pyruvate. Glycolysis generates two ATP molecules per glucose molecule,which is much less than in the aerobic respiration but still enough to preventthe formation actomyosin complex in the muscle (See Fig 3.1). However, theformation of lactic acid reduces the tissue pH from neutral towards pH 5.5-6.

Eventually the low pH inhibits the glycolysis and the ATP generation ceaseswhich results in formation of actomyosin from the components actin andmyosin which are kept dissociated by ATP. Formation of actomyosin resultsin muscle contraction and it is observed as rigor mortis.

Fig 3.1. Thepost mortem glycolysis generated protons and ATP. The ATP forces theequilibrium between actin + myosin and the actomyosin towards the dissociated state.When pH has dropped too much the ATP generation through glycolysis ceases and theequilibrium shifts towards formation of the actomyosin complex, which results in musclecontraction, i.e. rigor mortis. After some time (Table 3.2) the actomyosin complex ishydrolysed by proteases (cathepsins and calpains).

The time course of this most mortem metabolism and the final pH depends onthe animal species (Table 3.2). The final pH is considered important for theshelf-life. This pH is not only dependant on the animal species but also on thecondition of the animal before slaughtering. An animal that has been stressedhas a lower blood glucose level and the post mortem metabolism can thencease due to glucose limitation rather than pH inhibition and the result is ameat with higher pH. Since the dominating spoilage flora on refrigerated freshmeat is Pseudomonas (and other Gram negative psychrotrophic rods) andthese organisms are quite sensitive to pH below about 5.5-6, the final pH ofthe meat is considered important for the shelf-life.


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Table 3.1. Typical pH of meat from different animals and lenth of rigor mortis.

Animal type Rigor mortis final pH

Cow 10-20 h 6 - 5.5Swine 4-8 h 6Chicken 2-4 h 6.4 - 6Fish min-h (longer on ice) 6.8 - 6.4

The meat contains many nutrients for the microorganisms (Table 3.3) whichonly grow on the exudate from damaged tissue. Furthermore, it is only on thesurface of meat the microorganisms grow, unless the meat has beenmechanically perforated or minced. Therefore, the microbial count isexpressed as cells/ cm

2or cfu/ cm

2, where cfu means colony forming units on

agar plates.

Table 3.2 .Example of microbial nutrients in meat exudateComponent oncentration g/Kg

Lactic acid 9Creatine 5Inosine 3Carnosine 3Amino acids 3Glucose-6P 1

Nucleotides 1

Glucose 0.5

Fresh meat is usually stored at refrigerator temperature which gives a shelflife around one week, however longer for beef, but this shelf life dependsstrongly on other factors like the hygiene during slaughter and handling of themeat. It is often assumed that also a low pH after rigor mortis is important.Under these conditions the microflora at the time of spoilage is dominated byGram negative psychrotrophic rods of the genera Pseudomonas,Achromobacter, Alcaligenes, Acinetobacter och Flavobacterium. Theseorganisms are often obligate aerobes. Many investigations reportPseudomonas, and especially P. fragi as common spoilage flora on freshcold-stored meat. There are also reports which state that this type ofmicroflora on meat is universal and not dependent on which animal the meatcomes from. The flora is always dominated by bacteria, only small amountsof yeasts and molds are developing under these conditions.

During storage, the bacteria initially grow exponentially, sometimes after alag phase which is caused by a shift of domination microflora. The cell

concentration increases from about 103

cells/cm2

on a meat of highesthygienic quality towards 10

7- 10

8cells /cm

2. Then the spoilage becomes


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apparent through bad odour, and sometimes discolorisation and slimeformation. Typical growth curves on refrigerated pork and chicken are shownin Fig 3.2 It is apparent that the shelf life of such products depends on thegrowth rate, which is mainly determined by the temperature, and the initialamount of bacteria, which is strongly related to the hygiene during and afterslaughter.

Fig 3.2. Example of microbialgrowth measured as "totalaerobic count" during storage of

fresh pork and chicken meat atrefrigerator temperature.

According to one hypothesis, the shelf-life of fresh meet depends on theavailability of glucose at the surface. As long as glucose is available, this isthe main energy source for the bacteria, but when it is exhausted, otherorganic compounds, e.g. amino acids provide the energy. When aminoacidsare used as energy source, ammonia is split off by oxidative deamination and

produces bad odour. This is supported by the data shown in Fig 3.3 whichshows how the glucose gradually is exhausted at the surface when themicroflora approaches the spoilage stage. It can also be an explanation of whymeat from stressed animals has a lower shelf-life, since short intensive stress

before the slaughter may reduce the blood glucose concentration.

Fig 3.3. Glucose concentrationgradients and microfloradevelopment during cold storing offresh meat. At N=32*107 cm-2 themeat was classified as spoilt and thiscoincides with glucose exhaustion atthe surface.

0

400

Glucose(g/g)

0

400

0 20

Distance from surface (mm)

N*10-7=

2.7

6.3

32

110 280

odr

slem

grisktt

kyckling

Tid (d)0 2 4 6 8 10

1

2

3

4

5

6

7

8

log N/cm2

chicken

slime

Days

odour

pork


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Carbon dioxide and vacuum packagesVacuum packaging of meat, both fresh and cured meat, dramatically prolongsthe shelf-life. It was originally believed that the main mechanisms of vacuum

packaging is that oxygen is removed and that this hampered the main spoilageflora. However, storing meat under nitrogen atmosphere does not improve theshelf-life. Fig 3.4 shows that the microflora develops slower, but thefermentative metabolism which dominates under anaerobic conditions

produces more off-flavour, unless the dominating microflora is composed oflactic acid bacteria. The figure also shows that storing the meat under CO2atmosphere significantly reduces the rate of microbial growth. When the CO2

packed meat was opened and subjected to air, the microbial growth rateimmediately increased.

Fig 3.4. Influence ofthe gas atmosphere onthe growth rate ofmicroorganisms onrefrigerated fresh pork

meat. Some of the CO2stored samples wereopened and furtherexposed to air, asindicated in the CO2-

plot.

When the composition of the microflora was investigated under theseconditions it became clear that the atmosphere exerts a selecting pressure, seetable 3.4. In air the dominating microflora usually is Pseuomonas. Theseorganisms are obligate aerobes or use nitrate respiration in absence of oxygen.In nitrogen atmosphere different species from the Enterobacteriacae familydominate. These organisms possess a strong fermentation capacity with ill-tasting products from the mixed-acid fermentation or 1,3 butandiolfermentation pathways. The CO2 not only reduces the rate of growth on themeat, but it also exerts a selective pressure which favours growth ofLactobacillus, which with their lactic acid fermentation have less impact on

the spoilage than thePseudomonas .

0 8 16 24 323

4

5

6

7

8

9

CO2

Luft

Luft

Kvve

Luft

Tid (dagar)

logN / cm2

airN2

air

air

CO2


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Table 3.4. Dominating spoilage flora on cold stored pork in different atmospheres.O2%

N2%

CO2%

Pseudomonas Entero-bacteriacae

Aeromonas Brochothrix Lactobacillus

20 80 +

100 +80 20 +

80 20 + +

10 90 + +

100 +

The selective pressure of CO2 is explained by the different inhibitory effectthis gas has on various microorganisms. Pseudomonas belongs to the mostCO2 sensitive bacteria while lactic acid bacteria are very resistant to this gas.Most molds are very sensitive while yeasts are very resistant to CO2.

Fig 3.5 Relative sensitivity ofmicroorganisms to inhibition of growth

by carbon dioxide.

When fresh meat is vacuum packed after slaughter, which is often the case formeat that is to be stored for tendering, CO2 is released from the tissues duringthe first day and since the plastic film of the vacuum package has a low gas

permeability and the gas headspace is removed by the vacuum, the partial

pressure of CO2 raises rapidly and exerts a protecting function. Also the shelf-life promoting effect of vacuum packing of cured meat products is similar butin that case it is the metabolic activity of the microflora which produces theCO2. Table 3.5 lists some properties of bacteria which contribute to theselection pressure in vacuum packed fresh and cured meat.


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Table 3.5 Some characteristics of the organisms that dominate thespoilage flora on cold-stored fresh and cured meat in differentatmospheres.

Organism Properties

Pseudomonas Fast growingAerobicVery CO2-sensitiveSensitive to low aw

Enterobacteriaceae FacultativeIntermediate CO2-sensitivity

Aeromonas FacultativeIntermediate CO2-sensitivity

Brochothrix thermosphacta FacultativeRelatively CO2 resistant

Resistant to low aw

Lactobacillus Very CO2-resistantIndifferent to oxygenResistant to low aw

The inhibitory effect of CO2 seems to be synergistic with low temperature instorage of meat as shown in Fig 3.6. This may partly be due to the increasingsolubility of CO2 at declining temperature. Even if CO2 dissolves in water and

partly is hydratized and dissociates to bicarbonate, it is the gaseous CO2molecule which has the inhibitory effect. This also means that the effect isstrongly pH dependent and declines with increasing pH.

Fig 3.6. Time needed to reach 106 cells cm-2 on pork meat stored at different temperatures

in air or in CO2.

C


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The antimicrobial effect of CO2 on many spoilage organisms has been utilised

also for direct packaging of food in gaseous atmosphere. These so called

"controlled atmosphere" packages contain mainly carbon dioxide as growth

inhibiting compound but also some oxygen to avoid anaerobic metabolismand decolourization of the haeme in meat.

Vacuum packing of food is applied also for other reasons than to provide

microbial inhibition via CO2. One common reason for vacuum packing is to

prevent oxidative rancidification or other oxidising reaction with molecular

oxygen (e.g. peanuts), or to prevent evaporation of flavour compounds (e.g.

coffe). When cheese is packed in vacuum tight plastic films it is likely that a

mold inhibiting CO2 atmosphere develops, but on the other hand, molds are

obligately aerobic so the lack of oxygen is also a mold-protecting mechanism.

Fish.The post mortem metabolism is important also in the fish. An importantreaction is the degradation of ATP which results in a transient accumulationof inosine monophosphate (IMP). This compound contributes to the sensoricappreciation of "fresh fish" taste. IMP is also utilised as a flavour improvingadditive in the food industry, in analogy with the meat flavour enhancingeffect of glutamine.

Fig 3.7. During the post mortemmetabolism in the fish tissue inosinemonophosphate (IMP) is transientlyaccumulated.

This metabolism has been utilised to develop a "fish-freshness" biosensor inJapan (Fig 3.8). Since the absolute level of the IMP varies much between fishsorts and even between individuals, it is not sufficient to analyse only theconcentration of IMP. Instead the ratio IMP/(IMP + inosin + hypoxanthine) isused as a fish-freshness index. The enzymatic biosensor measures the oxygenconsumption catalysed by xanthine oxidase. If only xanthine oxidase is

present in the analysis, the oxygen consumption represents the concentration

of hypoxanthine. If also the nucleotide phosphorylase is present, the oxygenconsumption represents the concentration of hypoxanthine + inosine. By

ATP

ATPaseADP

MyokinaseAMP

AMP-deaminaseIMP

PhosphomonoesteraseInosine

Nucleoside phosphorylase

hypoxhantine + ribose-P


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including also the 5'-nucleotidase the oxygen consumption also includes theIMP.

Fig 3.8. Principle of a "fish-freshness" biosensor based on analysis of the degradation ofIMP degradation. The oxygen consumption catalysed by xanthine oxidase is analyses withor without the enzymes nucleotide phosphorylase and 5'-nucleotidase and a index thatrepresents the concentration of IMP in relation to the sum of the metabolites is calculated.

The microbial spoilage of refrigerated fresh fish has large similarities withthat of fresh meat.Pseudomonas is often dominating in the spoilage flora (Fig3.9). A similar organism, Shewanella putrifaciens (previously calledPseudomonas putrifaciens orAlteromonas putrifaciens) is another spoilageorganism specifically associated with marine fishes. It has the capacity to

produce both hydrogen sulfide from cysteine and trimetylamine (TMA) byanaerobic respiration with TMAO as electron acceptor. Due to this capacity to

produce bad odour the fish may be spoilt at 10 times lower total microflora ifShewanella putrifaciens dominates.

Fig 3.9. Distribution ofspoilage organisms onrefrigerated fresh fish.

Aeromonas is mainlyassociated with fresh-water fishes andShewanella withmarine fishes.

CH2-P O

N

N

OH

N

N

OH OH

OH

OH

O

IMP

InosineHypoxanthine

1 5-nucleotidase

2 nucleotidephosphorylase

3xanthineoxidase

1 = IMP + I +Hx2 3

= I +Hx2 3

3 =HxEnzymer = analys

Index =IMP

IMP + I +Hx


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MilkMilk is a very good substrate for microbial growth. However, it is protected

by several antimicrobial mechanisms which favour the development of lacticacid bacteria if the temperature is not too low. The lactoperoxidase system isone of these antimicrobial systems in milk (Fig 3.10). Milk contains theenzyme lactoperoxidase and small concentrations of its substrate thiocyanate.The milk is contaminated with lactic acid bacteria during the milking. These

bacteria are catalase negative and therefore the hydrogen peroxide, whichalways is produced as a by-product in the metabolism, is not removed bycatalase as in other microbial systems. Instead, the lactoperoxidase uses thehydrogen peroxide to oxidise the thiocyanate to hypothiocyanate. Thiscompound is strongly oxidising and reacts with sulfhydryl groups in transport

proteins in the bacterial membrane, especially in Gram negative bacteria,

while the lactic acid bacteria are relatively resistant. The lactoperoxidasesystem has been reported to have an antimicrobial function also in tears andother body-fluids.

Fig 3.10. The lactoperoxidase system. The lactoperoxidase in milk uses the hydrogenperoxide to oxidise thiocyanate to the strongly oxidising hypothiocyanate which oxidisestransport proteins in bacterial membranes. Especially Gram negative bacteria are sensitiveto the hypothiocyanate.

When the milk leaves the udder it becomes infected by about 100 so callled

udder cocci per milliliter. During the further handling in the cow house the

milk is infected with several types of microorganisms as shown in Table 3.6

Table 3.6 The initial milk contamination microflora

Infection Source

E. coli

Enterococcus Feces

MicrococcusBacillus sporesMold sporesYeasts

Air

LactococcusLactobacillusGram-negative rods

Milking equipment

oxidase catalase

LPSCN

- HO-S-protein

OSCN-

thiocyanate

hypothiocyanate

HS-protein

H2O2

H2O

O2


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If the milk is stored at room temperature the "lactic streptococci", i.e.Lactococcus spp. will first dominate the microflora and protect it from mostof the other microorganisms by means of lactic acid production. EventuallyLactobacillus, which can grow at lower pH than the other bacteria (below 5)will dominate. This fermented milk is similar to yoghurt and it was previously

produced on the farms (Swedish filbunke). If the milk is stored furtherproteolytic molds will finally raise the pH and it will be further destroyed byputrification by Clostridium andBacillus. These reactions do not take place inrefrigerated milk.

When the milk is cooled after milking and stored refrigerated on the farm,psychrotrophic gram negative rods (Pseudomonas and similar) will dominate.These bacteria will not make it sour as does the lactic acid bacteria. If stored

too long the milk is spoilt by ammonia, peptides and free fatty acids. Thispsychrotrophic microflora, which itself is very heat sensitive, is known toproduce comparatively heat resistant proteases and lipases which may createproblems in the later storage. When the milk reaches the dairy it is pasteurisedwhich efficiently eliminates the psychrotrophic Pseudomonas flora and mostother bacteria. However, some of the more heat resistant organisms, mainlyLactobacillus and Micrococcus will survive, and the bacterial endosporesfromBacillus are not influenced at all by the pasteurisation.

After the pasteurisation the milk becomes re-infected with the dairy

equipment microflora. This may restore the psychrotrophic Pseudomonasflora or at bad hygiene even theEnterobacteriacae flora. The final spoilage ofthe refrigerated milk therefore differs depending on the contamination flora.Members of the Enterobacteriacae family may spoil the milk withfermentation.Bacillus spores my germinate and spoil the milk by proteolysis.This is especially common in fatty products like cream. Also proteolysis andlipolysis by enzymes from the earlyPseudomonas flora may contribute to thefinal spoilage of milk. However, the old days souring of milk by lactic acid

bacteria is not the common fate of refrigerated pasteurised milk.

EggThe egg is infected on the surface when the hen lays the egg. This flora isdominated by Pseudomonas, Staphylococcus, Micrococcus and fecal

bacteria. It is not uncommon that the hen is infected with Salmonella andduring the 1990ths many reports on Salmonella infected egg yolks appearedin England. The surface microflora is usually not infecting the interior of theegg due to a number of defence mechanisms, which are illustrated in Fig 3.11.If this protection fails and the egg becomes invaded by bacteria it is usuallyPseudomonas fluorescens which dominates (80%). These infections can be

detected by illumination of the egg with UV-light.


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Fig 3.11. The egg is protected against bacterial infections an multiple ways: The shell and

the two membranes provide mechanical hinders for the bacteria. The high pH in the eggwhite is non-optimal for many bacteria. The egg white contains several protectionmechanisms: Lysozyme ruptures cell walls of many bacteria. Albumin, conalbumin andavidin make several nutrients unavailable by strong complex formations.

3.2 Fruits and vegetables

Fruits and vegetables do have a high water activity but they develop anotherspoilage scenario than meat, fish and milk. Many of these products are

protected mechanically by the pectins which constitute a "glue" between thecells and gives rigidity. When fruits and berries ripen, endogeneous pectinasesstart to hydrolyse the pectin and this also makes the products more susceptibleto microbial attacks. Another common protection is the low pH of some ofthese products. This group of foods also has a much lower concentration offree amino acids and other nutrients than meat, fish, and milk. For thesereasons it is usually not the Pseudomonas and other spoilage bacteriamentioned above which dominate in the spoilage. Instead it is often pectinase

producing organisms, which mostly means molds, that initiate the spoilage offruits and vegetables. In the later phase, when the pectinolytic organisms have

opened up the defence structure, also yeasts participate in the spoilage.

One of few bacteria involved in spoilage of vegetables is the plant pathogenErwinia carotovora. This organism has been subject to studies of the corumsensing phenomenon which plays a central role in the ecology of manyorganisms. In this case the corum sensing is based on accumulation of N-acylated homoserine lactones (AHL) which accumulates around the cells (Fig3.12). When the concentration of AHL is high enough this compound inducesthe pectinase synthesis. The strategic advantage of not producing the

pectinase constitutively is obvious, since the plants have their defence

systems which generate antimicrobial chemicals when the plant is attacked.

Noprotect

ion

inner keratin

1-10m pores in shell

outer mucin layer

Con-albumin: Fe2+ complexingAvidin: Biotin complexingLysozyme: kills G+ bacteria

Albumin: viscous, high pH

(pH9,5), riboflavin + pyridoxincomplexing


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Only by delaying the pectinase synthesis until the number of bacteria is largeenough, can the hydrolysis of the pectins be fast and efficient enough. Oncethe pectinases have damaged the structure of the fruit/vegetable, otherorganisms follow and contribute to the soft rot. Due to the often low pH,molds and yeasts, rather than bacteria are common in the spoilage of these

products.

Fig 3.12. Erwinia carotovora utilises corum sensing to invade plants. They start byhydrolysing the protecting pectin layer with extracellular pectinases. When the plantrecognises a microbial attack it defends itself by producing antimicrobial ( )compounds. Instead of initiating this defence response at low concentration ofErwiniacells, they first accumulate acylated homoserine lactones (AHL) and when theconcentration is high enough this is a signal for induction of the pectinase ( )

production. By delaying the attack until many bacterial cells have accumulatedErwiniagains increased virulence.

It is estimated that only about 20% of the fruits and vegetables are spoilt bymicroorganisms. The endogenous metabolism of the products, which leads to

over-maturation plays a major role for the spoilage. Furthermore, drying alsocontributes to the spoilage. To reduce and better control the endogenousmetabolism, fruits and to some extent also vegetables are stored in modifiedatmospheres (Controlled Atmosphere, CA-storage). Common principles are toincrease the CO2-concentration, which also has a microbial inhibition effect,and to reduce the oxygen concentration by adding nitrogen gas. Many fruits

produce ethylene gas, which acts as a maturation hormone, and for someproducts absorption of the ethylene is included in the CA storage. Addition ofethylene or cessation of the absorption is then used to initiate the ripening.Table 3.7 gives an example of a modified atmosphere for fruits. The exactcomposition is optimised for each product.

AHL

AHL

AHL

AHL

AHL

pectinolytic

bacteria

plant cell


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Table 3.6. Example of modified atmosphere for storage of fruitsO2 : 0 - 5 %

CO2 : 2 - 10 %

N2 : 90-95 %

Relative humidity: 90-95%

3.3 Cereals

Grains on the field usually have a primary microflora of 103

- 106

bacteria g-1

.Lactic acid bacteria, coliform bacteria and Bacillus spores dominate. Aweather dependent flora of fungal spores is also present. At humid conditionsthe mold spore count can be 10

5g

-1. Different species ofAspergillus and

Penicillium usually dominate. If the grains are soaked in water the bacterialflora will dominate. Regulations set a maximum water concentration of 13%

for storage of grains for human food and then no significant microbial activityis expected due to the low water activity. If the water content exceed 15%mold growth begins. Even if the grains are kept dry enough according to theregulations, local humid zones may appear in the silos, e.g. due to watercondensation on walls. Under these conditions mold growth and mycotoxinformation may appear.

During the milling of the grain most of the microflora follows the hull butsome microorganisms are transferred to the flour. Typical microbial counts

are 10

2

-10

3

bacteria plus about 100

mold spores per gram sifted flour andabout ten times more in course flour. At correct dry storage of the flour thereis no microbial activity, but as soon as water is added a vigorous growthstarts.

The surface of the bread becomes sterilised in the oven and a dry hard breadsurface protects the bread against mold growth. If the bread is cut before

packing the surfaces are usually infected and if the bread is kept too moist in aplastic bag mold growth will spoil it. The inner part of a bread is usuallyheated to 95-99C which means it is essentially sterile with respect to

vegetative cells and mold spores. There is however a rare bakery problemcalled ropiness, which is caused by polysaccharide formation by Bacillussubtilis. The organism has then survived the baking in spore form and if thetemperature is kept at 30-45 C too long and the bread has not become dryenough during the baking the B. subtilis spores germinate and grow very fastand produce the polysaccharides.

During storage of the bread, spoilage is entirely caused by molds which havecontaminated the bread after the baking. To reduce the rate of mold growth

propionates are often used a preservatives in industrial baking. Dry bread(knckebrd) is not subject to any microbial spoilage, provided it is stored


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dry. Under such conditions the very slow spoilage is eventually caused byrancidification.

3.4 Preserved foodsThe spoilage of preserved food depends on the type of preservation. Ingeneral, if the preservation prevents microbial spoilage, the ultimate fate isusually spoilage by rancidification, which usually is a very slow process.

Dried products. In the drying process the water activity is reduced to so lowlevels that no microorganisms are active. If the storage conditions are not dryenough, mold formation may occur, but otherwise the shelf-life is limited byrancidification processes, which depend very much on the fat composition of

the product. During spray-drying the food is exposed temperatures that killthe most sensitive bacteria, but endospores, mold spores and more heatresistant vegetative bacteria asEnterococcus, Lactococcus, Micrococcus, andLactobacillus may survive. When such products (e.g. dry milk, soups, sauces,etc.) are reconstituted with water they are usually very susceptible to fastmicrobial spoilage and considerable risks for food poisoning.

Table 3.8. Summary of common spoilage floras on different types of food

Cured meat products are usually protected by the low water activity createdby salt addition. If the products are fermented they are also protected by thelactic acid and the competitive effects of the lactic acid bacteria. These

products are often further protected with nitrite. A common bacterium invacuum packed cured meat products, is Brochothrix thermosphacta. Thisorganism is similar to Lactobacillus (CO2 resistant and tolerant against low

aw) which usually dominates vacuum packed meat products, but it is a severe


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spoilage organism since it produces stinking metabolites. Also the low-awtolerant Micrococcus andLactobacillus are common in these products.

Salted fish and fish preserves are also protected mainly by the low wateractivity and chemical preservatives. In salted fish products mainly halophilicstrains ofPediococcus, Micrococcus, and yeasts grow and they do this at avery low rate with slow spoiling. Usually these products are to be cold storedand the main shelf-time limitation is usually rancidification of the fat.

Table 3.9.Properties of common food related organisms

Organsim Properties Products

Gramneg. rods:- Pseudomonas

- Shewanellaputrefaciens

- Psychrotropic- Aerobic- Sensitive to low aw

- Sensitive to low pH- CO2-sensitive

- H2S-producer

Refrigerated fresh:-Meat-Fish

-Milk

Fish

Lactic acid bacteria:-Lactobacillus-Lactococcus-Pediococcus

EnterococcusB. thermpsphacta

- O2-indifferent- Resistant to low aw

- Resistant to low pH- CO2-resistant

Fish preservesVacuum packedFermented foodSmoked/salted/dried meat/fishPickles

Grampositive cocci:

-Micrococcus-Staphylococcus

- Facultative

- Resistent to low aw- Resistant to low pH- Heat resistant- Lipo-/proteolytic

Fish preserves

Vacuum packedFermented foodSmoked/salted/dried meat/fish

Spore formers:-Bacillus-Clostridium

- Extremt vrmeresistenta- Mesofila- Starkt fermentativa

Heat sterilised foodReconstituted dried food.Pre-cookedMilk:B.cereus

Enterobacteriaceae:-E.coli-Enterobacter m.fl.

-Erwinia

- Strongly fermentative

- Pektinase-active

MilkPre-cooked foodVegetables

Molds - Aerobic- CO2-sensitive

- Pektinase-active- Resistant to low aw

- Resistant to low pH- Lipo-/proteolytic

VegetablesFruitDried food

Yeasts - Facultative- CO2-resistant

- Resistant to low aw

- Resistant to low pH

VegetablesFruitLow-pH preservesSweet products


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38

Chapter 4. Foodborne pathogens

Most cases of so called food poisoning are caused by microorganisms. Only a

few per cent of the food poisoning cases are reported to be caused by toxic rawmaterials like toxic mushrooms or plants or contamination by toxic impuritieslike heavy metals. The remaining cases of food poisoning can be divided intomicrobialfood intoxication, when microorganisms have produced toxins in thefood and microbialfood borne infections, when pathogenic microorganisms inthe food are ingested and infect the human body.

Intoxications and infections caused by microorganisms in food and wateraccount for a large number of fatal cases and large economic loss in thesociety. Food borne pathogens causes millions of death cases every year,especially in poor countries and it is especially children that are the victims. Itis difficult to estimate the true statistics behind the food borne diseases, sincemost cases are never confirmed by clinical analysis. This is especially true for"mild" but common diseases likeBacillus cereus intoxication and Clostridium

perfringens infections, since they are usually confirmed by analyses only inlarge outbreaks. On the other hand, statistics on the severe Clostridiumbotulinum intoxication is probably reflecting the true cases, at least in theindustrial world.

Fig 4.1 Number of cases with food borne diseases reported to the Swedish Institute forInfectious Disease Control according to the law for report on certain diseases (Averagenumber per year during 1997-2005).


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4. Foodborne pathogens 39

Only some of the microbial food poisoning diseases are reported to authoritiesaccording to law. See Fig 4.1. Other sources of statistics that also includeorganisms that are not covered by obligatory reporting gives a similar picture,

namely that Campylobacter, Salmonella and Norovirus (earlier calledcalicivirus) are among the most frequent causes of food borne illness, but italso shows that Clostridium perfringens and Staphylococcus often occur in theoutbreaks (Fig 4.2).

Fig 4.2 Statistics of food borne diseases in Sweden for a 5 year period. Calicivirus =

Norovirus .Source: Vr Fda, nr 5, 1999.

4.1 Microbial food intoxications

Staphylococcus aureus. The probably most common microbial foodintoxication is caused by certain strains of Staphylococcus aureus. Thisorganism is also known as a common pathogen causing infections in woundsand blood, but these infections are not considered to be transferred via food. S.aureus produces a series of toxins and other virulence factors (Table 4.1) but it

is mainly the enterotoxins that cause food poisoning after ingestion of food onwhich S. aureus has grown and produced the enterotoxins.

Table 4.1 Some virulence factors ofS. aureus

Toxins Exoenzymes

Membrane damaging toxins (several) CoagulaseEpidermolytic toxin StaphylokinaseToxic shock syndrom toxin ProteasesPyrogenic exotoxin PhospholipaseEnterotoxin ( 6 serotypes) Lipase

Hyaluronidase

Cases Outbreaks


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S. aureus is a common inhabitant on animals and humans where it grows onmucose membranes, for instance in the nose, even on healthy individuals, andit is frequently found in pus and wounds. The common source of food

contamination is therefore human hands. This organism is very resistant to lowwater activity (Table 2.2) which means that they can grow on salted andrelatively dry products. It does not grow under refrigerator conditions, andwithout growth no toxin is produced. It has low competitive power comparedto many other bacteria, like lactic acid bacteria and Pseudomonas. For thisreason, a small amount ofS. aureus is usually accepted in food ( e.g. 102 - 103g-1) before it is classified as not acceptable (Swedish: otjnligt) which meansthe product must be withdrawn from the market.

The intoxications are associated with a large number of foods, often food thathas been cooked which eliminates competing microorganisms and food that ishandled by human hands: Chicken, ham, salads, pizza, kebab, sauses, paseriesetc. The enterotoxins are very heat stable and contaminated food may thereforestill be poisonous after re-heating when all vegetative cells have been killed.

The disease caused when eating S. aureus enterotoxis is characterised by aviolent nausea with vomiting, diarrhoea and convulsions. It is one of the fewcases when the eating of infected food results in an almost immediate illness,within one or a couple of hours. The patient usually recovers in 1-2 days and

the disease is not associated with further complications.

Bacillus cereus. This organism is a facultatively anaerobic endospore formerthat is ubiquitously present in Nature. Therefore vegetables are usuallycontaminated with this organism. It is also frequently present in milk, probablysince the dusty air in the barn contaminates the milk and the subsequentpasteurisation has no effect on the endospores, while most competing organismare killed. B. cereus produces three enterotoxins which cause relatively milddiarrhoeal illness with an incubation time of 6-24 hours, and an emetic toxin,

cereluid. The haemolysins are inactivated in the stomach and this type ofdisease is actually an infection where the toxin is produced locally byB. cereusgrowing in the intestine. The cereluid is a heat stable protein and this disease isconsidered to be a true intoxication. It has a shorter incubation time, 0.5-6hours, and is especially associated with rice dishes. B. cereus is assumed to bea very common agent of food poisoning, but both diseases are usuallyproceeding fast with little complications and therefore isolated outbreaks arenormally not identified and the statistics becomes unsure. B. cereus is not socompetitive but after heating of a product the spores may become the

dominating organisms and if the food after that is kept too long in thetemperature range 15-45C the spores may germinate and grow and produce


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the toxin. Like mostBacillus this organism is typical mesofilic with respect totemperature, but certain strains are reported to be psychrotrophic and maygrow down to about 4 C.

Clostridium botulinum. The most well-known and feared microbialintoxication is botulism, which is caused by one of several toxins of Cl.botulinum. This organism is an obligately anaerobic spore forming bacteriumthat is very common in soil and water. The toxin is classified according toserotype A-F, where type A, B, E, and F are toxic to humans. Cl. botulinumtype E is commonly found on fishes and this toxin is relatively heat labile,destroyed by boiling, while type A is more heat resistant.

The endospores make also heat treated food potentially dangerous sincesurviving spores may grow out. The botulin toxin is a very toxic protein that isproduced during growth of the vegetative cells in food. Cl. botulinum does notgrow at temperatures below 4C, at pH below 4.5, or in presence of oxygen.The toxin acts as a neurotoxin paralysing the central nervous system. It is oneof the most potent toxins known with a lethal dose of about 10-6 g. After anincubation time of 18-36 hours, the illness sometimes starts with nausea and isfollowed by the effects on the CNS caused by blocking of the acetyl cholinerelease at the nerve synapses: double-seeing, difficulties to swallow and finallyparalysing of the breathing. At this stage the mortality is high. In US statistics

during 1950 - 1970 the number of fatal cases was almost as high as the numberof reported cases. After that an anti-toxin became available but mortality is stillconsiderable. Fortunately, the number of cases is low, in Sweden the average isless than one/year.

The few cases of botulism in Sweden are associated with home preserved(marinaded or smoked) fish and home preserved meat. The precautions thatmust be taken to avoid botulism in association with food preservation are lowpH (often vinegar), high salt concentration and storage below 4C. In

commercial preservation nitrate also plays an important role. This is furtherdescribed in Chapter 6.

The so called infant botulism has another mechanism. It is caused by a Cl.botulinum infection of the intestines where the spores germinate, grow andproduce the toxin. This disease is only associated with babies under one yearage who have not obtained the normal competitive intestinal microflora, andthe infection origin has exclusively been honey which often (10%) containsspores of Cl. botulinum. For this reason authorities recommend not to give

honey to babies.


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Mycotoxins. Intoxications by fungal toxins, mycotoxins, are not found in thestatistics on food borne diseases. The reason is that these diseases, contrary tothe other diseases discussed here, do not cause acute symptoms. Most reports

on mycotoxins describe their effects as cancerogenic or liver or kidneydamaging, with symptoms emerging long time after consumption of the food.An exception is patulin, which is associated with intestinal illness but it is alsoa suspected carcinogen.

There are hundreds of mycotoxins described in the literature. Biochemicallythey are typical secondary metabolites produced by moulds. It means they aremainly produced late in or after the growth phase. Most mycotoxins areresistant to temperatures used in cooking. Fig 4.3 shows the chemical structureof some mycotoxins. For some of the mycotoxins (e.g. aflatoxin

food micro bio loy

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