potential of high isostatic pressure and pulsed electric ... · different fields of work. i would...

Potential of high isostatic pressure and pulsed electric fields for the processing of potato and pea proteins – structural and techno-functional characterization in

model solutions and plant tissue

vorgelegt von Dipl.-Ing.

Anne Kathrin Baier aus Heppenheim an der Bergstraße

von der Fakultät III – Prozesswissenschaften der Technischen Universität Berlin

zur Erlangung des akademischen Grades

Doktor der Ingenieurwissenschaften - Dr.-Ing. -

genehmigte Dissertation

Promotionsausschuss Vorsitzende: Prof. Dr. Dipl.-Ing. habil. Cornelia Rauh Gutachter: Prof. Dr. Dipl.-Ing. Dietrich Knorr Gutachter: Prof. Dr. rer. nat. habil. Sascha Rohn Tag der wissenschaftlichen Aussprache: 07.08.2015

Berlin 2016 D 83

A

Zusammenfassung

Ziel dieser Arbeit war das Potential isostatischen Hochdrucks und gepulster elektrischer Felder für

die Produktion hochwertiger Pflanzenproteine zu bewerten. Die Charakterisierung der in

Proteinlösungen und pflanzlichem Gewebe von Kartoffel und Erbse hervorgerufenen

Veränderungen erfolgte anhand struktureller und techno-funktioneller Merkmale sowie durch die

Untersuchung von Diffusions- und Extraktionsvorgängen.

Die Anwendung isostatischen Hochdrucks stellt eine schonende Alternative zu konventionellen

thermischen Haltbarmachungsverfahren dar. Insbesondere für die Pasteurisation von

Kartoffelproteinlösungen bietet die Technologie deutliche Vorteile, da strukturelle und

funktionelle Änderungen bei Drücken bis zu 600 MPa im Gegensatz zu einer thermischen

Behandlung zwischen 60 und 80 °C minimal ausfielen. Hochdruck bewirkte des Weiteren eine

Modifizierung der Proteine und Stärke in Erbsen, die sich klar von denen einer Hitzebehandlung

unterschied. Druck führte zu einer Aggregation der Erbsenglobuline während hohe Temperaturen

vor allem die Albuminfraktion PA2 beeinflussten. Mithilfe der Hochdruckbehandlungen konnten

Proteinkonzentrate mit besonderen funktionellen Eigenschaften sowie Mehle mit veränderter

Stärkeverdaubarkeit gewonnen werden.

Gepulste elektrische Felder ermöglichen eine Permeabilisierung biologischer Membranen ohne

gleichzeitige Hitzeschädigung empfindlicher Lebensmittelinhaltsstoffe. Lokale pH-Änderungen

oder verstärkte Oxidationsvorgänge im elektrischen Feld modifizierten das Protein im

Kartoffelgewebe bereits bei einer Feldstärke von 1 kV/cm und verbesserten die

Schaumeigenschaften der hergestellten Proteinkonzentrate (24 % höheres Schaumvolumen). Die

dafür verantwortlichen strukturellen Veränderungen konnten jedoch nicht vollständig aufgeklärt

werden. Hochspannungsimpulsbehandlungen von bis zu 10 kV/cm erhöhten die Effizienz von

Erbsentrocknung und -rehydratation sowie die Ausschleusung von Raffinose-äquivalenten

Oligosacchariden. Die Erbsenproteine wurden durch ein elektrisches Feld dieser Intensität oder

seine Nebenreaktionen nicht beeinflusst. Folglich kann diese Technologie genutzt werden um den

Stofftransport im Erbsengewebe zu verbessern oder die Haltbarkeit der Proteinlösung durch

Inaktivierung vegetativer Mikroorganismen zu verlängern ohne die Proteingewinnung oder

-qualität zu beeinträchtigen.

Hochspannungsimpulse und isostatischer Hochdruck können somit einen wertvollen Beitrag zur

Prozessierung pflanzlicher Proteine liefern. Weitere Forschung ist erforderlich um ihr Potential für

diesen Lebensmittelsektor vollständig auszuschöpfen und konkrete Anwendungsmöglichkeiten für

innovative Pflanzenproteinprodukte aufzudecken.

B

Abstract

The aim of this thesis was to evaluate the potential of high isostatic pressure and pulsed electric

fields for the production of high quality plant proteins. Induced changes in protein solutions and

plant tissue of potato and pea were analyzed by means of structural and techno-functional

characterization as well as by investigation of diffusion and extractions procedures.

The application of high isostatic pressure provides a gentle alternative to conventional heat

preservation. Especially for the pasteurization of potato protein solutions the technology offers

significant benefits as structural and functional changes due to pressures up to 600 MPa were

minimal in contrast to thermal treatments between 60 and 80 °C. High pressure furthermore led

to modifications of proteins and starch in peas differing from those of a thermal processing.

Pressure led to an aggregation of pea globulins, whereas high temperatures mostly affected the

albumin fraction PA2. Protein concentrates with unique functional properties and flours with

modified starch digestibility could be produced by the application of high pressure.

Pulsed electric fields can permeabilize biological membranes without causing extensive heat

damage of sensitive food ingredients. Local pH changes or enhanced oxidation activity in the

electric field modified proteins in potato tissue already at an electric field strength of 1 kV/cm and

led to the recovery of protein concentrates with improved foaming behaviour (24 % higher foam

volume). However, the responsible structural alterations could not be completely clarified. Pulsed

electric field treatments up to 10 kV/cm enhanced the efficiency of pea drying and rehydration as

well as the release of raffinose equivalent sugars. Pea proteins were not affected by an electric

field of this intensity and its side reactions. Thereby, this technology can be used to improve mass

transport within pea tissue or to extent shelf-life of protein solutions by inactivation of vegetative

microbial cells without altering protein recovery or quality.

Pulsed electric fields and high isostatic pressure are thereby suitable to make a unique

contribution to the processing of vegetable proteins. Further research is needed to completely

exploit their potential for this food sector and to exactly figure out application possibilities for

innovative plant protein products.

C

Acknowledgements

First of all, my sincere gratitude goes to Prof. Dr. Dietrich Knorr for this interesting research topic,

inspiration and motivation during the last years and the opportunity to collect experience in many

different fields of work. I would also like to thank Prof. Dr. Cornelia Rauh for being the chair of my

defence committee and for giving me the possibility to finish this thesis at her department. My

very special thanks go to Prof. Dr. Sascha Rohn for his willingness to evaluate this thesis and for

the pleasant atmosphere within the application and realization of research projects.

Part of this research was supported by funds of the Federal Ministry of Food and Agriculture

(BMEL) based on a decision of the Parliament of the Federal Republic of Germany via the Federal

Office for Agriculture and Food (BLE) under the innovation support programme. Thanks to all

partners from the project LeguAN for the fruitful cooperation. Particular thanks go to Annika

Weckmüller for the protein analysis via Kjeldahl method. I would also like to express my thanks to

Prof. Dr. Harshadrai Rawel and Dr. Christophe Schmitt for their support in the establishment of

protein analytics. Christophe is furthermore acknowledged for the supply of potato protein

isolate. I am thankful for the possibility to use the zetasizer equipment of the department of Food

Technology and Material Science.

I would like to thank Ariane Weiler, Christian Hertwig, Sara Bußler, Mareike Wiggers, Benedict

Purschke, Alejandro Valdez, Melanie Bergmann, and Phuong-Vy Nguyen for their committed work

that made a major contribution to this thesis and to the success of research projects. Thanks go to

all colleagues from the department of Food Biotechnology and Food Process Engineering for the

wonderful time and the great working atmosphere. Special thanks go to Irene Hemmerich,

Doreen Schiller and Mike Richter for their analytical support and to Sophie Uhlig for her help in all

administrative issues. I particularly thank Erik Voigt for proof reading and his great contribution to

the linguistic quality of this thesis.

I am grateful to my parents for their never-ending support during my PhD and the provision of an

anytime-daycare centre that made completion of the thesis possible. Daniel, thank you so much

for your love, the time you spent helping me with experiments and technical concerns and your

patience during moments of self-doubt. Finally, I would like to thank Emilie for being the best

motivation to finish the written part of this thesis.

Index

D

Index

Zusammenfassung .......................................................................................................................... A

Abstract .......................................................................................................................................... B

Acknowledgements ........................................................................................................................ C

Index ............................................................................................................................................... D

Preface ............................................................................................................................................ 1

Chapter I Trends and aspects of vegetable protein supply............................................................ 5

Sustainable protein supply – a global challenge ............................................................................ 7

Plant-based diets – also a health issue......................................................................................... 11

Protein recovery from domestic non-animal sources .................................................................. 16

Chapter II Proteins as techno-functional ingredients ................................................................... 33

Food proteins on a mission - the main functional properties used in industry ........................... 36

Structure-functionality relationships of proteins ......................................................................... 42

Modification and preservation of protein functionality .............................................................. 47

Chapter III The use of emerging technologies to alter protein structure .................................. 61

Changes in potato protein structure after thermal and high pressure treatments ..................... 64

Influence of high pressure and elevated temperature on pea proteins ...................................... 80

Changes in protein structure induced by pulsed electric fields (PEF) .......................................... 95

Chapter IV High pressure and pulsed electric fields as alternatives for protein modification and

preservation ................................................................................................................................ 105

Foaming properties as an indicator for techno-functional alterations ...................................... 106

High pressure, high temperatures and pulsed electric fields for shelf-life increase of protein

solutions ..................................................................................................................................... 117

Chapter V Innovative processing and its effect on vegetable tissue ...................................... 131

Cell structure and cell disintegration – influence of innovative and conventional processing . 132

Release of cellular compounds during thermal, high pressure and pulsed electric field treatment

.................................................................................................................................................... 137

Index

E

Influence of thermal and non-thermal pre-treatments on the efficiency of mass transport.....141

Chapter VI Emerging technologies and their potential in protein recovery ............................ 155

The influence of pulsed electric fields on potato protein extractability and quality ..................156

Heat, high pressure and pulsed electric fields and their influence on yield and properties of pea

proteins .......................................................................................................................................165

Chapter VII Heat and high pressure to modify the properties of pea flour .............................. 177

Functional properties of modified pea flours .............................................................................178

Influence of heat and high pressure on enzyme activity in pea flour .........................................185

Changes in the enzymatic digestibility of pea protein and starch after heat and pressure

application ...................................................................................................................................193

Chapter VIII The potential of high pressure and pulsed electric fields for protein processing –

conclusion and future perspective ................................................................................................. 211

Annex A Material and Methods ................................................................................................ 221

Material .......................................................................................................................................222

Buffer systems .............................................................................................................................223

Heat, high pressure, pulsed electric field and mechanical treatments ......................................225

Mass transfer processes ..............................................................................................................230

Tissue properties .........................................................................................................................234

Quantification of single components ..........................................................................................236

Protein characterization ..............................................................................................................244

Techno-functionality tests ..........................................................................................................251

Evaluating impacts on sample shelf-life ......................................................................................253

Enzymatic digestibility .................................................................................................................256

Data evaluation and statistical analysis ......................................................................................257

Annex B Indices ........................................................................................................................ 261

List of figures ...............................................................................................................................262

List of tables ................................................................................................................................273

Abbreviations ..............................................................................................................................275

Index

F

Annex C Personal information .................................................................................................. 277

Curriculum vitae ......................................................................................................................... 278

Publications / Presentations ...................................................................................................... 280

Eidesstattliche Erklärung ............................................................................................................ 282

Preface

1

Preface

A growing world population as well as economic development and altered consumption patterns,

especially in developing countries, led to an increased global demand for animal protein in the

recent years. Providing that this demographic trend will continue in the coming decades the

needs for dietary protein can soon no longer be covered by the livestock industry. The

requirement for long-term, sustainable supply strategies promotes research in the field of

alternative protein sources. Plants receive special attention within these raw materials, as many

of them are already part of the human diet and therefore possess a high consumer acceptance.

Beside the supply with nitrogen and essential amino acids dietary proteins have to fulfil certain

functional requirements to find broad application in industrial food production. Its ability to

stabilize interfaces in foams and emulsions, form gel networks or film structures are important

criteria when choosing a protein as techno-functional ingredient. An effective recovery of high

quality protein from vegetable sources is still a big challenge in the related industrial sector. A

high microbial load, the need to inactivate endogenous enzymes or to remove antinutritives often

entails extensive processing of plant proteins that further reduces their functional quality.

Implementation of these new protein sources therefore requires innovative technological

concepts that achieve food safety while maintaining nutritional and functional properties.

High isostatic pressure and pulsed electric fields came into focus of the food industry in the recent

years as they offer a non-thermal and gentler processing of temperature-sensitive products. The

first high pressure units found their way into industrial food production more than 20 years ago.

They are mainly used as alternatives to conventional heat pasteurization and sterilization as

sensorial and nutritional properties can be preserved to a greater extent. Nevertheless, high

pressure also offers the opportunity to modify macromolecules like proteins and starch and

thereby achieves specific functional properties differing from those obtained by traditional

processing steps. A detailed introduction to high pressure processing of foods can be found for

instance in Smelt (1998), Hendrickx and Knorr (2002) and Rastogi et al. (2007). Pulsed electric

fields can permeabilize biological membranes due to attraction of charges by the respective

electrodes. Accordingly, this technology is applied to improve mass transfer processes or to

inactivate vegetative cells in heat labile solutions. The dependence on certain intrinsic factors like

cell geometry and size allows a targeted realization of specific process goals. The mechanism of

action, process parameters, equipment types and application possibilities are well described by

Barsotti et al. (1999), Barsotti and Cheftel (1999) and Toepfl et al. (2005).

The aim of this thesis is to investigate and to identify the potential of these two emerging

technologies for protein processing with regard to recovery concepts, shelf-life increase and

Preface

2

modification of functional properties. Potatoes and peas were chosen as representative plant

materials available on the regional market. Both vegetables accumulate starch as energy carrier

for germination, but they differ in tissue properties like cell size and water content and in protein

structure and composition. As a pulse traditionally cultivated in Central Europe, peas are an eco-

friendly alternative to imported soybeans or products made of them. The plant’s ability to fix

environmental nitrogen is a further benefit of legume cultivation. The protein content of potatoes

is comparably low but the high turnover of tubers, especially in starch recovery, leads to large

amounts of insufficiently used protein. Innovative technologies may help to strengthen crop

rotation with legumes and contribute to a holistic usage of the potato tuber. Thermal treatments

will be performed as reference process.

The thesis is subdivided in eight chapters. The first two sections introduce to the demand for

alternative protein sources, as well as to the close process-structure-function relationship of

protein products. The influence of heat, pressure and pulsed electric fields on the structure of

proteins in model solutions will be investigated in chapter III. How these results may affect the

usage of the technologies for shelf-life increase of functional modification will be discussed in

chapter IV. The subsequent two sections are dedicated to the improvement of mass transfer from

potato and pea tissue, in general and with regard to protein recovery. Chapter VII will deal with

the influence of different pre-processes on the properties of pea flour. Finally, potential

application fields for emerging technologies in protein processing will be evaluated. A detailed

description of the experiments performed will be given in the annex.

References

Barsotti, L. & Cheftel, J. C. (1999). Food processing by pulsed electric fields. II. Biological aspects. Food Reviews International, 15(2), 181-213.

Barsotti, L., Merle, P. & Cheftel, J. C. (1999). Food processing by pulsed electric fields. I. Physical aspects. Food Reviews International, 15(2), 163-180.

Hendrickx, M. E. G. & Knorr, D., Eds. (2002). Ultra High Pressure Treatments of Foods. Food Engineeering Series. Springer Science and Business Media, New York.

Rastogi, N. K., Raghavarao, K. S. M. S., Balasubramaniam, V. M., et al. (2007). Opportunities and challenges in high pressure processing of foods. Critical Reviews in Food Science and Nutrition, 47(1), 69-112.

Smelt, J. P. P. M. (1998). Recent advances in the microbiology of high pressure processing. Trends in Food Science & Technology, 9(4), 152-158.

Preface

3

Toepfl, S., Heinz, V. & Knorr, D. (2005). Overview of pulsed electric field processing of foods. In: Emerging Technologies for Food Processing. Elsevier, Oxford, UK.

Chapter I – Trends and aspects of vegetable protein supply

5

Chapter I

Trends and aspects of vegetable

protein supply


6

Proteins are an essential component of human and animal organisms as they form a major

structural component of all body cells. Additionally, they are involved in metabolic processes of

the entire organism, for instance in form of enzymes, membrane carriers or hormones. The

synthesis of proteins for their different functions is carried out by the conversion of dietary

protein. The constant intake of high quality food protein is therefore a requirement for good

health and a constant issue in the food and health industry. It ensures the supply with essential

amino acids and with non-specific nitrogen for the synthesis of nutritionally dispensable amino

acids and other physiologically important nitrogen-containing compounds. Pathways of protein

metabolism are illustrated in Figure I.1. Excess nitrogen is secreted via the urea cycle.

Consideration of personal protein and amino acid needs in the everyday diet is therefore

recommendable to not overly stress liver and kidneys.

Figure I.1: Schematic representation of the metabolic demands for amino acids (according to WHO et al., 2007).

Nowadays, at times when low carb diets dominate the front pages of women’s and fitness

magazines the advisable daily protein intake is intensively discussed and affects the work of many

scientists from the food and nutrition area. Advantages mentioned in this context are positive

effects of a protein rich diet on body composition and blood lipid profile during weight loss in

comparison to a high carbohydrate (Layman et al., 2003) or standard diet (Farnsworth et al.,

2003), as well as the role of protein for the short-term satiety and hence for the maintenance of

tissueprotein

replation ofgrowth andfasting loss

free amino-acidsubstrate pool

ureasynthesis

metabolicdemand

uppergastro-

intestinaltract

lowergastro-

intestinaltract

ureasalvage

surplusnitrogen

urinarynitrogen

sweatnitrogen

faecalnitrogen

dietaryprotein

protein losses(skin, hair,…)


7

body weight (Anderson & Moore, 2004). However, long term effects of this eating habit are

unknown due to a lack of statistically relevant data (WHO et al., 2007).

The institute of medicine of the national academics recommended in 2005 a daily allowance of

0.8 g of protein per kg of body weight for an adult based on meta-analysis of nitrogen balance

studies (National Academies of Sciences, 2005). This means a slightly increase in the

recommended protein intake since 1985 when the FAO proposed a safe protein intake of 0.75 g

per kg body weight for grown-up male and female (Young & Pellett, 1994). Recommended values

for children and adolescents are higher relating to their body weight depending on their age and

gender.

Alternative protein sources from plants, insects or microorganisms come more and more into

focus of government and industry as it is foreseeable that the increasing protein demand

predicted for the next decades cannot be satisfied with existing agricultural and industrial

concepts. In addition, an increased nutritional awareness and animal welfare contribute to

changes in the consumption pattern, especially in industrialized regions. The following chapter

introduces to the challenges of sustainable protein supply, to health aspects of a plant based diet

and to the alternative protein sources currently discussed in food research.

Sustainable protein supply – a global challenge

In 2005, the daily protein intake in the world averaged 85 g per person. The large deviation

between different geographic and economic regions is illustrated in Figure I.2. People in the

European Union consumed 105 g protein per person per day, whereas in Southern and Central

Africa the average protein consumptions amounted 49 and 39 g per person per day, respectively

(FAO, 2013). These differences underline that supply with adequate amounts of protein and

essential amino acids still cannot be ensured for 13 % of the world’s population. Insufficient

protein supply over a long time period, especially during growth phase, implicates serious health

issues. Protein malnutrition of children can lead to growth failure, a weakened immune system

and even to their death (Chen et al., 1980).

Human protein requirements can be covered by the consumption of protein deriving either from

animal or vegetable origin. Currently 61 % of the global protein supply is covered by plant foods.

Regional differences are not only observed in the protein intake but as well in the sources the

protein supply derived from (see as well Figure I.2). The consumption of animal products is

affected by traditions and religious influences; not least it is an indicator for the economic wealth

of a region (Smil, 2002). The developed countries cover their protein consumption to more than


8

Figure I.2: Dietary protein consumption and percentage coming from livestock products (according to FAO, 2013).

50 % from animal protein, while in Burundi, Malawi and Sierra Leone more than 95 % of the

protein derived from vegetable sources.

The global production in livestock and fishery steadily increased in the recent decades (see Figure

I.3a), on the one hand due to a growing world population, on the other as a response to the

increasing demand for animal products. Economic growth, rising incomes and urbanization

promote the consumption of livestock products in the developing countries, where the average

annual growth of meat, milk and egg consumption amounted 2.6, 3.1 and 2.1 % (FAO, 2009). In

the developed countries the intake of livestock products is more than three times higher and

increased by approximately 0.6% between 1995 and 2005. Especially in countries with high levels

of gross domestic product per capita, contrary trends towards vegan and vegetarian diets or

excessive consumption of animal products can be observed. A survey in the EU 25 in 2005

revealed that more than half of the respondents eat meat and meat products more than four

times a week, whereas 2 % declared to strictly abstain from meat (Figure I.3b).

The growing demand for livestock products requires huge amounts of the global land and crop

sources. 30 % of the ice-free terrestrial surface is occupied by livestock, directly as grassland and

indirectly by feed production (Steinfeld et al., 2006a). Trends in livestock production also

influence livestock feeding. The use of traditional fibrous, energy-rich roughages is declining,

while protein rich products together with feed additives became the first choice in intensive

> 50 % of protein from livestock

10-50 of protein from livestock< 10 % of protein from livestock

> 100 g protein / person /day

60-100 g protein / person /day< 60 g protein / person /day

No data available


9

livestock breeding (Steinfeld et al., 2006b). Cereals are, with 60 % by weight, the main feed crops,

followed by oilcake and tubers with 17 % and 11 %, respectively (FAO, 2009). In 2005, 1250

million tons of concentrated feed were used world-wide, containing approximately 77 million tons

of protein that would be also suitable for human nutrition (Steinfeld et al., 2006a).

Figure I.3: Trends in the consumption of livestock products: a) development of the global production and per capita consumption of livestock goods and fish (FAO, 2013); b) meat consumption in the EU 25 per weak (European Commission, 2005).

A multiple of feed protein is necessary for the production of animal protein due to inefficiently

conversion of nutrients. On average, 10 g of vegetable protein are needed to generate 1 g of

0

20

40

60

80

100

120

140

160

0

200

400

600

800

1000

1200

1400

1600

1961 1964 1967 1970 1973 1976 1979 1982 1985 1988 1991 1994 1997 2000 2003 2006 2009

per c

apita

con

sum

ptio

n in

kg

/ yr

prod

uctio

n in

Mill

t

fish and seafood hen eggs meat dairy products

livestock and fish production

2%9%

35%

27%

27%

Never

Once a week

Two or three times a week

Four or five times a week

More than five times a week

a)

b)


10

animal protein (Reijnders & Soret, 2003), strongly depending on type of protein and livestock

breeding (see Table I.1). Their higher basal metabolism, large body mass and long gestation and

lactation periods make ruminants the least efficient energy and protein converters (Smil, 2002). In

contrast to animals, plants do not depend on the consumption of other organism to receive the

amino acids necessary for protein synthesis. Enzymatic conversion of nitrogen from surrounding

nitrate sources enables plants to synthesize amino acids by themselves.

Table I.1: Environmental evaluation of animal and vegetable protein production

Protein conversion efficiency in %

Fossil energy requirement

in kcal/kcal of protein e

Water footprint in L/g of protein h

Beef cattle 4 - 11 a, b, c, d 40 112

Lamb 4 a 57 63

Pork 9 - 16 a, b, c, d 14 57

Poultry 18 - 33 a, b, c, d 4 34

Hen eggs 26 - 29 a, c 39 29

Milk 25 - 31 a, c, f, g 14 31

Cereals ---

2.2

21

Pulses --- 19

Oilcrops --- 16

aWedin et al. (1975); bPimentel and Pimentel (1982); cFlachowsky (2002); dSmil (2002); ePimentel and Pimentel (2003); fWang et al. (2007); gHuhtanen and Hristov (2009); hHoekstra (2012)

Reducing livestock production in favour of growing vegetable human food seems to be an obvious

solution to combat world hunger. However, comprising the economic motives of the producers,

limitations in livestock production would probably lead to a reduction in crop production.

Malnutrition in poor regions is often a result of lacking purchasing power, not of an inadequate

food supply. Extensive cultivation of feed crops can even provide a buffer for times of food

shortage due to unexpected crop failure, political crisis or natural disasters. Nevertheless, the

livestock sector does influence demand and prices of the whole food sector to a large extent in

detriment of economically weak consumers. (FAO, 2009)

Intensive livestock production extensively contributes to serious environmental issues. The

agricultural revolution in the last three centuries made the food industry increasingly dependent


11

on fossil energy sources. Production of animal protein requires a higher use of fossil fuel mainly

due to inefficient conversion of feed protein. The world agricultural sector contributes to 20 % to

the greenhouse-gas emissions, whereof 80 % are caused by livestock production (McMichael et

al., 2007). 27 % of the water footprint of humanity can be traced back to animal products. The

water footprint is an indicator for the water use in relation to consumer goods and consists of the

volume of water that is consumed from the global fresh- and rainwater sources as well as of the

volume required to dilute polluted water to quality standards. Feed crop cultivation makes with

98 % the largest contribution to the water footprint of livestock breeding (Mekonnen & Hoekstra,

2010). Transportation over long distances is usually necessary and makes feed cultivation even

less ecological. The smaller water consumption of traditional roughages compared to modern

feed concentrates results in a smaller water footprint of grazing systems compared to intensive

industrial livestock breeding (Hoekstra, 2012).

The global food market requires reorganization towards responsible and sustainable handling of

terrestrial commodities. Moderate meat intake, replacement of animal foods by vegetable

alternatives and shift towards more expensive, but more sustainable livestock systems could help

to preserve the global energy and water resources. Production systems with high efficiency like

insects or algae have to be taken into consideration to supply the growing population with

sufficient amounts of protein. Available sources have to be used and shared more equally.

Primarily countries with immoderate energy and protein consumption have to change their eating

habits and regulate overproduction of food. Accurate consumer education and provision of

attractive product alternatives are important tools to change buying and dietary patterns in the

developed countries. Rethinking in the population and changes in product demands would also

influence the food industry in regard to more sustainable portfolios and production concepts.

Plant-based diets – also a health issue

Besides the general quantity of protein delivered by the human diet, quality and composition of

the protein absorbed are of great importance to guarantee the sufficient supply with essential

amino acids. In the past, protein from animal sources was often considered more valuable as its

composition is similar to that required in the human body. Young and Pellett (1994) compared the

nutritional value of different plant proteins and identified deficiencies of plant proteins in

comparison to those deriving from animals regarding the supply of essential amino acids (see

Table I.2) and the true digestibility of the protein, that ranged from 94 to 97 % for meat, milk and

eggs, to 86 and 78 % for cereals and beans, respectively. However, the authors stressed that

regarding the high protein consumption recorded in the developed countries the adequate supply


12

with all essential amino acids could be ensured despite the renouncement of meat and other

animal products. A mixture of different vegetable foods is recommended to balance deficiencies

of single protein sources. The opinion that in developed countries a meat-free, plant-based diet

supplies sufficient amounts of protein and amino acid is shared by Millward (1999) and McDougall

(2002). Messina and Mangels (2001) recommended slightly higher protein intakes for vegan

children than for non-vegans to balance lower digestibility rates. A diversified vegan diet can meet

the protein requirement of all ages, whereas adequate intake of iron, zinc, calcium, vitamin B12

and riboflavin is a critical issue in the vegan diet, especially during growth phase that has to be

balanced by carefully composed diets and possibly by fortified foods and supplements (Sanders &

Reddy, 1994; Messina & Mangels, 2001).

Table I.2: Survey of the amino acid content of different food protein sources (Young & Pellett, 1994)

Food Source Lysine Sulphur amino acids Threonine Tryptophan

mg/g protein

Legumes 64 ± 10 25 ± 3 38 ± 3 12 ± 4

Cereals 31 ± 10 37 ± 5 32 ± 4 12 ± 2

Nuts, seeds 45 ± 14 46 ± 17 36 ± 3 17 ± 3

Fruits 45 ± 12 27 ± 6 29 ± 7 11 ± 2

Animal Foods 85 ± 9 38 44 12

Nevertheless the interest in vegetarianism is steadily increasing. Trends in the number of

vegetarians and vegans can only be estimated due to a lack of reliable and comparable data. A

major change in meat consumption happened as a consequence of BSE outbreak at the end of

2000. Data Collection at the beginning of 2001 resulted in 8 % of vegetarians in Germany, which

means a tenfold increase since 1983 where a percentage of 0.6 % German vegetarians was

reported (Vegetarierbund Deutschland, 2013). Within a National Food Consumption survey in

2008, 1.6 % of the population described themselves as vegetarians; approximately 0.1 % of the

population followed a vegan diet (MRI & BMELV, 2008).

Beside environmental concerns and animal welfare, health aspects are the main reasons for a

meat-free diet (see Figure I.4). Sabate (2003) described a paradigm shift in the Western

population. In contrast to meat-based diets, the benefits of plant foods are thought to

overbalance possible nutritional deficits. Vegetarianism is associated with lower risk of

cardiovascular diseases, type 2 diabetes and prevalence of obesity (Burr & Sweetnam, 1982;

Dwyer, 1988; Kestin et al., 1989; Fraser, 2009), as these health problems are strongly influenced


13

by dietary patterns (Hu et al., 1999; Fung et al., 2001). A vegetarian diet is often accompanied

with a higher intake of fruits, vegetables, nuts and vegetable oils that are all considered as health

protecting foods (Ness & Powles, 1997; Sabate, 1999).

Figure I.4: Reasons cited for being a vegetarian, agreement in % (Statistic Brain, 2012).

Studies also revealed the positive effects of a protein rich diet based on vegetable food on the risk

of coronary heart diseases (Halton et al., 2006) and type 2 diabetes (Halton et al., 2008), whereas

high intakes of red and processed meat are considered as risk factors for type 2 diabetes (Aune et

al., 2009). Processed meat is also associated with an increased risk of coronary heart disease

(Micha et al., 2010). Processing of meat, as well as of other foods, is often connected to an

increase of saturated fatty acids, which are regarded as promoting factors for high cholesterol

levels and coronary heart diseases (Ulbricht & Southgate, 1991; Kromhout et al., 1995).

More than 90 % of all deaths in Germany can be attributed to these noncommunicable diseases

(see Figure I.5). Per capita government expenditure on health increased by 80 % to 3698 US$ at

average exchange rate within the last ten years, and the percentage of health expenditures that

occurs from the five major noncommunicable diseases (cardiovascular disease, cancers, endocrine

and metabolic diseases, respiratory diseases, mental health and neurological disorders) rose from

27 % in 2002 to over 50 % (WHO, 2011a).

In many cases, the risk to suffer from one of these diseases is directly related to a rising body

mass index (BMI). Since 1980 the average age-standardized BMI of German male and female

increased from 25.4 and 24.7 to 27.2 and 25.7, respectively. In 2008, 62.8 % of men and 46.6 % of

women had a BMI over 25, whereof 23.1 % and 19.2 % were obese with a BMI higher than 30.

Overweight and obesity are the fifth leading risk for global deaths. 65 % of the world's population

lives in countries where more deaths are caused by overweight and obesity than by underweight

and malnutrition (WHO, 2013). Further risk factors for noncommunicable diseases are current

0 10 20 30 40 50 60

Weight maintenance

Weight loss

Animal welfare

Food-safety concerns

Natural approaches to wellness

Environmental concerns

Improve of overall health


14

tobacco smoking, physical inactivity, raised blood pressure, blood glucose and cholesterol, with

the latter four being often connected to an unhealthy diet as well.

The World Health Organization advises people to limit their energy intake from total fats and

sugars and to increase the consumption of fruit, vegetables, legumes, whole grains and nuts. The

support of the food industry is required in promoting healthy diets by reducing fat, sugar and salt

in processed foods and by proposing a broad range of available and affordable, nutritious and

healthy products (WHO, 2013).

Figure I.5: Contribution of noncommunicable diseases to the overall mortality in Germany (% of total deaths, all ages, WHO, 2011b).

Although its consumption exceeds the recommended value, energy from protein only contributes

to 12 % to the daily energy intake of the average European (FAO, 2013). This can be explained by

the lower energy density of approximately 4.1 kcal per g compared to lipids or alcohol with

9.3 kcal or 7 kcal per g, respectively (FAO, 2003). Carbohydrates provide a similar energy density,

but are consumed to a much larger extend. Animal protein does not differ from the vegetable one

regarding the energy content, but as foods are not consumed as single nutrients, protein from

livestock products is often accompanied by higher amounts of lipids raising the overall calorie

content. Replacing animal protein by protein-rich plant products can thereby help limiting the

daily energy intake.

Currently, the share of vegetable protein at the overall protein intake is with 43 % and 39 % in

Europe and Germany considerably lower than the global average of 61 % (FAO, 2013). Figure I.6

shows the contribution of different vegetables to the overall vegan protein supply. Approximately

two third of the protein supply are covered by cereals, whereof in Europe protein from wheat

45%

26%

4%

3%

13%

4%5% Cardiovascular diseases

Cancers

Respiratory diseases

Diabetes

Other noncommunicable diseases

Injuries

Communicable, maternal, perinataland nutritional conditions


15

amounts more than 85 %. The consumption of starchy roots in Europe and Germany, which

corresponds to the consumption of potatoes contributes to the protein supply to a comparably

large extend. These data may be traced back to the long history of potato cultivation and to a

large variety of convenience products, snacks and ready to eat meals that are more popular in

these parts than in other regions of the world. The contribution of peas, beans and lentils to the

protein intake in Europe, and particularly in Germany, is very low compared to the rest of the

world, where pulses are an important part of the protein supply. Disfavour of the characteristic

taste, the flatulence causing properties and the laborious preparation were mentioned in a survey

as main reasons for the low consumption of pulse dishes (Klemcke et al., 2013).

Figure I.6: Composition of the vegetal protein supply calculated from the daily protein consumption in 2009 (FAO, 2013).

A long-term establishment of meat alternatives and a conversion to a more plant based diet

requires the creation of a manifold product offer as well as the consideration of specific needs in

the population. Advanced product development takes into account the age-dependent

preferences and needs of the consumers as well as the challenges for people bound to a specific

form of diet due to an allergy or health disorder.

Protein from oilcrops is predominantly consumed in form of soy products and peanuts, which

both possess an allergenic potential. A food allergy occurs as an immune response to intake of a

certain food protein mistakenly identified by the body as a harmful invader. The consequences

range from mild to life-threatening and include allergic reactions affecting skin, respiratory system

0

20

40

60

80

100

World Europe Germany

cont

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ion

to th

e ve

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supp

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%

Cereals Starchy roots Pulses Oilcrops Vegetables Fruits Others


16

and intestinal tract (Sampson, 2004). More than 17 million Europeans suffer from food allergy.

Children and young people are particularly concerned. Allergies in children have doubled in the

last 10 years and the number of hospital admissions of children with diagnosis related to food

allergies had increased by seven (EAACI, 2011). Beside soy and peanut protein, those of bovine

milk, eggs, tree nuts, fish, shellfish and wheat are counted among the big-8 food allergens.

Intolerance to certain cereals, including wheat, is one of the most common chronic health

disorders in western countries. Their consumption causes an inflammatory reaction in the small

intestine due to hypersensitivity of the intestinal mucosa to cereal gluten. This autoimmune

disorder, known as celiac disease, requires a life-long abstinence of gluten-containing foods. The

exact number of celiac disease patients is unknown due to a high number of undiagnosed cases,

but studies estimated that one of 130 to 300 European people is affected (Easton, 2003). Creation

of low-allergenic vegetable foods on basis of other plants than cereals could help people

concerned to compose a balanced, diversified diet.

This subchapter has overviewed the health benefits of a plant based diet and the predominant

supply of the human protein requirement by vegetable products. The realization of dietary

references in terms of good public health and relief of the medical scheme calls for a variety of

attractive wholesome products. Regional plant sources and eco-friendly process designs should be

chosen in regard to environmental compatibility to assure a long-term, healthy and sustainable

protein supply of the population.

Protein recovery from domestic non-animal sources

Additional to the assurance of all metabolic processes in the tissue, plants accumulate proteins in

protein bodies and storage vacuoles to build reserves of carbon, nitrogen and sulphur for periods

of growth, seed and fruit set (Herman & Larkins, 1999). Consequently particular high protein

concentrations are found in embryo cells like seeds and tubers to ensure rapid growth after

germination. These protein build-ups are often accompanied by other energy carriers like starch

or lipids. To guarantee an economic and holistic usage of the whole fruit, all major components as

well as valuable minor compounds should be regarded in the recovery process. In the past, food

industry often concentrated on the main component by weight, leading to many production

concepts for starch and vegetable oil with protein rich residues and press cakes as by-products.

In 2010, approximately 10 million tons of starch were recovered by the European starch industry

from 22 million tons of raw material. Potatoes, wheat and corn are the main starch suppliers and


17

contribute to the initial material in equal shares. In Germany, potatoes were with almost 3 million

tons and more than 60 % ratio the main starch source (Stärkeindustrie, 2013).

Potatoes can be cultivated in moderate climate and give with 42 to 60 tons per hectare an

exceptionally high crop yield. Potato tubers consist of 20 % dry matter, whereof starch accounts

up to 80 % (Alting et al., 2011). The protein content of potatoes is with 1 % of coagulable protein

relatively small, but the high turnover of potatoes within the starch sector implies an amount of

edible protein that exceeds 200 000 tons per year. Smaller amounts of protein could also be

obtained from process water of French fries production.

The steps usually applied in industry to recover starch from potato cells are illustrated in Figure

I.7. After removal of foreign material the tissue is disintegrated in saw-tooth raspers to make the

starch granules accessible. Sulphur dioxide is added in form of sodium hydrogensulphite or

sodium disulphite to avoid enzymatic browning. The fruit juice, containing most of the protein, is

separated from starch and fibres in two-phase decanters. The starch is extracted from fibres in jet

extractors, refined, concentrated and dried (Bergthaller et al., 1999). Intensive washing

procedures result in high quantities of strongly polluted waste water.

Figure I.7: Simplified flow chart of potato starch production according to Witt (1996) and Bergthaller et al. (1999).

coagulation

starch refining

counter currentextraction

fruit juiceseparation

rasping

washing

dewatering & drying

potatoes

starch proteinpulp


18

Potato fruit juice has a dry matter content up to 5 %, composed of all soluble, physiological active

cell ingredients, proteins, sugars, minerals, organic acids and other small compounds (Knorr,

1977). Potato proteins present in fruit juice are divided in three groups, namely patatin, protease

inhibitors and other proteins, including lectins and several endogenous enzymes. Patatin, a

glycoprotein with a molecular weight around 40 kDa and an isoelectric point between 4.5 and 5.2,

accounts for 38 % of the protein present in fruit juice. The heterogeneous group of protease

inhibitors forms with the majority of the protein and consists of several sub-groups with different

molecular sizes (Alting et al., 2011).

Approximately half of the protein, predominantly the heat sensitive patatin, can be recovered by

combining heat treatment and isoelectric precipitation. Therefore sulphurous, sulphuric or

hydrochloric acid are added till a pH value of 4.8 and the temperature is raised to 110 to 120 °C by

steam injection (Bergthaller et al., 1999). These radical conditions are connected to a loss of

techno-functionality and limit the applicability of the coagulated protein to the feed industry. The

protein concentrate has an off-colour varying from pale yellow to greyish-green or brown

dependent on the precipitation procedure (Meuser & Smolnik, 1979).

Solanic, a subsidiary enterprise of the Dutch AVEBE group, applies mild recovery steps including

adsorption processes to gain various protein products with different functional properties from

potato fruit juice. Their applications range from sports nutrition over meat analogues to

confectionery and ice-cream. The company benefits from the unaffected consumer attitude

towards potato protein as it is vegan, gluten and lactose free and provides a low allergenic

potential.

Oilseeds are not named according to botanical classification, but by their oil content that has to

amount at least 15 %. Different plant species can generate oil bearing components as their

production is mainly regulated by surrounding temperature and moisture. Most of these plants

accumulate as well high amounts of protein in their storage bodies (see Table I.3). Several seed

oils are recovered in industrial scale for the food industry, but in these parts rapeseed, sunflower

seeds and soybeans are the most important oil providers (see Figure I.8). In Europe, these three

crops account for 98 % of the oilseeds harvested (FEDIOL, 2013). Their pronounced percentage of

protein and its enrichment in the remainders of oil production make them also an interesting

source for recovery of vegetable food protein.

Rapeseed and sunflower seed proteins both consist of water soluble albumins with a molecular

weight of 12 to 14 kDa and globulins with a higher molecular weight around 300 kDa soluble in

salt solutions. Soybeans belong to the botanical family Leguminosae. Their protein composition is

with two major and two minor fractions similar to those of other legumes species. In rapeseeds


19

albumins and globulins are present in almost equal shares, in sunflower seeds and soybeans the

ratio is 25 % to 75 % and 10 % to 90 %, respectively (Krause et al., 2007). A special characteristic of

the rapeseed globulin is it isoelectric point of 7.2 in comparison to values in the range of 5 for

other vegetable proteins (Kroll et al., 2007). A related particularity in the net charge or the

solubility profile can donate rapeseed protein special application possibilities in the food sector.

Table I.3: Lipid and protein contents of selected oilseeds of interest for the food industry

Oil content in g/100 g dry matter

Protein content in g/100 g dry matter

Cotton a 30 53

Flaxseed b 40 – 44 20 - 25

Groundnut a 50 25

Rapeseed c 40 – 45 20 – 25

Sesame b 42 – 56 20 – 25

Soybeans a 20 40

Sunflower seed d 44 – 51 17 – 19

aCheftel et al. (1992); bKochhar (2002); cKrause et al. (2007); dGupta (2002)

Process design in oil mills is adapted to the properties of the raw material. Due to their higher oil

content rape and sunflower seeds are commonly de-oiled in screw extrusion presses prior to

solvent extraction, while soy oil is directly extracted with hexane. Thermal pre-treatments are

often performed to disintegrate tissue cells, kill endogenous enzymes and facilitate oil release by

viscosity reduction (Turtelli Pighinelli & Gambetta, 2012). High temperatures and nonpolar

organic solvents lead to denaturation of the protein and to changes in its functionality.

Approaches for a simultaneous recovery of oil and protein resulted either in insufficient product

yields due to formation of stable emulsions in watery extraction medium (Wäsche, 2002) or in

hindered industrial applicability because of complex micelle technology (Leser et al., 1989; Ugolini

et al., 2008).

The production of vegetable oils in the EU 27 nearly quadrupled in the last 30 years (see as well

Figure I.8). On the one hand this can be traced back to the large increase in per capita

consumption of vegetable oil in the 1990s (FAO, 2013), on the other it is a result of the rising


20

popularity of biofuel, won by ester interchange of lipids with methanol. In 2010 and 2011, 54 % of

the European plant oils were used in the food industry; more than 30 % were converted to

biodiesel (FEDIOL, 2013). Independently from the application of the recovered oil a huge amount

of press cake or extraction meal accrues during seed processing. Traditionally pelletized for the

feed industry, the rising masses of protein rich extraction meals require more economic and

ecologic application forms. At present, no food protein production of industrial relevant scale is

known for the meals of European oil mills. The Canadian Companies Burcon and BioExx market

functional food proteins based on canola, a variation of the rape plant.

Figure I.8: Production of oil and defatted meal from different oilseeds in the EU 27 (FEDIOL, 2013).

Beside the effective usage of protein rich waste resources, strengthening of domestic protein rich

plants provides an alternative to the import of vegetable protein from other regions of the world.

Already centuries ago, the vegetarian diet of roman gladiators was enriched with high energy

foods like beans and lentils, giving them a stately, even slightly overweight figure (Susanna, 2013).

In the Middle Ages, pulses were an important staple food for the peasant population giving them

strength for the physically onerous work on the fields (Rösener, 1985). High crop yields and high

protein contents made the modest plants attractive suppliers of nutrients. Nowadays, various

types of pulses are still the main sources for the protein supply of the poorer people in developing

countries.

0

5

10

15

20

25

30

35

40

45

1980 1990 2000 2010

prod

cutio

n in

Mill

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Rapeseed Sunflower seed Soybeans Others Oil Meal


21

Legumes are of less importance for the European food market due to reasons already mentioned

previously in this chapter. The insufficient competiveness of pulses reduces their attractiveness in

the agricultural sector. In 2010, 1.4 million tons of field peas and 1.3 million tons of fava beans

were harvested in the EU 27 (UFOP, 2013). Together with soybeans and lupines they account for 1

to 7 % of the arable crop area in different European countries in contrast to 15 to 25 % in

countries outside of Europe (Von Richthofen, 2006).

Soil degradation and desertification in crop areas due to intensive cultivation in monocultures

often have to be compensated by rainforest deforestation (Aiking, 2011). Crop rotation with

legumes improves soil quality and possesses benefits for the following wheat cultivation.

Symbiosis with nitrogen-fixing bacteria lowers the needed amount of nitrogen fertilizers, the grain

yield can be increased and costs for pesticides and tillage can be reduced (Von Richthofen, 2006).

Enhanced cultivation of pulses thereby provides not only an additional vegetable protein source,

but also advantages for agriculture and environment.

Table I.4: Biochemical composition of selected legume seeds compiled by Gueguen (1983) from several references

Cultivar Starch Lipids Protein Fibre Albumin Globulin Glutelin

in % of dry matter in g/100 g protein

Faba bean (Vicia faba minor) 47.0 1.8 34.5 2.4 20 60 15

Pea (Pisum sativum) 48.6 2.0 24.4 n.s. 21 66 12

Lentil (Lens culinaris) 52.8 1.1 23.9 3.8 n.s. n.s. n.s.

Lupine (Lupinus albus) --- 12.5 39.5 13.4 10 - 20 80 - 90 ---

Soybean (Glycine max) --- 19.6 42.8 5.5 10 90 ---

Besides a high protein content, pulses provide starch or lipids as energy carriers depending on

their subfamily and significant amounts of fibre (see Table I.4). The major proteins in pulses are

water or salt soluble. Prolamins and glutelins constitute only a small portion of the total protein.

The albumin fraction contains enzymes, protease and amylase inhibitors and lectins. The 11 S

legumin and the 7 S vicilin present in hexameric and trimeric structure, respectively, compose the

globulin fraction. Their isoelectric points are both in the range of pH 5. In peas, an additional


22

globulin named convicilin can be found differing from vicilin in its molecular weight and the

presence of sulphur-amino acids. The proportion of proteins of different solubility as well as the

ratio of legumin to vicilin influences the functional and nutritional properties of the legume

variety and its protein (Gueguen, 1983).

Dry milling and subsequent air classification of legumes enable separation into protein rich and

starch rich flours without usage of water. Depending on the raw material, protein contents up to

70 % can be achieved in the light fraction. Higher protein concentrations require watery

extraction at alkaline or strong acidic conditions followed by isoelectric precipitation or

ultrafiltration (Boye et al., 2010). The label NUTRALYS® stands for pea proteins with high purity

designed for the food market by the French starch manufacturer Roquette.

Leafs form a nearly inexhaustible source of vegetable protein. They consist to approximately 80 %

of water and contain 2 to 4 % of protein (Cheftel et al., 1992). Good sources of protein are the

foliated by-products from bean, pea, potato and sugar beet processing (Pirie, 1978). Traditionally,

leafs are dried and used as winter fodder for ruminants, but separation and fractionation of the

contained protein promises manifold applications. Grinding and pressing of the leaves results in

extracts that contain up to 60 % of the protein and only small amounts of fibre. The protein can

be effectively won in large scale by heat precipitation (Pirie, 1969), but the nutritional value of the

heat sensitive product is decreasing at the same time (Duckworth & Woodham, 1961).

Reasonable handling of leaf protein can result in a palatable food product, but it can also be

applied as non-ruminant feed as the included pigments intensify the colour of poultry and egg

yolk (Cheftel et al., 1992).

Table I.5 shows the amino acid requirements for an adult without any additional demands due to

illness, pregnancy or lactation. For this target group rapeseeds, soybean and leafs provide a

complete amino acid profile, while the nutritional value of potato, sunflower and peas and faba

bean protein is limited by leucin, lysine and the sulphur-containing amino acids, respectively. As

already mentioned before, supplementation with other vegetable protein sources could balance

these deficiencies, for example combination with sulphur-rich cereal proteins. Additional factors

influencing the nutritional quality, like digestibility of the protein or presence of antinutritional

components like enzyme inhibitors, are not regarded in this comparison as they strongly depend

on the degree and way of processing. Sarwar (1997) reported protein digestibility corrected

amino acid scores ranging from 49 to 100 for soy products differing in protein purity and

pretreatment.

Same applies to evaluating the techno-functional properties of protein products. Fivefold higher

solubility, higher oil adsorption and slightly improved foaming properties can be obtained from


23

potato protein precipitated without heating (Knorr, 1977). An overview on oilseed proteins given

by Kroll et al. (2007) presented improved foaming and water binding properties but decreased oil

adsorption and emulsification for sunflower protein won by membrane processes compared to

isoelectric precipitation. Properties of rapeseed products are strongly influenced by the presence

of phytic acid.

Table I.5: Nutritional comparison of selected plant proteins

Adult AA requirementsa in mg / 100 mg protein

Limiting amino acid scoreb in %

Potatoc Rapeseedd Sunflower seede Soybeanf Field

peag Faba beang Leafh

His 1.5 285 201 165 133 155 185 n.s.

Ile 3.0 102 122 132 170 144 142 157

Leu 5.9 89 118 104 125 116 125 149

Lys 4.5 172 132 84 124 159 139 124

Met + Cys 2.2 96 189 170 109 91 86 123

Phe + Tyr 3.8 135 172 193 192 182 185 247

Thr 2.3 147 198 138 174 142 147 209

Trp 0.6 n.s. 183 185 n.s. 127 122 283

Val 3.9 114 119 122 105 120 123 154

aamino acid pattern recommended by the world human organization (WHO et al., 2007); bLAA calculated according to Iqbal et al. (2006); data obtained from cpotato fruit juice (Bartova & Barta, 2009); dmean of three rape grist and one press cake (Krause et al., 2007); emean for seven cultivar (Earle et al., 1968); fcommercial meal (Bandemer & Evans, 1963); gfield pea cv Australian, faba bean cv Alfred (Mariscal-Landin et al., 2002); hmean of twenty leaf protein preparations from different cultivar (Pirie, 1969)

Nevertheless, an overview of the functional properties of some plant proteins is given in Figure

I.9. Although these data can just give a rough indication it can be noticed that proteins from

legumes exhibit good adsorption properties in relation to soy protein. Protein extracted from

potatoes shows excellent surface activity. Unfortunately, no comparison of the foaming behaviour

from potato and soy was available, but properties of potato protein were on a similar level as hen


24

egg white powder (Ralet & Gueguen, 2001) and milk protein (van Koningsveld et al., 2002), both

known as good foaming agents. For oilseed proteins no clear application area can be announced

due to high deviations in the detected properties.

Figure I.9: Functional properties of selected domestic protein alternatives in comparison to soybean (=1). Data obtained from Krause et al. (2007), Holm and Eriksen (1980), Fernandez-Quintela et al. (1997), Aluko et al. (2009).

Beside these representatives of rooted plants, other non-animal protein sources come into

consideration for sustainable human food supply. Algae, particularly the green and blue-green

species, contain similar protein amounts like leafs, usually 40 to 60 % of the dry matter. The

nutritional and techno-functional properties are comparable to those of terrestrial plant proteins.

Due to their easy cultivability, their high growth rate and the possibility to adapt cultivation

parameters, algal protein recovery possesses enormous economic feasibility (Alting et al., 2011).

Additional to cyanobacteria, protozoal protein can be won in significant amount by continuous

fermentation of other bacteria or yeasts, like Pseudomonas or Candida varieties. Protein contents

vary from 63 % to 80 % of dry matter. Problems can arise from the proportional high amounts of

nucleic acids, which have to be reduced in thermal or alkaline process steps to improve

digestibility (Cheftel et al., 1992). Beneath the mould fungi, those forming mycelia seem to be the

most promising protein source. Mycoprotein of Fusarium venenatum is marked under the brand

QuornTM as a meat alternative (Finnigan, 2011).

0.0

0.5

1.0

1.5

2.0

2.5

water oil capacity stability volume stability

adsorption emulsion foaming

func

tiona

lity

in re

latio

n to

soy

prot

ein

[-]

Sunflower seed Rapeseed Potato Field pea Faba bean


25

Insects are commonly used for nutritional purposes in many parts of the world. More than 1900

species are reported to be edible with beetles, caterpillars, bees, ants, crickets, grasshoppers and

locusts among them. They are of high nutritional value due to high amounts of protein,

unsaturated fats, vitamins, minerals and fibre. As cold-blooded organisms they possess a high

feed conversion efficiency that can exceed those of ruminants by more than the tenfold. Rearing

of insects can be performed without extensive land use and under usage of organic waste. Insects

never belonged to the food culture in Western countries what hinders their establishment in food

use as a whole organism. Addition of isolated components like proteins or lipids to enhance the

nutritional value of common products could reduce consumer restraint. At the moment, no

extraction procedures are implemented for a cost-effective protein recovery from insects. Further

research in this field is required to make this protein source a possible alternative for the food

market. (van Huis et al., 2013)

Vegetable protein alternatives are a topic of increasing interest regarding the consumers but as

well the field of food research. A rising number of vegetarian restaurants and supermarkets as

well as discussions about a common ‘veggie day’ reflect changes in people’s attitude towards

meat products. More than 15 000 results were found on google scholar on the 20th of June 2013

for the search criteria ‘plant protein environment’ and ‘plant protein sustainability’. The search

item ‘plant protein health’ even exceeded 170 000 results. Previous and future support programs

of the German government or the European Union like ‘Legume-supported Cropping Systems for

Europe’ or ‘Horizon 2020’ promote the cultivation of protein rich crops and the development of

domestic vegetable protein sources for food and feed (BMELV, 2012). Intensive research and

economic promotion are necessary to change to vegetable proteins in the long term. Innovative

processing concepts and emerging recovery techniques are required for an effective and ecologic

usage of alternative protein sources. Existing procedures have to be optimized to function in large

scale productions. Marketing strategies have to enhance the attractiveness of livestock

alternatives to increase the sales of new protein products. In conclusion, there is still a long way

to go to a sustainable global protein supply, but initial steps have been taken as this topic is

receiving the deserved attention from government, science and industry.

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Alting, A. C., Pouvreau, L., Guiseppin, M. L. F., et al. (2011). Potato proteins. In G. O. Phillips & P. A. Williams: Handbook of Food Proteins. Woodhead Publishing, Philadelphia.


26

Aluko, R. E., Mofolasayo, O. A. & Watts, B. A. (2009). Emulsifying and foaming properties of commercial yellow pea (Pisum sativum L.) seed flours. Journal of Agricultural and Food Chemistry, 57(20), 9793-9800.

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Bergthaller, W., Witt, W. & Goldau, H. P. (1999). Potato starch technology. Starch-Starke, 51(7), 235-242.

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Chen, L. C., Chowdhury, A. K. M. A. & Huffman, S. L. (1980). Anthropometric assessment of energy-protein malnutrition and subsequent risk of mortality among preschool aged children. American Journal of Clinical Nutrition, 33(8), 1836-1845.

Duckworth, J. & Woodham, A. A. (1961). Leaf protein concentrates 1. Effect of source of raw material and method of drying on protein value for chicks and rats. Journal of the Science of Food and Agriculture, 12(1), 5-&.

Dwyer, J. T. (1988). Health-aspects of vegetarian diets. American Journal of Clinical Nutrition, 48(3), 712-738.

EAACI (2011). 17 million Europeans allergic to food; allergies in children doubled in the last 10 years. Zurich, Sitzerland / Venice, Italy, European Academy of Allergy and Clinical Immunology.


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Earle, F. R., Vanetten, C. H., Clark, T. F., et al. (1968). Compositional data on sunflower seed. Journal of the American Oil Chemists Society, 45(12), 876-879.

Easton, J. (2003). Celiac disease is far more common than thought, The University of Chicago Medicine - Communications.

European Commission (2005). Eurobarometer Special N°229 - Attitudes of consumers towards the welfare of farmed animals.

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Chapter II – Proteins as techno-functional ingredients

33

Chapter II

Proteins as techno-functional

ingredients


34

When sufficient food is available to fulfil nutritional requirements, consumers tend to select food

by means of hedonistic criteria. Whereas odour and flavour may be strongly affected by minor

ingredients like aromatics or esters, the texture of a product is mainly influenced by the

interactions among the major food components water, carbohydrates, lipids and proteins

(Kinsella, 1982). The majorities of our food products are colloidal or disperse systems of at least

two components different in composition or aggregation state. Generally the phases are not or

only partly soluble with one another and have to be stabilized by other food ingredients.

Especially macromolecules like proteins are of great importance for this purpose.

Figure II.1: Summer menu of star restaurant VAU in Berlin downloaded on 26th June 2013.

Figure II.1 shows the summer menus offered by starred chef Kolja Kleeberg as an example for

food products offered in many restaurants. Almost every course is based on meat or fish

products. The ability of proteins in muscle fibre to bind water significantly influences meat texture

and thereby contributes to its quality. Lipid droplets in green sauce, velouté or vinaigrette are

prevented from separation by milk, wheat or egg protein. Some sorbets may contain beaten egg

white or gelatine for shelf-life increase, but mousses and meringues are more common examples

for the usage of proteins to stabilize gas bubbles in the food matrix. Formation of protein

networks are the precondition for the gel structure in cheese and the viscoelastic dough of bread


35

and tart. Of course, proteins do not only contribute to the creation of haute cuisine, but as well

fulfil these requirements in private households and in industrial food production.

In this context people often refer to proteins as a functional ingredient. This often leads to mix-up

with functional food, a product containing additional ingredients for health promotion or disease

prevention. No clear definition for functionality exists but distinction between the nutritional and

biochemical properties of proteins seems reasonable. Akiva Pour-El (1981) defined functionality

as ‘any property of a food or food ingredient except its nutritional ones that influences its

utilization’. This includes all physicochemical properties that affect the protein’s behaviour during

preparation, processing, storage and consumption and therefore mainly contribute to product

quality (Hall, 1996). An overview on important food proteins and their application areas is given in

Table II.1.

Table II.1: Major functional proteins (Zayas, 1997)

Source Protein Properties and applications

Cereals Wheat gluten, maize zein Bakery products, whipping agents

Egg Egg white albumin, whole egg Emulsifying and foaming agents, gelation

Fish Muscle, collagen Gelling, surimi products

Meat Muscle, collagen, blood protein Gelling, emulsification, water holding

Milk Whey protein, casein, whole /skim milk powders Emulsifying, viscosity

Oilseed Isolates /concentrates / flours of soybean, groundnut, sesame Bakery, texturing of meat products

The following chapter will focus on these techno-functional properties and will not consider the

nutritional value and enzymatic activity of proteins during processing, which are as well of

importance for their application in food. A protein’s techno-functionality is strongly dependent on

size, shape and conformation of the protein which is strongly influenced by product composition,

e.g. pH value or ionic strength, and protein processing history. Knowledge about all these

interactions may help to targeted select proteins for specific food purposes (Hall, 1996).


36

Food proteins on a mission - the main functional properties used in

industry

The techno-functional properties of proteins can be grouped either by their dependence on the

molecule’s nature or by their interplay with other molecules present. They will be discussed

according to the order given in Table II.2.

Table II.2: Functional protein properties and their relation to structural attributes

Required molecular character (according to Zayas (1997))

Responsible interaction (according to Cheftel et al.

(1992a))

Solubility

Hydrophilic Protein-water

Water retention

Gelling

Protein-protein Aggregation Not available

Film formation Not available

Fat binding Hydrophobic Not available

Emulsification Amphiphilic Surface properties

Foaming

Protein products are usually available in a dry form. In this state they possess only limited

functionality. Therefore, the first steps in making the protein a functional ingredient are often

hydration and solvation. Many other properties are dependent on the protein’s solubility making

this attribute extremely important for the overall protein applicability (Vojdani, 1996). Some

proteins are naturally of inferior solubility or denatured during ungentle processing. Preserving a

high solubility by using mild recovery strategies is thus a main goal in the production of high

quality protein products.

The main steps necessary to bring a protein into solution are shown in Figure II.2. Dry protein

products contain structural water engaged in hydrogen bonds of the native protein structure.

These water molecules cannot be removed without irreversible protein destruction and do not

participate in reaction or solvation processes (Cheftel et al., 1992a). When the powder is exposed


37

to water or vapour, molecules are adsorbed by hydrogen bonds with the amino acids forming a

monolayer, followed by multilayers due to water-water interactions. Sometime during water

adsorption the protein starts to swell. Depending on the protein’s nature and the availability of

water this process can be interrupted at a certain extend or continue until the molecule is

surrounded by enough water to form a stable solution (Chou & Morr, 1976).

Figure II.2: Sequence of protein-water interaction for dry protein according to Chou and Morr (1976).

The terms swelling, water holding, water retention, water binding, hydration capacity, water

adsorption, water impeding all describe the molecules’ ability to bind water. The incorporation of

water influences texture, juiciness and mouth feeling of a product and therefore its acceptability

by the consumer. Solid foods can contain up to 95 % of water and as the appearance of free water

is regarded as a deficiency in product quality, ingredients are required that help to keep water

into the product structure during long-term storage, mechanical impacts during transport and

temperature fluctuations (Barbut, 1996). Usually, no distinct water binding properties are

ascribed to proteins of high solubility as they tend to go into solution already in a small excess of

water, which makes a determination of the hydration capacity inaccurate.

Proteins go into solution until a maximum concentration is reached and an equilibrium between

molecules present in the liquid and solid phase is achieved. The degree and speed of the solvation

process is strongly influenced by environmental conditions. Depending on whether energy is

adsorbed or released during solvation, heating or cooling of the solvent can accelerate the

process. A moderate temperature increase has a positive effect on protein solubility as long as

thermal denaturation is avoided. Temperatures around 40 to 50 °C are suitable to enhance the

solvation process for most proteins. A protein’s solubility is minimal at its isoelectric point, when

swelling

dry proteinwater molecule

adsorption via polar site binding

swollen particles or mass

solvation

solution

liquid water condensation

multilayer water adsorption


38

it possesses a neutral net charge. Increasing the net charge by changing the solvent’s pH

promotes protein-water interactions and leads to repulsion of molecules with the same net

charge, which prevents them from aggregation. Presence of neutral salts also influences water

interactions as ions interacting with charged parts of the protein may form a double layer

enhancing or decreasing electrostatic repulsion. In this context, it is often referred to the so called

Hofmeister series ordered by kosmotropic and chaotropic salts. The latter are ‘structure breakers’

that denature proteins by decreasing hydrophobic interactions that stabilize their three

dimensional structure (Vojdani, 1996).

Traditionally, vegetable proteins are grouped by their solubility in different media into water

soluble albumins, globulins soluble at a certain ionic strength and prolamins and glutelins that can

be solved in alcohol and strong alkali, respectively (Osborne, 1924). The last two exclusively

appear in the seeds of cereal grains. They are rich in proline and glutamic acid and their molecular

interactions are responsible for the viscoelastic properties of dough during bread making (Cheftel

et al., 1992b). The ability to form a protein network by intermolecular forces, known as gelling

capacity, is often required during food processing and important for the formation of

characteristic rheological structures.

Gelation requires a well-ordered protein matrix able to hold large amounts of water

(Hermansson, 1979). Attractive forces between protein molecules have to be strong enough to

form a stable network. Repulsive forces counteract with the attraction to avoid exclusion of water

from the matrix (Matsumura & Mori, 1996). Usually, a partial denaturation of the protein is

necessary initiated by heat, pH change or pressure application. Proteins exposed to heat, strong

alkali or solvents might gelatinize without further processing (Cheftel et al., 1992a). Primary

aggregates of several proteins that remain dispersed undergo secondary aggregation to form a gel

network (van der Linden & Foegeding, 2009). Intermolecular links are based on formation of

covalent bonds, mainly disulphide bridges, and non-covalent interactions in varying degrees

(Foegeding & Davis, 2011). Therefore, macroscopic properties like texture, water and fat holding

capacity may differ depending on matrix structure, type of interactions or presence of other

macromolecules in the network.

Aggregates with altered functional properties may be obtained if secondary aggregation of

denatured proteins to larger clusters is inhibited, for instance by electric repulsion. Depending on

pH, protein concentration, type and quantity of salts and heating temperature, semi-flexible

fibrils, flexible strands, dense spherical particles or fractal aggregates can be produced. Heat

denatured protein suspensions that do not separate in the steady-state are heat stable, which can


39

be an advantage for their further application, for example as thickening, agents, to enrich foods

with proteins or encapsulate valuable compounds. (Nicolai & Durand, 2013)

Edible films made of protein can be applied on various foods to reduce moisture loss, restrict

absorption of oxygen, decrease migration of lipids, as a physical protection during mechanical

handling and transport or as an alternative packaging material (Kester & Fennema, 1986). They

can be produced either from protein solutions by surface film formation or casting a defined layer

thickness on a non-sticky surface (Wittaya, 2012), or from certain proteins with thermoplastic

behaviour at low moisture content by extrusion processes or compression moulding (Hernandez-

Izquierdo & Krochta, 2008).

Interactions of proteins with lipophilic substances are of high importance for food production as

well. Analogous to water binding, fat can be incorporated in the protein matrix and its release

during processing and storage should be avoided. The amount of lipids that can be bound by

proteins is strongly dependent on the type of fat, its droplet size and distribution as well as the

presence of emulsifying agents (Barbut, 1996). The ability to hold fat in the food matrix also

influences nutritional and sensorial attributes of the product as lipids are often carriers of fat

soluble vitamins and flavours.

In most food systems lipids are dispersed in an aqueous phase forming an emulsion. An emulsion

is thermodynamically unstable as its two phases are naturally immiscible. The free energy of the

emulsion is higher than the sum of energy of the separate liquids. Consequently, energy input

usually brought in by mechanical dispersion of the discontinuous phase is necessary for

generating an emulsion (Hill, 1996). Adsorption of amphiphilic substances at the interface lowers

the tension at the phase boundary and thereby the free energy of the whole system. As food

lipids are not solely composed of nonpolar components, the interfacial tension between

vegetable oils or milk fat and water is lower compared to the one between homopolar alkanes

and water (Chiralt, 2005).

An even higher interfacial tension has to be transcended producing water-air dispersion as it is

required for many foods possessing a light, airy texture. In its simplest form foam consists of gas

bubbles surrounded by a liquid or solid continuous phase. The latter is usually obtained by phase

transition of liquid foam and possesses an excellent long-time stability. In most cases, air is

induced into the food system, but also nitrogen and carbon dioxide are of importance, particularly

in the beverage sector. Foams are regarded as “bubbly” if the volume of the gas phase lies below

a close packing level and “polyhedral” if above (Dickinson, 1992). Usually, bubbly foams are

produced during input of the gas phase, changing to polyhedral foams during drainage of the


40

liquid. In polyhedral arrangement, contact points of several lamellae form plateau borders to

which the liquid phase drains as a consequence of differences in Laplace pressure (Walstra, 2003).

Due to their amphiphilic character proteins markedly contribute to the shelf-life of emulsions and

foams. Proteins may diffuse to the interface or can be convectively transported to it during

processing, followed by a relatively slow arrangement at the phase boundary (Dalgleish, 1997).

The molecular structure of the protein is changed during adsorption, when hydrophobic regions

are orientated to oil droplets or gas bubbles, while the hydrophilic parts are extended into the

aqueous phase, known as “loop-train” model (Wierenga & Gruppen, 2010). Proteins can cover a

larger area of the interface than could be expected by their molecular volume, indicating that

unfolding of its three-dimensional structure had occurred (Clarkson et al., 1999b). This expansion

particularly happens in early stages of interface occupation and the protein can be refolded and

compressed when further protein molecules are adsorbed (Wierenga & Gruppen, 2010). Local

inhomogeneities in the interfacial tension lead to Marangoni convection of the bulk phase which

additionally compresses and expands protein molecules (Clarkson et al., 1999b).

Determination of enzyme activities after their adsorption at interfaces revealed the durability of

these structural alterations. Conformational changes mostly concern the protein’s tertiary

structure, changes that are reversible after desorption from the interface, and affect the

secondary structure to only a small extent (Clarkson et al., 1999a). The irreversibility of the

structure loss depends on the type of protein and increases with less protein present at the

interface and the duration until the molecules are desorbed (Clarkson et al., 1999b). Wierenga

and Gruppen (2010) stated that the changes proteins undergo at the interface are smaller than

long-time believed. Circular dichroism showed that even random coil proteins like caseins do not

completely unfold during adsorption.

The viscoelastic properties of the amphiphilic film formed around the phase border markedly

influence the stability of the dispersion. Lower drainage rates were reported for protein stabilized

foams than for those stabilized by small surfactants as proteins contribute to the interfacial

rheology to a greater extent by building a network through hydrogen bonds, hydrophobic,

electrostatic or covalent interactions (Wilde & Clark, 1996; Wilde, 2000; Chiralt, 2005). Surfactants

may displace proteins from the interface and thereby negatively influence the shelf-life of foams

and emulsions (Dalgleish, 1997). As food systems are normally multi-component systems,

whereof many have surface-active properties, product composition requires specific attention

when evaluating and selecting stabilizing agents. The predominant surfactant will form a

monolayer at the interface or when different surfactants do not impact each other they may form

a mixed monolayer or a multilayer depending on their character (Chiralt, 2005). Insufficient


41

presence of surfactants leads to a shift in size distributions towards higher diameters as the

interfaces cannot completely be occupied (McClements, 2004). Bridging effects may even

destabilize the system when one protein molecule is adsorbed at different interfaces (Hill, 1996).

Other molecules present like polysaccharides, salts and acids may also influence product

properties by increasing the viscosity of the aqueous phase or by changing protein conformation

and net charge.

Figure II.3: Destabilisation mechanisms of foams and emulsions.

Emulsions and foams are sensitive to changes in environmental conditions, but usually must be

stable for a long period and stand processing steps like heating, chilling, freezing, drying or

mechanical stress (McClements, 2004). Destabilization is for both kinds of dispersion a

consequence of gravitational and flow forces, intermolecular and interparticular attraction and

repulsion, which contribute to the collapse of the system to different extents (Chiralt, 2005). The

important destabilization mechanisms are illustrated in Figure II.3. They are hard to distinguish as

they usually strongly influence each other.

Separation of gas or lipids and water due to density differences mostly follows Stoke’s law.

Viscosity increase of the continuous phase and downsizing of the discontinuous phase can lower

the rate of phase separation. Emulsions containing droplets smaller than 1 μm do not cream as

the Brownian motion is stronger than the movement due to gravitational forces (Hill, 1996). In

creaming

aggregation coalescence

drainage

disproportionation


42

meat products lipid droplets bigger than 100 μm are stabilized due to their limited mobility in

these highly viscous products.

Droplets and bubbles may aggregate due to the predominant attraction force between their

surfaces (Chiralt, 2005). When the thickness of the interfacial film is decreased the flocculated

parts may coalesce forming larger oil and gas packages (Damodaran, 2005). Especially in foams

coalescence is forwarded as liquid drains from the interfacial films due to gravitation or

differences in Laplace pressure within the lamella. A limited solubility of the discontinuous in the

continuous phase allows wandering of gas or oil from smaller to bigger bubbles and droplets,

known as disproportionation (Damodaran, 2005) or Ostwald ripening (Chiralt, 2005), respectively.

The consequent decrease in the surface to volume ratio reduces the sum of interfacial tension

and free energy of the system.

Destabilization mechanisms are strongly dependent on the system’s pH and ionic strength. A

higher adsorption rate at the interface was observed for proteins with low net charge and a

higher packaging density of protein is possible due to the absence of electrostatic repulsion

(Wierenga & Gruppen, 2010). Electrostatic repulsion of the surfactants helps to overcome the van

der Waals attraction between oil droplets and to stabilize the emulsion to a greater extent than

steric repulsion from the interfacial layers (McClements, 2004). pH values far away from the

isoelectric point and low ionic strengths favour a uniform net charge of the protein molecules and

thereby the repulsive forces between single droplets (Hill, 1996). As the ionic strength of a food is

usually predetermined by its recipe and usually exceeds 0.01 M, the present salts neutralize the

proteins net charge making steric repulsion the dominant force preventing aggregation. Higher

surface shear elasticity was measured close to the isoelectric point making the interfacial layer

more flexible and therefore more stable. Not to forget the contribution of acids and salts to the

protein conformation and its ability to unfold. The relationship of protein structure and its

functional properties will be further discussed in the following subchapter.

Structure-functionality relationships of proteins

A strong relation exists between a protein’s techno-functionality and its spatial molecular

structure. Even small alterations in composition and conformation can lead to huge changes in

the functional behaviour. Genetic varieties of β-lactoglobulin merely differ in two amino acid

residues, but possess clearly dissimilar adsorption and emulsification properties (Wilde, 2000).

Understanding the relationship between structure and functionality could help to predict the

behaviour of protein products in various applications and different media. Therefore, detailed


43

knowledge of the meaningful structural parameters and their impact on certain functional

properties is required. The main molecular characteristics influencing protein functionality were

compiled by J.E. Kinsella (see Table II.3).

Table II.3: Molecular protein characteristics affecting its functional properties (Kinsella, 1982)

Amino acid composition (major groups present)

Amino acid sequence (distribution of segments / polypeptides)

Secondary / tertiary conformation (compact /coil)

Surface charge /hydrophobicity / polarity

Size, shape (topography)

Quaternary structures

Secondary interactions (presence of intra and inter-peptide)

Disulphide /sulphhydryl content

Environmental conditions (pH, redox, salts, temperature)

The three-dimensional structure of protein molecules is traditionally partitioned into four

sublevels, illustrated in Figure II.4. Many of the structure attributes listed in Table II.3 are directly

related to the primary structure of the protein. The amino acid sequence built according to

genetic information of the biological cell dictates the further folding of the molecule leading to a

unique spatial structure. Amino acids can be grouped into different classes by the nature of their

residues. Proportion and distribution of certain classes may strongly influence functional

behaviour of the molecule. Aspartic acid and glutamic acid can bind 6 to 7 molecules of water per

charged residue. A high content of these amino acids leads to enhanced water binding.

Sulphhydryl groups can contribute to network formation by oxidizing to disulphide bridges

(Kinsella, 1982).


44

Figure II.4: The four sublevels of the protein structure (redrawn according to common illustrations in literature).

In α- and β-caseins serinophosphate and carboxyl groups are located at the N-terminal of the

molecule, while the rest of the sequence possesses predominantly hydrophobic amino acids. This

asymmetric distribution in the amino acid pattern gives casein a detergent-like character and

excellent surface properties (Damodaran, 1994). Apart from proteins where certain amino acids

Primary structure

Secondary structure

positivelycharged

Arg – His – Lys – Asp – Glu – Ser – Thr – Cys – Pro – Asn – Gln – Ala – Val – Ile – Leu – Met – Gly – Phe – Tyr - Trp

aromaticnegatively

charged hydrophobicpolar uncharged

β-pleated sheet α-helix

Tertiary structure

Quaternary structure


45

predominate the primary structure, knowledge of the amino acid pattern is of limited use for

predicting functional properties. α-lactalbumin and lysozyme, for example, have similar amino

acid compositions, but differ markedly in their physical attributes (Kinsella, 1982). The

polypeptide chain is folded to reduce the free energy to the lowest level possible. α-helices and β-

pleated sheets formed via hydrogen bonding are ordered in a three-dimensional arrangement.

The general organization of globular proteins is similar, but their tertiary structure differs

markedly in the proportion of alpha-helices, plated sheets and random coil (Kinsella, 1982).

According to Damodaran (1994) the main functional properties can all be related either to the

hydrodynamic properties or the surface properties of a protein. Size, shape and flexibility of the

molecule influence the hydrodynamic properties visible in its viscosity as well as in its thickening,

gelation and texturization ability. Surface-related attributes are determined by hydrophilic,

hydrophobic and steric properties of the molecule and are reflected by the protein’s solubility,

wettability, fat and flavour binding, as well as by its interfacial activity. Especially the latter

characteristic is often used as an indicator for protein quality or its alteration during different

processing steps.

The three steps to stabilize an interface are the diffusion of protein to the interface, adsorption

and refolding at the interface and protein-protein interactions to form a continuous viscoelastic

film (Kinsella, 1981). Proteins with lower molecular weight show better diffusion to the interface

which promotes a rapid reduction of the interfacial tension (Grunden et al., 1974). Unfolding of

the protein structure to occupy the interface is enabled by hydrophobic interactions with the

nonpolar discontinuous phase. The hydrophobicity of amino acids or peptides can be determined

by their solubility in media of different polarity or their retention times in reversed phase

chromatography. The proportion of hydrophobic amino acids in the primary structure may give

hints for the intensity of hydrophobic interactions, but it is generally accepted that the surface

hydrophobicity correlates better with the functional properties than the absolute hydrophobicity

(Nakai et al., 1996).

The three-dimensional structure of the protein follows the thermodynamic requirement to

minimize its free energy. In polar solvent charged residues are orientated to the surface, whereas

the hydrophobic residues tend to be buried inside the molecule. Due to steric constraints given by

the polypeptide chain, some of the hydrophobic residues have to be orientated to the surface

forming hydrophobic patches. In many food proteins these cavities occupy 40 to 50 % of the

protein surface (Damodaran, 1994). The presence of hydrophobic patches influences the

thermodynamic stability of a protein. Proteins with high surface hydrophobicity are more

sensitive to thermal and interfacial denaturation than the ones whose hydrophobic residues are


46

completely buried in the interior of the molecule (Damodaran, 1994). Hydrophobic protein-

protein interactions are as well influenced and therefore their solubility in polar media.

Good correlations between surface hydrophobicity and emulsion and foaming properties were

reported by several authors (Kato & Nakai, 1980; Kinsella, 1981; Nakai, 1983; Lee & Kim, 1987).

Wierenga and Gruppen (2010) assumed that in contrast to surfactants, of which 100 % are

adsorbed at interfacial contact, not every protein molecule arriving at the interface will be

adsorbed. The affinity of the protein to the interface will increase with rising hydrophobicity

making an absorption more likely. A flexible protein structure also favours a rapid adsorption at

the interface. A low proportion of disulphide bridges enables a higher flexibility of the protein to

refold at the interface. Proteins do not have to be intact to possess good interfacial properties. A

small degree of hydrolysis may enhance the emulsification properties of β-lactoglobulin, because

the structure is less rigid than the one of the native protein (Dalgleish, 1997).

Certain amino acids inhibit formation of a helical structure due to bulky or highly charged side

chains. The rigid pyrrolidone ring of proline prevents free rotation of the chain and a lack of

substitute hydrogens inhibits formation of hydrogen bonds. Consequently, proline and

hydroxyproline are located at the bends or folds of the polypeptide chain. Same applies for iso-

leucin, serine and charged amino acids, which also tend to disrupt helical structures. These folds

form the random areas of the molecule allowing flexible occupation of interfaces (Kinsella, 1982).

Comparison between the non-structured, flexible random-coil β-casein, the highly ordered, rigid

globular lysozyme and the less ordered globular beef serum albumin revealed same diffusion

rates, but faster adsorption at the air-water interface with less structured conformation as caseins

unfold and spread rapidly at the interface (Graham & Philipps, 1975). Results of the foam stability

were vice versa. Fast adsorption often leads to a high foam volume, whereas proteins with slow

adsorption tend to make finer foams with higher long-time stability (Kinsella, 1981). Stability of

foam is a result of the rheological properties of the protein film. Proteins able to form viscous

films with high shear viscosity are more suitable to stabilize disperse systems. Excessive rigidity of

the molecule prevents the protein conformation to adapt to local shocks (Kinsella, 1981).

Although the relationship of protein structure and functionality has been understood in many

aspects, a quantitative prediction of the functional behaviour by means of the conformation is still

not possible. Functional properties of certain products are usually analyzed in model systems in a

laboratory scale and cannot directly be transferred to their behaviour during industrial processing.

(Damodaran, 1994). Nevertheless, knowledge about structural changes during processing may

help to understand the influence of these technological steps on protein functionality and can

serve as a tool to systematically select adequate modification methods.


47

Modification and preservation of protein functionality

The previous chapter highlighted the close relationship between a protein’s structure and

conformation and its functional properties. This is of great importance in food technology as

processing concepts often require application of some physical treatments or adaption of

environmental conditions like ionic strength or pH value with regard to desired product

composition. Bakery products or meringues for example start as wet protein foams that undergo

heating and drying procedures during former product manufacturing. High product quality

requires maintenance of the foaming properties during the impact of these external influences

(Foegeding & Davis, 2011).

The three-dimensional structure of a native protein is spontaneously folded with respect to a

decrease in the molecule’s free energy. Non-covalent forces like hydrogen bonds, electrostatic or

hydrophobic interactions favour folding of the molecule, whereas the conformational entropy of

the polypeptide chain opposes this process (Damodaran, 2006). Stabilization of the protein

conformation by a rather small free energy beneath 100 kJ/mol explains its sensitivity towards

environmental influences. Even small changes in temperature, pressure or ion concentration may

lead to a shift in the equilibrium between folded and unfolded state and hence lead to

dissociation of oligomers, unfolding of tertiary and uncoiling of secondary structures (Sikorski,

2002). Any of these structural alterations that do not involve a cleavage of the polypeptide chain

are regarded as a denaturation of the protein (Cheftel et al., 1992c). The exposure of amino acids

usually buried in the interior of the molecule may change the surface hydrophobicity and the

isoelectric point of the protein and cause a transformation of intramolecular to intermolecular

bonds (Grinberg et al., 1993; Sikorski, 2002). Dependent on the degree of deconformation a

denaturation may be reversible or irreversible.

The process of denaturation is often considered as at two-state mechanism with a distinct native

and denatured state, but also three-step models assuming a molten-globule intermediate exist

(Damodaran, 2006). Due to a higher degree of flexibility a protein can assume several

configurations upon denaturation what makes the denatured state hard to define and quantify.

Usually changes in physical, chemical or functional properties are used as indications. Functional

changes that possibly occur are differences in solubility and water binding, viscosity increase,

improved protease digestibility or loss of biological function in case of enzymes (Cheftel et al.,

1992c).

Heating and cooling steps are an integral part of many unit operations in food processing and

preservation and almost always involve a denaturation of the food’s protein. Usually heat

denaturation leads to rupture of non-covalent bonds. Hydrophobic interactions are of


48

endothermic nature. Consequently, existent bonds are stabilized and their formation is favoured

at elevated temperatures. Exothermic hydrogen bonds and electrostatic interactions are

weakened upon temperature increase, but, as they are usually abolished in watery solutions at

physiological ionic strength, their contribution to conformational changes is often negligible

(Damodaran, 2006). The temperature at which the free energies of native and denatured state

are in equilibrium is regarded as the denaturation temperature. Most of the globular proteins, like

whey and legume proteins, have denaturation temperatures in the range of 70 to 80°C. The

denaturation process usually spreads over a certain temperature range as different structural

domains unfold at different temperatures (Sikorski, 2002). Beside its dependence on extrinsic

factors the heat sensitivity of a protein is determined by some structural attributes. A high

number of cross-links and a large proportion of alpha-helices contribute to thermal stability

(Kumar et al., 2000). Proteins possessing less ordered secondary and tertiary structures, like

bovine caseins, exhibit excellent temperature stability. Low protein concentrations often favour a

reversible denaturation as protein-protein interactions are less probable.

Prolonged heating and very high temperatures can lead to irreversible changes within the primary

structure independent from the protein concentration. Rupture of peptide bonds, derivatization

of amino acids, cysteine and cystine destruction, deamidation of asparagine and glutamine,

release of hydrogen sulphide from disulphide bridges may be the consequence (Wang &

Damodaran, 1990; Cheftel et al., 1992c). Formation of sensory-active compounds, especially in

combination with reducing sugars, is often a desired effect of extensive heating (Sikorski, 2002).

Some proteins as well exhibit cold denaturation, mainly as a consequence of weakened

hydrophobic interactions (Damodaran, 2006). Therefore, proteins with a high proportion of

hydrophobic amino acids are particularly sensitive towards cooling. Enzyme inactivation,

dissociation of oligomers upon cooling as well as aggregation and precipitation during freezing are

reported (Cheftel et al., 1992c).

Changes in protein conformation can as well be induced by altering the surrounding pressure. A

system will respond to a rise in pressure according to the principle of Le Chatelier and reduce its

volume by chemical reaction or closer packaging of the molecules (Heremans, 1982). The volume

of a protein is given by the sum of the constitutive volume of the atoms, the volume of void

spaces due to imperfect packaging of the residues and the volume decrease resulting from

hydration of peptide bonds and charged amino acid residues (Kauzmann, 1959). Compression of

cavities under pressure leads to a volume reduction but also to a disruption of the hydration shell,

which opposes the volume decrease. Due to these contrary effects only a marginal compressibility


49

can be determined for many proteins. For some fibrous proteins the compressibility factor might

even be negative as hydration changes overbalance void compression (Damodaran, 2006).

Hydrogen bonds are not accompanied by a large volume change and are therefore pressure stable

(Messens et al., 1997). Some authors even reported a negative volume change upon their

formation (Heremans, 1982). However, changes between existent hydrogen bonds under

pressure are possible resulting in alterations of the secondary structure (Van Eldik et al., 1989).

Shifts between α-helices and β-sheets may occur, but a clear favouritism of one form could not be

determined (Wong & Heremans, 1988). Charged groups are usually surrounded by a diffuse layer

of ions connected to a volume increase. This electric double layer is disrupted upon

pressurization. Hydrophobic interactions are pressure sensitive due to a positive reaction volume

upon their formation. Tests in model systems revealed that unfolding of aromatic side chains is

favoured under pressure whereas exposure of aliphatic residues is restricted (Messens et al.,

1997).

The quaternary structure is mainly stabilized by pressure sensitive hydrophobic and electrostatic

interactions and the formation of oligomers is accompanied by a large volume increase. Therefore

dissociation into monomers may already happen under moderate pressures up to 200 MPa

(Messens et al., 1997). Unfolding of tertiary and secondary structure occurs at higher pressures

exceeding 200 MPa or 300 MPa, respectively (Hendrickx et al., 1998). The primary structure of a

protein is pressure stable up to 1000 – 1500 MPa, as the volume changes due to bond exchanges

and alterations of bond angles are nearly zero (Van Eldik et al., 1989; Mozhaev et al., 1996;

Hendrickx et al., 1998). Nevertheless the reactivity of thiol groups is enhanced due to protein

unfolding and formation of disulphide bridges contributes to the aggregation and gelation under

and after pressure application (Messens et al., 1997).

Due to their various impacts on different bonds stabilizing the protein conformation pressure and

temperature cannot be regarded independently. Assuming denaturation as a two-state process

an ellipsoidal phase boundary can be drawn for most proteins (see Figure II.5). At moderate

temperatures the pressure stability is maximal. At lower temperatures transition into the

denatured state occurs at lower pressures (zone I). Thermal stability first increases at moderate

pressures (zone II) and is again lowered when pressure is further enhanced (zone III). The form of

the phase boundary suggests different denaturation mechanisms at different pressure-

temperature conditions (Zipp & Kauzmann, 1973; Tauscher, 1995; Heremans et al., 1997).


50

Figure II.5: General scheme of pressure-temperature phase diagrams of proteins and pressure effects on protein structure redrawn according to Messens et al. (1997).

Beside physical effects proteins might undergo denaturation in the presence of certain chemicals

and solvents, for instance ethanol or acetone (Damodaran, 2006). Ions of some alkaline earth

metals like calcium and magnesium or of heavy metals like copper and iron form stable complexes

with proteins (Cheftel et al., 1992c). Sometimes they are even integral part of the protein

molecule and their removal leads to destabilization. Organic detergents like urea, guanidine as

well as tensides may cleave hydrogen bonds or hydrophobic interactions (Cheftel et al., 1992c).

The majority of organic solvents cause protein denaturation by destruction of hydrophobic

interactions. Aromatic and aliphatic amino acids possess a higher solubility in a nonpolar than in

an aqueous environment. They are exposed to the molecule’s surface causing unfolding of the

tertiary structure and subsequent aggregation. Heating in an organic solvent leads to irreversible

disruption of hydrogen bonds (Damodaran, 2006).

Most proteins are stable in a certain pH range with a maximum stability at their isoelectric point,

when their net charge is zero. Extreme pH values may cause electrostatic repulsion between

charged groups that cause unfolding. In many cases the native structure can be retained when the

pH is reset to optimum value (Cheftel et al., 1992c). Prolonged exposure to extreme acid or basic

conditions can cause hydrolysis of certain peptide bonds or crosslinking based on β-elimination

(Sikorski, 2002).

Application of pulsed electric fields (PEF) for mass transfer improvement or inactivation of

vegetative microbial cells got into focus of food research in the recent years. Due to its non-

thermal character this technology provides as well great potential for protein processing.

Nevertheless, an impact on protein conformation can currently not be excluded as reliable and

comparable research data are insufficiently available. A reversible enhancement of thiol reactivity

in ovalbumin with increasing PEF intensity was reported by Fernandez-Diaz et al. (2000), whereas

Native structure

Temperature

Pres

sure

I III

II OligomericStructure

Intermediate State

Monomere Structure

Aggregation


51

no marked unfolding was found for β-lactoglobulin in a similar PEF system (Barsotti et al., 2001).

Ten exponential decay pulses of 12.5 kV/cm slightly influenced denaturation temperature and

gelation rate of β-lactoglobulin and egg white protein (Perez & Pilosof, 2004). An increased

surface hydrophobicity was reported for soybean protein exposed to electric field strengths

higher than 20 kV/cm (Li et al., 2007; Xiang, 2008). Contrary literature data are available with

regard to enzyme inactivation due to PEF treatments (Ho et al., 1997; Van Loey et al., 2001;

Martín-Belloso & Elez- Martínez, 2005). Breakage of electrostatic interactions in the electric field

and subsequent oligomer dissociation and unfolding of the protein structure are proposed by

some authors (Manas & Vercet, 2006). Up to now it remains unclear to which extent local pH

shifts (Meneses et al., 2011), release of metals from the electrode surface (Morren et al., 2003)

and oxidation due to formation of radicals (Sato et al., 1996) contribute to the denaturation

effect. Usually, oxidation of food components is initiated by radicals and reactive forms of oxygen

generated by the impact of UV light or ionizing radiation or even by the activity of endogenous

enzymes. Elimination of a hydrogen atom may form radical proteins that tend to polymerize with

other radical proteins or food ingredients (Sikorski, 2002).

The high sensitivity of proteins towards various technological impacts requires tailor-made

processing and recovery concepts with respect to the individual conformational stability.

Unfortunately, these demands may not always be realizable as economic concerns as well as the

usage of other food ingredients have to be considered as well. Proteins from oilseeds have usually

undergone solvent extraction of lipids and desolventization previously to protein recovery which

negatively influences the properties of the protein product (Wolf, 1970). Temperature increase or

strong alkaline extraction conditions to improve diffusion of protein from the plant tissue may

alter protein conformation and contemporaneously its functionality (Hall, 1996).

Concentration of extracted protein may be performed using membrane processes or precipitation

by adjustment to the isoelectric point. Depending on the spreading width of molecular weights or

isoelectric points of the present protein fractions both techniques result in more or less efficient

product yields. Choose of the respective concentration technology enables as well a

differentiation in separate protein fractions. Figure II.6 shows the solubility profiles of pea

proteins won by ultrafiltration or isoelectric precipitation (IP). Pea albumins cannot be

precipitated in a pH range around 4.5 and are consequently not present in the IP concentrated

sample. Higher protein yields using membrane filtration were as well reported for the

homologous soy protein (de Moura et al., 2011). Both proteins products show the typical U-

shaped solubility curve progression of many globular proteins, but the solubility of the membrane

concentrated sample is shifted to higher values, as the albumins stay in solution over the entire

pH range. Precipitated proteins are more difficult to solubilize, as firstly strong protein-protein


52

interactions have to be overcome. Similar observations were made by other working groups

(Alamanou & Doxastakis, 1995; Chew et al., 2003; Boye et al., 2010). Additional heating during

protein precipitation may further alter the functional properties (Knorr et al., 1977; de Ogara et

al., 1992).

Figure II.6: Solubility profiles of pea protein concentrates won by isoelectric precipitation and freeze drying or ultrafiltration with a 10 kDa cut off and subsequent freeze or spray drying.

A reduction of water activity is essential to obtain a storable and transportable product. A

promising alternative to cost and time-efficient lyophilization is spray drying which preserves the

native properties to a greater extent than time-consuming drying in warm air or drum drying

above 100 °C. In the case of pea protein, negligible differences in the solubility profile were

determined. As many proteins contain an immense microbial load deriving from the raw material

or possess endogenous enzyme activity, inactivation steps are often necessary. Emerging

technologies are interesting alternatives to conventional heat treatments as they might preserve

the native protein to a greater extent.

Beside their essential usage in protein recovery technological steps can be applied to specifically

modify protein properties. One of the simplest ways to improve functionality is mild heat

treatment that may cause unfolding or aggregation without subsequent protein precipitation

(Foegeding & Davis, 2011). Thermal pre-treatment improved the foaming properties of soy

protein (Horiuchi et al., 1978) and the egg white proteins ovalbumin and lysozyme (Hagolle et al.,

2000). Heating of whey protein isolate to 70 °C for one and five minutes enhanced stability of the

0.0

0.2

0.4

0.6

0.8

1.0

1.2

3.0 4.0 5.0 6.0 7.0

solu

ble

prot

ein

in g

/ g

prot

ein

pH value

IP precipitation + freeze drying ultrafiltration + freeze drying ultrafiltration + spray drying


53

afterwards built foams in comparison to the control, while prolonged heating impaired foam

stability (Zhu & Damodaran, 1994). Contrary results of heat treatments to 55 and 80 °C in regard

to overrun and foam stability in dependency on sample pH were reported by Phillips et al. (1990).

Emulsion properties of sodium caseinate were improved by a 5 minute heating (Jahaniaval et al.,

2000), whereas the emulsion properties of whey protein and 11 S globulins in Vicia faba L. were

declined (Galazka et al., 1999; Raikos, 2010).

Besides thermal processing, pressure application provides the opportunity for a targeted protein

modification. An increased foamability of whey protein at pH 7.0 with increasing pressure was

reported by Ibanoglu and Karatas (2001), whereas Pittia et al. (1996) found no changes in foaming

properties of pure β-lactogloblin after pressure treatment. Negative effects of pressure

treatments were observed regarding emulsions formed and stabilized by pure β-lactogloblin

solutions (Galazka et al., 1995; Galazka et al., 1996) and varying effects of high pressure on the

emulsion properties of legume proteins can be found in literature (Galazka et al., 1999; Molina et

al., 2001; Chapleau & de Lamballerie-Anton, 2003; Torrezan et al., 2007). This compilation of

research data reveals the thin line between a functional improvement and over-processing to

contrary effects. Parameters of heat and pressure treatments have to be chosen conscientiously

taking into account the individual stability of the protein and the influence of extrinsic factors like

pH or sample purity.

Additional to physical modification possibilities, protein properties can be altered using chemical

or enzymatic means. Crosslinking by transglutaminase builds up protein networks for product

structuring. Enzymatic hydrolysis may enhance protein flexibility and thereby its functionality.

Improved solubility and enhanced interfacial properties in caseinates as well as higher foam

stability in soy protein can be achieved by targeted protein hydrolysis (Were et al., 1997; Flanagan

& FitzGerald, 2002). Introduction of acetyl or succinyl group may as well alter protein

functionality. Succinylation of soy protein led to improved emulsion and foaming properties

(Franzen & Kinsella, 1976). Nevertheless, physical modifications are usually preferred as they get

along with shorter processing times and the avoidance of chemical additives.

The present chapter pointed out the close relationship between the structural and functional

properties of proteins and the strong impact of technological steps on the quality of the protein

product. Due to ecologic concerns and the demand for sustainable global protein supply a

reduction of existing protein needs for functional usage is required and new protein sources gain

more importance. Prediction models for the interplay of technology, structure and functionality

could help to optimize protein recovery and processing concepts, especially in regard to

innovative techniques still at the beginning in industrial food application.


54

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Chapter III – The use of emerging technologies to alter protein structure

61

Chapter III

The use of emerging

technologies to alter protein

structure


62

The aim of this chapter is to identify the structural changes the proteins undergo in solutions with

varying purity and composition when subjected to high pressures, high temperatures or pulsed

electric fields. Experiments were performed according to Figure III.1. Treatment conditions were

chosen according to common parameters found in literature depending on the purpose of the

process. Detailed information on performed processes and analyses are given in Annex A pages

225 ff and 244 ff.

Figure III.1: Flow chart of the experiments and analyses performed with protein in solution.

Preliminary tests were conducted to determine the protein solubility at certain pH values and to

be able to adjust the respective protein concentration. Protein solubility was very high for both

potato samples used (see Table III.1). Potato protein concentrate won in laboratory scale

possesses a protein content of only 55 % and thereby a high amount of accompanying substances

which potentially influence sensitivity towards different processes. In a 1 % protein solution

approximately 2 % of soluble solids were found reflecting the high solubility of all components

that were available in the basic material potato fruit juice and that were also concentrated during

protein recovery. Conductivity of the solution is correspondently high as potatoes are rich in

minerals, especially potassium. The commercially available protein isolate contains all protein

dissolvation

centrifugation10 000 g; 10 min

high temperature

centrifugation500 g; 5 min

centrifugation10 000 g; 5 min

high pressure pulsed electric fields

protein

solublesolids

soluble protein particle sizedistribution

surfacehydrophobicity

SDS PAGE

turbidity

fast and total accessible

thiol groupscharacterization of

aggregatesfast and total accessible

thiol groups

solubleprotein

conduc-tivity

pH adjustmentHCl or NaOH

tap water


63

fractions with a molecular weight higher than 35 kDa, mostly patatin. A protein content of 92 %

reflects the high purity of the isolate and allows a targeted adjustment of desired medium

properties in the solution. Unfortunately no detailed process scheme for the protein isolate was

available.

Table III.1: Characterization of the protein rich basic materials and the 1 % protein solutions made of them

Potato Pea

Isolate Concentrate Concentrate Flour

(RT grinding)

Flour (cold

grinding)

Protein content in g/g product 0.92 0.549 0.760 0.520 0.474

Soluble protein in g/g protein 0.99 ± 0.07 0.96 ± 0.03 0.46 ± 0.02 0.67 ± 0.02 0.44 ± 0.02

Average particle diameter in nm 6.5 ± 0.3 6.2 ± 1.2 127.7 ± 26 10.9 ± 0.8 11.8 ± 2.4

Soluble solids in °Bx 1.15 ± 0.02 2.08 ± 0.8 1.4 ± 0.04 1.7 ± 0.04 n.d.

Conductivity in mS/cm 0.92 ± 0.01 4.02 ± 0.08 1.94 ± 0.12 1.65 ± 0.15 n.d.

Protein concentrate or protein enriched flour were recovered from peas cultivar Salamanca either

by aqueous extraction or dry air classification. Protein solubility in tap water amounted

approximately 0.67 g/g protein for pea flour grinded at room temperature. A solubility of

approximately 66 % at neutral pH was also reported for native legume proteins and protein

isolates (Gueguen, 1983). Noticeable is the markedly lower protein solubility of the concentrate in

relation to the flour. Freezing and subsequent lyophilization might have affected protein structure

and its interactions with the solvent. This assumption is supported by data obtained for cold

grinded flour of a different cultivar that was frozen in liquid nitrogen before milling to avoid

temperature increase due to energy dissipation. Both samples show reduced protein solubility

indicating sensitivity of the protein towards freezing. Freeze dried samples also had a 10fold

higher particle diameter possibly as a result of aggregate formation during this process step and

incomplete solubilization during sample preparation. Conductivity in solutions made of

concentrate was slightly higher than in those made of flour due to incomplete separation of salts

built within alkalizing and neutralization. Ultrafiltration units with higher flow capacity would have

probably led to a more efficient separation of low molecular substances and a higher purity of the


64

protein samples. Two batches of protein-enriched flour were supplied during realization of these

experiments differing in protein content and protein solubility. Batch one contained 32.8 %

protein with a water solubility of approximately 0.6 gram per gram protein. Specifications of

batch two are given in Table III.1. The impact of these differing starting materials on the process

effect will be discussed in the following subchapters.

Changes in potato protein structure after thermal and high pressure

treatments

Changes in protein structure and solubility induced by temperatures up to 80 °C and pressures up

to 600 MPa were investigated in dependence on the sample composition. Temperatures

exceeding 60 °C and pressures higher than 400 MPa are suitable to inactivate vegetative

microorganisms and thereby to extend shelf-life of the protein products. Lower treatment

intensities can achieve conformational changes and thus provide the potential to modify a

protein’s functional properties (see chapter IV).

Figure III.2: Influence of thermal treatments at ambient pressure on solubility and absorbance at 605 nm of potato protein solutions pH 7; sample: self-made protein concentrate; initial protein concentration: 10 g/L; treatment time: 10 min protein quantification via Biuret method.

The influence of thermal treatments on solubility and absorbance of 1 % (w/w) protein solutions

(pH 7) prepared from potato protein concentrate are shown in Figure III.2. A slight increase in the

absorbance and a small reduction in protein solubility were already observed after application of

0.1

1

10

0

0.5

1

20 °C 40 °C 50 °C 60 °C 70 °C 80 °C

abso

rban

ce a

t 605

nm

[-]

prot

ein

in so

lutio

n in

rela

tion

to th

e un

trea

ted

sam

ple

[-]

absorbanceprotein in solution


65

50 °C. Treatments at 60 °C enhanced the absorbance by more than factor 50 and decreased

solubility in tap water to around 50 %. Further temperature increase did not markedly intensify

the absorbance but further reduced the proportion of soluble protein to approximately 20 %. This

is in agreement with results presented by Pots et al. (1999a) who reported formation of

aggregates in neutral solutions made of patatin, the major potato storage protein, already at

temperatures of 50 °C. The proportion of aggregates further increased with rising temperature

and treatment time. The rates of endothermic reactions are raised at higher temperatures.

Further temperature increase enhances the rate of unfolding and aggregation by a multiple once

the denaturation temperature of a protein is exceeded (Cheftel et al., 1992a).

Figure III.3: Particle size distributions in potato protein solutions pH 7 treated at different temperatures; sample: self-made protein concentrate; initial protein concentration: 10 g/L; treatment time: 10 min; centrifugation for 5 min at 500 g.

No linear relationship between protein solubility and the absorbance of the samples was

observed. Smaller aggregates can also scatter incident light but may not be completely removed

during a 5 minute centrifugation. This suggestion is confirmed by the particle size distribution

determined via dynamic light scattering, which shows an increase of the average particle diameter

by the 10fold for thermal processing at 60 °C and further enlargement by factor 100 after 80 °C

treatment (see Figure III.3). Pots et al. (1999b) reported a growth in the apparent Stokes-Einstein

radii of patatin at temperatures higher than 55 °C and no additional increase in particle size during

subsequent cooling to ambient temperature.

0

5

10

15

20

25

30

35

1 10 100 1000 10000

num

ber i

n %

diameter in nm

20 °C 40 °C 50 °C 60 °C 70 °C 80 °C


66

Absorbance and solubility were altered to a lower extent by high pressure compared to thermal

treatments. The absorbance changed by factor 25 at maximum and solubility was reduced to 75 %

for the highest treatment intensity investigated (see Figure III.4). Pressure treatments performed

at 40 °C had a higher impact than those conducted at 20 °C. Proteins are known to be more

sensitive to a pressure-induced unfolding at elevated or very low temperatures (Winter et al.,

2007). Tertiary interactions in patatin are already weakened at temperatures higher than 28 °C

(Pots et al., 1998a) which may sensitize the structure to a pressure induced unfolding.

Figure III.4: Influence of high isostatic pressure on protein solubility and absorbance at 605 nm of potato protein solutions pH 7; sample: self-made protein concentrate; initial protein concentration: 10 g/L; treatment time: 10 min; protein quantification via Biuret method.

Unfortunately, no comparative literature for pressure treatments of potato protein was available,

but pressure-induced aggregation was reported for several other proteins (Funtenberger et al.,

1995; Chapleau & de Lamballerie-Anton, 2003; Patel et al., 2005; Qin et al., 2013). No changes in

particle size were determined which confirms the minor impact of high pressure on potato

protein (see Figure III.5). It has to be noticed that samples were centrifuged at low intensities

prior to dynamic light scattering to remove very large aggregates that do not underlie Brownian

motion. Based on the results of this analysis, presence of few very large aggregates after pressure

treatment can therefore not be excluded.

0.1

1

10

0

0.5

1

0.1 MPa 200 MPa 400 MPa 200 MPa 400 MPa 600 MPa

20 °C 40 °Cab

sorb

ance

at 6

05 n

m [-

]

prot

ein

in so

lutio

n in

rela

tion

to th

e un

trea

ted

sam

ple

[-]



67

Figure III.5: Particle size distributions in potato protein solutions pH 7 exposed to different pressure-temperature combinations; sample: self-made protein concentrate; initial protein concentration: 10 g/L; treatment time: 10 min; centrifugation for 5 min at 500 g.

High pressure and high temperature may both lead to unfolding of the protein conformation and

exposure of hydrophobic residues to the surface (Johnston et al., 1992; Galazka et al., 1999;

Zhang et al., 2003; Puppo et al., 2004; Wang et al., 2008). This analysis is not only valuable to

determine structural alterations, but might as well indicate changes in functional behaviour as

surface hydrophobicity can be directly correlated to a protein’s ability to adsorb at interfaces and

thereby influences the formation of foams and emulsions. Depending on the technology applied

differences in the exposure of aromatic and aliphatic amino acid residues may occur. Therefore,

distinction between both types of hydrophobic side chains was made using the fluorescence dyes

ANS (1-anilino-8-naphthalene sulphonate) and CPA (cis-parinaric acid). Results of both

measurement series were in good correlation showing a homogeneous exposure of both types of

side chains (see Figure III.6).

A strong increase in surface hydrophobicity by more than factor 4.5 was observed for samples

exposed to 70 and 80 °C, whereas only small changes were found in samples subjected to lower

temperatures. The native patatin molecule is highly structured. Circular dichroism analysis

revealed 33 % α-helices, 45 to 50 % β-strands and 15 % random coil (Pots et al., 1998a; van

Koningsveld et al., 2002a). Already temperatures exceeding 50 °C lead to an increase in random

parts and to a shift in tryptophan-fluorescence. These changes are only partially reversible during

0

5

10

15

20

25

30

35

1 10 100 1000 10000

num

ber i

n %

diameter in nm

20 °C 20 °C, 200 MPa 20 °C, 400 MPa

40 °C, 200 MPa 40 °C, 400 MPa 40 °C, 600 MPa


68

Figure III.6: Surface hydrophobicity of the soluble protein fraction of potato protein solutions pH 7 subjected to following treatments: top: thermal treatments at atmospheric pressure; bottom: pressure-temperature-combinations; sample: self-made protein concentrate; initial protein concentration: 10 g/L; treatment time: 10 min; protein concentration during fluorescence analysis: 1 g/L for aromatic hydrophobicity, 0.1 g/L for aliphatic hydrophobicity; protein quantification via Biuret method.

cooling (Pots et al., 1998a). Surface hydrophobicity measurements only reflect changes in the

soluble protein fractions as fluorescence analyses are only possible in clear and centrifuged

samples. Possibly, changes in the surface hydrophobicity also occurred after 60 °C but enhanced

hydrophobic protein interactions may have led to an aggregation of these proteins and their

removal from the solution. Pressure treated samples showed only small alterations in surface

surf

ace

hydr

opho

bici

tyin

dex

in m

V *

g pr

otei

n/ μ

mol

dye

0

4

8

12

16

20

20 °C 40 °C 50 °C 60 °C 70 °C 80 °C

aromatic hydrophobicity aliphatic hydrophobicity

0

4

8

12

16

20


20 °C 40 °C


69

hydrophobicity. Analogue to treatments at 60 °C, fractions unfolded at 400 and 600 MPa may

have been removed during centrifugation. Nevertheless, as changes in protein solubility due to

high pressure were quite small, the influence of this technology on the surface hydrophobicity of

potato protein is small compared to thermal processing.

Composition of the soluble proteins after different treatments is shown in Figure III.7. Three main

fractions can be differentiated via SDS PAGE. A small band in the range of 85 kDa can be identified

as lipoxidase. The pronounced fraction around 40 kDa is patatin, the main storage protein present

in potatoes. Patatin usually exists as a dimer, which dissociates into its subunits in the presence of

SDS (Racusen & Weller, 1984). The broad distributed fractions with low molecular weight are

composed of different proteins all possessing protease inhibitor activity. This heterogeneous

group can be further differentiated by its specific enzyme inhibitor activity and its molecular

weight ranging from 4 to 40 kDa, with the majority having a size around 20 kDa (Alting et al.,

2011). Polyphenoloxidase with a molecular weight between 60 and 70 kDa (Partington & Bolwell,

1996) could not be found. As enzymatic browning definitely occurred during potato

manufacturing, polyphenoxidase might have been removed somewhere during further

manufacturing of the fruit juice.

Band intensity of patatin and protease inhibitors were maintained after pressure application,

while their intensity decreased for the 60 °C samples and was completely invisible after treatment

at 70 and 80 °C. This is in accordance with the solubility which was most affected at the highest

temperatures investigated. Approximately 20 % of protein remained in solution after

centrifugation but probably will have formed large aggregates that were not able to enter the gel

network. Bands belonging to lipoxidase disappeared for treatments at 70 and 80 °C as well as

600 MPa. This is in agreement with inactivation results presented for this enzyme elsewhere (see

Figure VII.7, Figure VII.8 and Figure VII.9). Nevertheless, no direct correlation can be drawn from

band intensity to enzymatic activity as conformational changes might already affect activity

without leading to protein aggregation.

The protein composition shows a relatively homogeneous removal of the main protein fractions

during thermal treatments. Pots et al. (1999a) reported that further aggregation at the same

conditions occurred for patatin remaining in solution after a first heating step, which indicates a

homogeneous and consistent heat sensitivity of patatin.

To get deeper insight in the mechanism leading to precipitation of potato protein during

processing, experiments were also conducted with commercial protein isolate mainly composed

of patatin. The influence of medium composition was investigated by varying pH value and ionic


70

Figure III.7: Non-reducing SDS PAGE of the soluble protein fraction of potato protein solutions pH 7 subjected to following treatments: top: thermal treatments at atmospheric pressure; bottom: pressure-temperature-combinations; sample: self-made protein concentrate; initial protein concentration: 10 g/L; treatment time: 10 min; A: Lipoxidase (85 kDa); B: Patatin monomer (41 kDa); C: Protease inhibitors (19-25 kDa).

strength. Solubility of the isolate was only slightly affected by high temperatures at neutral pH.

More than 85 % of the protein can still be found in the supernatant after centrifugation of all

thermal and pressure treated samples (see Figure III.8). This is in accordance with Pots et al.

(1998a) who observed no precipitation after thermal treatment of isolated patatin at

temperatures up to 90 °C. Usually, a combination of heat with acid treatment or addition of

A

B

C

A

B

C

60 °C 70 °C 80 °C20 °C 40 °C 50 °C

20 °C 40 °C

0.1 MPa 400 MPa400 MPa 200 MPa 600 MPa200 MPa


71

Figure III.8: Protein solubility of potato protein solutions pH 7 and pH 6 subjected to following treatments: top: thermal treatments at atmospheric pressure; bottom: pressure-temperature-combinations; sample: commercial protein isolate; initial protein concentration: 10 g/L; treatment time: 10 min; protein quantification via Biuret method.

kosmotropic salts is applied to recover protein from potato fruit juice. A treatment pH of 6, which

roughly corresponds to the natural pH of potato fruit juice, led to increased precipitation of

protein for both technologies. In pressure treated samples, the proportion of soluble protein was

reduced by 46 %, while thermal treatments reduced protein solubility by more than 90 %. A

decrease in the protein’s net charge at pH values closer to the isoelectric point may lead to

prot

ein

in so

lutio

n in

rela

tion

to th

e un

trea

ted

sam

ple

[-]

0.0

0.2

0.4

0.6

0.8

1.0

1.2

20 °C 40 °C 50 °C 60 °C 70 °C 80 °C

pH 7 pH 6

0.0

0.2

0.4

0.6

0.8

1.0

1.2


20 °C 40 °C


72

enhanced protein interactions. This difference in pH sensitivity may be important for food

applications as the majority of products possess at least a slight acidic pH value.

Results of dynamic light scattering are shown in Table III.2. Only slight changes after heat

treatments were found at treatment pH 7. Surprisingly, a distinct growth in particle diameter was

also found in samples pressure treated at neutral pH, although the soluble protein content was

not markedly changed. Hence, the formation of soluble aggregates can be assumed. These

aggregates may be of industrial relevance due to their particular functionality (Nicolai & Durand,

2013). Adjustment of pH 6 in the samples shifted the particle size distribution to higher values,

probably due to an acid induced formation of aggregates. Thermal and pressure treatments

further increased the particle size. A marked reduction in the average particle size was observed

after processing at 60 °C and 200 MPa at 40 °C. Treatment conditions may have led to particle

dissociation, the formation of aggregates separable during gentle centrifugation or a combination

of both.

Protein solutions prepared from commercial isolate possessed a slightly higher surface

hydrophobicity compared to the self-made concentrate (see Figure III.9). An increase in

hydrophobicity was already observed after treatment at 60 °C. This might either arise from the

negligible precipitation in heat treated isolate and the maintenance of unfolded protein or from

Table III.2: Average particle diameter of thermal and high pressure treated protein solutions made of commercial isolate at pH 7 and pH 6; initial protein concentration: 10 g/L; treatment time: 10 min; solutions were centrifuged for 5 min at 500 g

Particle diameter in nm

20 °C 40 °C 50 °C 60 °C 70 °C 80 °C

pH 7 6.8 ± 2.6 7.2 ± 1.3 20.7 ± 7.5 9.9 ± 0.6 22.1 ± 7.6 29.7 ± 1.0

pH 6 82.3 ± 1.3 94.8 ± 4.4 105.4 ± 23.1 6.3 ± 0.6 244.8 ± 28.0 189.5 ± 9.4

20 °C 40 °C


pH 7 6.3 ± 2.1 6.4 ± 0.5 76.5 ± 0.6 5.7 ± 0.7 135.4 ± 3.6 120.8 ± 6.1

pH 6 84.4 ± 17.2 137.6 ± 4.6 160.8 ± 10.6 6.3 ± 0.2 218.9 ± 11.1 208.2 ± 5.8


73

Figure III.9: Aromatic surface hydrophobicity of the soluble protein fraction of potato protein solutions pH 7 and pH 6 subjected to following treatments: top: thermal treatments at atmospheric pressure; bottom: pressure-temperature-combinations; sample: commercial protein isolate; initial protein concentration: 10 g/L; treatment time: 10 min; protein concentration during fluorescence analysis: 1 g/L; protein quantification via Biuret method.

higher temperature sensitivity due to differences in protein recovery. A production scheme was

not available for the isolate, but certain technological steps, like pasteurization or drying at

elevated temperatures, may alter the protein conformation and lead to exposure of hydrophobic

patches. Proteins with a high surface hydrophobicity also possess a decreased thermal and

interfacial stability (Damodaran, 1994).

surf

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74

Surface hydrophobicities determined in samples treated at 20 °C were similar for both treatments

pHs. Pots et al. (1998b) reported only small conformational changes of patatin in the pH range of

6 to 8. Unfolding occurred at higher temperatures at pH 6, but the authors also observed protein

precipitation at this pH value. In this thesis, the surface hydrophobicity of the soluble fraction was

even slightly reduced after thermal treatments at pH 6, probably due to aggregation of

hydrophobic protein fractions and their removal during centrifugation. Treatments at 80 °C led to

an increase in hydrophobicity indicating a higher extent of unfolding that affected even proteins

which remained in solution.

Analogous to the results of the concentrates no changes in surface hydrophobicity were detected

for pressurized samples, affirming the higher stability of patatin towards this technology.

Differentiation between aromatic and aliphatic hydrophobicity was waived as first results

indicated no differences between both analyses.

As a result of the fractionation and isolation procedure, bands for lipoxidase and protease

inhibitors are almost invisible in SDS PAGE and the patatin band is broadly distributed due to the

predominance of this protein fraction in the sample (see Figure III.10). Thermal treatments at

pH 7 led to additional bands in the range of 45 and 50 kDa and between 100 and 150 kDa. This is

in relatively good agreement with the results of Pots et al. (1999b) who found aggregates with

molecular weights of 43, 82, and 108 kDa after heating of patatin at pH 7. A protein band with a

molecular weight between 100 and 150 kDa was also visible after high pressure treatment but it

was considerably less pronounced. This confirms again the high pressure stability of the patatin

molecule. At pH 6, the band intensity of patatin was slightly altered by processing at 60 °C and

600 MPa, whereas it was completely removed in samples exposed to 70 and 80 °C. This

observation confirms the higher temperature stability of this protein at slightly acidic pH values.

Treatment temperatures higher than 60 °C or pressurization at 600 MPa led to removal of the

lipoxidase band at both pH values.

One can conclude that potato protein is less sensitive towards pressure application than towards

heat exposure. High pressure promotes the dissociation of oligomers as this change in quaternary

structure is often connected to a high volume decrease (Schade et al., 1980; Weber & Drickamer,

1983). Loss of the patatin’s dimeric structure under pressure cannot be followed in this

electrophoresis due to the presence of dissociating SDS. Almost all globular proteins show a

positive compressibility indicating the presence of internal cavities that make the molecule more

flexible (Gekko & Hasegawa, 1986). Nevertheless, no correlation between the protein’s

compressibility and their pressure sensitivity can be found as, for example, the whey proteins α-

lactalbumin and β-lactoglobulin possess a similar compressibility, but differ markedly in their


75

Figure III.10: Non-reducing SDS PAGE of the soluble protein fractions of potato protein solutions pH 7 and pH 6 exposed to following treatments: 60 °C, 80 °C at ambient pressure and 600 MPa at 40 °C; sample: commercial protein isolate; initial protein concentration: 10 g/L; treatment time: 10 min; A: Lipoxidase (85 kDa); B: Patatin monomer (41 kDa); C: Protease inhibitors (19-25 kDa).

behaviour under pressure. The existence of four disulphide bonds in α-lactalbumin in comparison

to two disulphide bonds and one free thiol group in β-lactoglobulin is often mentioned as the

main reason for the higher pressure stability of the former protein (Messens et al., 1997). No

noticeable high number of these stabilizing bonds was reported for potato proteins, as the

protease inhibitors have two disulphide bonds (Bauw et al., 2006) but patatin only possesses one

accessible thiol group (Welinder & Jorgensen, 2009). Possibly, the absence of disulphides enables

a higher structural flexibility and rapid refolding upon pressure release. A lower pH during

treatment may enable the formation of permanent structural changes.

Solubility changes after different treatments observed in neutral solutions of protein concentrate

are similar to those obtained for solutions of patatin isolate at pH 6, not at pH 7. Solutions made

of concentrate and isolate differ markedly in their purity and in their conductivity. These findings

suggest that not only the pH value, but as well other environmental parameters like the ionic

strength affect the degree of protein precipitation after treatment. To reveal the mechanism that

is responsible for the formation of large aggregates, solubility tests in different media were

conducted. Phosphate-citrate buffer was used as basic solution and addition of different reagents

was performed to specifically affect certain molecular interactions. Sodium dodecyl sulphate

(SDS) breaks hydrophobic interactions and hydrogen bonds, whereas urea is known to primarily

pH 7

600 MPa20 °C 80 °C60 °C 600 MPa20 °C 80 °C60 °C

pH 6

C

B

A


76

interact with the latter one. Electrostatic interactions between protein molecules can be

weakened by addition of salts, acids or alkali. Reducing agents like dithiothreitol (DTT) can cleave

intra- and intermolecular disulphide bonds (Liu & Hsieh, 2008). Unfortunately DTT reduces as well

the copper ions responsible for Biuret reactions, so that a determination of soluble protein was

not possible for reaction mixtures containing this chemical. Analyses were carried out with

protein concentrate pH 7 and isolate pH 6 treated at the highest process intensities investigated

within this thesis. As no treatment induced precipitation occurred in isolates at neutral pH, these

samples were not investigated with the technique mentioned above.

Changes in absorbance and protein solubility in the presence of different reagents are shown in

Figure III.11. Mixing with buffer already decreased the absorbance of samples treated at 80 °C

indicating that the formed aggregates are very sensitive to environmental changes. However, as

solubility was not affected, differences in the absorbance might arise from size rearrangement of

the formed aggregates, not from their solubilization. Addition of sodium chloride increased the

absorbance of pressurized samples, but led to no changes or slight improvement in protein

solubility for isolate and concentrate, respectively. This might as well arise from changes in the

already formed aggregates. As already mentioned, no direct conclusion on protein solubility can

be drawn from the absorbance of a sample.

DTT did not improve the absorbance in comparison to the pure buffer. Even an enhancement of

the absorbance was detected for pressure treated samples. As DTT is most effective at higher

temperatures, the exposure of pressurized protein to 70 °C might have additionally led to thermal

aggregation and intensified precipitation. This is in accordance with Pots et al. (Pots et al., 1999b),

who stated that formation of disulphide bonds is not of importance in the primary aggregation of

patatin.

Presence of SDS led to almost complete resolubilization of temperature induced aggregates. SDS

is known to completely unfold and elongate the protein structure necessary for instance in

electrophoretic characterization. As a certain particle diameter is required to scatter incident

light, the absorbance of all SDS containing samples is almost zero. Hydrophobic interactions are

stabilized by applying higher temperatures due to their endothermic nature (Damodaran, 2006).

Therefore, heat treatment probably induced the formation of additional hydrophobic bonds.

Solubility of pressure treated samples was as well improved by SDS, but not to the same extent.

Additionally, an effect of urea on pressurized samples was observed, that was not existent for

heat treated protein. This leads to the assumption that proteins exposed to high temperatures

mainly aggregate via hydrophobic interactions, whereas in pressure-induced aggregates,

hydrogen bonds and hydrophobic effects are involved to a similar extent. This is also confirmed by


77

the results of the surface hydrophobicity where an exposure of hydrophobic groups was only

observed after thermal processing. Formation of hydrogen bonds is not connected to a high

volume increase (Messens et al., 1997). Therefore, aggregation via these hydrophilic interactions

is also likely to occur under pressure.

Figure III.11: Stability of aggregates formed in potato protein solutions during treatments at 80 °C and 0.1 MPa or 40 °C and 600 MPa towards addition of different reagents: top: absorbance of the samples in respective reagents at 605 nm; bottom: proportion of precipitated protein in the samples mixed with respective reagents; not precipitated protein was quantified via Biuret method; sample: commercial protein isolate and self-made protein concentrate; initial protein concentration: 10 g/L; treatment time: 10 min.

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78

The main bonding mechanisms responsible for aggregation also explain the sample’s sensitivity

towards ions. Presence of salts intensifies both hydrophobic and hydrogen bonds, whereat the

latter to a higher extent (Anon et al., 2011). With increasing ionic strength the retardation

between particles decreases promoting the formation of large aggregates, known as the

Smoluchowski-Fuchs mechanism (Pots et al., 1999b). As the ionic strength and the pH value are

often altered during or after protein processing, the stability of the heated or pressurized protein

in varying media can be a key factor in selecting an appropriate technology.

Setting the pH value to 6 after thermal treatment reduced protein solubility by up to 90 % due to

subsequent formation of larger aggregates (Figure III.12). These results are similar to those

obtained for thermal treatments at pH 6 indicating that presence of protons during heating is not

necessary to achieve precipitation, but acidification promotes interactions between primary

aggregates. Pressure treated samples were as well slightly affected, but the content of soluble

protein stayed above 88 %. Samples pressurized at pH 6 showed a solubility reduction of almost

50 %. Consequently the presence of protons during pressure application is necessary to achieve a

considerable protein precipitation.

Addition of the same amount of sodium chloride did not markedly affect solubility. This indicates

that the presence of salts is not the main reason for an enhanced precipitation in protein

solutions prepared from concentrate. Pots et al. (1999b) reported that aggregation of heat

treated patatin is promoted by ionic strengths of at least 50 mM. The ion concentrations obtained

by addition of NaCl in this investigation were estimated to be below that value, whereas an initial

conductivity of 4 mS/cm as determined for solutions made of protein concentrate is a sign of an

ionic strength exceeding this critical value. Hence, it cannot be excluded that higher ionic

strengths at neutral pH would also lead to precipitation of heat treated potato protein.

Interactions with other components deriving from potato fruit juice still present in the

concentrate might also influence protein’s sensitivity towards processing. Baxter et al. (1997)

suggested that proline rich proteins, like patatin, interact with polyphenols to achieve

precipitation. As polyphenols are present in many food systems, it has to be clarified whether

these interaction only occur during heating or as well afterwards.

Nevertheless high pressure shows many advantages as preservation technique for potato protein,

as structure and solubility of the protein were less affected than by conventional thermal

treatments. Increasing the shelf-life of potato protein in solution may enable to skip energy

consuming drying processes in certain cases or inhibit excessive growth of present

microorganisms after resolubilization. The lower sensitivity of pressure treated protein towards


79

changes in product composition might expand the application possibilities of this protein in the

food area.

Figure III.12: Influence of post-process medium changes on the solubility of protein solutions subjected to following treatments: top: thermal treatments at atmospheric pressure; bottom: pressure-temperature-combinations; sample: commercial protein isolate; initial protein concentration: 10 g/L; treatment time: 10 min; HCl: addition of HCl to decrease pH value from 7 to 6; NaCl: adjustment of equimolar NaCl concentration at pH 7; protein quantification via Biuret method.

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80

Influence of high pressure and elevated temperature on pea

proteins

Pressure and heat were applied to pea flour and protein concentrate to figure out their influences

on protein structure and to highlight their potential as preservation or modification techniques.

Figure III.13 shows the influence of treatment temperatures up to 80 °C on the absorbance and

protein solubility in solutions made of protein concentrate won by watery extraction,

ultrafiltration and freeze drying.

Figure III.13: Influence of thermal treatments at ambient pressure on protein solubility and absorbance at 605 nm of pea protein solutions pH 7; sample: self-made protein concentrate; initial protein concentration: 10 g/L; treatment time: 10 min; protein quantification via Biuret method.

The increase in the absorbance and the loss in solubility with rising treatment temperature

indicate denaturation of proteins and their aggregation with one another or with other pea

components extracted and concentrated during protein recovery. Carbonaro et al. (1997)

reported an 80 % reduction in water solubility of protein from different legume varieties cooked

for 2 h. Only pH values exceeding 10 led to higher protein solubility. In these experiments the

protein content in solution was reduced to maximal 40 % at neutral pH and the absorbance

increased by factor 14. These changes are markedly higher than those induced by high pressure

(see Figure III.14). All pressure-temperature combinations tested kept more than 90 % of protein

into solution. The absorbance increased at most by factor 3.9. Analogous to results obtained for

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81

potato protein, the effect of pressurization was higher at 40 °C due to a pressure-temperature

synergism.

Figure III.14: Influence of high isostatic pressure on protein solubility and absorbance at 605 nm of pea protein solutions pH 7; sample: self-made protein concentrate; initial protein concentration: 10 g/L; treatment time: 10 min; protein quantification via Biuret method.

Figure III.15: Particle size distributions in heat treated pea protein solutions pH 7; sample: self-made protein concentrate; initial protein concentration: 10 g/L; treatment time: 10 min; centrifugation for 5 min at 500 g.

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82

Although overall treatment effects were smaller compared to those obtained for potato protein

concentrate, the general conclusion that the proteins possess high pressure stability and are more

sensitive to thermal denaturation is the same for both vegetable protein sources. These

suggestions are supported by the results of the particle size analysis shown in Figure III.15 and

Figure III.16. The average particle diameter was slightly reduced by treatments at 40 and 50 °C

and strongly increased by temperatures of 70 and 80 °C. Possibly, slightly elevated temperatures

led to further dissociation of small protein aggregates not completely solved during sample

preparation, while a further temperature enhancement induced the formation of new aggregates.

The latter is in agreement with the results of Carbonaro et al. (1997), who determined a shift in

the pattern of size exclusion chromatography to higher retention times for extracts from cooked

legumes. No changes were observed after high pressure treatments. As already mentioned

before, removal of large particles that do not undergo Brownian motion cannot be excluded.

Comparison of solutions made of protein-enriched pea flour showed an opposite behaviour

(Figure III.17). Protein remained into solution after thermal treatments, while high pressure led to

solubility reduction up to 35 %. In solutions made of protein enriched flour batch one a decrease

in the soluble protein content to 65 % after exposure to 60 °C was determined due to formation

of larger aggregates and removal of them during centrifugation. At higher treatment

Figure III.16: Particle size distributions in pea protein solutions pH 7 exposed to different pressure-temperature combinations; sample: self-made protein concentrate; initial protein concentration: 10 g/L; treatment time: 10 min; centrifugation for 5 min at 500 g.

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40 °C, 200 MPa 40 °C, 400 MPa 40 °C, 600 MPa


83

Figure III.17: Protein solubility and absorbance at 605 nm of pea protein solutions pH 7 subjected to following treatments: top: thermal treatments at atmospheric pressure; bottom: pressure-temperature-combinations; sample: air-classified pea flour batch two; initial protein concentration: 10 g/L; treatment time: 10 min; protein quantification via Biuret method.

temperatures as well as for high pressure processed samples, effects were similar to those

obtained for batch two. Differences in sample composition and purity may have altered the

aggregation and precipitation behaviour of the protein fractions.

The absorbance at 605 nm was enhanced for both technologies by more than the 60fold. The

highest absorbance values were determined after treatments at 60 °C and 200 MPa at 40 °C, not

for the highest treatment intensities investigated and samples with the highest decreases in


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84

solubility, respectively. Small differences in very turbid samples can hardly be detected correctly

due to the inaccuracy of the analysis and the high dilution stages.

Figure III.18: Particle size distribution of pea protein solutions pH 7 subjected to following treatments: top: thermal treatments at atmospheric pressure; bottom: pressure-temperature-combinations; sample: air-classified pea flour batch two; initial protein concentration: 10 g/L; treatment time: 10 min; centrifugation for 5 min at 500 g.

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85

Particle size distribution showed a lower initial particle diameter for protein solutions made from

flour compared to those made of protein concentrate. Incomplete solubilization of the protein

concentrate was already mentioned as a possible explanation. High pressure as well as

temperatures of 70 and 80 °C led to a 10fold increase in particle size (see Figure III.18). This does

not directly correlate to the sample’s absorbance as solutions made of concentrate showed a

similar average particle size without appearing turbid. An increase in particle size was not

observed after applying 60 °C but the sample’s absorbance increased to a similar extent as in

samples treated at higher temperatures. This again indicates that no direct correlation between

both analyses can be drawn and the changes in the absorbance are not only a result of altered

protein aggregate size.

The presence of other soluble compounds might as well contribute to this effect. Especially small

starch granules insufficiently separated during air classification and centrifugation might have

undergone gelatinization during treatments and have led to differences in light scattering.

Treated samples remained turbid after centrifugation which might be caused by a viscosity

increase due to gelatinized starch and insufficient separation of insoluble particles. This effect was

even more pronounced for pressure treated solutions (see Table III.3). This behaviour made

protein quantification with photometric methods inaccurate and decreases the validity of the

data. Nevertheless, the results obtained emphasize the importance of sample composition and

origin on the treatment impact.

Table III.3: Absorbance read at 540 nm of the centrifuged pea flour (batch two) solutions pH 7 subjected to heat or high pressure diluted 1:10 with deionized water (analogous to conduct of the Biuret analysis); initial protein concentration: 10 g/L; treatment time: 10 min; centrifugation for 10 min at 5 min at 10 000 g

Absorbance [-]

20 °C 40 °C 50 °C 60 °C 70 °C 80 °C

20.090 ± 0.014 0.090 ± 0.014 0.125 ± 0.007 0.195 ± 0.049 0.190 ± 0.042 0.255 ± 0.078

20 °C 40 °C


0.083 ± 0.021 0.207 ± 0.072 0.373 ± 0.246 0.140 ± 0.036 0.500 ± 0.144 0.743 ± 0.136


86

Contrary results were published on the precipitation behaviour of other legume proteins that all

possess a similar protein composition compared to pea protein, especially after high pressure

treatments. No changes in solubility after pressure application up to 700 MPa were reported by

Torrezan et al. (2007) for soy protein. Molina et al. (2001) determined an improved solubility of

soy protein isolate, and its isolated 11S and 7S fractions due to pressure treatments up to

600 MPa, whereas the solubility of protein solutions prepared from lupine flour were slightly

reduced by 20 % after pressure treatments exceeding 400 MPa (Chapleau & de Lamballerie-

Anton, 2003). These findings strengthen the observation that protein composition, its

concentration and solvent properties may alter the intensity of a treatment effect or even lead to

a different aggregation mechanism.

Structural analyses were conducted to reveal reasons for varying treatment effects and identify

the underlying aggregation mechanism. A thermally induced increase in surface hydrophobicity

was observed for samples exposed to 70 and 80 °C. (see Figure III.19). The decreased surface

hydrophobicity at 60 °C correlates with the decrease in protein solubility observed in solutions

prepared from pea flour batch one at this treatment intensity. In solutions made of protein

concentrate the aromatic hydrophobicity was slightly higher after treatment at 40 and 50 °C, but

lower at higher temperatures. The aliphatic hydrophobicity of protein concentrate solutions was

decreasing with rising temperature. Again, these decreases in hydrophobicity can be traced back

to a fortified aggregation of hydrophobic protein fractions and their removal during subsequent

centrifugation. Soluble proteins from cooked legumes differed in their amino acid composition

from the whole cooked seed (Carbonaro et al., 1997). The proportion of basic and acidic amino

acids in solution was higher than in the cooked legumes and the proportion of hydrophobic amino

acids was correspondingly lower. This indicates that protein fractions containing many aliphatic

and aromatic residues tend to become insoluble during heating.

The aromatic hydrophobicity of pressurized samples was increasing, the aliphatic decreasing with

rising treatment intensity. Similar results were reported by Yang et al. (2001) for whey protein

isolate. Investigations with model solutions revealed, that exposure of aliphatic residues is

connected to a volume increase, whereas unfolding of aromatic side chains causes a decrease in

volume (Messens et al., 1997).

In consequence, the exposure of aromatic side chains is favoured under pressure, whereas that of

aliphatic chains is retarded. An increase in aromatic hydrophobicity due to pressure was reported

for soy protein (Zhang et al., 2003; Puppo et al., 2004) and fava bean (Galazka et al., 1999). In

flour samples the hydrophobicity stayed rather constant which could either indicate diminished

unfolding or, more likely, separation of unfolded proteins by precipitation.


87

Figure III.19: Influence of thermal treatments at ambient pressure (top) and different pressure-temperature-combinations (bottom) on the surface hydrophobicity of the soluble fraction of pea protein solutions pH 7; aromatic and aliphatic hydrophobicity of samples made of self-made protein concentrate, aromatic hydrophobicity of samples prepared from air-classified pea flour batch one; initial protein concentration: 10 g/L; treatment time: 10 min; protein concentration during fluorescence analysis: 1 g/L for aromatic hydrophobicity, 0.1 g/L for aliphatic hydrophobicity; protein quantification via Biuret method.

Aggregates formed during treatment were characterized with the method already explained in

the previous subchapter. Figure III.20 shows the results of the absorbance and solubility analyses.

Both methods are in accordance and indicate that heat leads to aggregates primarily formed by

hydrophobic interactions, whereas in pressure-induced aggregates both hydrophobic and

pea flourprotein concentrate

aromatic hydrophobicity

aliphatic hydrophobicity

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88

hydrogen bonds are involved. Same binding mechanisms were already determined for potato

protein. As already mentioned, hydrogen bonds can be formed during pressure as their formation

involves no significant volume increase (Messens et al., 1997).

Figure III.20: Stability of aggregates formed in pea protein solutions during treatments at 80 °C and 0.1 MPa or 40 °C and 600 MPa towards addition of different reagents: top: absorbance of the samples in respective reagents at 605 nm; bottom: proportion of precipitated protein in the samples mixed with respective reagents; not precipitated protein was quantified via Biuret method; samples: self-made protein concentrate and protein-enriched flour batch two; initial protein concentration: 10 g/L; treatment time: 10 min.

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89

Addition of pure buffer or those containing sodium chloride led to an enhanced absorbance of

heat treated protein concentrate but did not alter its solubility. In the other samples, the

absorbance was lowered after mixing with pure buffer. In contrast, solubility of the heated flour

was declined by this environmental change. Again, sensitivity of hydrophobic and hydrophilic

interactions towards alterations in ionic strength might have induced the formation of large

secondary aggregates scattering light to a different extent. This dependency might also explain

the differences in precipitation behaviour between flours of batches one and two as the batches

primarily differ in the content of non-protein components. Heating in the presence of DTT led to a

fortified aggregation of the temperature sensitive protein concentrate previously only exposed to

pressure. Thermally treated flour batch two and pressure treated concentrate only precipitated to

a small extent. Therefore, small variations in absolute protein solubility already led to a

proportionately huge change in the relativized data.

As the aggregation mechanism triggered by heat and pressure were similar for pea protein

concentrate and flour, these analyses did not give further insight in the different sensitivity of the

two initial materials. Differentiation into the protein’s main fractions via non-reducing SDS PAGE

can potentially reveal changes in the composition between untreated and treated flour and

concentrate. Figure III.21 shows the electrophoretic pattern of samples prepared from flour batch

one. The protein fraction with the highest molecular weight determined can be correlated to pea

lipoxidase with a molecular weight around 90 kDa (Shand et al., 2007). Legumin is a hexamer that

dissociates into a 60 kDa monomer in the presence of SDS. It consists of basic and acidic subunits

of 20 and 40 kDa, respectively, that are linked via a disulphide bond (O'Kane et al., 2004). The

vicilin fraction usually exists as a trimer. It dissociates into subunits of 50, 33, 20 and 17 kDa

(Gueguen, 1983). The larger convicilin with a weight around 71 kDa differs from vicilin by a

strongly charged, hydrophilic N-terminal (O'Kane et al., 2004). The 26 kDa pea albumin 2 (PA2)

dissociates from a dimer with a weight of 48 to 53 kDa (Croy et al., 1984).

No intensive alterations in the protein composition were induced by a temperature of 40 °C,

whereas after 60 °C the band belonging to PA2 completely disappeared. Similar effects were

reported by Le Gall et al.(2005), where the albumin band intensity was drastically reduced after a

3 minute treatment of peas at 85 °C, indicating that this protein could no longer be extracted.

After heating to 80 °C the legumin band was as well less pronounced. The surface hydrophobicity

of the pure 11S protein of fava bean only increased when temperatures exceed 70 °C (Galazka et

al., 1999). Probably the 11S fraction of pea, legumin, accordingly starts to unfold and aggregate at

higher temperatures. An additionally visible band with higher molecular mass could be the

consequence of legumin aggregation.


90

Figure III.21: Non-reducing SDS PAGE of the soluble protein fraction in solutions pH 7 prepared from pea flour batch one after thermal treatments at ambient pressure and pressure treatments at 40 °C; initial protein concentration: 10 g/L; treatment time: 10 min; A: Lipoxidase (90 kDa); B: Convicilin (71 kDa); C: Legumin (60 kDa); D: Vicilin (17-50 kDa); E: PA2 (26 kDa).

According to Galazka et al. (1999) the pure fava bean 11S protein changed its surface

hydrophobicity already after pressure application of 200 MPa. This is confirmed by the present

results of pea protein, where the band intensity of legumin was already decreased after 200 MPa.

A complete loss in native structure determined via CD spectra was observed for 11S soy glycinin

after pressurization with 400 MPa (Zhang et al., 2003). Gels formed with pure legumin can be

dissolved by urea and mercapto-ethanol, but not with SDS, showing that hydrophilic interactions

and disulphide bonds contribute to gel stability (O'Kane et al., 2004). This indicates that legumin is

generally able to form a network via hydrogen bonds.

The 50 kDa band of vicilin also disappeared with increasing pressure and convicilin is diminished

in its intensity. The vicilin trimer is stabilized by electrostatic interactions that can be easily broken

by high pressure (Pedrosa & Ferreira, 1994). High pressure favours the presence of monomers as

dissociation of oligomers is connected to a volume reduction (Messens et al., 1997). Zhang et al.

(2005) reported denaturation of the homologous 7S soy conglycinin for pressures exceeding

300 MPa. Vicilin has almost no sulphur-containing amino acids and possesses no disulphide

bridges that could stabilize its three dimensional conformation under pressure (Wright, 1987).

Vicilin and convicilin contain 32 and 8 % hydrophobic, as well as 22 and 60 % charged amino acids,

200 MPa 400 MPa 600 MPa20 °C 40 °C 80 °C

AB

E

D

C

D

60 °C


91

respectively (O'Kane et al., 2004). The higher proportion of charged side chains may be the reason

for the higher pressure stability of convicilin.

The albumin PA2 was not changed in its intensity by high pressure. The protein contains three

cysteine residues whereof two are involved in disulphide bridges. This structural links are

probably responsible for the molecule’s pressure stability. The band intensity of lipoxidase was

reduced after 80 °C and 600 MPa. These conditions do not necessarily correspond to the

denaturation temperature or pressure of the enzyme as changes in the protein conformation

leading to an activity loss might already occur at lower treatment intensity.

In general, similar changes were observed in the PAGEs run with protein concentrate (Figure

III.22). The band intensities of lipoxidase and PA2 was stronger pronounced, in return the

globulins are present to a smaller extent. No band could be determined in the range of 60 kDa

corresponding to the legumin monomer. This might be a consequence of the overall worse

solubility of the concentrate. Albumins usually possess a better solubility at low ionic strength

compared to globulins. Hence, the presence of albumins might proportionally increase when the

overall solubility is reduced. Germination of pea seeds may have started during the 20 h of

soaking previous to protein extraction. An initiated degradation of the globulins may have

occurred in this step that alters the overall protein composition (see as well chapter V). PA2 can

still be found in the seeds after germination, indicating that it is not degraded within this process

(Croy et al., 1984). Nevertheless, as SDS PAGEs run with protein extracts from swollen peas did

not show these changes in protein composition (see chapter VI), alteration of the proteins during

concentration and drying is more likely. Sensitivity towards cold denaturation was reported for

the 11S globulins of soy protein (Cheftel et al., 1992a). The homologous legumin might

consequently as well be affected during freezing and lyophilization. Changes in the

electrophoretic intensity of PA2 and lipoxidase were similar to those obtained for pea flour. As

the globulins were in general less pronounced, almost no changes were visible after high pressure

treatment. This corresponds to the results of solubility measurement, where no marked changes

were determined after high pressure treatment of protein concentrate.

Although the aggregate characterization showed no influence of disulphide formation during

protein precipitation the content of accessible thiol groups was affected by both technologies (see

Figure III.23 and Figure III.24). Solutions made of protein concentrate showed a thiol content

twice as high as the one in flour. This is a consequence of the higher proportion of PA2, which

possesses more sulphur-containing amino acids than legumin and vicilin (Bhatty, 1982; Croy et al.,

1984), but it might also indicate a previous oxidation of thiols in the pea flour.


92

Figure III.22: Non-reducing SDS PAGE of the soluble protein fraction in solutions pH 7 prepared from pea concentrates after exposure to different treatments; top: thermal treatments at ambient pressure; bottom: pressure treatments at 20 and 40 °C; initial protein concentration: 10 g/L; treatment time: 10 min; A: Lipoxidase (90 kDa); B: Convicilin (71 kDa); D: Vicilin (17-50 kDa); E: PA2 (26 kDa).

Heat treatment caused a decrease in the content of accessible thiol groups with rising

temperature. This effect was more pronounced for protein concentrate than for flour, probably

due to the overall higher heat sensitivity of the concentrate. A temperature of 80 °C slightly

A

B

E

D

60 °C 70 °C 80 °C20 °C 40 °C 50 °C

AB

E

D

20 °C 40 °C



93

increased the content of accessible thiols. This contrary effect might be related to the

denaturation of the 11S globulin, which is connected to thiol-disulphide interchanges, dissociation

into acidic and basic subunits and a subsequent aggregation (Cheftel et al., 1992b).

Figure III.23: Influence of thermal treatments at ambient pressure on total and fast accessible thiol groups of pea protein solutions pH 7; top: samples prepared of self-made protein concentrate; bottom: samples made of air-classified pea flour batch two; initial protein concentration: 10 g/L; treatment time: 10 min protein quantification via Biuret method.

0

2

4

6

8

10

12

20 °C 40 °C 50 °C 60 °C 70 °C 80 °C

0

2

4

6

8

10

12

20 °C 40 °C 50 °C 60 °C 70 °C 80 °C

supernatantwhole sample

fast accessible thiol groups

total accessible thiol groups

acce

ssib

leth

iolg

roup

sin μm

ol/ g

pro

tein


94

Figure III.24: Influence of several pressure-temperature-treatments on total and fast accessible thiol groups of pea protein solutions pH 7; top: samples prepared of self-made protein concentrate; bottom: samples made of air-classified pea flour batch two; initial protein concentration: 10 g/L; treatment time: 10 min; protein quantification via Biuret method.

Heating causes unfolding of the protein structure that leads to an exposure of fast accessible thiol

groups to the molecule surface, which constitute more than 90 % of the thiol groups in the 80 °C

sample. The better the accessibility the faster is the chemical conversion of the thiol groups. In

pressure treated samples the proportion of fast accessible thiols remained constant indicating

supernatantwhole sample

fast accessible thiol groups

total accessible thiol groups

acce

ssib

leth

iolg

roup

sin μm

ol/ g

pro

tein

0

2

4

6

8

10

12


20 °C 40 °C

0

2

4

6

8

10

12


20 °C 40 °C


95

that the overall unfolding of the protein structure was less pronounced or that only protein

fractions with low thiol content, like vicilin and convicilin, were targeted. Opposite results were

reported from Zhang et al. (2003) who found an increase in the fast accessible thiols of pure soy

glycinin with rising pressure.

Changes in the total accessible thiol groups were similar for pea concentrate and pea flour. A

reduction in thiol content due to pressure was also reported for soy protein (Puppo et al., 2004;

Torrezan et al., 2007). The reactivity of thiols to form disulphide bonds is increased under

pressure (Messens et al., 1997). Comparing the whole sample with the clear supernatant obtained

after centrifugation it is obvious that the accessible thiols are less affected in the soluble part.

These differences indicate that proteins with oxidized thiol groups are more likely to be involved

in aggregation and precipitation phenomena. As the effect is mostly pronounced for pressurized

samples, it might as well be a result of concentrating the sulphur-rich albumin fraction after

removing the globulins.

The general differences in protein composition might be the reason for the varying sensitivity

towards heat or pressure. A higher concentration of unfolded and denatured proteins may affect

as well the aggregation behaviour of the other protein fractions. This behaviour, as well as the

general difference of certain protein fractions towards thermal or pressure treatment, can be

useful to obtain protein products with tailor-made properties. As albumins and globulins differ

markedly in their techno-functional behaviour as well as in their nutritional and allergenic

properties, a target application of the respective technology may lead to a pea protein product of

desired functional and nutritional quality. The influence of pressure and heat on foaming

properties as well as their potential as preservation methods will be discussed in chapter IV.

Changes in protein structure induced by pulsed electric fields (PEF)

Within this thesis, pulsed electric fields were applied to pea and potato tissue to obtain cell

disintegration and mass transfer improvement (see chapter V). Protein solutions were exposed to

electric pulses of the same intensity as potential changes in protein conformation and properties

can be determined more easily than in the complex tissue. PEF treatment might also be used to

inactivate vegetative microorganisms in homogeneous liquids and therefore contribute to a shelf-

life increase of protein products. However, inactivation experiments performed in the available

discontinuous PEF unit did not result in a marked reduction of colony forming units.

Consequently, the impact of the corresponding treatment intensities on protein structure was not


96

evaluated as no clear correlation of PEF pasteurization to protein quality would be possible (for

details see chapter IV).

Figure III.25 shows the soluble protein content in solutions prepared from self-made protein

concentrate. Protein quantification using Biuret reagent did not indicate any changes in protein

solubility after pulsed electric field application, while the absorbance measured with Bradford

reagent rose after treatment by up to 14 %. As an increase in protein content in the sample

cannot occur, changes in the determined protein content may indicate alterations in the protein

conformation or interactions with other food constituents. In Biuret assay, copper ions form a

complex with the electron pair of the peptide nitrogen (Creighton, 1984). The colour formation is

therefore relatively independent from protein conformation and amino acid composition. The

Bradford assay is based on the interaction of Coomassie brilliant blue with specific amino acids of

the protein chain. Although a linear relationship of protein concentration and absorbance exists,

the overall intensity of colour formation varies with the type of protein and its amino acid

composition (Pierce & Suelter, 1977; Van Kley & Hale, 1977; Congdon et al., 1993).

Figure III.25: Quantification of soluble protein via different photometric methods in potato protein solutions pH 7 subjected to pulsed electric fields; sample: self-made protein concentrate; initial protein concentration: 10 g/L.

Solutions of whey protein isolate subjected to heat or high pressure exhibit higher aromatic

surface hydrophobicity due to unfolding and exposure of aromatic amino acids (Figure III.26).

These samples showed an increase in the Bradford response as well, although no changes in the

BradfordBiuret

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 kV/cm 1 kV/cm 2 kV/cm 2 kV/cm

2.5 kJ/kg 2.5 kJ/kg 5 kJ/kg

prot

ein

in so

lutio

n in

rela

tion

to th

e un

trea

ted

sam

ple

[-]


97

soluble protein content occurred. These results reveal a possible relationship between protein

unfolding and the intensity of the Coomassie dye binding. Binding of Coomassie blue alters the

internal packing of the protein molecule and leads to an energy transfer between protein and dye

dependent on their distance (Katrahalli et al., 2010). It is likely that changes in protein

conformation alter this distance and thereby the intensity of the light absorbance. Collagen, a

highly structured protein molecule, shows almost no absorbance in the standard Bradford assay.

Addition of SDS leading to protein unfolding results in an enhanced absorbance intensity

(Duhamel et al., 1981). Other detergents that interact with the protein molecule like urea or

TritonTM-X 100 also alter the response of proteins toward the Bradford assay to various extends

(Friedenauer & Berlet, 1989). Unfolding of the protein structure may as well enhance the

accessibility of the dye to the binding site.

Figure III.26: Quantification of soluble protein via different photometric methods and aromatic surface hydrophobicity of the soluble protein fraction of whey protein solutions pH 7 subjected to pulsed electric fields; sample: commercial whey protein isolate; initial protein concentration: 10 g/L; protein concentration during fluorescence analysis: 2 g/L; protein quantification via Biuret method.

Although the differences in Bradford response were smaller than those induced in whey protein

with heat and pressure, potato protein might have undergone conformational changes in the

electric field that can also alter its functional properties. Loosening of electrostatic interactions,

forming protein’s tertiary and quaternary structure, within the electric field was postulated by

several authors (Manas & Vercet, 2006). Changes in protein folding due to PEF treatment were

BradfordBiuret surface hydrophobicity

0

10

20

30

40

50

0.0

0.4

0.8

1.2

1.6

2.0

20 °C 80 °C 40 °C 40 °C

0.1 MPa 0.1 MPa 400 MPa 600 MPa

surf

ace

hydr

opho

bici

ty in

dex

in m

V *

g pr

otei

n / μm

oldy

e

prot

ein

in so

lutio

n in

rela

tion

to th

e un

trea

ted

sam

ple

[-]


98

reported for several proteins (Fernandez-Diaz et al., 2000; Perez & Pilosof, 2004; Li et al., 2007;

Xiang, 2008), but electric field strengths applied in literature were markedly higher than those

used in these experiments The relatively high pulse number (up to 1200 pulses) and the high

overall treatment time (up to 5.5 min) may have nevertheless led to structural alterations as

changes determined in a PEF treated product do not only arise from the electric field strength

itself. Although no significant warming due to energy dissipation during treatment was observed,

occurrence of local hot spots cannot be excluded. Reactions at the electrode-solution interface

may lead to electrolysis of the solution, electrode corrosion and introduction of electrode

Figure III.27: Quantification of soluble protein via different photometric methods and aromatic surface hydrophobicity of the soluble protein fraction of pea protein solutions pH 7 subjected to pulsed electric fields; sample: air-classified pea flour batch one; initial protein concentration: 10 g/L; protein concentration during fluorescence analysis: 1 g/L; protein quantification via Biuret method.

material into the sample (Morren et al., 2003). In the electroporation cuvettes used, the contact

surface between sample and electrodes is proportional high compared to other treatment

chamber designs and electrode reactions may therefore contribute to product changes to a

relatively high extent. Formation of radicals and peroxides during PEF treatment of water was

reported (Sato et al., 1996). These reactive species could lead to protein oxidation. The patatin

molecules contains one accessible thiol group (Welinder & Jorgensen, 2009) and is therefore

sensitive to oxidation, especially in impure solutions as prepared from protein concentrate. Local

pH changes at the electrodes may alter the structure of pH sensitive proteins. Meneses et al.

BradfordBiuret surface hydrophobicity

0

1

2

3

4

5

6

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 kV/cm 5 kV/cm 10 kV/cm 10 kV/cm

35 kJ/kg 35 kJ/kg 125 kJ/kg

surf

ace

hydr

opho

bici

ty in

dex

in m

V *

g pr

otei

n / μm

oldy

e

prot

ein

in so

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rela

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to th

e un

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ted

sam

ple

[-]


99

(2011) revealed PEF induced pH shifts in neutral salt solutions to 3.3 and 10.9 at the anode and

cathode, respectively, via pH indicators and digital photography. Changing the field strength from

10 to 25 kV/cm and increasing the pulse number had only slight additional effects on the pH

values determined. Hence, a shift in pH might already occur at low field strengths in the range of

those applied in this thesis. Pots et al. (1998b) determined changes in the secondary and tertiary

structure of patatin in dependence of the medium pH by using circular dichroism. Foaming

properties of potato protein products that were brought to pH 3 and afterwards back to 7 differed

from those of samples maintained at neutral pH (van Koningsveld et al., 2002). These changes in

the functional properties can serve as indicators for a partially irreversible pH modification of

potato protein. This may explain why structural changes caused by local pH changes are still

detectable after the PEF treatment ended.

Protein solutions made of pea flour did not show any changes in protein solubility after PEF

treatment – neither with the Biuret nor the Bradford assay (see Figure III.27). In conclusion, the

applied electric field intensities did not irreversibly alter the protein conformation. Determination

of the aromatic surface hydrophobicity led to the same conclusion. Furthermore, non-reducing

SDS PAGE of the pea protein solution did not show any changes in protein composition (see Figure

III.28). Nevertheless unfolding without further aggregation or reversible changes within the

electric field or due to a change in pH cannot be excluded as all analyses were performed post

processing.

Figure III.28: Non-reducing SDS PAGE of protein solutions pH 7 prepared from pea flour and treated with pulsed electric fields of varying intensity; initial protein concentration: 10 g/L; treatment time: 10 min; A: Lipoxidase (90 kDA); B: Convicilin (71 kDa); C: Legumin (60 kDa); D: Vicilin (17-50 kDa); E: PA2 (26 kDa).

AB

E

D

C

D

0 kV/cm0 kJ/kg

10 kV/cm35 kJ/kg

10 kV/cm125 kJ/kg

5 kV/cm35 kJ/kg


100

Conformational changes induced to potato protein by the usage of electric fields may also lead to

modifications of the functional properties. Results of the foaming experiments are discussed in

detail in chapter IV. As no changes were determined for pea proteins in solution, PEF technology

possesses high potential as a preservation method for these protein products or as cell

disintegration technique for the raw tissue material by contemporaneously maintaining protein

quality.

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Chapter IV – HP and PEF as alternatives for protein modification and preservation

105

Chapter IV

High pressure and pulsed

electric fields as alternatives for

protein modification and

preservation


106

The previous chapter summarized the effects of heat, high isostatic pressure and pulsed electric

fields on pea and potato proteins in solution. Treatment effects differ in regard to unfolding and

exposure of afore buried side chains, degree of aggregation and, in case of pea protein, even led

to separation of distinct protein fractions. To what extent these differences form a basis for a

targeted functional modification or a more gentle preservation of protein solutions will be

discussed in the following sections.

Foaming properties as an indicator for techno-functional alterations

There is a broad variety of functional applications for proteins in the food industry. The common

animal proteins from milk and eggs are particularly appreciated for their excellent interfacial

properties. To enable a long-term replacement of these products by vegetable alternatives, the

ability of innovative proteins to form and stabilize foams and emulsions has to be maintained or

even improved to a high extent during processing. Changes in the interfacial properties of potato

and pea protein after exposure to heat, pressure and electric fields were characterized by means

of foaming properties. There are certain differences regarding the formation and destabilization

processes of foams and emulsions, so transferring the obtained results to the emulsification

properties cannot be done without any reservation, but observed changes might serve as an

indication for a techno-functional modification in general. Foams were characterized using the

foam analyzer DFA 100, a computer-controlled foaming and analyzing unit. Measurements were

performed in regard to foam stability. Therefore, all solutions were brought to the same initial

foam volume to achieve comparable original conditions for foam decay.

Figure IV.1 shows the half-life of foams prepared from protein-enriched pea flour subjected to

thermal processing. Stability slightly decreased after application of 40 and 50 °C. A clear reduction

in foam half-life to 60 % compared to 20 °C could be observed for thermal treatments at 60 °C.

Stability was further diminished to 43 and 47 % after heating to 70 and 80 °C, respectively. Similar

observations were made by Megha and Grant (1986) who reported decreased foam stability of

protein concentrates from pea flour after thermal treatments of more than 50 °C. Temperatures

exceeding 60 °C were accompanied by a strong increase in the absorbance and a marked

reduction in the band intensity of PA2 (see Figure III.21 and Figure III.22). The results might be an

indicator that this rather small protein fraction markedly contributes to foam stability.

Denaturation of legumin occurring between 70 and 80 °C did not alter foam stability to a visible

extent and therefore seems not to be of great importance for the interfacial properties.


107

Figure IV.1: Half-life of foams prepared from heat treated pea flour suspensions pH 7; treatment time: 10 min; protein concentration during treatment: 10 g/L; protein concentration during foam analyses: 0.4 g/L; dilution medium: deionized water.

High pressure processing as well led to a reduction in foam stability in dependence on treatment

intensity (see Figure IV.2). Higher pressures as well as a temperature increase of 20 K caused

stronger functional alterations. This is in agreement with structural observations presented in the

previous chapter. Application of 400 and 600 MPa at 40 °C decreased half-life of the foams to 55

and 48 %, respectively, which corresponds nearly to the stability loss found in heat treated

samples. In conclusion, PA2, still available to a similar extent after pressurization of pea samples,

cannot be the only fraction affecting foam stability. Loss of the 7S protein vicilin already occurred

at pressures of 200 MPa. Changes affecting this protein fraction must therefore have a high

impact on foaming behaviour as well. Vicilin was reported to have better foaming and emulsifying

properties than legumin (Dagorn-Scaviner et al., 1987; Koyoro & Powers, 1987; Cserhalmi et al.,

1998). Less cysteine residues and no disulphide bridges allow a higher structural flexibility of the

7S vicilin (Koyoro & Powers, 1987). Functional changes in solutions of the homologous soy

conglycinin after combined high pressure-temperature treatment were similar to those of whole

soy protein (Puppo et al., 2011). This protein fraction can therefore be regarded as responsible for

the product’s emulsification behaviour. Emulsions prepared from heat or pressure treated Vicia

faba L. protein showed increased droplet size compared to control samples (Galazka et al., 1999).

This was traced back to oligomer dissociation and further aggregation of the subunits.

0

5

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15

20

25

20 °C 40 °C 50 °C 60 °C 70 °C 80 °C

foam

hal

f-life

in m

in


108

Figure IV.2: Half-life of foams prepared from pressurized pea flour suspensions pH 7; treatment time: 10 min; protein concentration during treatment: 10 g/L; protein concentration during foam analyses: 0.4 g/L; dilution medium: deionized water.

Probably the effects on foam stability were not caused by changes of a single protein fraction, but

are a result of the enhanced aggregation in general. This is supported by the relatively good

correlation of absorbance increase and reduction of foam half-life (see Figure III.17). Size and

shape of the molecule influence the hydrodynamic properties of the protein and might thereby

alter its ability to form a visco-elastic and stable foam lamella (Damodaran, 1994).The impact of

denaturation and aggregation on foaming properties strongly depends on the size of the particles

formed (Wierenga & Gruppen, 2010). Primary aggregates can contribute to stabilization by

building a steric barrier in the lamella, while larger clusters have an opposite effect. Particles with

higher volume have a lower specific surface to occupy the interface. A larger radius also impedes

a close packaging at the interface and hinders molecular flexibility. Sedimentation of large protein

aggregates during lifetime of the foam would as well reduce the effective protein concentration in

the lamella.

Probably some of the proteins are available in monomeric form after processing which might as

well decrease their ability to act as a steric stabilizer. Unfortunately, dissociation into monomers

that happens under pressure and at elevated temperatures was not analyzed and electrophoresis

in the presence of SDS does not allow any conclusion as oligomers are dissociated under these

conditions anyway. Nevertheless, the presence of smaller protein units can be assumed, as

0

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10

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20

25


20 °C 40 °C

foam

hal

f-life

in m

in


109

usually loss of the quaternary structure already occurs at low treatment intensities and is a

precondition for the observed aggregation and precipitation of the protein.

Figure IV.3: Foam stability of the supernatants obtained from pea flour suspensions pH 7 after heating at atmospheric pressure and pressure treatments at 40°C; treatment time: 10 min; protein concentration during treatment: 10 g/L; protein concentration during foam analyses: 0.33 g/L; dilution medium: deionized water; detection time: 10 min.

To subtract the effect of large aggregates, foaming tests were also conducted with the

supernatant of centrifuged samples. Protein concentrations were adjusted according to the

results of the Biuret assay. Experiments were performed with flours batch one, which possessed a

slightly higher overall foam stability. This can be traced back to the lower protein content and the

heightened presence of other surface-active components in the solutions. As no half-life could be

calculated for the very stable foams, changes in stability are expressed as remaining foam volume

after 10 minutes detection time. The effects of high temperatures are even more drastic than in

uncentrifuged samples (see Figure IV.3), as foam stability in relation to 20 °C decreased to 25 and

22 % after exposure to 70 and 80 °C, respectively. Treatment at 60 °C did not cause alteration of

foam stability. In batch one there was a marked reduction in solubility after applying this

temperature that was not found for higher treatment intensities. Removal of aggregates and

simultaneous adaption of the protein concentration can obviously balance the effect of

processing. The results obtained are in good correlation with changes observed for the surface

hydrophobicity of soluble protein (see Figure III.19). No increase in the exposure of hydrophobic

side chains was found for 60°C samples, but a marked increase in fluorescence intensity was

0

20

40

60

80

100

20 °C 60 °C 70 °C 80 °C 200 MPa 400 MPa 600 MPa

foam

stab

ility

in %


110

found for samples subjected to 70 and 80°C. Unfolding and increase in surface hydrophobicity

lead to a more rapid protein adsorption at the interface and thereby contribute to foam

formation (Kinsella, 1981; Lee & Kim, 1987; Wierenga & Gruppen, 2010). That does not

necessarily involve an improved stability of the disperse system. Comparison of β-casein, bovine

serum albumin and lysozyme revealed that the rather flexible casein possessed the fastest

adsorption but the lowest foam stability, while the highly structured serum albumin showed

worse foaming but good stabilizing properties (Graham & Philipps, 1975). The increased

hydrophobicity of heated pea protein may therefore result in a faster protein adsorption, but

could also be a reason for the reduction in foam life.

The time to reach maximum foam height was considered to reveal whether processed pea

proteins adsorb faster at the interface (see Table IV.1). Foam formation in supernatants was

completed after a shorter time due to a higher adjusted gas flow rate. Only a minor influence of

heat and pressure on the foaming time of supernatants was detected. Higher values obtained

after processing at 200 MPa might arise from overall higher deviations in these samples.

Foamability of whole samples thermally and pressure treated at higher intensities decreased in

most cases according to the lower foam stability and the presence of aggregated protein. Similar

effects of heat treatments are reported by Megha and Grant (1986) and Obatolu et al. (2007) for

pea and yam bean, respectively. In contrast, an improved emulsification after pressurization is

reported for soy and lupine protein (Molina et al., 2001; Chapleau & de Lamballerie-Anton, 2003;

Wang et al., 2008). The slow adsorption could be an indicator for a hindered molecular flexibility

and a reduced ability to unfold at the interface. A diminished flexibility of a larger particle size also

contributes to a decrease in stability. Lifetime of the dispersion is dictated by the rheological

properties of the protein film. A viscoelastic lamella provides sufficient flexibility to respond to

temporary compression and expansion due to interfacial tension inhomogeneities (Wilde & Clark,

1996; Clarkson et al., 1999; Wilde, 2000; Chiralt, 2005). Excessive rigidity of the protein hinders

the film to adapt to these local changes (Kinsella, 1981).

After centrifugation, effects of pressure on foam stability were less pronounced compared to

using the whole sample. Similar to the 60 °C sample, removal of large aggregates occurred, which

affected as well protein composition and led to a deconcentration of the globulins. Negative

effects on foam formation and foam stability may be balanced by the preservation of PA2 during

pressure treatment. Legume albumins can produce higher overruns compared to the globulins

(Sathe & Salunkhe, 1981), which may be a result of their lower molecular weight and their higher

mobility to diffuse to the interface. In regard to foam stability there was no significant difference

between albumin and globulin fraction. Nevertheless a higher content of PA2 in those samples did

not improve foam formation or stability in regard to the control. Differences in the soluble protein


111

content and the dilution factor might also lead to an enrichment of non-protein substances in the

sample. Other surface-active components might displace protein molecules from the interface

and cause a decrease in destabilization by negatively affecting film rheology (Wilde & Clark, 1996;

Dalgleish, 1997; Wilde, 2000; Chiralt, 2005).

Table IV.1: Time to reach maximum foam height of 160 mm; sample: protein-enriched pea flour; treatment time: 10 min; protein concentration during treatment: 10 g/L; protein concentration during foam analyses: 0.4 g/L for whole samples, 0.33 g/L for supernatants; dilution medium: deionized water

Foaming time in s

20 °C 40 °C 50 °C 60 °C 70 °C 80 °C

Whole sample 91 ± 11 80 ± 3 82 ± 7 131 ± 26 115 ± 13 136 ± 36

Supernatant 20 ± 0.5 20 ± 0.2 26 ± 1.0 25 ± 0.5

20 °C 40 °C


Whole sample 92 ± 9 89 ± 10 92 ± 10 96 ± 6 110 ± 36 113 ± 22

Supernatant 26 ± 8.8 20 ± 0.3 21 ± 0.6

Unfolding and denaturation under heat and pressure might also alter the molecule’s

physicochemical properties. Proteins were diluted to the test conditions in deionized water. Due

to solubilization of surrounding carbon dioxide, a sample pH close to 5 was reached before

measuring. This value corresponds to the range were the globulins as well as PA2 possess their

isoelectric point (Gueguen, 1983; Croy et al., 1984). The low net charge prohibits repulsive forces

between the molecules and allows a rapid reduction of the surface tension and a high packaging

density of protein at the interface (Kim & Kinsella, 1985; Hill, 1996; Wierenga & Gruppen, 2010).

Elasticity and stability are therefore particularly high at this environmental condition. Irreversible

exposure of amino acids originally hidden in the molecule’s interior may alter the isoelectric point

and shift the optimal pH towards higher or lower values (Sikorski, 2002). However, in case of

pressure treatments there was no structural evidence for an unfolding of the soluble protein.


112

Finally, both technologies investigated led to a decline in foaming properties. It was not possible

to preserve or even improve foaming behaviour of pea protein by these treatments. The exact

mechanism that contributes to a decrease in foam life remains unclear, but it is very likely that

different treatment effects on several protein fractions contribute to these results. Figure IV.4

summarizes the simultaneously arising changes in structure and functionality. A close relationship

between aggregation and reduction in foam quality exists. This is confirmed by the observation

that removal of very large particles achieved a total or partial reconstitution of the initial foaming

properties.

Figure IV.4: Mechanistic proposal for the structural alterations after application of heat (60 °C, 80 °C) or pressure (p), and after centrifugation (RCF) and adjustment of remaining protein concentration, effects on foam stability (FS) and volume expansion during foam formation (FE).

Investigations with potato protein were performed with commercial isolate that does not

undergo drastic precipitation during treatment (see Figure III.8). Therefore, an influence of large

clusters on foam stability can be excluded. Foams were less stable than those made from pea

flour (Figure IV.5). This might, amongst other effects, be caused by the higher purity of the

(con-)vicilin

leguminPA2

FS ↓↓ FE ↓↓ FS ↓↓ FE ↓↓ FS ↓↓ FE ↓↓

FS ↓↓ FE ↓↓FS → FE → FS ↓ FE ↓

60 °C 80 °C p

RCFRCF RCF


113

samples, the absence of other substances contributing to functional properties and an altered

ionic strength. Although no marked changes in particle size and absorbance were recorded, heat

treatments had a negative impact on foam stability. The maximal reduction in foam half-life was

observed in samples exposed to 70 °C. The time until 50 % of foam volume had collapsed was

almost halved due to this treatment. Heating led to an exposure of aromatic side chains and

increased sensitivity of the protein towards hydrophobic intermolecular bonding as soon as an

acidic pH is adjusted (Figure III.9 and Figure III.12). Shifting the pH to values around 5 during

dilution with deionized water may have led to formation of larger aggregates post processing that

negatively influenced foam stability.

Figure IV.5: Half-life of foams prepared from solutions of commercial potato protein pH 7 subjected to different temperatures; treatment time: 10 min; protein concentration during treatment: 10 g/L; protein concentration during foam analyses: 0.6 g/L; dilution medium: deionized water.

Pressurized potato protein did not show an increase in surface hydrophobicity or a comparably

strong precipitation at acidic pH values. Consequently, there was no negative impact on foam

stability after applying this technology (see Figure IV.6). No general trend in the time needed for

foam formation was detectable and all changes were within the range of analytical deviations

(data not shown). For higher treatment intensities there was even a marked increase in foam life

up to 70 %. Unfortunately, this positive effect was not stable over time, which also explains the

relatively high deviations between multiple determinations. Longer standing times between

treatment and analysis might as well have inhibited a detection of changes during structural

analysis and hinders finding a conformational explanation for the altered foam stability. Improved

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20 °C 40 °C 50 °C 60 °C 70 °C 80 °C

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114

foam stability of egg white protein after heat and high pressure application was traced back to

enhanced protein-protein interactions (Van der Plancken et al., 2007). Increased foaming ability

and stability of pressure treated walnut protein correlated with the degree of unfolding and is

thought to arise from a thicker viscoelastic film around the air bubbles (Qin et al., 2013).

Aggregation of whey protein during high pressure treatments enhanced thickness of the lamella

and decelerates thereby drainage and foam decay (Kresic et al., 2006). Proteins are superior to

low molecular weight surfactants in regard to foam stability due to their ability to form networks

by intermolecular bonds (Wilde & Clark, 1996; Wilde, 2000; Chiralt, 2005). High pressure may lead

to hydrogen bonds between different proteins. This effect was as well observed for potato

proteins exposed to pressure at a treatment pH around 6 (see Figure III.11). Hydrogen bonds are

usually not stable in an aqueous environment. It is therefore possible that bonds between polar

protein chains were converted to protein-water interactions over time and the ability to form

structures with advanced rheological properties decreased to initial values with proceeding

duration after pressurization. Further investigations are therefore necessary to reveal whether

this effect on functionality might be preserved during concentration and drying of the protein to

obtain a marketable product.

Figure IV.6: Half-life of foams prepared from solutions of commercial potato protein pH 7 subjected to different pressure-temperature combinations; treatment time: 10 min; protein concentration during treatment: 10 g/L; protein concentration during foam analyses: 0.6 g/L; dilution medium: deionized water.

0

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115

Figure IV.7: Stability of foams prepared from PEF treated pea flour (batch one) suspensions pH 7; protein concentration during treatment: 10 g/L; protein concentration during foam analyses: 0.33 g/L; dilution medium: deionized water.

Pulsed electric field treatment induced no obvious changes in the structure of pea protein. The

foaming properties of suspensions prepared from pea flour were as well not affected by this

technology. Neither the stability of the foams (see Figure IV.7) nor the time to achieve maximum

foam height showed distinctive alterations (see Table IV.2). This supports the hypothesis that pea

protein is insensitive to the application of an electric field or that occurring changes are reversible

and the protein refolds within a short period of time after treatment. By contrast, the foamability

of potato protein was slightly improved after PEF treatment, an indicator for an increased

hydrophobicity or an improved molecular flexibility. This observation correlates with the changes

found in the Bradford: Biuret ratio after treatment (see Figure III.25) and supports the idea that

comparison of both methods can be used as a rapid indicator for conformational changes. The

stability of foams made of PEF treated potato protein does not show any marked changes in

relation to the control (see Figure IV.8). It was already mentioned that unfolding of the protein

structure does not necessarily contribute to foam stability as steric and rheological impacts

possess greater importance. Analogous to the pressure treated samples it remains the question

whether these structural and functional alterations are stable during processing and can be

transferred into a storable protein powder.

0

10

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30

40

50

60

70

80

90

100

0 5 10 15 20 25 30

foam

stab

ility

in %

detection time in min

0 kV/cm 5 kV/cm, 35 kJ/kg 10 kV/cm, 35 kJ/kg 10 kV/cm, 125 kJ/kg


116

Table IV.2: Time to reach maximum foam height of 160 mm; sample: protein-enriched pea flour and self-made potato protein concentrate; protein concentration during treatment: 10 g/L; protein concentration during foam analyses: 0.33 g/L for pea protein; 0.6 g/L for potato protein; dilution medium: deionized water

Foaming time in s

0 kV/cm

5 kV/cm

35 kJ/kg

10 kV/cm

35 kJ/kg

10 kV/cm

125 kJ/kg

Pea protein 20.3 ± 0 20.2 ± 0.1 20.2 ± 0.3 19.9 ± 0.1

0 kV/cm

1 kV/cm

2.5 kJ/kg

2 kV/cm

2.5 kJ/kg

2 kV/cm

5 kJ/kg

Potato protein 78.1 ± 3.2 73.9 ± 0.7 75.9 ± 0.5 73.0 ± 1.7

In conclusion, none of the technologies investigated was able to improve the foaming properties

of pea protein. The stability of modifications induced in potato protein needs further

consideration. Protein concentrations for all these experiments were determined empirically and

chosen according to the stability within 30 minutes detection time. Due to the short lifetime

compared to real food systems, protein concentrations would have to be many times higher in

practical use. The transferability to high protein contents and a more complex product

composition requires additional testing. The same applies to emulsification properties that cannot

directly be derived from the present results due to differences in the destabilization principle.

Repulsion between protein molecules by steric or electrostatic means is, for instance, a desired

effect in stable emulsions as it impedes association of oil droplets. The large aggregates formed in

some of the treatments might therefore be an advantage in this kind of disperse system. Beside

the technological influence on interfacial properties, their impact on product rheology or gelation

behaviour would also be of great interest for the food industry. Nevertheless, a preservation of

functionality seems to be possible for some protein-treatment combinations. This displays good

start conditions for successfully applying these technologies for shelf-life increase as further

discussed in the following subchapter.


117

Figure IV.8: Stability of foams prepared from PEF treated solutions pH 7 prepared from self-made protein concentrate; protein concentration during treatment: 10 g/L; protein concentration during foam analyses: 0.6 g/L; dilution medium: deionized water.

High pressure, high temperatures and pulsed electric fields for

shelf-life increase of protein solutions

All technologies applied to the protein samples are suitable to inactivate vegetative microbial cells

and thereby to achieve food safety and to increase product shelf-life. Thermal pasteurization at

temperatures ranging from 60 to 80 °C for up to a few minutes is the conventional treatment to

improve the storage stability of liquid products (Adams & Moss, 2000). Depending on the sample

composition, especially its pH value, storability for a few days up to several months at room

temperature or under cold-storage can be obtained. Nevertheless, in case of functional protein

products, heat treatment is often connected to a decrease in quality and to a loss of specific

functional properties. For instance, egg products for industrial use are usually pasteurized

beneath their denaturation temperature to maintain protein functionality but overcome

contamination with Salmonella. However, heat treatment of egg white at 57 °C for several

minutes leads to partial loss of foaming ability (Ternes, 1994). Application of high isostatic

pressure or pulsed electric fields may provide an alternative for the shelf-life increase of

temperature sensitive protein products as in some cases the structural and functional properties

can be preserved to a higher extent.

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30

foam

stab

ility

in %


0 kV/cm 1 kV/cm, 2.5 kJ/kg 2 kV/cm, 2.5 kJ/kg 2 kV/cm, 5 kJ/kg


118

The initial microbial load of the protein products obtained for or gained within this thesis was too

small to record inactivation kinetics for different process conditions. Consequently, targeted

addition of some cultivated microorganism to the samples was necessary. Escherichia coli, Listeria

monocytogenes and Salmonella strains are widely-used indicator organisms in the starch industry

(Christoph Pieper, Emsland Stärke GmbH, personal communication) and in pulse processing

(Gupta et al., 2011). Non-pathogenic surrogates of E. coli and L. innocua were taken as reference

organisms for investigations performed within this thesis. L. innocua is similar in physiology and

metabolism with L. monocytogenes and shows an equal resistance towards external impacts

(Francis & O'Beirne, 1998). It is therefore a widely used alternative to its pathogenic analogue in

basic and methodical research. Ringer solution was used as a model system and solutions made of

self-made potato and pea protein concentrates were investigated as representatives for protein-

rich products. Treatments were performed at an initial microbial load of approximately 109 colony

forming units per mL. Due to the rapid heating and cooling in the glass capillaries the impact of

these phases could be neglected when creating the kinetics (Mathys et al., 2007).

Figure IV.9 shows the inactivation of E. coli and L. innocua in different media at a treatment

temperature of 60 °C. The progress of the inactivation curve does not follow a first order reaction,

but process efficiency decreases with proceeding treatment time. This so-called tailing is traced

back to the presence of heat sensitive members in the population and prolonged survival of

organisms with higher resistance (Peleg, 2000). L. innocua possesses higher thermal sensitivity

and reached the detection limit in ringer solutions already after 120 s dwell time whereas E. coli

was not inactivated to this extent, even after 10 minutes of treatment. Assuming a linear

inactivation curve the reduction in colony forming units would correspond to D-values of 1.67 min

and 0.3 min for E coli and L. innocua, respectively.

Information available in literature for D60°C of E. coli strains are ranging from 0.4 minutes (Ahmed

et al., 1995) to a few minutes (Smelt, 1998) in different products. Similar resistance like in this

experimental series was observed by Juneja and Marmer (1999) with a D60°C of 1.9 min in lamb,

turkey and pork meat. D60°C values of a few minutes are reported for L. innocua or

L. monocytogenes in peptone agar, meat or pasteurized milk (Farber, 1989; Mackey & Bratchell,

1989; Murphy et al., 2000). Sensitivity towards external influences and progression of inactivation

curves are markedly influenced by interactions on molecular and cellular level (Peleg, 2000).

Comparison of results from different working groups is difficult due to the wide range of extrinsic

and physiological criteria affecting heat sensitivity (Adams & Moss, 2000). In many references

detailed information on strain, its preparation, medium and pH used or the equipment taken for

processing are incomplete. Nevertheless, it is generally accepted that psychrotrophs are less heat

resistant than mesophiles or thermophiles and gram-negative bacteria possess higher heat


119

sensitivity than gram-positive organisms (Adams & Moss, 2000). The latter can be traced back to

the thickness of peptidoglycan in the bacterial cell wall, which is composed of about 40 layers in

gram-positive and 1 to 5 layers in gram-negative bacteria (Lengeler et al., 1999). Listeria is a gram-

positive non-sporeformer growing over a broad temperature range, whereas E. coli is a gram-

negative mesophil (Adams & Moss, 2000). Consequently higher heat resistance is ascribed to the

former one. The dissenting observations made in these experiments might be traced back to the

age of the strain or the failure to reach maximum stability in the stationary phase of cultivation.

Heat resistance of L. innocua was markedly higher in protein than in ringer solutions. The

inactivation curve shows a lag time and no clear reduction in colony forming units of more than

one log cycle was observed till a dwell time of 120 s. Accumulated exposure to heat progressively

weakens the organism and finally leads to this kind of shoulder formation in the inactivation

kinetic (Peleg, 2000). Protein solutions prepared from concentrates undergo drastic aggregation

upon heating that was already observed at 60 °C (see chapter III). These aggregates may work as a

protection film and shield the organism from external influences. Probiotics are usually covered

with proteins or protein-polysaccharide mixtures by heat, rennet coagulation or use of

transglutaminase (Gouin, 2004; Heidebach et al., 2009; Nedovic et al., 2011). Targeted film

formation of soy protein is for example initiated by heat treatment leading to exposure of

hydrophobic and thiol groups and the subsequent formation of new intermolecular bonds

(Wittaya, 2012). A similar behaviour of pea and potato protein during heating donates them

potential as encapsulation material for the food industry. Presence of milk protein or gelatine has

shown to increase survival rates of probiotic strains during heating or spray drying (Desmond et

al., 2001; Lian et al., 2002; Picot & Lacroix, 2004). Higher resistivity of these encapsulated

organisms in model digestion can be traced back to the amphiphilic character of the protein

matrix that buffers extreme pH values to a certain extent (Heidebach et al., 2009). E. coli only

showed a slight increase in resistance in protein solutions. Detection of a shoulder was not

possible probably due to the higher overall resistance and the broader measurement distance.

At treatment temperatures of 50 °C, no inactivation for both organisms was achieved within

10 minutes of dwell time. A few seconds at 70 °C already reduced the number of colony forming

units of L. innocua below the detection limit so that no kinetic could be recorded. Very short dwell

times also give proportionally greater importance to the heating and cooling period. Nevertheless,

it can be concluded that thermal processing for 10 minutes at temperatures of 60 °C or higher, as

used for the investigations of structural and functional alterations, would lead to a marked

reduction of the microbial load and would therefore increase product stability. Further

experiments would be necessary to exactly determine the minimum process intensity necessary.


120

Figure IV.9: Inactivation of vegetative microbial cells by heat treatment at 60 °C in 1/8 ringer solution or solutions made of pea and potato protein concentrate with a protein concentration of 10 g/L; top: E. coli; bottom: L. innocua.

Already in 1899 Hite revealed the potential of high pressure to impede the microbial spoilage of

food (Hite, 1899). During the last 25 years this technology found its way into many areas of

industrial food preservation. Due to the diverse mechanism of action it is suitable as a

preservation alternative for temperature sensitive products. Typical examples are fruit and

-9

-8

-7

-6

-5

-4

-3

-2

-1

0

0 100 200 300 400 500 600

-9

-8

-7

-6

-5

-4

-3

-2

-1

0

0 20 40 60 80 100 120

treatment time in s

log

10(N

/N0)

[-]

treatment time in s

ringer solution pea protein solution potato protein solution


121

vegetable products like smoothies or guacamole, in which colour, flavour and vitamins can be

preserved to a large extent while increasing their shelf-life.

The effect of a 400 MPa treatment on the inactivation of E. coli and L. innocua is presented in

Figure IV.10. The detection limit was reached for both organisms in diluted ringer solution after

300 s of dwell time. L. innocua showed a slightly higher pressure resistance after 150 s of

treatment. It is not possible to draw any conclusion on the shape of the inactivation kinetic as too

little data points are arranged above the detection limit. Buzrul and Alpas (2004) reported an

upward concave curve progression for L. innocua in peptone solution. A 5 minute pressurization

at 345 MPa and 45 °C reduced the microbial load by 4 log cycles. Further processing for another

5 minutes led to an inactivation of 4.5 log cycles. D400 MPa values of 1 to 3 minutes are reported for

the related strain L. monocytogenes (Smelt, 1998).

Pressure application of 600 MPa already achieved a complete inactivation of all colony forming

units after 150 s dwell time. Further investigations would be interesting to show whether a

10 minute treatment at 200 MPa and 40 °C or at 400 MPa and 20 °C would be sufficient for

inactivation of the indicator organism as changes in protein structure and functionality were less

pronounced at these processing conditions. According to Carlez et al. (1993) the pressure

inactivation of L. innocua in beef muscle at 20 °C is markedly retarded compared to 4, 35 or 50 °C.

5 log cycles of inactivation can be obtained for L. innocua in PBS puffer after 15 minutes at

400 MPa and 25 °C (Wuytack et al., 2002). Varying results were found by the authors for different

strains of E. coli under the same conditions. The cell membrane of the organisms is considered to

be the primary side of pressure damage (Carlez et al., 1993). Hence, it is assumed that gram-

negative bacteria are less pressure stable due to the same reasons mentioned for heat treatment

(Carlez et al., 1993; Wuytack et al., 2002), but the existence of barotolerant mutants of E. coli is as

well reported (Hauben et al., 1997; Garcia-Graells et al., 1998).

No pronounced protection effect of plant proteins present during processing was observed. This

can be probably explained by the little aggregation tendency in these protein concentrates under

pressure and the absent film formation (see chapter III). A small shoulder in the inactivation

kinetic might not be recorded by the given experimental plan. A higher pressure resistance in milk

compared to buffer or meat products is reported for L. innocua (Styles et al., 1991; Patterson et

al., 1995). Colony forming units of E. coli in pasteurized liquid egg decreased after pressurization

for 10 minutes at 20 °C at 300 MPa and 450 MPa by 0.4 and 4.5 log cycles, respectively (Ponce et

al., 1998). Inactivation effects determined within these experiments in protein solutions were

slightly higher, probably due the higher treatment temperature.


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Figure IV.10: Inactivation of vegetative microbial cells by pressure treatment at 400 MPa and 40 °C in 1/8 ringer solution or solutions made of pea and potato protein concentrate with a protein concentration of 10 g/L; top: E. coli; bottom: L. innocua.

Moderate temperature increase is also a simple possibility to improve the process efficiency of

pulsed electric fields. Higher temperatures enhance the fluidity of the cell membrane and

facilitate rupture of the phospholipid bilayer (Toepfl et al., 2005). Nevertheless, none of the

parameter combinations applied in these experiments achieved more than 2 log cycles of

-9

-8

-7

-6

-5

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-3

-2

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0

0 100 200 300 400 500 600

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-8

-7

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-5

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0

0 100 200 300 400 500 600

ringer solution pea protein solution potato protein solution

treatment time in s

log

10(N

/N0)

[-]

treatment time in s


123

inactivation (see Figure IV.11). A slight increase in the reduction of colony forming units was

obtained with rising energy input, but neither an increase of treatment temperature nor of the

electric field strength led to a significant rise of the process efficiency. The inactivation rate was

slightly higher for E. coli, especially at lower energy inputs. This observation was made as well by

other working groups, who reported less log cycles of inactivation for L. innocua compared to

E. coli at the same process conditions (Dutreux et al., 2000; Aronsson et al., 2001; Picart et al.,

2002). Higher electric field strengths can achieve electroporation of smaller organisms as the

external potential currently applied to the membrane is directly proportional to the cell radius

(Barsotti & Cheftel, 1999). This explains the higher resistance of Listeria compared to other

organisms as the former one is rather small and its inactivation therefore requires higher

treatment intensities (Toepfl et al., 2005). Increasing the electric field strength from 30 to

40 kV/cm markedly enhanced inactivation of L. innocua in skim milk and liquid egg (Calderon-

Miranda et al., 1999b; 1999a) indicating that the critical field strength for this cell size is located

somewhere between these two values.

Nevertheless, similar findings were not made within the present dataset. Exponential decay

pulses were applied with the equipment used, where the voltage rose quickly to the maximum

level and then decayed to level zero. The rate of voltage decay strongly depends on the electrical

resistivity of the sample (Toepfl et al., 2005). Due to the high conductivity of the diluted ringer

solutions it was not possible to induce pulses with a sufficient duration to the samples and the

effective energy delivered to the system was very low. This might have led to the absent

inactivation effect. The inactivation effect on E. coli in simulated milk ultrafiltrate dramatically

decreased with increasing ionic strength (Vega-Mercado et al., 1996), whereas similar treatment

efficiencies were reported for different media with the same conductivity (Dutreux et al., 2000).

Doubling of the pulse width by otherwise constant treatment parameters led to a significant

improvement in the reduction of E. coli and L. innocua (Martin-Belloso et al., 1997; Aronsson et

al., 2001). Usage of different pulse moduli might help to improve process efficiency. The

inactivation obtained with the equipment used was not adequate for a shelf-life increase.

Therefore, investigations with protein solutions were skipped and shall be performed with a

different PEF unit.

In conclusion, high pressure has shown to be a suitable technique for the pasteurization of protein

solutions. Especially for the heat labile potato protein, pressure could be a gentle alternative to

high temperatures, as structural attributes and foaming properties can be preserved to a great

extent or may even be slightly improved. Additional experiments can reveal the most promising

pressure-temperature-time combinations. In case of pea protein pulsed electric fields should be

further investigated as a preservation method as this technology had no detectable influence on


124

the protein under the conditions tested within this thesis. Further adaption of the process

parameters could lead to better inactivation results and may nevertheless maintain the protein’s

native structure.

Figure IV.11: Inactivation of vegetative microbial cells in 1/8 ringer solution by pulsed electric field treatment of various intensities at different starting temperatures; top: E. coli; bottom: L. innocua.

30 kV/cm, 30°C 40 kV/cm, 30°C20 kV/cm, 30 °C 20 kV/cm, 40 °C 30 kV/cm, 40°C

-9

-8

-7

-6

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0

1

0 50 100 150 200 250

energy input in kJ/kg

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-8

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-1

0

1

0 50 100 150 200 250

detection limit

detection limit

log

10(N

/N0)

[-]

energy input in kJ/kg


125

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Chapter V – Innovative processing and its effect on vegetable tissue

131

Chapter V

Innovative processing and its

effect on vegetable tissue


132

In the following chapter the influence of different processes on tissue integrity and diffusion of

cellular substances is presented. Characterization of these parameters is an important

consideration to adequately discuss treatment effects on protein recovery in the consecutive

chapter VI.

Whole peas were treated with high temperature, high pressure (HP) and pulsed electric fields

(PEF) to modify cell structure and mass transport. Due to their high water content of

approximately 80 % and the relatively low proportion of protein, application of expensive thermal

and pressure treatments was regarded as uneconomic for potato processing. Whole potatoes

were consequently only subjected to pulsed electric fields.

Cell structure and cell disintegration – influence of innovative and

conventional processing

All technologies used provide the potential to irreversibly alter structure and properties of

biological membranes and complete cell complexes (Gonzalez & Barrett, 2010). Application of an

external electric field leads to an instantaneous increase of the potential applied on the cell

membrane. If a critical value is exceeded, the elastic properties cannot withstand the attraction

forces of the charges and pore formation is induced leading to a loss of the membrane’s

semipermeability (Barsotti & Cheftel, 1999). According to Crowley (1973) the critical potential

amounts approximately 1 V for bimolecular lipid membranes used as model systems for real

biological membranes. Angersbach et al. (2000) reported minimum field strengths of 0.4 to

0.8 kV/cm for permeabilization of cells with 50 - 120 μm size, which include potato cells.

Several characterization methods can be used to determine the degree of damaged cells after

treatment. Measuring the relative changes in the sample’s impedance is one fast and accurate

way to determine changes in membrane intactness. The method is based on β-dispersion, the

ability of intact membranes to function as an insulator at low frequency currents and allow high

frequency currents to pass through. Changes in the conductivity profiles in the relevant frequency

range can be related to changes in sample integrity (Angersbach et al., 1999; 2002).

Figure V.1 shows the cell disintegration indices (CDI) detected in potato tissue after PEF treatment

of different intensities. Both electric field strengths applied exceeded the values mentioned as

necessary for irreversible pore formation. Mean CDIs were ranging from 0.64 to 0.72 for all

parameters tested and are therewith in accordance with results reported for comparable

treatment conditions and the same raw material (Lebovka et al., 2002; Janositz, 2005).

Angersbach and Knorr (1997) used the ability to hold tissue moisture in a centrifugal field as an


133

indicator for cell intactness. Already a field strength of 0.4 kV/cm and an energy input of

0.36 kJ/kg led to significant moisture loss that was increasing with rising field strength and pulse

number. Results obtained in these experiments do not support the aforementioned findings as

the highest cell disintegration was found after application of 333 pulses at 1 kV/cm, with 2.5 kJ/kg

the lowest treatment intensity investigated. Nevertheless, it can be pointed out that all treatment

parameters led to an intense disruption of membranes and to a marked disintegration of potato

tissue.

Figure V.1: Cell disintegration indices (CDI) of potato tissues subjected to PEF treatments of different intensities in relation to untreated tissue calculated via electrical conductivity at frequencies of 5.5 kHz and 2.8 MHz. Columns with different letters deviate significantly (α = 0.05).

Due to their small size, cell disintegration indices of pea tissue could not be easily determined

with the available measuring device and performed test runs did not lead to satisfying results.

Sections of embedded tissue were analyzed under the microscope to nevertheless collect some

information on cell integrity after processing. Figure V.2 shows the microscopic pictures of

differently treated pea tissue. Toluidine blue, staining the lignified part of the cell wall bluish

green and the pectin-rich middle lamella reddish purple, was used for better visualization. In the

control sample, all cells are completely surrounded by a stained cell wall. A distinction between

different parts of the cell wall, as described by (O'Brien et al., 1964), was not possible as the

pictures appeared either bluish or purple. Cell walls of treated tissue appeared thinner or are

locally disrupted indicating that cells are at least partially disintegrated. The intercellular space

0.0

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1 kV/cm 2 kV/cm

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atio

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Figure V.2: Microscopic pictures of tissue sections taken from peas exposed to different physical treatments in a swollen state and re-dried in warm air at 50 °C. Top left: 20°C, top right: PEF treatment of 5 kV/cm and 125 kJ/kg; middle: PEF treatment of 10 kV/cm and 125 kJ/kg, bottom left: thermal treatment at 80 °C, bottom right: pressure treatment at 400 MPa and 40 °C; staining with toluidine blue.

covered a larger area, which also reflects rupture of the cell wall and a loosened connection of

single cells among each other. These observations indicate that opening of the cells is not only

due to a rupture of the membrane but also a consequence of disrupted cell walls. Unfortunately,

an evaluation of the intactness of the plasmalemma by means of these pictures is not possible.

Changes in the visual appearance of the cell wall under the microscope might also arise from an

altered staining intensity. Janositz et al. (2011a) attributed changes in staining of the middle


135

lamella either to a direct impact of the electroporation on cell wall polymers or to their

interaction with released cytoplasm after membrane disintegration. A decrease in lignin content

in asparagus after PEF treatment is also reported affecting the stability of the cell wall and

reducing the intermolecular forces between its different layers (Janositz et al., 2011b).

The mechanism of cell disintegration was already summarized for PEF treatment. Softening of

carrot tissue during cooking can be traced back to a turgor loss due to membrane disruption and

breakdown of middle lamella pectin by β-elimination (Greve et al., 1994a; Greve et al., 1994b).

Additional pressure treatment inhibited this temperature induced degradation of pectin (De

Roeck et al., 2008). Nevertheless, a loosening of cell to cell contact due to pectin solubilization

under high pressure is reported for the same initial material (Trejo Araya et al., 2007). Angersbach

et al. (2000) reported an increase in the damaged membrane area with rising pressure. This effect

was not only irreversible, but still rose hours after HP treatment, probably due to pore widening,

activity of degrading enzymes or a combination of both. Phospholipid membranes are sensitive to

pressure and temperature changes. High pressure leads to a phase transition into a gel state and

thereby influences membrane fluidity and permeability (Winter et al., 2007; Follonier et al., 2012).

Hartmann and Delgado (2004) mentioned cell wall stress and excessive mechanical strain of the

membrane as reasons for microbial cell damage after high pressure. Proteins incorporated in the

membrane are stabilized in their secondary structure by the surrounding bilayer, but dissociation

of multimers, subunits and unfolding of extramembrane domains also lead to loss of their

function in the cellular transport system (White & Wimley, 1999). Protein denaturation can occur

either by heat, cold or pressure changes and is regarded as a main reason for cell disintegration

after high pressure by several authors (Kato et al., 2002; Ulmer et al., 2002; Winter & Jeworrek,

2009).

Figure V.3: Photographs of agar plates used for germination tests of swollen peas subjected to different treatments. From left to right: ungerminated and germinated samples after 24 h, root development after 48 h; microbial growth on ungerminated samples after 48 h.


136

The treatment impact on pea tissue can also be observed regarding their ability to germinate and

develop a new plant. Control samples and peas heated to 40 °C formed small roots within 24 h

after placing them on water agar (see Figure V.3). After 48 h longitudinal growth of the seedling

and formation of small leaves at the seed’s surface were visible. All HP and PEF conditions tested

as well as temperatures higher than 60 °C led to an absence of germination (see Table V.1).

Table V.1: Germination ability of differently treated whole peas – (x) germination occurred within 48 h after treatment; (/) no germination detected within this time period; pressure treatments were performed at 40 °C, pulsed electric field treatments were conducted at ambient temperature; dwell time for thermal and high pressure treatments amounted 10 min, duration of the pulsed electric field treatment was determined by the pulse number

Thermal treatments 20 °C 40 °C 60 °C 80 °C

x x / /

High pressure 0.1 MPa 200 MPa 400 MPa 600 MPa

x / / /

Pulsed electric fields 0 kV/cm 5 kV/cm 10 kV/cm

x / /

The first step in germination is always the seed imbibition (Toole et al., 1956). Sufficient water

uptake was already realized during the preparation of the experiments and it cannot be excluded

that germination was already initiated before process application. Activation of endogenous

enzymes that break down the seed’s storage compounds occurs soon after imbibition and

promotes germination long before it is apparent to the eye (McDonald, 2013). Inactivation of

those enzymes by heat or pressure may have interrupted the process and thus impeded the

development of a visible seedling. Opposed observations about the influence of PEF on the

germination ability are published in several articles (Bokka et al., 2009; Dymek et al., 2012;

Songnuan & Kirawanich, 2012). Dymek et al. (2012) related the radicle emergence of barley seeds

after electroporation to a reduction in amylase activity as the gross metabolic activity of the seeds

was not changed. Similar effects are thinkable for pea seeds, but further investigations are

necessary to receive clarification.

Intensive microbial growth occurred on plates with peas unable to germinate. This might either

arise from an increased release of cell ingredients on the otherwise nutrient-free agar plate or

from diminished defence reactions. Root exudates are rich in indoles, benzoxazinones, terpenoids

and flavonoids which protect the germinated seed from microbial attack (Bais et al., 2006).


137

Inactivation of endogenous enzymes involved in plant’s stress responses may have led to an

absence of antimicrobial substances responsible for tissue protection. The antimicrobial effect of

legume hull extract can be traced back to its high polyphenol content (Kanatt et al., 2011). A

wash-out of these substances during processing could result in a missing plant defence. The

influence of the different processes on diffusion of tissue compounds will be discussed in detail in

the following subchapter.

Release of cellular compounds during thermal, high pressure and

pulsed electric field treatment

It is widely known, that disintegration of biological cells is essential for recovering their

intracellular stored components. Pore formation due to PEF leads to disruption of the cell barrier

and can thereby serve as an effective tool to recover valuable plant ingredients, for instance

colours suitable for food application like anthocyanins (Corrales et al., 2008; Puertolas et al.,

2013) or betanin (Kulshrestha & Sastry, 2003; Fincan et al., 2004). The enhanced diffusion can on

the one hand lead to an improved extraction; on the other hand an undesired release of

ingredients into the treatment medium may occur.

Figure V.4: Protein release into treatment medium in dependence of cutting geometry; pulsed electric field treatment with 1 kV/cm and 5 kJ/kg; calculation based on a protein content of 20 g/kg fresh potato; columns with different letters deviate significantly (α = 0.05).

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

whole potatoes potato cubes potato scraps

prot

ein

rele

ase

in m

g / g

pro

tein

untreated PEF-treated

a

a

b bb, c

c


138

Figure V.4 shows the diffusion of protein from potato tissue into the surrounding water during

PEF treatment. It is obvious that the protein release is rising with the degree of mechanical cell

disintegration. A higher specific surface is increasing the effective mass transfer area and

enhancing the diffusion rate. An additional protein release due to PEF at an electric field strength

of 1 kV/cm and an energy input of 5 kV/cm was not detected. 20 pulses at 1.5 kV/cm were

reported to increase the diffusion of glucose and fructose from potato slices up to 50 % (Jaeger et

al., 2010; Janositz et al., 2011a). Removal of sucrose in the same samples was less pronounced

leading to the assumption that a relationship between molecular size and PEF improved diffusion

exists. Raschke (2010) described in his thesis that protein release from yeast cells was effectively

enhanced after application of 10 kV/cm, whereas the diffusion of water molecules and DNA

already increased at lower electric field strengths. Treatment intensity can influence the number

and size of pores formed in the electric field (Toepfl et al., 2005). The more a critical field strength

is exceeded, the larger the size of the formed pores (Sugar & Neumann, 1984).

This coherence may be utilized to specifically extract low molecular substances or for a targeted

separation of cell ingredients with disparate size. In potatoes for example, the content of reducing

sugars may be decreased to hinder future Maillard processes during roasting and frying (Jaeger et

al., 2010). In legumes, oligosaccharides may be removed, whereas macromolecules like starch and

protein remain within the cell structure. Oligosaccharides belonging to the raffinose family cannot

be digested in the human small intestine, leading to flatulence during microbial fermentation in

the colon. A removal of these substances is therefore desired to increase the consumer’s

acceptance of legume based products. Figure V.5 shows the transport of protein and raffinose

equivalent sugars from whole peas into the PEF treatment medium. Both element groups are

present in the medium to a higher extent after electroporation. The release of protein is

enhanced by factor 14 and that of oligosaccharides by more than 40times. This leads to an almost

15 % reduction of the flatulence causing sugars while losing only 0.15 % of nutritionally valuable

protein. This difference might be caused by an inhomogeneous distribution of both components

in the pea seed and a pronounced release of oligosaccharides from cells of the pea hull or it may

again be an indication that especially the diffusion of substances with low molecular size is

improved. Prolonged incubation times or additional washing steps could be applied to further

improve the oligosaccharide removal after electroporation. Furthermore, the technology may be

applied to increase the efficiency of following process strategies, for instance a decomposition by

fermentative means. In addition, digestion studies could reveal to which extent the reduction in

oligosaccharide content affects the flatulence after consumption.

An enhanced release of protein and oligosaccharides was also observed for thermal and high

pressure treatments (see Figure V.6). Lower values were obtained for peas treated at 60 °C


139

probably due to an incomplete disintegration of the cell structure. Protein diffusion was almost

four times higher during pressure treatment indicating the presence of larger holes in the pea

tissue compared to both other techniques. A model of PEF disintegrated cells by Jemai and

Vorobiev (2002) suggested local pore induction in the membrane and the cell wall at the same

point due to breakthrough of opposite charges, whereas in thermal and pressure treated samples

random permeabilization in both cell barriers occurs. As a consequence, electroporation is more

suitable for a target extraction of small cell components as a more precise induction of a specific

pore size seems realizable.

Figure V.5: Release of protein and oligosaccharides from whole peas into treatment medium in dependence of the electric field strength applied; energy input: 125 kJ/kg.; time into treatment medium: 10 min; determination of protein content with Bradford reagent; quantification of oligosaccharides with an enzymatic test kit; columns with different letters deviate significantly (α = 0.05).

Highest values for protein diffusion were recorded for samples treated at 200 MPa, and with

increasing pressure the protein loss decreases. This can be traced back to general changes in the

protein solubility caused by high pressure that will be intensively discussed in chapter VI. The

same effect can also hinder increased protein wash-out in heat treated samples. Nevertheless,

the overall protein loss was beneath 0.07 % of the total extractable protein and therefore

negligible. As some of the pea proteins possess a biological function that negatively influences

their food application like protease inhibitor activity or allergenic potential, a targeted removal of

these protein fractions could enhance nutritional quality of the product. Detailed characterization

0

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oligosaccharidesprotein

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0.07 % of extractable protein

a a

c

b

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c


140

of the released protein is necessary to obtain information on its nature and properties and a

potential positive effect of its separation.

Figure V.6: Influence of heat and pressure on the release of protein and oligosaccharides from whole peas into the surrounding medium; top: thermal treatments at ambient pressure; bottom: high pressure treatments at 40 °C; treatment time: 10 min; release time: 15 min; quantification of protein with Bradford reagent; quantification of oligosaccharides with an enzymatic test kit; columns with different letters deviate significantly (α = 0.05).

oligosaccharidesprotein



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a a bd d

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0.1 MPa 200 MPa 400 MPa 600 MPa



a

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141

Influence of thermal and non-thermal pre-treatments on the

efficiency of mass transport

Disintegration of cells cannot only be used for an enhanced diffusion, but also to improve solid-

liquid separation. Removing potato fruit juice from starch and fibres by centrifugal forces is a

main step in potato starch processing. This step was simulated in lab scale using a hand press.

Figure V.7 shows the very small but significantly improved separation of fruit juice leading to a

smaller amount of press residue with lower moisture content. Similar results were reported by

several other authors for different plant materials (Bazhal et al., 2001; Praporscic et al., 2007;

Schilling et al., 2007). Disintegration of cell walls and membranes facilitates drainage of

intercellular liquids and enables an easier compression of cells by mechanical means.

Electroporation of the cells provides hereby qualitative, economic or time-related advantages

compared to conventional techniques as thermobreak, freezing-thawing or enzymatic treatments.

Significant differences between treatment intensities investigated were not detected, probably

due to the similar degree of cell permeabilization already determined via impedance

measurement.

Figure V.7: Influence of pulsed electric fields on the solid-liquid separation of potato tissue in a manual press. The moisture content in the press residue was determined using a moisture analyzer (Sartorius MA 35).

0.0

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eigh

t

press residuefruit juice

a

dddc

b bbb

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Moisture content in press residue in g / g fresh weight

0.51 ± 0.017 0.45 ± 0.010 0.45 ± 0.010 0.44 ± 0.007 0.44 ± 0.009


142

Subsequent drying of the press cake was only slightly improved by previous electroporation (see

Figure V.8). Inhomogeneities in the test material led to strong deviations in the drying rates of

multiple determinations. Stronger treatment effects and clear distinctions were found by many

other working groups for fresh fruits and vegetables (Ade-Omowaye et al., 2001; Lebovka et al.,

2007). Angersbach and Knorr (1997) presented a more homogeneous moisture distribution in PEF

treated potato cubes, an improved moisture transport to the product surface and a shift of the

drying front to outer cell layers. Tissue compression during solid-liquid separation might hinder

moisture transport within the intracellular space and cover positive effects of cell disintegration.

Nevertheless, drying of PEF treated press cake would be more economic compared to untreated

samples as the overall amount of water to be removed was lower after pressing.

Significant changes were determined regarding the drying rate of PEF treated whole peas (see

Figure V.9). As already visible in the drying curves of potato press cake, changes between treated

and untreated samples decrease with progression of drying and a decreasing moisture content. In

the falling rate period (second section) of the drying process moisture is transported to the

product surface through the capillary system. This diffusion within the tissue is getting slower the

closer the moisture content comes to the equilibrium state. In this drying stage pore formation by

PEF pre-treatment may not bring additional benefit or existing differences are too small to be

detected and thus negligible.

Figure V.8: Drying rates of potato press cakes in relation to its moisture content; press cakes were obtained from manual pressing of grinded potatoes previously treated with pulsed electric fields of different intensities; drying was performed for 300 min at 50 °C in a drying cabinet.

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0 kV/cm 1 kV/cm, 2.5 kJ/kg 1 kV/cm, 5 kJ/kg 2 kV/cm, 5 kJ/kg 2 kV/cm, 10 kJ/kg


143

Figure V.9: Drying rates of whole peas influenced by different treatments in relation to their moisture content; top: heat treatment; middle: pressure treatment at 40 °C, bottom: PEF treatment: treatment times amounted 10 min for heat and pressure and were dependent on the pulse number for PEF treatments. Peas were dried for 180 min at 50 °C in a moisture analyzer.

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144

Effects were less pronounced for the other technologies applied. Pressures higher than 200 MPa

were needed to increase the drying rate. Yucel et al. (2010) reported an improved drying of

carrots, apples and beans for pressures exceeding 100 MPa. An increased effective diffusion

coefficient during osmotic dehydration was found for potato samples pressurized at 400 MPa and

30 °C (Rastogi et al., 2000). In contrast, a negative impact of pressure treatments at 600 MPa and

70 °C was determined for the drying rate of green beans in a fluidized bed dryer (Eshtiaghi et al.,

1994).

Positive effects of hot water blanching on subsequent drying processes were obtained, for

instance, for green peas (Simal et al., 1996), pepper slices (Kaymak-Ertekin, 2002) and potato

chips (Leeratanarak et al., 2006). In these experiments, a 10 minute exposure to higher

temperatures was the least effective pre-treatment. The drying rate of peas treated at 80 °C fell

back to the rate of the untreated ones already for moisture contents of 0.9 g/gDM. Temperatures

at 60 °C even led to a reduction in the drying rate. Influence of pressure and temperature on

macromolecules stored in high concentration in the pea tissue might change water binding

properties that might interfere with the positive effects of the ruptured tissue structure.

Regarding the appearance of the warm-air dried peas the compact, shrunken optic of the treated

peas is noticeable (see Figure V.10). This is supported by differences in the specific seed volume

after drying (see Table V.2) which also influences light refraction and colour impression of

Figure V.10: Visual appearance of peas re-dried after exposure to different treatments: Top: pressure treatment; bottom left: heat treatment; bottom right: pulsed electric fields; treatment times amounted 10 min for heat and pressure and were dependent on the pulse number for PEF treatments; peas were dried for 6 h at 50 °C.

pressure

electric field strengthtemperature


145

Table V.2: Density of the peas after different treatments and warm air drying at 50 °C in relation to control (peas treated at 20 °C and atmospheric pressure without application of an electric field); dwell time for thermal and high pressure treatments amounted 10 min, duration of the pulsed electric field treatment was determined by the pulse number

Changes in the specific density [-]

0.1 MPa 40 °C 125 kJ/kg

40 °C 60 °C 80 °C 200 MPa 400 MPa 600 MPa 5 kV/cm 10 kV/cm

0.96 1.02 1.13 1.25 1.33 1.21 1.11 1.12

the peas’ surface. Breakage of cell walls and membranes may have caused a collapse of the tissue

structure during drying, which might also decrease the drying rate in the late drying progress.

Drive-out of gas inclusions of the intracellular matrix, as it is known for blanching steps can also

lead to shrinkage of the peas. Increase of product firmness due to degassing and absence of gas

inclusions in the intercellular space are also described for pressure treated samples (Basak &

Ramaswamy, 1998; Prestamo & Arroyo, 1998). Higher pressures may alter the diffusivity of

included gas, so it may diffuse out of the cell structure. Microscopic pictures revealed

accumulation of cell wall fragments in pressure treated tissue sections (see Figure V.11) not

visible in the control samples. A folding of the cell wall due to pressurization is also reported by

Trejo Araya et al. (2007).

Adequate water uptake is required to ensure sufficient tissue softening and starch gelatinization

when peas shall be consumed as a whole. According to the information on the label of

Figure V.11: Microscopic pictures of tissue sections taken from peas exposed to pressure treatments at 40 °C in a swollen state; left: 600 MPa; right: 400 MPa; treatment time: 10 min; drying for 6 h at 50 °C.


146

commercial dry peas either cooking for two hours or soaking for 12 h at room temperature

followed by one hour in boiling water are necessary to achieve desired product quality.

Rehydration curves at room temperature and at 100 °C are presented in Figure V.12 and Figure

V.13, respectively. All pre-treatments led to an accelerated water uptake at room temperature as

a result of tissue disintegration. Effects of pre-treatments on the rehydration rate were more

pronounced in comparison to the drying process. Diffusion into the tissue seems to be influenced

to a larger extent by pore formation and loosening of the cell structure in comparison to diffusion

out of the tissue. This effect might be due to a generally higher maximum diffusion rate caused by

a higher moisture gradient between tissue and rehydration medium in comparison to drying air.

Maximum treatment effects were obtained with pulsed electric fields, where the water content

exceeded 1.25 g/gDM already after 180 minutes of incubation in water. Afterwards, the water

content stayed almost constant. For high pressure and high temperature treated peas this phase

was reached after 240 and 300 minutes, respectively, whereas untreated and with 40 °C treated

peas needed more than 500 minutes to possess this water content. PEF controls took water up a

little bit faster, probably due to mechanical damage during sample handling. All samples in this

experimental series were stirred with a small metal rod to remove air bubbles from the treatment

chamber.

Improvements in water uptake were less pronounced at temperatures of 100 °C and mainly

appear in the first phase of rehydration. Cooking also leads to a cell disruption of untreated peas,

which might superimpose the effect of other cell disintegration techniques applied earlier in

processing. A reduction in cooking time can therefore not be achieved with the pre-treatments

tested.

Contrary results on the effect of blanching, HP and PEF on rehydration capacity were reported by

different working groups for several raw materials (Eshtiaghi et al., 1994; Rastogi & Niranjan,

1998; Ade-Omowaye et al., 2001; Taiwo et al., 2002; Amami et al., 2007; Gachovska et al., 2009;

Abe et al., 2011). Differences in product shape, drying technique and rehydration conditions may

strongly influence the overall rehydration activity and the contribution of pre-processing steps on

it. Shortening of rehydration times could increase the attractiveness of legume products as it

allows more flexibility and spontaneity in meal planning. Similar approaches already existed in the

former German Democratic Republic where pre-treatments reduced preparation times of pea

meals to 10 minutes.


147

Figure V.12: Rehydration of whole peas influenced by different technologies; top: heat treatment; middle: pressure treatment at 40 °C, bottom: PEF treatment; treatment times amounted 10 min for heat and pressure and were dependent on the pulse number for PEF treatments; peas were dried for 6 h at 50 °C; water uptake occurred for 24 h at room temperature.

moi

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mat

ter

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400 MPa, 40 °C

0 kV/cm

5 kV/cm, 125 kJ/kg

10 kV/cm, 125 kJ/kg


148

Figure V.13: Rehydration of whole peas influenced by different technologies; top: heat treatment; middle: pressure treatment at 40 °C, bottom: PEF treatment; treatment times amounted 10 min for heat and pressure and were dependent on pulse number for PEF treatments; peas were dried for 6 h at 50 °C; water uptake occurred for 2 h in boiling water.

moi

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400 MPa, 40 °C

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149

Table V.3: Characterization of the rehydration medium used for differently pre-treated peas: total soluble solids in °Bx and light absorbance and at 412 nm; treatment time: 10 min; drying for 6 h at 50 °C, rehydration in tap water for 24 h

0.1 MPa 40 °C

20 °C 40 °C 60 °C 80 °C 200 MPa 400 MPa 600 MPa

°Bx 0.50 0.55 2.35 2.00 2.75 2.70 2.60

E412 0.078 0.059 0.501 0.471 0.281 0.257 0.351

An intensive release of cell ingredients into the rehydration medium occurred (see Table V.3). The

brix value of the medium and thus the presence of soluble solids were markedly higher for

samples exposed to thermal and high pressure treatments. A high quantity of pigments was as

well released from the cell structure, which was shown by the increase in absorption at 412 nm,

the wavelength area, which corresponds to a yellow colour impression. Field peas are rich in

substances belonging to the carotenoid family and giving the food a yellow, orange or red colour.

These secondary metabolites are also discussed as health protecting food ingredients. Their wash-

out during rehydration is undesired and constitutes a major disadvantage of the cell disruption for

mass transport improvement. All rehydrated peas possessed almost the same specific volume

(see Table V.4), indicating that structural changes were covered by filling tissue cavities with

absorbed water. Hence, the pre-treatments did not affect the visual acceptance of the final

product.

Pulsed electric fields, high pressure and high temperature treatments affected the integrity of

potato and pea tissue and led to changes in the diffusion of cell components, especially those

with low molecular weight. The following chapter will reveal whether these effects may as well be

used to improve or modify the recovery of storage proteins.

Table V.4: Specific density of the peas after rehydration in relation to peas treated at 20 °C and atmospheric pressure; treatment time: 10 min; drying for 6 h at 50 °C, rehydration in tap water for 24 h

Changes in the specific density [-]

0.1 MPa 40 °C

40 °C 60 °C 80 °C 200 MPa 400 MPa 600 MPa

0.99 0.99 1.01 1.01 1.01 0.98


150

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Winter, R. & Jeworrek, C. (2009). Effect of pressure on membranes. Soft Matter, 5(17), 3157-3173.

Yucel, U., Alpas, H. & Bayindirli, A. (2010). Evaluation of high pressure pretreatment for enhancing the drying rates of carrot, apple, and green bean. Journal of Food Engineering, 98(2), 266-272.

Chapter VI – Emerging technologies and their potential in protein recovery

155

Chapter VI

Emerging technologies and their

potential in protein recovery


156

The impact of emerging technologies on cell disintegration and the consequential improvement of

mass transfer processes were already discussed in the previous chapter. Membrane and cell wall

rupture can also contribute to a more efficient recovery of storage proteins. This might either lead

to a higher protein yield or to a reduction in processing times which both would contribute to

higher production efficiency. Regarding the technological impact on protein solutions an in situ

protein modification already within the plant tissue is also thinkable. Analogous to chapter V,

potatoes underwent electroporation only whereas peas were subjected to heat, high pressure

and pulsed electric field treatments. Both vegetables accumulate starch as an energy carrier for

germination. Recovery and analyzing of stored protein is therefore in general easier compared to

that of plants containing high amounts of lipids.

The influence of pulsed electric fields on potato protein

extractability and quality

The influence of pulsed electric fields on potato protein solutions was already considered at the

end of chapter III. Slight unfolding of the protein structure was assumed to be the consequence of

PEF side reactions like pH shifts or enhanced oxidation. An improved separation of fruit juice from

potato tissue via manual pressing was presented in chapter V. The impact of the same treatment

intensities on protein extraction using solid-liquid separation and quantification of protein using

the Bradford assay is shown in Figure VI.1.

Protein content in the fruit juice decreased by 12 to 19 % after application of pulsed electric fields.

Previous results already revealed that no additional release of protein from potato tissue into the

treatment medium occurred as a consequence of electrical pore formation, although diffusion of

smaller molecules was enhanced during pressing. This confirms the suggestion that pores induced

by the treatment were too small to enable proteins to pass more easily. An improved separation

of water molecules and other low-weight substances may have led to a dilution of protein and

finally, to a lower protein content in the fruit juice. Nevertheless, the additional amount of fruit

juice won from PEF treated potatoes could not compensate the diminished protein extraction, so

that the overall amount of protein obtained in relation to potato dry matter was between 9 and

15 % lower after treatment. Significant changes were determined in comparison to the untreated

samples, but differences found between PEF intensities were not of statistical significance.

Nevertheless, the maximal decrease in protein yield was induced by a treatment at 1 kV/cm and

5 kJ/kg, with 666 pulses the highest pulse number applied. Therefore, further investigations were

conducted using these process parameters.


157

Figure VI.1: Protein content in potato fruit juice won by a manual pressing after pulsed electric field treatment of whole potato tubers and calculated protein yields in relation to tuber dry matter; protein quantification via Bradford analysis; columns with different letters deviate significantly (α = 0.05).

It was already mentioned that protein quantification via Bradford assay is fault-prone. Coomassie

Brilliant Blue specifically binds to certain amino acids, in particular basic and aromatic ones, which

makes the method dependent on primary structure and protein folding (Pierce & Suelter, 1977;

Van Kley & Hale, 1977; Congdon et al., 1993). On the one hand, this impedes estimation of the

factual protein yield, on the other hand differentiation between concentration changes and

structural modification without performing additional analyses is nearly impossible. Therefore,

comparison of the results with other quantification methods is necessary. Table VI.1 summarizes

protein yields obtained by watery extraction of lyophilized and grinded potato tissue that has or

has not been subjected to PEF before drying. Dry tissue cubes were thoroughly homogenized with

a porcelain mortar before extraction. So it can be assumed that cell disintegration of both

samples was maximal, and hence, extractability of soluble compounds was maximal. Protein

yields obtained, as well as the extent of treatment impact differed between the different

photometric analyses. The highest protein recovery was determined using the Biuret assay.

Around 90 mg/g dry weight corresponded well to the overall water soluble protein content in

fresh potatoes of approximately 1 %. Differences between PEF treatment and control were within

the range of analytical deviation. Protein quantification based on Biuret reaction is relatively

independent from protein composition and conformation (Creighton, 1984) and reflects very well

protein yieldprotein content in the fruit juice

0

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2

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prot

ein

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/ g d

ry m

atte

r

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a

c

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bbb

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158

the actual protein content in the extracts. The changes between 1 kV/cm and 0 kV/cm of -8 % and

+21 % determined by Bradford and UV method, respectively, must therefore arise from PEF

induced protein alteration. Unfortunately, it was not possible to find structural evidence for a

modification or interaction within this thesis, but several possible reasons for an altered assay

response are discussed hereinafter.

Table VI.1: Water extractable protein in mg/g dry matter determined via different photometric methods. PEF treatment: 1 kV/cm, 5 kJ/kg, 666 pulses, 2 Hz

Biuret Bradford UV280

untreated 90.4 29.1 41.8

PEF treated 89.8 26.8 50.5

Ratio PEF : untreated 0.99 0.92 1.21

The intensity of light absorbance at 280 nm primarily depends on the content of tyrosine and

tryptophan and to a smaller extent on the presence of phenylalanine and the number of

disulphide bonds (Aitken & Learmonth, 1996). Extinction coefficients differ for native and

denatured proteins due to changes in the exposure of chromophore side chains and the polarity

of their environment (Gill & von Hippel, 1989; Kelly et al., 2005). Unfolding of the protein’s

tertiary structure in the electric field might alter its absorbance in the UV light. This would explain

the observations made for protein solutions, but in contrast to those data, no increase in Bradford

response was determined for protein from treated potatoes. The composition of cell juice is even

more inhomogeneous than that of solutions prepared from self-made protein concentrate. Potato

fruit juice contains all soluble and physiologically active substances of the cell, inter alia proteins,

carbohydrates like pectin, lipids and minerals (Wilhelm & Kempf, 1981). During ultrafiltration of

the fruit juice the amount of liquid was reduced to approximately one fifth and the content of

substances smaller than 10 kDa must have decreased to a similar extent. Interactions of these

components with the assay might have prohibited an accurate measurement. Presence of nucleic

acids may lead to an overestimation of protein in the UV light (Aitken & Learmonth, 1996) but

only proteins are able to produce the anionic form of Coomassie Brillant Blue, detected within the

Bradford assay. Nevertheless, some detergents might stabilize its neutral form and an

interference with flavonoids may increase the absorbance at the corresponding wavelength

(Compton & Jones, 1985; Whiffen et al., 2007). The presence of detergents in fruit juice is rather

unlikely and improved extraction of polyphenols after PEF treatment as reported by other authors


159

(Corrales et al., 2008; Puertolas et al., 2013) would not alter the Bradford reaction into the

direction determined. Due to the high mechanical cell disintegration in freeze-dried powder,

maximum extractability of soluble compounds was assumed anyway.

Interactions with other potato constituents during or after treatment might be more distinct in

biological cells than in the model solution. Potato fruit juice naturally possesses a slightly acidic pH

value around 6, whereas protein solutions were treated at pH 7. This small shift in acidity might

have already altered the process impact or unfolding and reactivity of the protein. Subjecting to

local pH extremes close to the respective electrodes, as it was reported for treatment of liquids in

electroporation cuvettes, are not an explanation, since not all potatoes were positioned with a

similar distance to both electrodes. Nevertheless, pH shifts within the plant tissue might occur

due to charge separation within individual cells. pH changes may have a different effect when the

initial pH values was already lower or remained slightly acidic after the end of the treatment.

Complexation of potato protein with oxidized lipids and fatty acids during processing displays a

problem in economic protein isolation (Wilhelm & Kempf, 1981). Unfolding within the electric

field or temporary pH shifts might enhance lipid-protein interactions. A synergistic effect of low

pH and presence of fatty acids was observed for denaturation of egg albumin (Bull & Breese,

1967). Binding of protein to phospholipids or liposomes is dependent on the molecule’s net

charge and therefore on the actual pH value (Karel, 1973; Bergers et al., 1993). Beside

electrostatic forces, hydrophobic interactions and covalent bonds are responsible for the

complexation. Embedding of fatty acids into the hydrophobic cavities of human serum albumin as

well as formation of salt bridges to basic amino acids both involve arginine as a reaction partner

(Curry et al., 1999). Thereby, interactions with lipids might decrease the arginine availability for

Coomassie reduction and hence the apparent protein concentration. Pronounced oxidation of

fatty acids in PEF samples might occur due to formation of radicals or by facilitating enzymatic

reactions after cell disintegration. Significant changes in the acidity and oxidation parameters of

the lipid phase of egg liquid after PEF were reported by Marco-Moles et al. (2009), but no general

trend in lipid modification was observed. Reaction products of lipid peroxidation are manifold and

some have as well the basic amino acids arginine, lysine and histidine as their reaction partner

(Karel, 1973).

Beside lipids, plant phenols may bind to proteins by covalent or non-covalent interactions. The

latter are dominated by hydrophobic forces additionally stabilized by hydrogen bonds (Prigent et

al., 2003). Several isoforms of chlorogenic acid can be found in potato cells and their oxidation

products can react with active-hydrogen bearing groups of proteins and amino acids either at

their benzene ring or at their acrylic side chain (Friedman, 1997). These interactions are held


160

responsible for protein precipitation upon heating fruit juice (Pots et al., 1998a) or for the

differences in the pH solubility profile in comparison to isolated patatin (van Koningsveld et al.,

2002a). Patatin is even considered as an alternative to animal gelatin in removing astringent

polyphenols from red wine (Gambuti et al., 2012). Binding of phenols might have affected the

response of protein towards the Bradford or UV assay. A decrease in the absorbance of 10 to 15 %

was observed upon Coomassie binding to lysozyme in the presence of phenols (Prigent et al.,

2003). This was proposed to be the consequence of a reduced solubility after binding of phenols

to the protein, but it might be as well a result of an altered dye response. Binding of tannins to

saliva proteins led to chemical shift changes revealing that proline is an important binding side but

also that interactions as well occur with arginine and phenylalanine residues (Baxter et al., 1997),

amino acids which both contribute to the intensity of the Bradford assay. Application of an

electric field might intensify interactions between proteins and polyphenols. Phenylalanine side

chains are usually buried in the interior of the molecule. Protein unfolding and differences in

amino acid exposure during and after PEF might enhance the reactivity with phenols (Charlton et

al., 2002; Prigent et al., 2003). This could also be the result of a pH shift due to charge separation.

Patatin is less structured and shows different fluorescence spectra at pH 3 compared to neutral

values (Pots et al., 1998b). Additionally, formation of hydrogen bonds is more likely when

electrostatic repulsion is minimal (Appel, 1993). Protein precipitation due to an interaction with

tannin is maximal at pH values close to the isoelectric point (Stern et al., 1996).

Covalent attachment of flavonoids to soy protein or bovine serum albumin caused a decrease in

determined tryptophan, lysine and cysteine residues (Rawel et al., 2002a; Rawel et al., 2002b),

whereof the first two are involved in the Coomassie reaction. As light absorbance at 280 nm

mainly depends on tyrosine and tryptophan fluorescence, reduction in the availability of these

amino acids should have similar effects on both UV and Bradford method. Involvement of these

aromatic amino acids into the phenol binding can therefore not give an explanation for the

alterations in the PEF samples.

Pulsed electric fields may not only increase the intensity of protein-phenol attachment, but might

also change the binding mechanism. Radicalization of phenols may alter their binding affinity and

their preferred reaction ways (Appel, 1993). A shift to alkaline pH values promotes phenolic

oxidation to quinones (Ronlan, 1978; Kroll et al., 2003). Contact between polyphenols and

oxidizing enzymes is enabled by electroporation. Quinones are a reactive intermediate that can

easily experience further aggregation reactions. Charlton (2002) assumed that arginine cannot

form an independent binding side but strengthen the interaction between phenol and proline.

Larger molecules occupy a larger binding site and therefore promote an involvement of arginine.

Enhanced oxidation of polyphenols in the electric field or as a consequence of cell disintegration


161

may have led to binding of larger phenolic aggregates, changed the affinity of arginine to stabilize

the network and hence altered the Bradford response. Oxidized phenols also function as

substrate for lignin biosynthesis (Appel, 1993). Changes in phenol oxidation might therefore

contribute to an altered lignification of cell wall material after stress induction by electric pulses.

Figure VI.2: Protein yield determined via Bradford analysis obtained by extraction of freeze-dried potato cubes or solid-liquid separation and subsequent press cake extraction; PEF treatment: 1 kV/cm, 5 kJ/kg, 666 pulses, 2 Hz; percentages imply the changes due to PEF in relation to the control sample.

Differences in protein yield for untreated and PEF-treated potatoes can be determined with the

Bradford assay for various recovery concepts tested (Figure VI.2). The decrease in detectable

protein was more pronounced for the first experimental series shown in Figure VI.1 and for

investigations at pilot plant scale. This might have resulted from longer reaction times during

treatment and pressing in several batches. The yields using alkaline extraction of dry potato cubes

or press cake were always higher compared to neutral pH due to an overall better solubility at

high pH values. Although the extent of PEF effects was slightly different for both extraction

conditions, no general tendency could be observed. A comparison between different photometric

analyses was only conducted for tap water extracts from whole potatoes. Therefore, a loss of

protein after PEF cannot be excluded for other recovery methods used.

Protein was concentrated from fruit juice via ultrafiltration and subsequent spray drying in order

to check if alterations caused by pulsed electric field processing can be maintained during

0

5

10

15

20

25

30

35

40

45

control PEF control PEF control PEF control PEF control PEF control PEF

extraction of cubes pressing in lab scale pressing in pilot plant scale

prot

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yiel

d in

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/ g d

ry m

atte

r

- 6.6 %

- 8.1 %

- 9.1 %

- 11.6 %

- 14.2% - 12.1 %

fruit juicealkaline extractionwater extraction


162

recovery of a marketable end-product. Foaming properties were chosen as an indicator for

potential changes in functional behaviour. Temporary pH shifts due to charge separation in the

electric field provide one possible explanation for the PEF induced changes. Van Koningsveld et al.

(2002b) already reported that setting the pH to 3 and back to neutral values had lasting effects on

structure and functionality of patatin.

Figure VI.3: Changes in protein solubility determined with Bradford assay during pH-adjustment and re-setting to the initial value in potato fruit juice.

Figure VI.3 presents a solubility profile of potato fruit juice determined via Bradford analysis, so

that a simultaneous effect of solubility decrease and conformational changes is probable. The

decrease in the absorbance was partially maintained after resetting the pH value back to the

initial values around 6. Already 67 years ago, Jirgensons (1946) stated that changes observed in

potato protein after lowering the pH can only be abolished by an excess of hydroxide ions at

alkaline pH values. The protein decrease of 12 to 19 % detected in PEF treated potato fruit juice

can be found as well after adjustment to pH 4 and 5 and reset to pH 6. This pH range also

corresponds to the isoelectric point and solubility minimum of patatin (Alting et al., 2011). To

compare the effects of electric field exposure and temporary pH changes, fruit juice of untreated

potatoes was set to pH 4.5 for 5 minutes and back to pH 6 prior to concentration and drying.

Differences in foam formation between the three types of concentrates are shown in Table VI.2.

High standard deviations are caused by the applied whipping method as accuracy of the electric

mixer and visual assessment of foam and drainage is limited.

0

2

4

6

8

10

2 3 4 5 6 7 8 9

solu

ble

prot

ein

in g

/L

pH value [-]

pH adjustment pH adjustment and reset to pH 6


163

Table VI.2: Foam formation characteristics in 1 % protein solutions pH 7 prepared from self-made potato protein concentrate whipped with a handheld electric mixer; PEF treatment: 1 kV/cm, 5 kJ/kg, 666 pulses, 2 Hz; acid treatment: setting to pH 4.5 by HCL addition and reset to pH 6 with NaOH

untreated PEF treated acid treated

Foam expansion in % 240 ± 11 296 ± 27 276 ± 35

Foam density in mg/mL 185 ± 1 159 ± 3 171 ± 12

Concentrates won from PEF treated potatoes produced a 24 % higher foam volume and

consequently a lower foam density compared to the control samples. This might result from

faster adsorption of protein at the air-water-interface. Not every protein molecule diffused to the

interface is automatically adsorbed as it is the case for surface active detergents. An increase in

protein hydrophobicity increases the probability and rate of adsorption (Kinsella, 1981; Nakai,

1983; Dalgleish, 1997; Wierenga & Gruppen, 2010). Enhanced surface hydrophobicity might either

be caused by unfolding of the tertiary structure and exposure of buried amino acids or by

attachment of another functional group coming from other juice constituents. A positive effect on

foam formation and stability upon interaction with phenols is reported (Prigent et al., 2003)

although an increased hydrophilicity was reported when flavonoids were covalently attached to

proteins (Rawel et al., 2002a; Rawel et al., 2003). Contrary results are reported as well for

proteins in combination with lipids that may either act as foam promoter or killer (Karel, 1973).

pH variations led to a 15 % increase in foam volume. Decreasing the pH to 3 and back to 7 even

led to an overrun 2.5 times higher than that of the control (van Koningsveld et al., 2002b). This

can be traced back to the partial irreversibility of the pH dependent increase in random coil

structure (Pots et al., 1998b). The impact of pH shifts on foam formation was less pronounced

than the one of the pulsed electric field treatment. The extent of pH shifts was only estimated on

the basis of the pH dependent solubility and do not claim to meet the exact PEF conditions.

Furthermore, the PEF mechanism is probably more complex as pH alterations in both directions

occur and additionally, enhanced oxidation and radicalization may happen at the same time. The

exact composition of the obtained protein concentrates was not analysed. Differences in overall

composition, e.g. mineral content, might as well contribute to alterations in foaming behaviour.

No changes in foam stability were detected for concentrates with different pre-treatments (see

Figure VI.4). Unfolding of proteins does not directly contribute to foam stability. On the contrary,

foams with high overrun may be more instable as thinner protein lamellae are formed between

gas inclusions (Kinsella, 1981). A lower stability is also reported for the foams prepared after pH

shifts (van Koningsveld et al., 2002b). Protein concentrations used in these experiments were ten


164

times lower than those used in this thesis. So, the overall stability was lower and small

destabilizing effects might have been detected more easily. To improve foam stability, steric

concerns and the formation of a viscous interfacial film is of high importance (Kinsella, 1981;

Nakai, 1983; Wilde, 2000). Surface rheology of the protein lamellae seems not to have changed

due to the pre-treatments applied.

Figure VI.4: Foam stability in 1 % protein solutions pH 7 prepared from self-made potato protein concentrate whipped with a handheld electric mixer; PEF treatment: 1 kV/cm, 5 kJ/kg, 666 pulses, 2 Hz; acid treatment: setting to pH 4.5 by HCL addition and resetting to pH 6 with NaOH.

A lot of additional research is necessary to investigate the effect of pulsed electric field treatment

on potato protein and to implement it either as a tool for cell disintegration or target protein

modification. Clarification of a possible unfolding mechanism and the sensitive interactions with

other compounds available in fruit juice require more complex analytical methods and a

structured variation of different extrinsic factors. Nevertheless, promotion of protein reactivity by

application of an electric field offers many interesting research and application possibilities.

0

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60

70

80

90

100

0 10 20 30 40 50 60 70

foam

stab

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in %


untreated PEF treated acid treated


165

Heat, high pressure and pulsed electric fields and their influence on

yield and properties of pea proteins

Protein was extracted from wet-grinded flour of peas that were previously subjected to heat,

pressure or pulsed electric fields. Preliminary tests showed best extractability in alkaline buffer.

This is in agreement with the protein solubility profile and the common protein extraction

methods used for different legume species that are either performed under alkaline or strong

acidic conditions (Gueguen, 1983; Boye et al., 2010). Extraction in tap water was done as well as it

is a cheap and harmless extraction medium.

Figure VI.5 shows the results of protein extraction from peas treated with pulsed electric fields.

The intense mechanical cell disintegration during wet-grinding led to high protein yields, when

basic buffer was used for extraction. The protein content in peas averaged 0.26 g/g dry matter. 90

to 95 % of this protein could be extracted from untreated peas, which matches a satisfying

protein yield (Gueguen, 1983). Around 62 % of protein was extracted in tap water, which

corresponds to the water solubility of legume protein at neutral pH (Gueguen, 1983). The release

of ions from pea flour might have additively enhanced the ionic strength in the extraction medium

and improved the solubility of globulins. Nevertheless, for an economic protein recovery, the

usage of an alkaline medium is necessary.

Figure VI.5: Buffer and water extractability of protein from wet-grinded pea flour subjected to PEF treatments of 125 kJ/kg at room temperature; extraction time 30 min; columns with different letters deviate significantly (α = 0.05).

0.00

0.05

0.10

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0.30

0 kV/cm 5 kV/cm 10 kV/cm 0 kV/cm 5 kV/cm 10 kV/cm

buffer extractable water extractable

extr

acta

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prot

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/ g

dry

mat

ter

Biuret Bradford

b bb

a aa

c c c

d dd


166

PEF did not result in an improved protein yield. Even a slight decrease in protein extractability was

determined but changes between treated samples and the untreated control were not significant.

Analyses of whole peas revealed changes in cell wall appearance, pronounced diffusion of

substances into the surrounding medium and improved release and uptake of water into the

tissue. All these results indicate a cell disruption due to electroporation. The high mechanical

disintegration and the already maximal protein yield may have superimposed the PEF effects. A

potentially accelerated release of protein during extraction cannot be determined with these

analyses because a constant extraction time was chosen during experiments. Further

investigations are necessary to definitely exclude a positive effect of the treatment on the

economy of protein recovery.

Due to the different measurement principles, protein yields determined via Bradford assay only

amounted 60 % of those detected with the Biuret reaction. A small increase in absorbance up to

15 % was found for watery extracts of PEF samples. This might be the result of unfolding and

exposure of previously buried amino acids in the electric field. Results from chapter III on protein

solutions showed no comparable effects. Slight differences between pH 6.5 in pea tissue and pH 7

in model solutions might alter the sensitivity of the protein structure towards electric fields.

Enhanced light absorbance during measurements may also be the consequence of oligomer

dissociation and a connected alteration in dye binding. Carbonaro et al. (1997) stated that

arginine plays an important role in the association and dissociation of legume protein subunits.

Changes in quaternary structure are not detectable with SDS PAGE, where proteins are present in

monomeric form anyway, and could therefore not be determined with the analyses performed.

As similar increase in Bradford affinity, determined for potato protein solutions, did not result in

an altered protein functionality, the influence of PEF on the structure of pea tissue protein is

assumed to be negligible and was not further investigated.

Although it seems that no further increase in protein yield is possible by using emerging

technologies and that maximal recovery can already be achieved by conventional processing, the

influence of heat and pressure on protein extractability is worth investigating. Analyses of protein

solutions revealed specific impacts on composition and functionality (see chapter III and IV).

Figure VI.6 shows the buffer extractability from peas subjected to thermal and pressure

treatments. Pressures of 400 and 600 MPa significantly reduced protein yield, detected with the

Biuret method, to 81 and 62 %, respectively. The extractability of proteins from heat treated peas

was only slightly reduced to 93 % after exposure to 60 and 80 °C. These results reflect solubility

changes already observed for solutions prepared from pea flour in which greater changes were

induced by high pressure whereas thermally treated protein remained in solution.


167

Figure VI.6: Extractability of protein from wet-grinded pea flour subjected to thermal and high pressure treatments with 10 min dwell time; protein extraction in buffer at pH 9 within 30 min under continuous stirring; columns with different letters deviate significantly (α = 0.05).

These effects were even more pronounced considering the results of the Bradford assay.

Differences between both analytical methods could already be an indicator for changes in protein

conformation or composition. Decreased extractability of protein from cooked legumes was

accompanied by changes in the amino acid composition of the soluble fraction (Carbonaro et al.,

1997). The proportion of charged amino acids was enhanced whereas hydrophobic amino acids

were drastically reduced. This may lead to an altered Bradford response as both basic and

aromatic residues contribute to the colour intensity of the assay. The ratio Bradford to Biuret was

increased for samples treated at 60°C and reduced for those further heated to 80 °C indicating

that one of the protein fractions may possess denaturation parameters somewhere within this

temperature range.

Water solubility of protein from peas that were pressure treated at 400 and 600 MPa decreased

according to the worse alkaline extractability (see Figure VI.7). The ratio of water soluble to

alkaline soluble protein was diminished after heating or application of 200 MPa. The drastic

reduction in protein solubility after cooking of legumes was not observed at strong alkaline pH

values (Carbonaro et al., 1997). This indicates that the solubility decrease can be partially

compensated by a basic environment. The effects on extractability were again more distinct when

0.00

0.05

0.10

0.15

0.20

0.25

0.30

20 °C 40 °C 60 °C 80 °C 200 MPa 400 MPa 600 MPa

0.1 MPa 40 °C

buffe

r ext

ract

able

pro

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in g

/ g

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mat

ter

Biuret Bradford

a

aa

a

b

a

c

d

d, e

dd

e, f

d

f


168

the Bradford assay was used, except for samples extracted after a 60 °C treatment, which is

another indicator for an altered protein structure.

Figure VI.7: Protein extractability from wet-grinded pea flour subjected to thermal and high pressure treatments with 10 min dwell time; protein extraction in tap water within 30 min under continuous stirring; columns with different letters deviate significantly (α = 0.05).

The results indicate that further analysis of the protein composition is necessary. Figure VI.8

shows the electrophoretic pattern of the water extractable proteins. The general protein

composition is similar to the one obtained for solutions of pea flour (see Figure III.21). Five main

protein fractions can be found whereof the bands of vicilin distributed over a broader molecular

weight range. No marked changes in the protein composition were found for samples PEF treated

at 10 kV/cm, although the intensity of legumin seems more pronounced after treatment. Staining

of the PAGE was conducted with Coomassie Blue, the same dye as used in the Bradford assay.

Changes leading to an intensified absorbance in the assay may also alter the staining intensity in

the gel.

As already observed in protein solutions, the staining intensity of PA 2 was reduced drastically

when extracts were prepared from samples blanched at 80 °C. A reduced extractability of this

protein fraction from peas after heat treatment was also reported by Le Gall et al. (2005). Of

course, it is also possible that the albumin was extracted from the tissue, but had undergone

aggregation and was not further able to enter the gel network due to its large molecule size.

Unfortunately, samples treated at 60 °C were not run in the PAGE so that no differentiation

0.00

0.05

0.10

0.15

0.20

0.25

0.30

20 °C 40 °C 60 °C 80 °C 200 MPa 400 MPa 600 MPa

0.1 MPa 40 °C

wat

er e

xtra

ctab

le p

rote

in in

g /

g dr

y m

atte

r

Biuret Bradford

a a, b

b, ca, b, c

c

a, b, ca, b, c

d

e

d, e d, e

d, e

d, e

d, e


169

between both heating conditions could be made. A decrease in legumin, as it was found for

protein solutions, was not observed. It is possible that the overall lower availability of water in

pea tissue compared to solutions shifts the denaturation of legumin towards higher

temperatures. The thermal stability of dry protein is higher compared to solutions and the

denaturation temperature decreases with increasing hydration (Hagemann, 1988).

Figure VI.8: Non-reducing SDS PAGE of protein extracts from wet-grinded peas after pulsed electric field, thermal and high pressure treatments; extraction medium: tap water; extraction time: 30 min; A: Lipoxidase (90 kDa); B: Convicilin (71 kDa); C: Legumin (60 kDa); D: Vicilin (17-50 kDa); E: PA2 (26 kDa).

Similar observations can be made for pressure treated samples. In solutions, the intensity of the

vicilin band was already diminished after pressurization at 200 MPa. A marked reduction of

globulins in pea extract could be determined when the applied pressure reached 400 MPa. In the

600 MPa sample these fractions were completely absent. The highest treatment intensities tested

for heat and pressure also led to a complete removal of the lipoxidase band. In general, changes

induced in protein composition were similar to those detected in protein solutions, but sensitivity

of some protein fractions seems to be shifted towards higher treatment intensity.

Protein concentrates were won from extracts of differently pre-treated peas to reveal to what

extent changes in protein composition affect the functional properties of a dry, marketable

product. For this purpose alkaline extracts were neutralized, concentrated in membrane filtration

to approximately 50 %, frozen in liquid nitrogen and freeze dried to avoid any additional impact of

40 °C 80 °C

AB

E

D

C

D

200 MPa 400 MPa 600 MPa0 kV/cm 10 kV/cm


170

high temperature. The highest treatment intensities used, 80 °C and 600 MPa, were chosen as

representatives for the respective technology, as effects on protein yield and structure were most

pronounced in these samples. Details regarding the recovery of concentrates are provided on

page 233.

Table VI.3: Composition of protein concentrates won from swollen peas subjected to 20 °C (untreated), 80 °C (heat treated) or 600 MPa at 40 °C (pressure treated)

untreated heat treated pressure treated

Water content in g/g 0.05 ± 0.012 0.06 ± 0.015 0.04 ± 0.006

Protein content in g/g dry weight 0.67 ± 0.009 0.54 ± 0.001 0.49 ± 0.0

Composition of the products obtained is given in Table VI.3. Concentrates from pressure treated

peas contained less protein than the control samples, which was probably due to the overall

lower protein solubility, a reduced extractability of these fractions from protein tissue or a

removal during centrifugation. Surprisingly, concentrates of thermally treated raw material

possessed a lower protein content as well, although protein solubility at alkaline conditions was

only slightly affected due to this treatment. Temperatures of 80°C led to a gelatinization of starch

granules (see chapter VII) and to a leakage of amylose. An incomplete removal of carbohydrates

during centrifugation might have led to their enrichment in the protein concentrate. Further

analysis of the product composition is necessary to characterize the non-protein substances

present.

Figure VI.9 shows the solubility profile of the different concentrates. The overall solubility was

very high compared to information available in literature. This might have been caused by the

previous removal of less soluble proteins during wet recovery. The general progress of the curves

is consistent with that reported for native legume proteins (Gueguen, 1983; Carbonaro et al.,

1997). All products possessed a similar solubility at pH 3 and in a neutral environment. Their

distinct behaviour in between can be explained by the alterations in protein composition. The pea

globulins precipitate close to their isoelectric point in the range of pH 4 to 5. Solubility is therefore

minimal in this pH range. The albumin fraction PA2 possesses no net charge around pH 5 to 5.5

(Croy et al., 1984), but it remains soluble over the entire pH range. Aggregation and separation of

the globulins at 600 MPa and consequent concentration of PA2 led to a flatter progression of the

solubility curve. An extractability of approximately 70 % between pH 4 and 5 indicates that not all

globulins were lost during protein recovery. Probably some particles did not have the needed


171

diameter to be separated under these conditions. Hence, there was still a small decrease in

solubility, when the pH reaches the critical pH range of 4 to 5. The profile of heat modified protein

concentrate is slightly shifted towards higher solubility as well. Heating did not alter the alkaline

protein solubility and all protein fractions should have been available in the concentrate to a

similar extent as in the untreated sample. Certainly, some of them were present in an aggregated

or denatured state. This might have influenced their isoelectric point as well as their overall

precipitation behaviour. The presence of other pea substances, especially of gelatinized starch,

may have affected the sample viscosity and led to a hindered protein separation during

centrifugation.

Figure VI.9: Solubility of pea protein concentrates in phosphate-citrate buffer of different pH after 30 min of stirring; protein concentrates won from swollen peas subjected to 20 °C (untreated), 80 °C (heat treated) or 600 MPa (pressure treated); initial powder concentration 10 g/L; protein quantification via Biuret method.

Foam volumes were recorded over the entire detection time, as no calculation of half-life was

possible due to a high overall stability under these analytic conditions (see Figure VI.10). There

were only small differences in the stability of foams made of the distinct concentrates that were

in no relation to changes observed after treatment of protein suspensions (see Figure IV.1 and

Figure IV.2). Enhanced foam collapse was mainly related to the presence of large aggregates that

were removed somewhen in the recovery process. The results are therefore more comparable to

the changes found in centrifuged suspensions. Similar to the suspensions treated at 60 °C an

effective removal of bulky clusters regained initial foaming properties. Traces of gelatinized starch

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

3 3.5 4 4.5 5 5.5 6 6.5 7

solu

ble

prot

ein

in g

/ g

prot

ein

pH value [-]



172

may also have improved foam stability in heated samples and balanced out negative treatment

impacts on proteins. The pressure treated samples showed a similar decrease in stability as

detected for centrifuged pressurized suspensions, mainly caused by smaller aggregates from

vicilin and legumin not separated during centrifugation. The time needed to achieve maximum

foam height increased from 71 ± 1.2 s to 73.5 ± 0.5 s and 72.3 ± 0.4 s due to heat and pressure,

respectively. Effects on interfacial adsorption are thus negligible. Protein solutions were made in

tap water and cleared by centrifugation, where the overall solubility of untreated and heat-

treated concentrates amounted half the value of pressure treated ones. Probably the lower ionic

strength compared to buffer favours solubilization of the albumin and thereby leads to an

enrichment of this fraction in all samples. This would also explain the small differences between

the samples. SDS PAGE of the tap water solutions could provide clarity in regard to their

composition. Repetition of foaming experiments at lower protein concentrations could help to

point out differences in the functional behaviour more clearly.

Figure VI.10: Foam stability detected for solutions prepared from pea protein concentrates in tap water at pH 7; protein concentrates won from swollen peas subjected to 20 °C (untreated), 80 °C (heat treated) or 600 MPa at 40 °C (pressure treated); protein quantification in clear solutions via Biuret assay; protein concentration for foam analyses: 0.35 g/L; dilution medium: deionized water; testing unit: DFA 100.

The modified solubility in a slightly acidic pH range provides interesting application options for

pressure treated pea protein, for instance for protein enrichment of carbonated beverages or

fitness drinks. Compared to legume globulins the albumin fraction possesses a higher content of

0

20

40

60

80

100

0 5 10 15 20 25 30

foam

stab

ility

in %




173

sulphur-containing amino acids. Pressure treatment might thereby improve the nutritional value

of the protein product. Usage of high pressure for protein fractionation is furthermore a physical

alternative to separation via isoelectric precipitation. As pressurization of whole peas prior to

protein extraction leads to a drastic reduction in protein yield, advantages in regard to product

quality should be investigated more detailed and an application for the insoluble protein, for

instance in animal feed, has to be found to guarantee a holistic and sustainable utilization of the

raw material.

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176

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Chapter VII – Heat and high pressure to modify the properties of pea flour

177

Chapter VII

Heat and high pressure to

modify the properties of pea

flour


178

Changes induced in the tissue structure of whole peas or a modified protein conformation and

composition may also affect the properties of the flours produced from pre-treated peas. Changes

in the flour may occur in regard to the functional properties, the shelf-life or the nutritive value.

Fat and water binding as well as the gelation ability should indicate possible alterations in the

application properties of flour products. The activity of endogenous enzymes and the accessibility

of protein and starch to enzymatic digestion serve as indicators for the two other characteristics.

As effects on protein were more pronounced for thermal and pressure processing, the following

investigations focus on these two technologies. To completely evaluate the potential of both

emerging technologies, the effect of pulsed electric fields on pea flour would have to be analyzed

as well.

Functional properties of modified pea flours

Thermal or pressure treatments of whole peas may also affect the utilization of the respective

flours, for instance in pasta, bakery products or as thickening agents. Water and fat binding were

chosen as important characteristics for the flour as they are of high importance in the

aforementioned products. They influence the juiciness and mouth feel of the product and

consequently its sensorial quality. Release of water or fat during storage and transport is

undesired and markedly reduces the consumer acceptance (Barbut, 1996).

Figure VII.1 shows the water binding ability of flours prepared from differently pre-treated peas.

Approximately 1.2 gram of water per gram of dry matter were hold by the pea flour against a

centrifugal force of 10 000 g. Literature data for the water binding capacity of legume flours

extents to a wide range. Between 0.7 and 0.8 gram of water could be bound by flours made of

yellow peas, dehulled lentils, field peas, faba and mung beans (del Rosario & Flores, 1981; Sosulski

& Mccurdy, 1987; Ma et al., 2011). Higher values up to 2.4 gram per gram were reported for

lentil, winged bean, cowpea or soy (Narayana & Rao, 1982; Sosulski & Mccurdy, 1987; Abbey &

Ibeh, 1988; Nagmani & Prakash, 1997). Differences might arise from varying macroscopic

characteristics like the degree of milling, the homogeneity of compound distribution or from the

different overall product composition of the raw materials. Hulling, for instance, slightly decreases

the water binding properties of flours probably due to changes in the fibre content (Ma et al.,

2011). In general, results from different working groups are only of limited comparability due to

differences in the methods and the equipment used.

Commercial whole-wheat flour was analyzed as a reference as it may be a potential competitive

product in the food sector. 0.9 gram of tap water was adsorbed per gram of dry matter, which


179

corresponds to 75 % of the water quantity bound by the pea flour. Legume flour clearly contains

more protein than wheat flour that may interact with water by forming hydrogen bonds at polar

and charged amino acid residues. This is confirmed by the results of Dervas et al. (1999), who

reported a gain in water binding of wheat flour from 53 % to up to 66 % with increasing

replacement by lupine flour.

Figure VII.1: Water binding capacity of flours prepared from peas treated at different pressure-temperature combinations; peas were swollen for 20 h in tap water before treatments; treatment time: 10 min; drying of peas for 6 h at 50 °C; binding capacity was determined at ambient temperature against a centrifugation at 10 000 g for 15 min.

Temperatures of 40 and 60 °C as well as high pressure of 200 MPa only affected the water binding

properties of pea flour within the frame of the analytical deviations. A slight increase in water

adsorption was observed after pressure treatments at 400 and 600 MPa. A distinct growth in the

amount of bound water could be determined for flours from peas subjected to 80 °C. This is in

agreement with the findings of other authors who reported an increase in the water and fat

adsorption of flours after hydrothermal treatments (Narayana & Rao, 1982; Abbey & Ibeh, 1988;

Nagmani & Prakash, 1997; Ma et al., 2011). Protein denaturation as well as starch gelatinization

and swelling of crude fibres were mentioned as possible reasons for the increment in water

retention.

Nevertheless, this treatment effects cannot be observed regarding the fat binding properties,

where results ranged from 1.18 gram to 1.26 gram of absorbed oil for all pre-treatments applied

(see Figure VII.2). The lower values obtained for treatments at 40 °C might arise from flour

0.0

0.4

0.8

1.2

1.6

2.0

20 °C 40 °C 60 °C 80 °C 200 MPa 400 MPa 600 MPa

0.1 MPa 40 °C

wat

er b

indi

ng c

apac

ity in

g /

g dr

y m

atte

r


180

inhomogeneities or incorrect testing. Literature data are again widely spread ranging from 0.4 to

2.9 mL of adsorbed oil (del Rosario & Flores, 1981; Narayana & Rao, 1982; Sosulski & Mccurdy,

1987; Abbey & Ibeh, 1988; Nagmani & Prakash, 1997; Ma et al., 2011). Beside on the

aforementioned flour properties lipid binding is additionally dependent on the type of fat used

and on the presence or absence of an emulsifier in the sample (Barbut, 1996). Fat binding

properties of wheat flour were with one gram of adsorbed oil again lower than those of pea

flours, probably due to hydrophobic interactions between oil and protein. This comparison

reveals possible advantages of legume flour in the food industry as its higher protein content does

not only improve the nutritional value of a product, but might as well contribute to its storage

stability. Alterations of water and fat binding might as well enable a positive adaption of the

product recipe, for example towards higher water and lower energy contents.

Figure VII.2: Fat binding capacity of flours prepared from peas treated at different pressure-temperature combinations; peas were swollen for 20 h in tap water before treatments; treatment time: 10 min; drying of peas for 6 h at 50 °C; binding capacity was determined at ambient temperature against a centrifugation at 10 000 g for 15 min.

As changes in the protein conformation are possible explanations for the altered flour properties

after treatment, water and fat binding abilities of protein concentrates prepared from heat and

pressure treated peas were analyzed as well (see Figure VII.3). Oil adsorption was with 2.9 gram

per gram of dry weight higher in the protein concentrates than in the flour. The higher protein

content might enforce hydrophobic interactions. The porosity of the freeze-dried concentrates is

higher than those of pea flours, which enables the encapsulation of higher amounts of fat in the

0.0

0.4

0.8

1.2

1.6

2.0

20 °C 40 °C 60 °C 80 °C 200 MPa 400 MPa 600 MPa

0.1 MPa 40 °C

fat b

indi

ng c

apac

ity in

g /

g dr

y m

atte

r


181

product matrix. Fat binding is markedly influenced by structural attributes like presence of

cavities, pore and strand size (Barbut, 1996).

Figure VII.3: Tap water and fat binding of protein concentrates prepared from peas treated at 20 °C or 80 °C at ambient pressure or at 40 °C and 600 MPa; peas were treated after 20 h of swelling in tap water; treatment time: 10 min, binding capacity was determined against centrifugation for 15 min at 10 000 g.

Increased fat and water binding was reported when the protein content of a flour is enhanced by

air classification or by extraction and isoelectric precipitation (Sosulski & Mccurdy, 1987). These

findings were not confirmed for the water retention of pea concentrates for which the amount of

bound liquid was with 1.05 gram lower than for the flours. The relatively high water solubility of

the concentrates at neutral pH may have counteracted the water retention. An opposing

relationship between solubility and water binding was observed by several authors (Narayana &

Rao, 1982; Abbey & Ibeh, 1988; Nagmani & Prakash, 1997; Heywood et al., 2002; Ma et al., 2011).

High solubility in the added water makes accurate determination of bound water difficult as

protein already in solution might be removed during decantation of surplus water. Concentrates

won via membrane processing and gentle drying usually possess a higher solubility than those

recovered via isoelectric precipitation. Nevertheless, Boye et al. (2010) reported only slight

differences in the water holding capacity of yellow pea protein concentrates won by these two

concentration techniques.

Fat and water binding capacities remained nearly unchanged for heat processed concentrate, but

decreased by 20 % and 5 % for the high pressure sample, respectively. Concentrates from heat

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

20 °C 80 °C 600 MPa 20 °C 80 °C 600 MPa

water binding fat binding

bind

ing

capa

city

in g

/ g

dry

mat

ter


182

and pressure treated peas had lower protein contents compared to the untreated sample (see

Table VII.1). Pea protein possesses lower solubility after pressure application, which may have led

to an enrichment of small soluble compounds in the concentrate that do not markedly interact

with lipids. The specific volume of the HP concentrate is lower as well, which may have led to a

smaller binding surface and a reduced wettability with fat and water. The heat treated sample is

supposed to contain gelatinized amylose, which also contributes to the binding properties.

Table VII.1: Protein content and specific volume of protein concentrates prepared from peas treated at 20 °C or 80 °C at ambient pressure or at 600 MPa at 40 °C; peas were treated after 2 h of swelling in tap water; treatment time: 10 min

20 °C 80 °C 600 MPa

Protein content in g/dry weight 0.67 ± 0.009 0.54 ± 0.001 0.49 ± 0.0

Specific volume in cm³/g 11.9 ± 3.1 11.5 ± 1.3 8.2 ± 1.0

As the water retention of the concentrates was not affected by heat treatment, alterations in the

flour properties cannot be traced back to a modification of the protein structure. Changes in

water binding might arise from a thermal effect on the starch granules, the main ingredient in pea

flours. Figure VII.4 shows microscopic pictures of the flours prepared from pre-treated peas.

Destruction of the granules is already visible for 60 °C and 600 MPa samples. Temperatures of

80 °C led to a complete disruption and leakage of amylose. Gelatinization can be visually assessed

by the loss of birefringence (Banks & Muir, 1980).

Figure VII.4: Flour suspensions in a transmitted light microscope prepared from peas subjected to the following treatment conditions: Top: 20 °C (left), 60 °C (middle) and 80 °C (right) at atmospheric pressure; bottom: 200 MPa (left), 400 MPa (middle) and 600 MPa (right) at 40 °C; peas were swollen in tap water for 20 h previous to treatments; treatment time: 10 min.


183

The microscopic pictures of the flour suspensions do not allow a conclusion on changes of the

birefringent character, but the maltese cross was visible in all tissue sections, assuming that starch

gelatinization was incomplete during the processing of whole peas (see Figure VII.5).

Gelatinization of pea starch was reported to begin at temperatures higher than 60 °C (Biliaderis et

al., 1980; Ratnayake et al., 2001). The gelatinization temperature is highly dependent on the

amount of available water as extensive hydration of the amorphous regions facilitates melting of

starch crystallites. Low water contents lead to a shift of the gelatinization towards higher

temperatures (Biliaderis et al., 1980). Pea starch gelatinization is usually accompanied by an

increase in granule volume by factor 20 to 80 (Ratnayake et al., 2001). This effect is not visible in

the taken pictures and might be a result of restricted water content during heating. In swollen

peas, the available water is with approximately 1.3 gram per gram of dry weight rather limited.

The presence of other water binding substances like salts, sugars and proteins might further

decrease the amount of disposable water. Heat-moisture treatments (heating for several hours at

limited moisture content) does not alter size and shape of starch granules and leads to reduced

swelling and decreased amylose leakage compared to gelatinization at an excess of water (Hoover

& Vasanthan, 1994; Ratnayake et al., 2002). Nevertheless, granule disruption and dissociation of

the helical structure occur during this treatment (Gunaratne & Hoover, 2002).

Figure VII.5: Microscopic pictures of tissue sections taken from peas exposed to 10-minute treatments in a swollen state and re-dried in warm air at 50 °C. Left: 80 °C; right: 600 MPa at 40 °C.

Heat and pressure processing of peas also altered the gel forming properties of their respective

flours (Figure VII.6). The strength of gels made from pea flours was more than five times higher

than the one of gels made from whole-wheat flour. This can be traced back to the different starch

composition of legumes compared to cereals and the higher ratio of amylose to amylopectin

(Sosulski et al., 1989). The higher amylose content of pea starch enables network formation at

lower concentrations forming products with different structure and mouth feeling (Stute, 1990b;


184

1990a). Results have to be interpreted carefully due to worse reproducibility and reliability of the

method but a clear decrease in gel strength was found for heat treated flours. One reason might

be the incomplete gelatinization due to limited water availability. Heat-moisture treatment of

maize flours led to a reduction of dough elasticity and a decrease in bread volume and crumb

softness (Miyazaki & Morita, 2005).

Figure VII.6: Firmness of gels made of 20 % (w/v) pea flour suspensions heated to 100°C for 5 min and cooled down to ambient temperature for 3.5 h; flours were made from peas treated at different temperature-pressure combinations for 10 minutes in a swollen state and re-dried for 6 h at 50 °C; gel firmness was calculated via force-displacement curves of the texture analyzer.

High pressure treatments also led to a reduction in gel firmness, but affected flour properties to a

smaller extent. Swelling of starches also happens under pressure as uptake of a certain water

amount into the granules is connected to a reduction in bulk volume (Douzals et al., 1996).

Starches modified by pressure undergo restricted granule expansion and exhibit decreased

amylose leakage compared to thermal gelatinization (Stolt et al., 2000). Consequently, gels from

wheat starch formed under pressure possessed a higher density than those induced by heat

application (Douzals et al., 1998). Hydrogen bonds are known to be stabilized under pressure

which might suppress unwinding of the amylose helix (Knorr et al., 2006). Amylose can stabilize

the amylopectin structure, which could as well explain why starches with high amylose content

possess higher pressure stability (Stolt et al., 2000). Analogously to the heat treatment, the

product’s water content is of great importance for the treatment impact as the degree of

gelatinization decreases with the availability of water (Rumpold, 2005).

0

10

20

30

40

50

60

70

80

20 °C 40 °C 60 °C 80 °C 200 MPa 400 MPa 600 MPa

0.1 MPa 40 °C

gel f

irmne

ss in

N/ m

m


185

Starches can be classified by their polymorph structure into those with higher (type A) and lower

(type B) packing density of the double helix (Bogracheva et al., 1998). Starches of the B type are

reported to be more sensitive towards heat-moisture treatments (Hoover & Vasanthan, 1994;

Ratnayake et al., 2002), but they usually possess higher pressure stability due to an already

existent incorporation of water molecules into the looser granule structure (Stute et al., 1996).

Legume starches belong to type C where granules of both starch types A and B are present. 56 to

62 % of A type granules are reported for pea starch (Gernat et al., 1990; Cairns et al., 1997). The

special composition of legume starches explains their sensitivity towards both high pressure and

thermal treatment and might be another reason for the different functional properties in

comparison to whole-wheat flour.

For an adequate evaluation of, i.a. the baking properties of pea flour, binding tests should be

repeated at elevated temperatures to see whether changes are maintained at these conditions or

whether the effects are superimposed by overall thermal effects during product manufacturing.

As preliminary result it can be concluded that high pressure does not alter flour properties to a

large extent whereas heat treatment leads to changes in water binding capacity. This influence

may be important when choosing a technology for other process objectives like enzyme

inactivation, reduction of microbial contamination or improvement of digestibility. Two of these

quality aspects will be discussed in the following subchapters.

Influence of heat and high pressure on enzyme activity in pea flour

Beside the growth of undesired microorganisms, reactions catalyzed by endogenous enzymes can

lead to alteration of the product characteristics and thereby reduce its shelf-life. Enzymatic

reactions affect product quality by changing colour or texture, by causing off-flavours or by

degradation of nutritionally valuable food ingredients. Enzymes are a special class of globular

proteins with molecular weights ranging from 6 to 250 kDa (van Oort, 2010). They enable

biological reactions by lowering the energy required to bring a substrate into the transition state.

Therefore, the substrate is bound close to or in the so-called active site of the enzyme by covalent

bonds, hydrogen bonds, hydrophobic or electrostatic interactions (Karlson, 1984). Although the

enzyme is composed of up to a few thousand amino acids, only a few of them form the active site

(van Oort, 2010). Its individual composition and conformation endows the enzyme with the high

specificity for substrate and catalyzed reaction (Bayindirli, 2010). Even small conformational

alterations of the active site may lead to changes in the enzyme’s activity or substrate specificity

(Tsou, 1986). The complex conformation of the large protein molecule can ensure the flexibility of

the active site and stabilize the native structure by forming intramolecular bonds, but


186

conformational changes due to external influences may also affect the active site and contribute

to enzyme inactivation (Ruttloff et al., 1977). Upon heating, the native protein molecule unfolds

to random coiled structures that do not possess catalytic activity (Adams, 1991). Secondary

reactions like aggregation, deamidation, hydrolysis, oxidation and interactions with other

compounds further lead to irreversible inactivation (Volkin & Klibanov, 1989; Adams, 1991).

Thermal blanching with water or steam is therefore the most common way to inactivate enzymes

in fruits and vegetables (Selman, 1994).

Due to its high heat stability peroxidase (POD) is widely used as an indicator for a successful

blanching process (Hemeda & Klein, 1991). The enzyme is present in many plants and

microorganisms and consists of several isoforms of different stability (Wong, 1995). It catalyzes

oxidation reactions involved in plant defence and detoxification mechanisms, but in vegetable

foods it may cause negative colour and flavour changes. Many legumes contain high levels of

lipoxidase (LOX) activity causing the oxygenation of polyunsaturated fatty acids and their esters

and glycerides (Siedow, 1991; Gökmen et al., 2002). These reactions generate highly reactive

products that may further diminish the nutritive value of the food and often possess aromatic

character. Limiting lipoxidase activity is therefore as well of great importance for controlling

quality deterioration in vegetables (Gökmen et al., 2005). Peroxidase and lipoxidase were thus

chosen as indicator enzymes for the temperature and pressure effect on enzyme solutions, whole

peas and the flours made of them. Temperatures and pressures were chosen according to the

preliminary experiments performed with whole peas.

Inactivation kinetics were recorded for both enzymes in pure solutions under optimum

conditions. Logarithmic display revealed that inactivation curves nearly follow a first order kinetic.

Longer dwell times to achieve inactivation of several log cycles were not performed due to the

declining accuracy of the analytical methods and missing practical relevance of longer processing

periods.

The thermal inactivation of soybean lipoxidase and horseradish peroxidase is shown in Figure

VII.7. The former one is sensitive to temperatures of 60 °C or higher. Exposure to this temperature

for 150 s already led to an activity reduction of one log cycle. Longer dwell times further reduced

enzyme activity. At 80 °C the inactivation occurred more rapidly. Almost two log cycles of

inactivation were obtained after 150 s of processing. Higher thermal stability was reported by

other authors for extracts from soybeans or soy milk. Inactivation of 0.4 and 1.4 log cycles after a

ten minute heat treatment of 67 to 69 °C were reported for extract and milk, respectively (Wang

et al., 2008). 10 minutes in boiling water reduced the activity of extracts from defatted soy flour

by 2.5 log cycles (Kong et al., 2008). The differences in stability may be a result of different


187

heating systems used in different research groups and the higher purity of solutions from isolated

enzymes compared to extracts.

Figure VII.7: Activity in pure enzyme solutions after exposure to different temperatures at ambient pressure in relation to samples treated at 20°C; top: lipoxidase from soybeans; bottom: peroxidase from horseradish.

An unclear inactivation trend was determined for treatment temperatures of 40 °C. Lipoxidase

activity decreased to 80 % after 5 minutes but remained almost unchanged after 10 minutes

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holding time. This behaviour may be caused by the performance of the thermal processing. To

minimize the influence of heating and cooling, enzyme solutions were treated in small glass

capillaries sealed in the flame of a Bunsen burner. Deviations or mistakes during sealing may

heated parts of the sample to temperatures higher than 40 °C and have already affected the

temperature sensitive enzyme. The same treatment intensity applied to samples in plastic

reaction tubes did not cause enzyme inactivation. It can therefore be assumed that lipoxidase is

stable within a ten minute treatment at 40 °C.

Peroxidase showed higher thermal stability. An activity loss of 20 % at maximum was obtained at

a temperature of 60 °C within the chosen timescale. 80 °C led to a reduction of one log cycle after

450 s treatment time. 0.5 log cycles of inactivation after 10 minutes at 80 °C and almost no effect

of 60 °C were reported as well by Chang et al. (1988) for horseradish peroxidase. These

observations also confirm the high temperature stability of peroxidase and its suitability as a

blanching indicator.

Besides high temperatures, high pressures can be applied to alter the conformation of the

enzyme molecule and thereby achieve its inactivation (Seyderhelm et al., 1996). Analogously to

thermal treatments, different enzymes possess different pressure stabilities and the parameters

required have to be chosen according to the most stable enzyme that should be inactivated.

Pressurization at 600 MPa at 40 °C for 10 minutes led to a 2 log cycle reduction of lipoxidase (see

Figure VII.8). The inactivation effect is therefore comparable to those of a thermal treatment at

60 °C. Application of 200 MPa did not affect enzyme activity and the 60 % reduction in activity

caused by a 400 MPa treatment is not sufficient for a marked shelf-life improvement. Pressures of

at least 600 MPa were reported as necessary to obtain more than one log cycle of lipoxidase

inactivation (van der Ven et al., 2005; Wang et al., 2008). Exposure of soybean lipoxidase to

600 MPa and 40 °C for half an hour led to irreversible changes in the protein’s secondary structure

and to a destruction of the native enzyme (Heinisch et al., 1995). Lower pressure intensities

resulted in insufficient inactivation or required combination with higher processing temperatures

(Ludikhuyze et al., 1998; Tedjo et al., 2000; van der Ven et al., 2005; Wang et al., 2008).

Almost no peroxidase inactivation was achieved by the pressure treatments. Higher pressure

sensitivity was reported by Tedjo et al. (2000) who detected a 50 % reduction in activity after a

15 minutes treatment of horseradish peroxidase at 240 MPa and 55 °C. Nevertheless, the high

pressure conditions investigated are not adequate to markedly enhance product shelf-life in

regard to quality losses caused by peroxidase. Structural analysis revealed that the protein

molecule possesses a rigid core giving the enzyme its high pressure stability (Smeller et al., 2003).

This finding makes peroxidase also a suitable indicator enzyme for pressure treatments.


189

Figure VII.8: Activity in pure enzyme solutions after pressure treatments at 40°C in relation to untreated control; top: lipoxidase from soybeans; bottom: peroxidase from horseradish.

The results obtained for pure solutions may be an indicator for the stability of the enzymes

towards heat and pressure, but one cannot easily deduce the degree of inactivation in complex

tissue from these data. The sensitivity of enzymes towards processing strongly depends on the

source of the enzyme, the isoforms present as well as on environmental conditions like ionic

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strength, pH value or presence of other plant compounds. The experiments were therefore

conducted with whole peas as well. Dwell times of 10 minutes were performed to be in

accordance with other experiments presented in this thesis.

Activity of lipoxidase in pea flour made of treated peas compared to flour from unprocessed peas

is shown in Figure VII.9. Swelling, treatment at 20 °C and redrying of the peas already led to an

activity reduction of 20 %. Lipoxidase is located primarily in the cotyledons of the peas (Eriksson,

1967), so that a wash-out of the enzyme during water uptake is rather unlikely due to long

transport distances within the tissue. Soaking in water for 20 h might already have initiated the

germination process, which can either enhance or decrease lipoxidase activity in plants (Siedow,

1991). 6 h of warm air drying at 50 °C may cause lipoxidase inactivation, although the

temperature applied is rather low. The inactivation of, for example, lipase during drying was more

dependent on the moisture content available than on the drying temperature applied (Luyben et

al., 1982).

Figure VII.9: Lipoxidase activity in extracts from peas treated at different pressure-temperature combinations in relation to activity in untreated, unswollen peas; peas were treated after 20 h swelling in tap water; treatment time: 10 min; drying for 6 h at 50 °C.

In peas treated with 40 and 60 °C the activity was higher again and even exceeded the initial value

of unswollen peas. The overall protein extractability decreases with increasing process

temperature. Results shown previously in this thesis showed no homogeneous aggregation of

protein during treatments, but loss of certain protein fractions depending on the technology

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applied. Relating enzyme activity to the content of protein in the extracts may therefore falsify

the activity impression. Nevertheless, relating enzyme activity to the dry matter of the flour led to

similar activity results as the relation to the protein content. Alteration of the peroxidase

structure can result in a loss of catalytic activity, but may form a conformation with lipid oxidation

ability (Adams, 1991). However, the inactivation results detected for peroxidase do not support

this hypothesis. Slight temperature elevation might cause partial unfolding of the enzyme leading

to a better accessibility of the active site and a higher enzyme activity. Interaction with other

tissue components might have prevented refolding and preserved the increased activity during

drying. The exposure to temperatures higher than the environmental ones might have also

caused a stress response in the plant, leading to promoted lignification. As lipoxidase is, amongst

others, involved in this secondary metabolic pathway (Siedow, 1991), stress might lead to

enhanced production of this enzyme. However, results presented in chapter V showed a loss in

the peas’ germination ability after exposure to 60 °C. This loss is probably caused by membrane

rupture leading to cell death, which would impede further metabolic processes.

In general, lipoxidase is more stable towards processing in peas compared to pure solutions. No

inactivation was obtained after a 60 °C treatment, and application of 80 °C or 600 MPa had with -

2.5 and -1.3 log cycles, respectively, slightly lower inactivation effects. A strong reduction of

lipoxidase activity in peas at 80 °C, but no marked effects at 60 °C were reported by other authors

(Günes & Bayindirli, 1993; Indrawati et al., 2000; Gökmen et al., 2002; Gökmen et al., 2005). After

pressurization at 200 and 400 MPa at 40 °C activities determined were close to that of the 20 °C

sample. Unfolding of the enzyme due to slight temperature increase might be prohibited under

pressure or the positive effect is compensated by a slight inactivation. At constant temperatures,

the inactivation rate in green beans increased with higher pressures (Indrawati et al., 1999b), but

at room temperatures a pressure of at least 500 MPa was necessary to achieve lipoxidase

inactivation (Indrawati et al., 1999a). Although the effectiveness of a pressure treatment was

higher at very low (4 °C) or elevated temperatures, an effective inactivation required more

intensive processing with pressures higher than 400 MPa or temperatures above 40 °C (Indrawati

et al., 2000; Wang et al., 2008).

Stability differences between tissue and enzyme solutions may be a result of pH variations.

Isolated lipoxidase from soybean was treated in borate buffer at pH 9, whereas the natural pH of

pea cells is close to 6. Accordingly, pea lipoxidase possesses a different pH optimum (Gökmen et

al., 2002). However, contrary results of a pH change were reported for pressure and temperature

stability of soybean lipoxidase (Ludikhuyze et al., 1998). In swollen pea tissue the amount of

available water is limited. In the absence of water, enzymes can withstand much higher

temperatures than in solutions, where an excess of water molecules is present (Adams, 1991). Dry


192

heating at 90 °C for 45 minutes did not markedly alter LOX activity in pea flour, whereas a

treatment of the extracts with 60 and 80 °C already reduced activity by more than 2 log cycles

after 30 and 10 minutes, respectively (Henderson et al., 1991). This was traced back to a complex

formation with other macromolecules and a more tightly and ordered bonding of the surrounding

water molecules to tissue enzymes.

Analogously to lipoxidase, the heat stability of peroxidase was slightly higher in pea tissue (see

Figure VII.10), probably due to pH differences and water availability as well. The activity was

reduced by more than one log cycle after 10 minutes at 80 °C. Gökmen et al. (2005) reported only

slight changes in the peroxidase activity of peas after 10 minutes at 60 or 65 °C and a complete

inactivation after 4 minutes blanching at 80 °C. Günes and Bayindirli (1993) detected 30 % rest

activity after 10 minutes at 80 °C for the same raw material. Inconsistency between the results

might result from differences in the blanching process or the analytical methods used. In these

experiments, almost no effect on peroxidase activity was determined after treatments at 20 and

60 °C, whereas 10 minutes at 40 °C reduced the activity by almost 50 %. Similar results were

obtained for pressure treatments at this temperature, even at those intensities where no effect

on the stable peroxidase was expected. The activity in all these samples was determined one

week later than the control and the flours made from peas treated at 20, 60 or 80 °C. Longer

storage might have reduced overall enzyme activity.

Figure VII.10: Peroxidase activity in extracts from peas treated at different pressure-temperature combinations in relation to activity in untreated, unswollen peas; peas were treated after 20 h swelling in tap water; treatment time: 10 min; drying for 6 h at 50 °C.

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Temperatures around 40 °C might as well have been regarded as a stress signal by the seeds that

caused changes in enzyme activity. The ascorbat peroxidase activity in leaves exposed to 44 °C for

one hour was reduced by approximately two thirds (Panchuk et al., 2002). Exposure to other

stress forms also reduced the peroxidase activity in pea seedlings (McCue et al., 2000). A higher

consumption of phenols for antimicrobial or antioxidative purposes was given as one possible

explanation. Horseradish peroxidase becomes quickly inactivated during the oxidation of phenols,

either by reaction with an excess of hydrogen peroxide, occlusion in the polymeric oxidation

products or destruction of the enzyme’s heme by free radicals (Mao et al., 2013). External stress

might have led to an increased formation of reactive oxygen intermediates and also affected the

enzymes regulating their levels in the plant cells (Mittler, 2002; Caverzan et al., 2012).

Pressures of 400 and 600 MPa only slightly reduced peroxidase activity compared to a pure

thermal treatment at 40 °C. Krebbers et al. (2002) reported 75 % of rest activity in green beans

after 60 s at 500 MPa and ambient temperature. Rest activities ranging from 50 to 70 % were

found by Quaglia et al. (1996) in green peas for pressures between 400 to 700 MPa, while

900 MPa decreased the activity to 20 % at a maximum temperature of 43 °C. Consequently, very

high pressure intensities are necessary to markedly reduce peroxidase activity in legumes and to

prevent the product from quality loss due to enzymatic oxidation. Regulation of peroxidase in the

seeds is of importance as its activity is often connected to a loss of ascorbic acid and other

antioxidants during storage (Quaglia et al., 1996; Krebbers et al., 2002; Gökmen et al., 2005).

Pressure treatments of the tested intensities may diminish lipid oxidation but cannot replace

blanching as a tool to achieve long-time stability of peas, for instance under cold storage.

Nevertheless, in case high pressure is applied to alter functionality of the flours, a reduced

oxidation of lipids can be seen as an additional benefit.

Changes in the enzymatic digestibility of pea protein and starch

after heat and pressure application

Flours from differently processed peas were subjected to a simulated digestion procedure to

investigate, whether a thermal or pressure pre-treatment does affect the digestibility of the main

macro-nutrients in peas, proteins and starch. The conditions during these investigations were

kept as similar as possible to the human digestion, but several simplifications were necessary due

to economic and practical concerns. A scheme of the digestion procedure is shown in Figure VII.11

and will be explained in detail in the following paragraph. All enzymes connected to the digestion

of lipids were not considered in this experimental plan.


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The digestion of food starts with addition of saliva in the human mouth. Human saliva possesses a

neutral to slight acidic pH value and contains a huge number of different electrolytes and several

enzymes. α-amylase, that hydrolyses bonds between α-linked polysaccharides, causes a viscosity

decrease of the chyme (Rehner & Daniel, 2010). Its activity in the human saliva may vary due to

external influences (Nater et al., 2006; Rohleder et al., 2006) and was set to 100 U/mL in the

present experiment. It is the only saliva enzyme of importance in quantitative terms (Rehner &

Daniel, 2010). Hence, amylase was the only enzyme used in the first step of the simulated

digestion. The retention time of the chyme in the mouth is very short, so samples were given to

the next digestion step already after 30 s of amylase incubation time.

Figure VII.11: Buffers, solutions and enzymes added during the different steps of the simulated digestion.

In the stomach, the chyme is mixed with hydrochloric acid and pepsin. Their concentrations in the

gastric juice are different between individuals and during the day (Alvarez et al., 1935). The

average gastric juice has an acid concentration of 0.5 % leading to pH values of 2-4 in the

stomach. This was achieved by mixing the sample with hydrochloric acid of the respective

concentration in the ratio of 1:1. The pepsin concentration varies between 50 and 300 mg/L

α-amylase

phosphate buffer pH 7

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(Carter, 2014). Pepsin from porcine gastric mucosa with a concentration of 250 mg/L was added,

resulting in a final concentration of 12.5 mg/L in the chyme-gastric juice mixture. The slow

peristaltic movement of the stomach was simulated by simple rotation on a shaker plate. One

main function of the stomach is to deliver small portions of chmye to the duodenum (Rehner &

Daniel, 2010). Consequently, the retention time in the stomach extends over a period of 0.5-6 h

(BKK, 2014). In this experiment the reaction time was set to 1 h.

The great variations in stomach retention lead to inhomogeneities in the gastric chyme, especially

in regard to pH value and degree of digestion, when entering the duodenum. The pH is balanced

to neutral ranges by secretion of bicarbonate ions from the pancreas. In the experiments 150 mM

bicarbonate solution was added in a ratio of 1:1. Macromolecules are further hydrolysed by the

addition of α-amylase, trypsin, chymotrypsin, proelastase and procarboxypeptidase (Rehner &

Daniel, 2010). The latter two were not used within the experiments due to simplification of the

procedure.

The digestion with enzymes bound in the brush border of the small intestine directly follows.

Fragments of starch were digested to monosaccharides by maltase, isomaltase and sucrase

(Rehner & Daniel, 2010). The digestion of peptides into small fractions is more complex due to the

high number of existent primary structures in food proteins. More than 20 different proteolytic

enzymes are available in the epithelial cells (Rehner & Daniel, 2010). Dipeptidyl peptidase was

chosen as representative for the simulated digestion. This enzyme cleaves dipeptides from the n-

terminal chain, when proline or alanine residues are located at position two of the chain (Sigma-

Aldrich, 2014). The degradation of starch and protein in the small intestine were followed over a

time period of 3 h. The effect of digestion is reflected in an increase of accessible amino groups

and reducing sugars. These photometric methods require a clear, centrifuged sample, so that only

soluble parts of the flour could be analyzed.

The enzymatic degradability of protein in the first digestion steps is shown in Figure VII.12. The

initial number of amino groups in the untreated control was slightly higher compared to flours

previously subjected to heat or pressure. This can be traced back to the overall higher solubility of

native pea protein, whereas treatments at 80 °C or 600 MPa reduced protein solubility at neutral

pH values (see chapter VI). Protein that did not go into solution within 30 s of mixing was

separated during subsequent centrifugation and not determined by the analytical method. As

addition of amylase in the mouth does not lead to cleavage of peptide bonds, the slight increase

in amino groups can be traced back to an improved solubilization after further 30 s of

homogenization with buffer and enzyme.


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The number of accessible amino groups nearly doubled during 60 min of gastric digestion. Pepsin

hydrolyses proteins into oligopeptides and has a strong acidic activity optimum close to pH 2

(Rehner & Daniel, 2010). Nevertheless, considering the long incubation time, the effect of this

enzyme is rather small. Pepsin exhibits a higher activity in the degradation of animal protein,

especially of collagen (Rehner & Daniel, 2010). Hence, its meaning for the digestion of vegetable

proteins, like those from pea flour, is rather small. The effect of acid protein hydrolysis in the

stomach is said to be generally overestimated (Rehner & Daniel, 2010), but the protein solubility

might have been altered due to the very low pH value. Legume proteins possess best solubility at

either alkaline or very acidic pH values (Gueguen, 1983). An enhanced solubility might have led to

a higher presence of protein and therefore of accessible amino groups in the supernatant.

Figure VII.12: Influence of thermal and pressure treatments of whole peas on the protein digestibility in the respective dry pea flour. Peas were swollen for 20 h in tap water and subjected to 80 °C for 10 min (heat treated), 600 MPa at 40 °C for 10 min (pressure treated) or not further treated (untreated); drying of whole peas for 6 h at 50 °C; digestion according to Figure VII.11 and annex page 256.

No marked changes in the hydrolysis of untreated and pre-treated samples were determined. The

increase in amino groups compared to the mouth was highest for pressure treated samples, but

did not result in an overall improvement of digestibility in the following digestion steps. Improved

pepsin hydrolysis after pressurization was reported for whey protein isolate and β-lactoglobulin

(Chicon et al., 2008; Zeece et al., 2008), but the higher affinity of the enzyme towards animal

proteins might also influence the effectiveness of previous processing. An increased hydrolysis of

soybean protein was found as well by Penas et al. (2004), but pressures used in these experiments

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were 200 MPa at maximum. Unfolding of the protein structure at low pressure intensities might

have improved accessibility for the enzyme, whereas aggregation at higher pressures might have

counteracted this process advantage. An enhanced digestion after thermal treatment, as is was

detected for cooked mungbeans (Kataria et al., 1989), was also not found. Possibly, the intensity

of a 10 minute treatment at 80 °C is too small compared to usual legume cooking times of 1 h in

boiling water.

A more frequent sampling would enable a more detailed investigation of the enzymatic

proteolysis and could reveal positive effects, for example on the enzyme kinetics. A more rapid

pepsin hydrolysis would enable protein degradation already of the first chyme portions reaching

the duodenum. Assuring cleavage into oligopeptides of all proteins could help to minimize the

product’s allergenic potential, as a certain chain length is needed to cause an immunological

reaction (Rehner & Daniel, 2010).

Figure VII.13: Protein digestibility in pea flours made of heat and pressure treated whole peas. Peas were swollen for 20 h in tap water and subjected to 80 °C for 10 min (heat treated), 600 MPa at 40 °C for 10 min (pressure treated) or not further treated (untreated); drying of whole peas for 6 h at 50 °C; digestion according to Figure VII.11 and annex page 256.

Digestion in the duodenum with trypsin and chymotrypsin and in the small intestine with

peptidase resulted in an equal number of amino groups for all samples (see Figure VII.13).

Legumes are known to possess trypsin inhibitors, whose activity can be strongly reduced by heat

application. A slight to marked improvement in digestibility was therefore reported for legume

products after ordinary cooking (Rehman & Shah, 2005; Martin-Cabrejas et al., 2009). Again, the

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treatment intensity applied might not be sufficient to achieve this goal. Inactivation of trypsin

inhibitors in soybeans required processing times of 30 and 22 min at 100 or 110 °C, respectively

(Liu, 1997). The enzyme is also very resistant to pressure application in the range of 200 to

800 MPa due to a high number of disulphide bonds (van der Ven et al., 2005; Yin et al., 2008).

Therefore, an improved digestion due to trypsin inhibitor inactivation is unlikely for the treatment

intensities used in this investigation. The trypsin inhibitors in the related soybean do only slightly

affect the activity of pepsin or chymotrypsin (Kunitz, 1947). Changes in the activity caused by heat

or pressure might therefore as well be masked by the activities of the other enzymes. As already

mentioned, a huge number of peptidases would be necessary to simulate realistic conditions of

the small intestine. Some alterations of certain amino acids, for example due to the formation

disulphide bonds, might not be determined with the experimental plan developed. A more

comprehensive digestion plan would be necessary to definitely exclude advantages or

disadvantages of both treatments. At the present stage, the pre-treatments did not alter the

enzymatic digestibility of pea protein – neither in a positive nor in a negative way.

Besides protein, starch is the other macromolecule in legumes that is relevant for the product’s

energy content and nutritional value. Its degradation during processing and digestion can be

followed via the increase in the reducing sugar content. Figure VII.14 shows the digestibility of pea

starch from pre-treated seeds in the first four stages of the simulated digestion. The initial

content in reducing sugars was twice as high in flours from pressure treated peas in comparison

to those from heated and control samples. Gomes et al. (1998) also found a marked increase in

the reducing sugar content of wheat and barley flour after a ten minute exposure to pressures

from 400 to 600 MPa. This increase can be either a result of pressure activation of endogenous

amylases or of a better accessibility of the starch to those enzymes after pressure induced

gelatinization. Buckow (2006) reported a decreased activity of α- and β-amylase with rising

pressure at non-denaturing temperatures. The higher proportion of digested starch in pressure

treated peas can therefore be traced back to a better accessibility of the starch granules to

enzymatic conversion. This hypothesis is also confirmed for the digestion in the mouth, where

saliva α-amylase was added. The increase in reducing sugars in pressure and heat treated samples

amounted approximately 1.5 times of that of the untreated samples. The digestibility of starch is

positively correlated to its degree of gelatinization (Holm et al., 1988; Chung et al., 2006; Sandhu

& Lim, 2008), an effect only observed in the peas exposed to intensive processing (see Figure

VII.4). An absent raise in the initial starch degradation after heating might be caused by thermal

inactivation of endogenous enzymes at a temperature of 80 °C.

A further progress of starch digestion was observed in the stomach. This degradation is still

caused by the saliva amylase, which remains active to a certain degree in the strong acidic


199

environment of the gastric juice (Rehner & Daniel, 2010). High standard deviations might arise

from differences in time of sample taking and inhomogeneities of the temperature distribution

under the heating jacket. The highest increase was observed in the pressure treated peas,

whereas a similar hydrolysis was found after heat treatment and treatment at ambient

temperature. Anew addition of α-amylase led to further starch digestion in the duodenum. Again,

the effectiveness of digestibility was highest for the pressure treated samples. This result is rather

unexpected as previous observations indicated a higher degree of gelatinization after heat

treatment than after pressurization.

Figure VII.14: Starch digestibility in pea flours exposed to heat and pressure treatments. Peas were swollen for 20 h in tap water and subjected to 80 °C for 10 min (heat treated), 600 MPa at 40 °C for 10 min (pressure treated) or not further treated (untreated); drying of whole peas for 6 h at 50 °C; digestion according to Figure VII.11 and annex page 256.

A marked increase in starch digestibility in different legumes can be obtained by ordinary cooking

(Bravo et al., 1998; Rehman & Shah, 2005). It was already mentioned before that conventional

cooking implies higher treatment intensities in respect to temperature and time than the thermal

treatment applied within this experiment. A limited amount of available water may also diminish

the positive effect of a heat treatment. Contrary effects on digestibility were reported for heat-

moisture-treatments of different sample materials (Lehmann & Robin, 2007; Chung et al., 2009; Li

& Gao, 2010). Therefore, smaller effects in comparison to literature data may be the result of

shorter processing at lower temperatures or the restricted amount of water in the soaked seeds.

In general, the comparability of results between several working groups is limited due to

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differences in the treatment conditions, the digestion method and enzymes used as well as in the

composition of the sample.

Several further explanations can be found for the higher digestibility of the pressurized sample.

The rate of enzymatic activity is negatively correlated to the molecular weight of amylose and

amylopectin (Sandhu & Lim, 2008). Hence, the already proceeded starch degradation in

pressurized flour might further improve the effectiveness of the digestion procedure. The

increased water retention of flour from heat treated peas could also affect enzymatic activity (see

Figure VII.1). Less availability of water during digestion increased the reaction constant for the

enzymatic starch degradation by factor 2.5 to 4, independent from the degree of gelatinization

(Singh et al., 2010). Starch from pressurized peas may also be more susceptible to acid hydrolysis

than the other samples and thus be easier digested in the stomach. Processing might also have

led to the formation of a protein barrier around the starch granules that affects digestibility (Singh

et al., 2010). This effect may be more pronounced for heat aggregated proteins. In conclusion, the

best amylase digestibility was found for pressure treated samples. This technological effect might

lead to a faster viscosity decrease of the chyme, altering, for instance, mouth feeling or the

difficulty level of swallowing of a product. It offers as well the opportunity to improve a target

industrial enzymatic hydrolysis of starch to maltose.

The number of reducing sugars increased during digestion in the small intestine in all samples

(Figure VII.15). Degradation curves for pressure processed and control samples follow a similar

progress – although the one of the untreated pea flour is on an overall lower level. The highest

enzymatic digestibility was found for heat treated samples. Hydrolysis into small saccharides in

this section of the simulated digestion is enabled by interaction of the pancreatic α-amylase and

the intestinal enzymes, separating smaller sugar subunits. Thermally gelatinized starch seems to

be more susceptible for a hydrolysis by these enzymes. Takahashi et al. (1994) found similar

maltase digestibility of corn starch after 600 MPa or 100 °C processing. Selmi et al. (2000)

reported a slower digestion of corn starch and an incomplete digestion of wheat starch after

pressure application of 600 MPa compared to heat treatment at 80 °C using amyloglucosidase.

High pressure seems to specifically promote the activity of α-amylase, whereas the digestion into

monosaccharides is markedly enhanced for heat gelatinized pea starch. The improved digestibility

of legume starch after different processing was also traced back to a decrease in amylase inhibitor

activity (Hoover & Zhou, 2003). Both, the lectin-like and the proteinaceous inhibitor forms

investigated in beans and cereals, respectively, consist of non-covalently bound subunits (Franco

et al., 2002). High pressure may have led to an inactivation of these inhibitors, as it favours

dissociation into monomeric structures (Penniston, 1971; Messens et al., 1997). Inhibitors

affecting mammalian amylase are usually highly specific and do not influence the activity of plant,


201

bacterial or fungal enzymes (Marshall & Lauda, 1975). Due to a deficiency in human or animal

products, the intestinal digestion had to be simulated using enzymes of vegetable and microbial

origin. This could explain why starch degradation into monomers was highest for heat treated

samples, as the rate of hydrolysis might be more dependent on the degree of gelatinization than

on inhibitor activity.

Figure VII.15: Influence of thermal and pressure treatments of whole peas on the starch digestibility in dry pea flour. Peas were swollen for 20 h in tap water and subjected to 80 °C for 10 min (heat treated), 600 MPa at 40 °C for 10 min (pressure treated) or not further treated (untreated); drying of whole peas for 6 h at 50 °C; digestion according to Figure VII.11 and annex page 256.

Starch that is not hydrolyzed into glucose within 120 minutes of digestion is classified as resistant

starch (Englyst et al., 1992). These starches cannot be enzymatically degraded due to deficient

gelatinization, physical inaccessibility or retrogradation, are not adsorbed by the intestine and

function in the human body as dietary fibre (Berry, 1986; Englyst et al., 1992). Positive effects

ascribed to resistant starch are the reduced glycemic response, an improved blood lipid profile,

prebiotic effects, increased satiety, reduced energy intake, improved bowel health and

micronutrient adsorption (Fuentes-Zaragoza et al., 2010). According to the classification of Englyst

et al. (1992), heat treated pea flour contains less resistant starch than the other samples. Further

observation of the enzymatic hydrolysis would be necessary to reveal whether all samples would

somewhen reach the same end value. Nevertheless, the results are also in agreement with the

techno-functional results, as resistant starch possesses a lower water binding capacity (Fuentes-

Zaragoza et al., 2010). The pressure processed pea starch unifies the advantages of a high α-

0.0

0.2

0.4

0.6

0.8

1.0

0 50 100 150 200

redu

cing

suga

rs in

mm

ol /

g dr

y m

atte

r

retention in small intestine in min



202

amylase digestibility with a high content in resistant starch. Legumes naturally contain high

amounts of slow digestible and resistant starch (Bravo et al., 1998; Polesi et al., 2011), but for

other starch sources or highly processed foods, pressure treatment might be a promising option

to reduce the amount of digestible starch and thereby the energy content of the food and its

influence on the glycemic index.

In conclusion, high pressure is a promising tool to modify the digestibility of the starch fraction in

peas without affecting protein digestion. Contemporaneously, the techno-functional properties of

native pea flour can be maintained to a higher extent in comparison to a thermal treatment. The

activity of endogenous enzymes, negatively affecting protein quality, can be reduced, though not

to a similar degree as by a blanching process. Pressurization can thereby contribute to the

production of pea products and flours with unique functionality, suitable for novel application

possibilities.

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Chapter VIII – The potential of HP and PEF for protein processing – conclusion and perspective

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Chapter VIII

The potential of high pressure

and pulsed electric fields for

protein processing – conclusion

and future perspective


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The aim of this thesis was to evaluate the potential of innovative food technologies for the

processing of vegetable proteins. The influence of pulsed electric fields, high isostatic pressure

and heat on proteins of potatoes and peas was investigated on the basis of protein solutions as

well as in complex plant tissue. The results pointed out several benefits of the emerging

technologies in comparison to conventional thermal processing.

Pea protein in solution or in the plant matrix did not show any changes after exposure to a pulsed

electric field. Solubility, protein composition or foaming properties were not influenced by pulsed

electric field treatments at the applied intensities (see chapter III and IV). Electroporation of the

membrane can thereby be used to achieve other process benefits without negatively affecting

protein recovery or quality. Promising results obtained within this thesis are enhanced drying and

rehydration rates of whole peas and a leaching of flatulence causing sugars during treatment

while simultaneously losing no marked amounts of protein (see chapter V). Using higher

treatment intensities to inactivate vegetative microorganisms in protein solutions and accordingly

reduce the microbial load for the following processing steps is another application possibility

worth pursuing. Unfortunately, no marked reduction in the microbial activity was achieved with

the available equipment due to the high conductivity of the sample solutions and the rapid

reduction of the electric field. So the impact of these treatment intensities on protein quality

could not be sufficiently investigated.

In contrast to pea protein, the conformation of potato protein was altered in a pulsed electric

field (see chapter III). This can probably be traced back to irreversible structural changes induced

by local pH shifts. Nevertheless, a clear impact of the structural modification on the foaming

properties was not found. When treatments were applied to whole potato tubers, a permanent

alteration of the protein was caused that persisted subsequent pressing, concentration and drying

steps and led to the production of a protein concentrate with improved foamability (see

chapter VI). Enhanced interaction with other potato ingredients, for instance lipids or

polyphenols, in the electric field may have caused an irreversible modification of the protein

structure. Further research is necessary to clarify the underlying mechanism on a molecular level.

The influence of high pressure on the solubility, surface hydrophobicity and extent of aggregation

in potato protein solutions was markedly lower than that of a thermal treatment (see chapter III).

Especially when the pH value or ionic strength was varied or the sample was of less purity, high

pressure was clearly superior in preserving protein quality and delivering consistent processing

effects. A strong reduction in colony forming units of vegetative indicator microorganisms was

achieved by treatments at 400 MPa and 40 °C for a dwell time of 10 minutes, in ringer as well as

in protein solutions (chapter IV). High pressure is therefore a suitable alternative for the


213

pasteurization of potato protein solutions. As potato products also undergo enzymatic browning

reactions and possess trypsin inhibitor activity, these indicators for a successful pasteurization

should also be investigated before an implementation of high pressure preservation.

Both, heat and pressure treatments, led to the formation of aggregates in pea protein solutions,

negatively affecting foam stability (see chapter IV). Depending on the initial product, concentrate

or protein enriched flour, and its protein composition, aggregation was accompanied by a marked

reduction in protein solubility. Non-reducing SDS PAGE revealed that thermal treatments induce

structural changes of the pea albumin PA2, whereas high pressure affected the pea globulins

legumin and vicilin (chapter III). Similar electrophorese profiles were obtained when extracts from

treated whole peas were analyzed. This offers the possibility for a protein modification within the

plant tissue and the recovery of concentrates with an altered solubility profile (chapter VI).

Differences in starch gelatinization between heat and pressure further enable the production of

pea flour of particular techno-functional and nutritional quality (see chapter VII). A sufficient

inactivation of the endogenous pea enzymes peroxidase and lipoxidase by high pressure was not

achieved. Therefore, pressurization does not offer an equivalent alternative to conventional

blanching in regard to avoiding enzymatic quality losses.

Options for an integration of both technologies into existent processing concepts are presented in

Figure VIII.1 and Figure VIII.2. The flow chart for potato processing is based on the general

technological steps performed in starch manufacturing producing protein rich potato fruit juice as

a by-product (Witt, 1996; Bergthaller et al., 1999). Pulsed electric fields can be applied in the early

steps of manufacturing prior to grinding of the tubers. Technological concepts are already

available to treat whole potatoes with a capacity sufficient for the starch industry (Loeffler, 2006).

A reduction in the tubers’ cutting resistance (Janositz, 2005) and an improved separation of fruit

juice can save process time and costs. Modification of the protein can lead to a protein product

with improved foamability. The influence of these alterations on other techno-functional aspects

and the protein’s nutritional value need to be further investigated. Understanding of structural

changes on a molecular level would help to carry out a complete evaluation of the product

modifications induced. Furthermore, the effect of an electric field treatment on the recovery and

properties of starch, the main potato ingredient, has to be analyzed. At present, no effect on

starch quality is reported in literature for the parameters applied for cell disintegration and

protein alteration, but Han et al. (2009) found changes in the granular structure for higher PEF

treatment intensities. Further experiments should be performed to exclude a negative impact of

the treatment parameters chosen.


214

Figure VIII.1: Possible ways to implement pulsed electric fields and high pressure into existent potato protein processing strategies.

High pressure preservation could be applied prior to the drying step to reduce the microbial load

and the enzymatic activity in the protein concentrate and contemporaneously maintain protein

quality. To keep process efficiency maximal, the protein should be concentrated via membrane

techniques prior to pressurization. Determination of the maximum protein concentration suitable

for a processing without functional degradation is therefore necessary. Additionally, the minimum

process intensity necessary in regard to pressure, temperature and time has to be figured out to

make the preservation step as cost-efficient as possible.

The integration of emerging technologies into pea processing is more complex due to a high

number of possible production chains. Application of high pressure and pulsed electric fields

require the intake of water in the tissue structure. The minimum moisture content needed for the

treatments has to be determined to avoid dispensable swelling and drying. Tissue

permeabilization by pulsed electric fields can increase the diffusion of flatulence causing sugars

into the surrounding medium. Additional washing and incubation may even enhance the removal

washing

pulsed electricfields

rasping

fruit juiceseparation

membraneconcentration

spray drying

high pressure

potatoes

starch +pulp

proteinsolution

proteinconcentrate

reduced cuttingresistance

improved solid-liquid

separation

increasedfoamability

enhanced shelf-life


215

Figure VIII.2: Possibilities to implement pulsed electric fields and high pressure into different pea process options.

of small molecules. Detailed investigations have to figure out the approximate size of formed

pores and quantify the loss of other low molecular weight substances, especially those with

positive health effects. The rates of the following drying and rehydration procedures of whole

peas would be increased as well. This may on the one hand save processing costs for the industry,

on the other hand save time for preparation of meals in private households. A similar approach

swel

ling

pulse

del

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lds

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peas

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grin

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peas

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grin

ding

prot

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ate

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ein

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216

already existed in the former German Democratic Republic, where pea products with reduced

swelling and cooking times were commercially available (MWFK, 2014). The higher flexibility and

spontaneity thus obtained may increase the attractiveness of legumes for the consumer.

Similar benefits can also be realized by high pressure treatment of whole peas. Furthermore, a

modification of the starch digestibility is attained, leading to differences in the available amount

of energy and in the glycemic index. Mechanical disintegration of the seeds can lead to flours with

slightly improved water binding and modified gelation properties. Wet grinding of the treated

peas directly after processing is then recommended to increase the specific surface prior to the

subsequent drying process. Air classification may additionally be applied to obtain flour fractions

with different protein and starch content. Compared to a thermal treatment, changes in the

techno-functional behaviour due to pressure are rather small. When pre-treatments are applied

to reduce microbial and enzymatic activity, minor functional alterations after pressurization can

also be regarded as an advantage of this technology.

The wet flour from pressurized peas may as well be used to recover a protein concentrate with

higher albumin content and with higher solubility at slight acidic pH ranges compared to protein

products from untreated peas. A raised percentage of sulphur-containing amino acids will also

contribute to an improved biological value of the product. The impact of a different composition

of the concentrate on its allergenic potential still has to be investigated to exclude negative

effects for the consumer. In contrast to this thesis, the concentrated protein should be spray-

dried instead of applying a lyophilization to reduce process costs. Previous results did not show

marked differences between the concentrates obtained by both drying techniques, probably due

to the very short exposure to heat during spray drying. The globulins aggregated during pressure

treatments are separated from soluble protein along with other insoluble components like starch

and fibre. A suitable application has to be found for this product stream to make the overall

processing concept economically advantageous. This fraction might, for instance, be used for a

protein-enrichment or to improve the properties of solid foods, like extruded or bakery products.

Both technologies may also be applied to concentrated protein solutions. The recovery of starch

and other insoluble pea components will thereby not be affected by the treatments.

Permeabilization of microbial cell membranes in the electric field may lead to an increase of the

product stability by avoiding rapid growth of dormant microorganisms when the protein

concentrate is inserted in products with high moisture content. Here again, high pressure

treatment will lead to a fractionation into insoluble globulins and an albumin-enriched fraction.

Detailed analysis with protein solutions differing in concentration and composition have to be

performed to clarify the mechanism of protein interactions and to enable a target aggregation of


217

the pea globulins. Application possibilities for both concentrates have to be identified for a

holistic usage of all product streams.

Knowledge about the treatment intensities used allows a rough estimation of the processing costs

arising from the application of emerging technologies. Costs for warming or cooling of the

Table VIII.1: Estimated costs for pulsed electric field treatment on industrial scale

Tissue disintegration Protein solutions

Whole potatoes Whole peas Preservation

Capacity [kg/h] 50 000 2 000 4860

Generator power [kW] 80 80 150

Investment costs [M €] 0.75 0.75 0.45

Depreciation period [a] 5

Electric field strength [kV/cm] 1 10 25

Treatment temperature [°C] ambient ambient 35

Specific energy input [kJ/kg] 5 125 110

Energy costs [ct/kg] 0.02 0.60 0.50

Depreciation charge [ct/kg] 0.06 1.56 0.39

Wear parts [ct/kg] 0.06 1.56 0.39

Total costs [ct/kg] 0.15 3.71 1.28

Ratio product to treatment medium [-] 0.2 0.35 1

Product dry matter content [kg/kg] 0.22 0.45 0.01

Total costs for final dry product [€/kg] 0.03 0.25 1.28

Information about treatment intensity and equipment for preservation of liquids are according to Toepfl (2013). Investment costs for tissue integration in a continuous PEF unit suitable for starch manufacturing shall be around 750 000 € (personal communication with Stefan Töpfl, Deutsches Institut für Lebensmitteltechnologie, Quakenbrück, Germany). Calculations are based on an estimated process efficiency of 62.5 %, on a yearly operation duration of 4800 h and an energy price of 10.5 ct/kWh. Costs for wear parts were expected to amount 20 % of investment costs.


218

samples were not considered in the calculations. As already mentioned, continuous pulsed

electric field equipment, adequate for implementation in potato starch manufacturing, is

commercially available. High capacities and a comparably low energy input lead to processing

costs of a few cents per kg of product, even regarded on a dry weight basis (see Table VIII.1).

Disintegration of pea tissue was conducted with higher energy inputs in laboratory scale. Using

the same PEF equipment with a maximum generator power of 80 kW would limit the capacity to

2 t/h and would result in correspondingly higher processing costs. A higher generator

performance would, in return, cause higher investment costs. However, the estimated costs of

25 ct/kg dry pea exceed what is reasonable for a removal of oligosaccharides and a reduction of

preparation times. The minimal energy input necessary should be figured out to decrease energy

costs and to be able to increase the processing capacities. Furthermore, the moisture content and

the ratio of peas to treatment medium should be optimized to enhance process efficiency.

The same applies for the high pressure treatment of whole peas (see Table VIII.2). Optimization in

regard to dwell time and unit utilization is necessary. The increase in value due to a modification

of protein and starch can hardly be estimated, what hinders efficiency analysis. Nevertheless, it

can be assumed that additional costs of 80 ct/kg for pea flour are markedly too high, especially in

regard to an implementation in staple foods like bread or pasta. Currently, a high pressure

modification would only be feasible for foods with special purposes, for instance, products for

weight loss or diabetics that usually obtain higher prices. Costs for processing of protein solutions

with the aim of decontamination or modification before drying are extremely high for both

technologies applied, mainly due to the low protein concentration used in the experiments within

this thesis. Although the price for high quality plant proteins is with a few €/kg relatively high,

process efficiency has to be increased by enhancing the protein concentration during processing.

This thesis presented the great potential of high pressure and pulsed electric field treatment for

an implementation in protein processing, but also revealed the need for further research. Some

techno-functional relations, material flows of important ingredients and mechanisms of molecular

interactions still need to be clarified and more detailed considerations of economic aspects and

industrial feasibility are required. Investigations should be extended to the recovery and

processing of other innovative protein sources like leaves, insects or microorganisms. A functional

modification of animal proteins with the aim of material savings seems as well reasonable to

reduce the usage of foods from the livestock sector. Novel agricultural and technological

approaches are needed to increase the consumption of vegetable foods and proteins in the long

term. Innovative protein production cannot only be achieved with conventional processing

strategies, but requires the application of emerging concepts and technologies. High isostatic


219

pressure and pulsed electric fields may thereby deliver a valuable contribution to the production

of high quality proteins and the switch to a more sustainable food production.

Table VIII.2: Estimated processing costs for a high pressure treatment in an industrial unit of the type wave 6000 model 300 (Hiperbaric, Burgos, Spain)

Whole peas Protein solutions

Modification Preservation

Vessel size [L] 300

Vessel filling ratio [%] 50

Intensifier power [kW] 6 x 45

Investment costs [M €] 1.476

Depreciation period [a] 5

Pressure [MPa] 600 600 400

Starting temperature [°C] ambient ambient 30

Dwell time [min] 10 10 7.5

Cycle time [min] 13.8 13.8 10

Energy consumption [kWh/cycle] 16.1 16.1 11

Energy costs [ct/kg] 1.1 1.1 0.75

Depreciation charge [ct/kg] 9.2

Wear parts [ct/kg] 6.4

Total costs [€/kg] 0.17 0.17 0.12

Ratio product to treatment medium [-] 0.35 1 1

Product dry matter content [kg/kg] 0.45 0.01 0.01

Total costs for final dry product [€/kg] 0.80 16.7 12.1

Calculations are based on the information supplied by the manufacturer (November 2009), on an estimated pump efficiency of 62.5 %, on a yearly operation duration of 4800 h and an energy price of 10.5 ct/kWh.


220

References

Bergthaller, W., Witt, W. & Goldau, H. P. (1999). Potato starch technology. Starch-Starke, 51(7), 235-242.

Han, Z., Zeng, X. A., Yu, S. J., et al. (2009). Effects of pulsed electric fields (PEF) treatment on physicochemical properties of potato starch. Innovative Food Science & Emerging Technologies, 10(4), 481-485.

Janositz, A. (2005). Auswirkung von Hochspannungsimpulsen auf das Schnittverhalten von Kartoffeln (Solanum tuberosum). Diploma thesis, Berlin, Technische Universität Berlin.

Loeffler, M. J. (2006). Generation and application of high intensity pulsed electric fields. In J. Raso & V. Heinz: Pulsed electric fields technology for the food industry - Fundamenatls and applications (27-72). Springer Science and Business Media, New York, USA.

MWFK (2014). Die Küche des Atomzeitalters: Tempoerbsen und Kuko-Reis. Source: http://www.mwfk.brandenburg.de/sixcms/detail.php/534701. Accessed on: April 2014.

Toepfl, S. (2013). Process design by innovative techniques. Source: http://2013.ifoodconference. com/fileadmin/user_upload/2013/PDF/iFood_Toepfl_2013_04_16.pdf. Accessed on: April 2014.

Witt, W. (1996). Stärkegewinnung. In H.-D. Tscheuschner: Grundzüge der Lebensmitteltechnik (363-372). B. Behr's Verlag GmbH & Co, Hamburg, Germany.

Annex A – Material and Methods

221

Annex A Material and Methods


222

Material

Waxy potatoes cultivar Belana were purchased from a local supermarket (Metro Berlin-

Marienfelde, METRO Cash & Carry Deutschland GmbH, Düsseldorf, Germany) in package sizes of

10 kg. Potatoes were stored in the basement at temperatures around 16 °C, low air moisture and

little light irradiation until use. The chemical composition was not investigated.

Dry yellow peas cultivar Salamanca were provided by Norddeutsche Pflanzenzucht (Hans Georg

Lembke GmbH, Holtsee, Germany). Endosperm and shell fraction had water contents of 0.129 and

0.091 g/g and protein contents of 0.274 and 0.147 g/g dry matter, respectively (personal

communication with Annika Weckmüller, Universität Potsdam). This results in a protein content

of approximately 0.26 g/g dry matter for the whole peas. Peas were stored in plastic bottles at

room temperature until use.

Figure A. 1: Production scheme of protein-enriched pea-flour.

Protein enriched pea flour produced according to Figure A. 1 was purchased at Institut für

Getreideverarbeitung (IGV, Potsdam, Germany). Protein contents of 32.8 and 51.5 % were

determined in batches one and two, respectively, using the Kjeldahl method.

Commercially available potato protein isolate (SOLANIC 206P, Mat: 102425667, Batch: 145498)

was provided by Nestlé Research Center (NRC, Lausanne, Switzerland).

Self-made protein concentrates were produced according to page 233.

shelling

milling in two steps1. < 2.0 mm2. < 0.5 mm

pulverization

air classificati on

peas

protein-enriched

flour

protein-depleted

flour


223

All protein concentrates and protein enriched flours were vacuum-packed and stored at

temperatures between 2 – 6 °C until use.

Buffer systems All buffers were stored at room temperature until use if not otherwise noted. A pH meter

(Digital-pH-meter, Knick Elektronische Messgeräte, Berlin, Germany) was used to adjust the

correct pH values.

TRIS-HCl buffer for protein extraction and thiol group determination:

To determine protein extractability, a 0.05 M TRIS-HCl buffer covering the pH range 7.2 – 9.0 was

prepared. 25 mL of 0.2 M TRIS solution (Carl Roth GmbH & Co KG, Karlsruhe, Germany) and

2.5 mL of an equimolar hydrochloric acid solution were filled up to 100 mL with deionized water.

A 0.2 M buffer was used for the quantification of thiol groups. 12.11 g of TRIS were solved in

approximately 400 mL of deionized water. A pH value of 8 was adjusted with 1 M hydrochloric

acid and the overall volume filled up to 500 mL. For protein unfolding 1 % SDS (Carl Roth GmbH &

Co KG, Karlsruhe, Germany) was added in a mass ratio.

0.05 M sodium acetate buffer (pH 4.5) for oligosaccharide extraction:

0.05 M solutions of acetic acid (96 % (v/v), Merck KGaA, Darmstadt, Germany) and sodium

acetate (water free, Merck KGaA, Darmstadt, Germany) were mixed to a final pH of 4.5. The ratio

between acetic acid and acetate was approximately 1:0.8.

0.01 M phosphate buffer (pH 7.0) for surface hydrophobicity:

0.01 M solutions were prepared from sodium hydrogen phosphate and sodium dihydrogen

phosphate (both Merck KGaA, Darmstadt, Germany). Solutions were mixed to finally give a pH of

7.0. The ratio between sodium hydrogen phosphate and sodium dihydrogen phosphate amounted

approximately 3:2.

0.05 M citrate-phosphate-buffers (pH 3 to 7) for solubility test and foam analyses:

0.05 M solutions of citric acid (Merck KGaA, Darmstadt, Germany) and sodium hydrogen

phosphate (Merck KGaA, Darmstadt, Germany) were mixed in an appropriate ratio (see Table A.

1). Citrate buffers were stored at 2 - 8 °C due to the hazard of spoilage with mould fungi.

Citrate-phosphate buffers of higher molarity were prepared according to the same procedure.


224

Table A. 1: Approximate ratios for phosphate citrate buffer preparation of different pH values

pH value 3 4 5 6 7

Ratio sodium phosphate : citric acid 1:2 1.25:1 2:1 3.5:1 10:1

Buffers and reagents for SDS-Pages

SDS sample buffer (2x concentrated) pH 6.8:

All compounds listed in Table A. 2 were mixed in the denoted ratio. Aliquots of 500 μL were

frozen and stored at -20 °C. The sample buffer was given to the protein sample in a ratio 1:1

before analysis.

Table A. 2: Composition of the sample buffer for the non-reducing SDS page

Compound Amount Concentration (2x)

0.5 M TRIS-HCl (solution 1) 2.5 ml 0.126 M

10 % (w/v) SDS (solution 2) 4 ml 4 %

Glycerol (99 %) 2 ml 20 %

0.1 % (w/v) Bromphenol blue (solution 3) 0.5 ml 0.005 %

Deionized water fill to 10 ml

Solution 1 was made by diluting 3 g of TRIS (Carl Roth GmbH & Co KG, Karlsruhe, Germany) into

30 mL of deionized water. The pH value was set to 6.8 with 4 N hydrochloric acid (≙ 14.4 %) and

filled up to 50 mL with deionized water.

For preparation of solution 2 and 3, 1 g of sodium dodecyl sulphate (SDS; Carl Roth GmbH & Co

KG, Karlsruhe, Germany) and 0.01 g of Bromphenol blue (Sigma-Aldrich, St. Louis, Missouri, USA)

were each diluted in deionized water to give an overall volume of 10 mL.

SDS electrode buffer (10x concentrated) (running buffer) pH 8.3:

The running buffer contained 25 mM TRIS, 192 mM Glycine (Merck KGaA, Darmstadt, Germany)

and 0.1 % (w/v) SDS. 29 g of TRIS and 144 g of glycine were diluted under stirring in deionized

water. 10 g of SDS were added under gentle stirring to avoid foam formation and filled up to 1 L in

a volumetric flask. The running buffer is stable for 6 months at room temperature and has to be

diluted 1: 10 with deionized water before usage.


225

Fixative:

1 L of fixative were prepared by mixing the following reagents in the denoted ratio % (v/v)

40 % Ethanol (96 %)

10 % Glacial acetic acid (100 %)

50 % Deionized water

Staining solution:

One tablet of PhostGelBlue (Pharmacia, Uppsala, Sweden) was dissolved in 300 ml deionized

water and filtrate through folded filter. The staining solution can be used several times

Destaining solution:

1 L of destaining solution was prepared by mixing the reagents in the denoted ratio % (v/v)

10 % Ethanol (96 %)

7.5 % Glacial acetic acid (100 %)

82.5 % Deionized water

Utilized destaining solution was used again in the first and second destaining step of another

analysis. The solution was stored in glass beakers containing a piece of foam plastic for dye

adsorption after the first usage.

Heat, high pressure, pulsed electric field and mechanical treatments

Protein samples were allowed to dissolve for 30 min under continuous stirring. During this time,

the pH was controlled and, if necessary, adjusted to the desired value with 0.1 M hydrochloric

acid. A pH meter (Digital-pH-meter, Knick Elektronische Messgeräte, Berlin, Germany) was used

for measuring clear solutions and pH test paper (pH indicator strips (pH 2.0 - 9.0), Merck KGaA,

Darmstadt, Germany) for turbid suspensions. The suspensions were centrifuged for 10 min at

10 000 g (F14-6x250y, Sorvall RC 6+, Thermo Fisher Scientific, Waltham, Massachusetts, USA)

before the treatments.

Peas were swollen before the treatments for 20 ± 0.25 h with tap water in a ratio of 1: 3 (w/w) at

room temperature. Swollen peas were separated from liquid with a sieve and paper tissues.


226

Broken or incompletely swollen peas, distinguished by the wrinkled hull, were sorted out and not

used for further experiments.

Potatoes were manually cleaned from dirt. No further pre-treatments were conducted.

Thermal Treatments

For the heat treatment of liquid samples, a self-constructed capillary heating system was used

(see Figure A. 2). Samples were transported by a peristaltic pump (SCI Q 323, Watson Marlow,

Falmouth, UK) with a flow rate of 1.25 mL/s. Liquid was pumped through two metal heater spirals

whereof the first was placed in a water bath adjusted to treatment temperature and the second

one was further heated in an oil bath (Polystat cc3, Huber GmbH, Offenburg, Germany) filled with

silicon oil (M40.165.10, Huber GmbH, Offenburg, Germany). Settings of the oil bath necessary to

obtain the target temperature were determined empirically (see Table A. 3). After passing the

second heating element, samples were filled into test tubes, capped with screw closures and left

in the water bath for dwell times of 10 minutes.

Figure A. 2: Continuous heating system for the thermal treatment of protein solutions.

For the treatment of small sample volumes below 2.5 mL, solutions were pipetted directly into

the preheated test tubes. Temperature control was performed in an additional tube filled with tap

water. Heating times amounted 45 to 90 seconds for target temperatures of 40 to 80 °C and were

taken into account during processing to ensure dwell times of 10 minutes at the final

temperature.

Swollen peas were weighed and given in pressure stable plastic bags. Tempered tap water was

added in a ratio of 1:1 (w/v) and the temperature kept at the desired value for 10 min in a water

bath (C20, Lauda, Lauda-Königshofen, Germany). Temperature was decreased to ambient

temperature with running tap water and bags were dried to avoid contamination of the

treatment medium. After 15 min, the bags were opened and peas separated from the treatment

medium with a sieve. Peas were manually dried with paper tissue and delivered to further

processing and analyses. Treatment media were frozen and kept at -20 °C until analyses.

feed testtube

ambienttemperature

must value

water bath oil bath


227

Table A. 3: Settings of oil bath temperature for thermal treatment of liquids in a capillary heating system

Treatment temperature [°C] 40 50 60 70 80

Temperature of the oil bath [°C] 40 52 65 76 86

For the thermal inactivation of microorganism and enzyme solutions, a sample volume of 125 μL

was filled into sterile AR-glass capillaries of 10 mm length, 1.0 mm inner and 1.3 mm outer

diameter (Kleinfeld Labortechnik GmbH, Gehrden, Germany). The capillaries were pre-cooled in

an ice bath and heated in a thermostat (Huber GmbH, Offenburg, Germany) with silicon oil

(M40.165.10, Huber GmbH, Offenburg, Germany) as heating medium set at the desired treatment

temperature. After the defined heating time, the samples were rapidly cooled in an ice bath

again.

High pressure

High pressure treatments were conducted in a pilot plant system with indirect pressure

generation (High Pressure Single Vessel Apparatus U4000, Institute for High Pressure Physics,

Warsaw, Poland) and a vessel volume of 750 mL. A 1:1 mixture of deionized water and 1,2-

propanediol (Sigma-Aldrich Corporation, St. Louis, Missouri) was used as pressure-transmitting

medium. To avoid temperature inhomogeneities during treatment, vessel and pressure-

transmitting medium were tempered before experiments with a connected water bath (DC10-

K20, Thermo Haake GmbH, Karlsruhe, Germany). Initial sample and medium temperatures

necessary to achieve certain treatments conditions were figured out by recording temperature

profiles with a Thermo-Egg (Knoerzer et al., 2010). Treatment parameters used are shown in Table

A. 4. For treatments at 40 °C, the water bath was set to 41 °C around 3 h before treatments.

Table A. 4: Temperature settings for high pressure treatments in the single vessel unit U4000

Treatment temperature [°C]

Pressure [MPa]

Medium temperature [°C]

Sample temperature [°C]

20 200 12 4

400 4 4

40 200 38 27.5

400 32 20

600 25 20


228

Samples were treated in bags made of pressure-stable polyamide/polyethylene foil. Vacuum-

sealing (PlusVac 23, Komet Maschinenfabrik GmbH, Plochingen, Germany) was conducted to

minimize the presence of air bubbles with different compression characteristics.

Liquids were additionally sealed into small plastic bags (Whirl Pak, Nasco, Fort Atkinson,

Wisconsin, USA); small sample volumes as well as samples intended for inactivation experiments

were filled into pressure stable plastic tubes (CryoTubes, Nunc A/S, Roskilde, Denmark) before

sealing.

Treatment of solids made the addition of a treatment medium necessary. Swollen peas and tap

water were filled into bags in a ratio of 1:1 (w/v).

Bags were given into the high pressure unit directly before treatment. After pressure treatments,

bags were cleaned from the pressure-transmitting medium and after a total time of 15 min, the

bags were opened and peas further processed analogous to the thermal treatment.

Pulsed electric fields (PEF)

Pulsed electric field treatments were performed discontinuously in self-constructed PEF units of

the TU Berlin. Treatment parameters are summarized in Table A. 5. Energy inputs were referred

to the mass between the electrodes, independent of the sample to water ratio. Pulse frequency

amounted approximately 2 Hz for all experiments. Sample temperatures before and after

treatments were recorded.

For protein solutions and microbial samples, a unit enabling treatments of small volumes ranging

from 400 μL to 500 mL was used. A power supply (HCK 800M-20000, FuG Elektronik GmbH,

Rosenheim, Deutschland) with a maximum voltage of 20 kV charged three capacitors (Ceramite

Y5U 6800Z, Behlke, Kronberg, Germany) with an overall capacity of 19.5 nF. A high voltage switch

(HTS 160-500 SCR, Behlke, Kronberg, Germany) was controlled by a frequency generator

(AFG 320, Tektronix, Inc., Beaverton, Oregon, USA) to enable the pulsating discharge of the

capacitors. A high voltage sensor (P6015 A, Tektronix, Inc., Beaverton, OR, USA) and an

oscilloscope (TDS 220, Tektronix, Inc., Beaverton, Oregon, USA) enabled monitoring of the

treatment efficiency. Samples were treated in electroporation cuvettes with 4 mm electrode

distance and a volumetric capacity of 750 μL (VWR International GmbH, Darmstadt, Germany).

For treatments at elevated temperatures, samples were pre-heated to the desired temperature in

a thermomixer (MKR 13, HLC BioTech, Bovenden, Germany).

Plant tissue was treated with pulsed electric fields in treatment chambers with higher holding

capacity. Three capacitors connected in series with a respective capacity of 1 μF were charged by

a high voltage generator (ALE802, lambda Emi, Neptune, United States) with a maximum voltage


229

of 25 kV. Current, voltage and pulse width were monitored by a TDS 220 oscilloscope (Tektronix,

Beaverton, USA). Discharging of the capacitors at the desired voltage was enabled by a tungsten

spark gap with adjustable electrode spacing.

Table A. 5: Intensities chosen for PEF treatment of different sample materials

Electric field strength [kV/cm]

Energy input [kJ/kg]

Pulse number [-]

Protein solutions 1 1.5 666

5 35 666

10 35 167

10 125 600

20 145 167

Peas 5 125 666

10 125 167

Potatoes 1 2.5 333

1 5 666

2 5 167

2 10 333

Peas were treated in a rectangular treatment chamber made of polyoxymethylene with fixed

stainless steel electrodes of 10 mm distance and a sample volume of 200 mL. 100 g of swollen

peas were filled up with tap water tempered to 20 °C. After treatment was finished, peas

remained in the treatment chamber and medium until 10 min had passed. PEF-treated peas were

handled equally to thermal and high pressure treated ones.

Potatoes were treated in a polypropylene tank with a base area of 370 by 270 mm (Kaiser und

Kraft, Stuttgart, Germany). The ground electrode was fixed on the bottom of the chamber; the

voltage electrode was removable (Gemmel-Metalle, Stuttgart, Germany). Two polypropylene

blocks were used to fixate the electrode distance of 50 mm. 22 to 23 potatoes with an overall

weight of 2 ± 0.05 kg were treated in 10 kg of tap water with an initial temperature of 20 °C.

Potatoes remained in the treatment chamber for a total time of 10 min until they were fetched

manually and dried with paper tissue.


230

In some cases, samples of several PEF treatments were unified to achieve sufficient material for

extensive analyses.

Mechanical sample disintegration

Grinding of potatoes for subsequent solid-liquid separation was performed in dependence of the

capacity of the pressing system in a customary food processor or with a centrifugal mill (see pages

230 ff).

Swollen and dried whole peas were grinded in a centrifugal mill with a rotor with 24 teeth and a

sieve insert of 4 mm mesh size (Retsch Centrifugal ZM 1, Hahn, Germany). The mill was fed

manually with a constant flow of material. Milling of dry peas was performed in two stages.

Freeze dried concentrates, potato cubes or press residue were homogenized manually with a

porcelain mortar.

Mass transfer processes

Solid-liquid separation

The separation of potato fruit juice from the residual material is one main step in potato starch

and protein recovery. Industrially realized with a decanter centrifuge, this process step was

simulated with pressing systems in laboratory and pilot plant scale.

Laboratory scale:

Around 500 g of potatoes were cut in cubes of approximately 1 cm³ and grinded in a customary

food processor (Braun Multisystem K3000, Kronberg, Deutschland) with a scraper disc of

approximately 2 mm pore size. Potato pieces remaining above the insert were cut in smaller

cubes with a kitchen knife. Pressing was performed in a manual press (Hafico HP 2, Schwanke

Tinkturenpressen, Neuss, Germany), using a cotton dish towel for particle separation. The

pressing cycle consisted of 5 pressing periods of 2 min each at pressure levels of 0, 50, 100, 150

and 200 bar. Pressing was stopped after 10 min by prompt release of the piston.

Pilot plant scale:

Around 20 kg of potatoes were cut in cubes with a cube dicer (FMC Mod365, Chicago, USA) and

afterwards grinded in a centrifugal mill (Stephan Microcut MCH 20K, Nazareth, Belgium) with a


231

rasp insert of 0.7 mm. 20 g (0.1 % (w/w)) of sodium metabisulphite were added to avoid

enzymatic browning. Potato mash was applied in two layers on a baling press (Triumph 1,

Hollmann, Düsseldorf, Germany) using the appendant pressing cloth. Pressure build-up was

performed after 2 min of potato juice flow and pressure levels of 50 and 100 bar were both kept

for 2 min as well. Total pressing time was 6 min.

Quantification of potatoes, press cake and fruit juice was performed gravimetrically. Fruit juice

was centrifuged at 10 000 g for 5 min (Biofuge pico, Heraeus, Osterode, Germany) and the

supernatant frozen in liquid nitrogen until further analyses. Results of the first pressing of an

experimental series were discarded due to comparably high losses during milling and pressing.

Transport of moisture

Warm air drying:

Peas were re-dried at 50 °C for 360 ± 5 min on aluminum trays in a drying cabinet (Heraeus

Instruments, Osterode, Germany). Samples were vacuum-packed and stored at temperatures

between 3 and 6 °C until use.

Freeze drying:

Potato cubes, press residue and protein extracts were frozen in liquid nitrogen and freeze-dried

for approximately 48 h (Gamma 1-16 LSC, Software Version 2274, Martin Christ

Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). Must values for pressure in the

dryer and temperature of the product plate amounted 0.009 mbar and 10 °C, respectively. Dried

samples were stored vacuum-packed at temperature from 2 to 6 °C.

Drying rate:

Drying rates for differently pre-treated samples were determined to evaluate the influence of

structural changes on drying efficiency.

Potato press residue was dried at 50 °C for 300 min in a drying cabinet (WK 1 180, Weiss Technik

AG, Altendorf, Switzerland). Approximately 30 g of press residue were weighed in petri dishes

with a diameter of 145 mm. The weight loss was monitored on a laboratory scale (Sartorius AG,

Göttingen Germany) in time intervals of 15 and 30 min during the first 60 and following 240 min,

respectively.


232

The drying rate of peas was determined with a moisture analyzer (Sartorius MA35, Sartorius AG,

Göttingen, Germany). 5 randomly selected peas were dried at 50 °C for 3 h. Mass changes were

recorded by a connected PC and the software Termite. Due to the limited running time of the

moisture analyzer a re-start of the drying process after 90 min was necessary.

Rehydration:

Water uptake into dry peas was investigated for water temperatures of 20 and 100 °C (following

preparation according to the label of commercial dry peas). Water uptake at 100 °C was

monitored for 2 h in intervals of 15 min. Weight increase at 20 °C was determined every 30 min

during the first two hours. After 2 and 5 h the interval was increased to 60 and 90 min,

respectively. The last weighing was then carried out after 24 hours.

For each sample, 50 peas were weighed and transferred into small bags made of 150 x 150 mm

pieces of fly screen. The bags were closed with cable ties. 100 mL glass beakers filled with

approximately 50 mL of tempered tap water served as sample containers. To maintain a

temperature of 100 °C, a water bath was used for heat supply. To determine the mass of adhering

water on the surface of the peas and the small bags, the samples were dipped into the water for

three seconds at the beginning of the experiment and weighed (LE 323 S, Sartorius AG, Göttingen

Germany). To remove the excess of adhesive water similarly for all samples, the small bags,

containing the peas, were shaken vigorously three times after removal from the water and then

patted dry with paper. After weighing, the peas were immediately transferred back to the beaker

for further swelling. The amount of hydration medium was kept constant by refilling losses with

tempered tap water. Dry matter of the dried and rehydrated samples was determined according

to page 236.

Extraction processes

Detection of protein extractability:

For extractability tests, either tap water or 50 mM TRIS-HCL buffer pH 9.0 were used as solvents.

1 g of wet grinded flour or 0.2 g of dry flour or press residue were weighed on a precision scale

(Sartorius, Göttingen, Germany) and extracted with 10 mL of solvent for 30 min under continuous

stirring (350 rpm, Variomag, H & P Labortechnik, Munich, Germany). The extracts were

centrifuged at 10 000 g for 5 min (Biofuge pico, Heraeus, Osterode, Germany) and the clear

supernatants were frozen in liquid nitrogen and stored at -18 °C for protein analyses.


233

Production of protein concentrates:

Protein was recovered from plant tissue either by solid-liquid separation (see page 230) or by

protein extraction with solvents on water basis. Concentrates were produced according to Figure

A. 3.

Figure A. 3: Recovery of protein concentrate via wet processing from peas and potatoes.

Peas were swollen for 20 h in the triple amount of tap water. Swollen peas were ground in a

centrifugal mill (see page 230). Around 300 g of pea flour were suspended in 2 700 g of tap water

and the pH value adjusted to 9 with a few millilitres of 1 M sodium hydroxide. Extraction was

carried out for 30 min under continuous stirring and insoluble parts were removed by

centrifugation at 10 000 g for 10 min (F14-6x250y, Sorvall RC 6+, Thermo Fisher Scientific,

Waltham, Massachusetts, USA). The supernatant was neutralized to pH 7 with 1 M hydrochloric

acid.

Protein extracts were purified and concentrated using an ultrafiltration module with a molecular

weight cut off of 10 kDa (Vivaflow 200 VF20P0, Sartorius AG, Göttingen, Germany). The filtration

was set up according to the instructions. A peristaltic pump (SCI Q 323, Watson Marlow,

Falmouth, UK) with a flow rate ranging from 100 to 140 rpm was used for liquid transport and

pressure build-up. Maximum pressure at the retentate side was 2.5 bar. Ultrafiltration took

approximately 5 h to decrease the volume of the protein extract from 2 500 mL to 500 mL. The

concentrated extract was frozen in liquid nitrogen, crushed with a mallet and freeze-dried for 48 h

(see page 231).

spray drying

solid-liquidseparation

grinding

potatoes

potatoprotein

peas

extraction

centrifugation

membrane concentration

freeze drying

wet grinding

peaprotein


234

Potato protein was recovered from potato fruit juice, obtained in pilot plant scale. The juice was

centrifuged at 10 000 g for 10 min to remove remaining starch granules and concentrated via

ultrafiltration (Proscale, Merck KGaA, Darmstadt, Germany) to approximately 20% of its initial

volume. A membrane of modified polyether sulfone with a 10 kDa cut off was used (Pellicon 2,

Biomax membrane cassette, Merck KGaA, Darmstadt, Germany). The applied feed pressure was

6 bar at maximum. The concentrated fruit juice was dried in a laboratory spray drier (Mini Spray

Dryer B-191, Büchi Labortechnik GmbH, Essen, Germany).The respective temperatures of air inlet

and product outlet amounted 120 and 80 °C at most.

Extraction of oligosaccharides:

Extraction of oligosaccharides was performed according to the method recommended by

Megazyme with some modifications. 1 g of wet milled pea flour was weighed in glass test tubes

with screw closure. 5 mL of ethanol (96 % (v/v), Carl Roth GmbH & Co KG, Karlsruhe, Germany)

were added and the mixture incubated for 5 min at 85 °C to inhibit enzyme activity. The

suspension was transferred into 100 mL glass beakers and 50 mL of 50 mM sodium acetate buffer

were added. After 15 min of continuous stirring, aliquots of the mixture were centrifuged at

10 000 g for 5 min (Biofuge pico, Heraeus, Osterode, Germany). The supernatant was stored at

-20 °C until analyses.

Enzyme extraction:

For determination of lipoxidase and peroxidase activity in pea flour, 1 g of flour was extracted

with 10 mL of cold deionized water in 10 min of continuous stirring. The samples were centrifuged

for 10 min at 10 000 g at 12 °C (F21-8x50y, Sorvall RC 6+, Thermo Fisher Scientific, Waltham,

Massachusetts, USA) and for another 5 min at 10 000 g at ambient temperature (Biofuge pico,

Heraeus, Osterode, Germany). The clear supernatants were directly used for the assay of

enzymatic activity.

Tissue properties

Impedance measurement

The degree of disintegrated cells in potato tissue was determined via impedance measurement.

Cylinders of potato tissue with a length of 20 mm and a diameter of 10 mm were prepared with a

kitchen knife and a plastic cylinder serving as sample container. Stainless steel electrodes were

placed on opposite sides of the cylinder for voltage transfer. Changes in sample conductivity were


235

measured with impedance analyses equipment (Biotronix GmbH, Hennigsdorf, Germany). The cell

disintegration index (CDI) was calculated with the following equation:

where Kl and K’l are the electrical conductivity of untreated and treated material, respectively, at

a frequency of 5.5 kHz and Kh and K’h are the electrical conductivity of untreated and treated

material, respectively, at 2.8 MHz. The cell disintegration index varies from 0 for intact cells to 1

for completely disintegrated cells.

Germination ability

To evaluate the influence of pre-treatments on the germination ability, 10 randomly chosen peas

were kept on water agar (1 % (w/v), Oxoid, Hampshire, England) for 48 h and the germination

ability was assessed by visual criteria.

Specific density

The specific density of the peas after drying was determined via seed displacement. Rapeseed was

chosen for filling empty spaces between the peas due to its small particle size and its

homogeneous bulk density. This method is very common for the characterization of bakery

products (Street, 1991). Liquid displacement is unsuitable for volume detection due to the

immediate interactions with the pea tissue and the subsequent volume increase.

100 g of peas were added to 500 mL of rapeseed. The volume change was detected in a

measuring cylinder. The specific density of the samples was calculated as weighted mass divided

by volume.

Microscopy

Tissue sections of untreated and treated peas were analyzed under the microscope to illustrate

treatment effects on a cellular level. Histological sections were cut and microscoped at the

Zentraleinrichtung Elektronenmikroskopie (ZELMI) of the TU Berlin. The cutting thickness of the

sections amounted 10 μm. For a better visualization, the cell walls were stained with toluidine

blue.

Embedding of pea tissue in cold-curing resin (Technovit 7100 - the sliceable, Heraeus Kulzer GmbH

& Co KG, Wehrheim Germany) was performed to prepare uniform and thin specimens. All


236

fixation, dehydration and embedding steps were performed in enclosed polyethylene embedding

moulds (Histoform S) according to Table A. 6.

Table A. 6: Preparation steps for the embedding of pea tissue

Reagent Residence time

Fixation 0.1 M phosphate buffer pH 7 containing 1 % glutaraldehyde overnight at 4 °C

washing with deionized water 3x à 15 min

Dehydration 50 % ethanol 30 min

70 % ethanol 2x à 60 min

95 % ethanol 60 min

95 % ethanol overnight

Embedding 96 % ethanol and basic solution 1 1 to 2 h

100 mL of basic solution 1 plus 1 g of hardener overnight

Polymerization 15 mL of prepared solution (basic solution 1 plus hardener) and 1 mL of hardener number 2 1 to 2 d

Integrity of starch granules in pea flour was analyzed in water suspensions using a reflected-light

microscope (Eclipse E400, Nikon Instruments, Inc., Melville, New York, USA). A microscope camera

(PL-A662, Pixellink, Ottawa, Canada) and a corresponding software package (Capture SE, Pixellink,

Ottawa, Canada) were available to take and edit microscopic pictures.

Quantification of single components

Moisture content

The moisture content in all samples was quantified via drying and following differential weighing.

A moisture analyzer (Sartorius MA35, Sartorius, Göttingen, Germany) was used to determine the

moisture content of potato press residue after solid-liquid separation. Approximately 1 g of

residue was placed on an aluminium dish and dried at 120 °C until weight changed less than 0.1 %

of the initial weight.


237

The moisture content of peas, flours and protein samples was determined via drying in a drying

oven (Heraeus Instruments UT6060, Hanau, Germany). 1 to 2 g of sample material was dried in

glass bowls for 48 h (± 4 h) at 105 °C. The weight changes were determined after 30 min of cooling

in an exsiccator filled with Silica gel as drying agent (Carl Roth GmbH & Co KG, Karlsruhe,

Germany). Samples were dried for another 60 min and weighing was repeated until the decrease

was smaller than 0.002 g.

Total soluble solids (Brix)

The refractive index of a substance indicates how fast light travels through this material in

comparison to the speed of light in vacuum. Charges of each atom present in the material radiate

electromagnetic waves amending those of the light, usually leading to a shorter wavelength than

the original. Higher concentrations of dispersed substances increase those interferences. Based

on this principle, the concentration of soluble solids in a liquid can be estimated.

Measurements were performed at 20 °C in a digital refractometer (PFM 80, Bellingham and

Stanley, Turnbridge Wells, UK). Results were given in °Brix corresponding to % of soluble solids.

Protein

Proteins in solution were quantified via photometrical analyses. The protein content in isolates

and flours was determined by nitrogen quantification by the digestion method according to

Kjeldahl. Kjeldahl analyses were performed at the Department of Food Chemistry in Potsdam. A

conversion factor of 6.25 was used to calculate the total protein content from liberated nitrogen.

Biuret test:

In strong alkaline solutions, copper (II) ions interact with the electron duplet of the adjacent

peptide nitrogen forming a tetra-coordinated cupric ion complex (Creighton, 1984, see Figure A. 4

left side). The violet complex possesses an absorbance maximum at 540 to 560 nm. The intensity

of the colour formation is directly proportional to the number of peptide bonds and thus to the

protein concentration. Tartrate is added to stabilize the copper ions.

The Biuret reagent does not contain the homonymous molecule (2-Imidodicarbonic diamide,

((H2N-CO-)2NH)) but due to its peptide-like structure (see Figure A. 4 right side) the Biuret

molecule undergoes the same complexation in the presence of copper ions and gives the same

response to the test as proteins.


238

Figure A. 4: Complex forming of copper ion with the n-terminal amino acids of bovine serum albumin (proposed by Peters and Blumenstock (1967), left side); Biuret molecule (according to Sigel and Martin (1982), right side).

The Biuret reagent was made with deionized water containing 9.4 mM copper sulphate (Merck

KGaA, Darmstadt, Germany), 28.56 mM potassium-sodium tartrate (Merck KGaA, Darmstadt,

Germany) and 831 mM sodium hydroxide. Therefore, 2.35 g of copper sulphate pentahydrate

(equivalent to 1.5 g of anhydrous copper sulphate) and 6.0 g of potassium-sodium tartrate were

diluted in 500 mL deionized water while warming cautiously. 300 mL of 10 % (w/v) sodium

hydroxide were added and the solution filled up with water to a total volume of 1 000 mL. The

reagent was stored at temperatures of 4 to 6 °C. Small colour changes occurred within storage

time.

Figure A. 5: Calibration curve for protein quantification via Biuret complexation made with bovine serum albumin as standard.

NH2

O

NH

O

NH2

O

R

N

NH

NH

O

NH

O

OHCH3

NHO

NH2

OOH

Cu2+

...

Histidin

Threonin

Asparticacid

y = 0.054x + 0.0068R² = 0.9997

0

0.1

0.2

0.3

0.4

0.5

0.6

0 2 4 6 8 10 12

diffe

renc

e in

abs

orba

nce

[-]

protein concentration in g / L


239

For protein quantification, 200 μL of adequate diluted samples were mixed with 800 μL of Biuret

reagent. Complexation either occurred within 45 min at room temperature or within 20 min at

37 °C (MKR 13, HLC BioTech, Bovenden, Germany). The absorbance was determined in semi-micro

cuvettes (VWR International Ltd., Darmstadt, Germany) at a wavelength of 540 nm (Lambda 25

Perkin Elmer, Waltham, Massachusetts). Blank values were prepared using deionized water or the

respective extraction solvent with regard to the dilution factor instead of the sample.

A calibration curve was made with bovine serum albumin (Fluka, Buchs, Switzerland) in deionized

water containing protein concentrations from 2 to 10 g / L. Several calibration curves were

prepared due to different people conducting analyses and different sets of pipettes used.

Exemplary, a calibration curve is shown in Figure A. 5.

Protein quantification via dye-binding (Bradford, 1976):

The method is based on the complex formation between proteins and the triphenylmethane dye

Coomassie Brilliant Blue. Three forms of Coomassie exist, giving different colour impressions. A

‘red’ form with a positive net charge, a neutral ‘green’ form and a ‘blue’ anion have absorbance

maxima of 470, 620 and 595 nm, respectively (see Figure A. 6).

Figure A. 6: Resonance forms of free ionic forms of Coomassie Brilliant Blue according to Chial et al. (1993); left side: red form; right side: blue form, centre: green form.

CH3

CH3

NH+

NH+

CH3

CH3

CH3

ONH+

SO

O

O-

SO

O

O-

CH3

CH3

N

NH+

CH3

CH3

CH3

ONH+

SO

O

O-

SO

O

O-

CH3

CH3

N

N

CH3

CH3

CH3

ONH+

SO

O

O-

SO

O

O-


240

Under acidic conditions, Bradford reagent is usually of brownish colour, as more than one

structural form of the molecule is present. Binding to protein converts the molecule to its blue

form, leading to a strong increase in the absorbance at 595 nm. Complexation involves

electrostatic attraction between the anion and the protein’s amino groups, van der Waals forces

and hydrophobic interactions. Amino acids responsible for dye binding are primarily arginine

residues, to a lesser extend aromatic and other basic amino acids (Compton & Jones, 1985). Due

to the dependency on certain amino acids the intensity of the colour formation is also determined

by the specific properties of the protein rather than just by its concentration.

Figure A. 7: Calibration curve for protein characterization with commercial Bradford reagent using bovine serum albumin as standard.

A commercially available reagent was used for Bradford assay (Protein Assay, BioRad Laboratories

GmbH, Munich, Germany). The reagent is composed of Coomassie Brilliant blue G250, methanol

and phosphoric acid and was stored at 4 to 6 °C until use. The reagent was used according to the

standard protocol described in the provided instruction manual with some modifications.

Bradford reagent was diluted with deionized water in a ratio of 2.2:10, thoroughly mixed and

filtered (Whatman No 1, Buckinghamshire, United Kingdom). 1 000 μL of diluted reagent were

added to 100 μL of adequate diluted sample to finally give a 20 % (v/v) concentration of original

Bradford reagent. After 15 min of reaction time the absorbance was determined in semi-micro

cuvettes (VWR International Ltd., Darmstadt, Germany) at 595 nm (Lambda 25 Perkin Elmer,

Waltham, Massachusetts). The calibration curve prepared with bovine serum albumin (Fluka,

Buchs, Switzerland) in deionized water is ranging from 20 to 100 mg/L (see Figure A. 7).

y = 0.0058x + 0.0105R² = 0.9988

0

0.1

0.2

0.3

0.4

0.5

0.6

0 20 40 60 80 100 120

diffe

renc

e in

abs

orba

nce

[-]

protein concentration in mg / L


241

Free amino groups

Free amino groups were quantified according to Church et al. (1983). In the presence of SDS in an

alkaline milieu accessible amino groups form a complex with ortho-phthaldialdehyde (OPA) und

mercaptoethanol, which strongly adsorbs light with a wavelength of 340 nm (see Figure A. 8).

Figure A. 8: Complex formation for quantification of free amino groups.

A solution of 100 mM sodium tetra borate (Merck KGaA, Darmstadt, Germany) and a solution of

5 % (w/w) SDS (Merck KGaA, Darmstadt, Germany) were prepared in deionized water. 40 mg of

OPA (Sigma-Aldrich, St. Louis, Missouri, USA) were pre-mixed with 1 mL of methanol (VWR

International GMbH, Darmstadt, Germany) and transferred afterwards into a 50 mL volumetric

flask containing 25 mL of sodium borate solution, 10 mL SDS solution and 100 μL

mercaptoethanol (Fluka, Buchs, Switzerland). The mixture was gently homogenized and filled up

to 50 mL with deionized water. The reagent is not storable and has to be freshly prepared for

each day of analysis. During measurements, the reagent was stored in lightproof 15 mL plastic

tubes.

Figure A. 9: Calibration curve prepared with cysteine for quantification of accessible amino groups.

OPA free amino group mercaptoethanolO

O

OH

O

R

NH2

+ +SH

OH

OHS

RN

+ 2 H2O

y = 0.1867x + 0.005R² = 0.9999

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

diffe

renc

e in

abs

orba

nce

[-]

accessible amino groups in mM


242

100 μL of sample were mixed with 1000 μL reagent. After 6 min reaction time the absorbance was

measured in UV suitable semi-micro cuvettes (VWR International Ltd., Darmstadt, Germany) at a

wavelength of 340 nm (Lambda 25 Perkin Elmer, Waltham, Massachusetts). Samples were diluted

in deionized water, which was used as well to prepare a blank sample. A calibration curve was

made using cysteine (Merck KGaA, Darmstadt, Germany) in a concentration range of 0.5 to 4 mM

(see Figure A. 9)

Reducing sugars

Quantification of reducing sugars was performed according to the method described by Bernfeld

(1955). Oxidation of reducing sugars will lead to the reduction of 3,5-dinitrosalicylic acid to

3-amino-5-nitro-salicylic acid (see Figure A. 10). This reaction can be monitored detecting the

absorbance at 540 nm.

Figure A. 10: Deoxidation of 3,5-dinitrosalicylic acid to 3-amino-5-nitro-salicylic acid in the presence of reducing sugar.

A colour reagent was prepared containing 48 mM 3,5-dinitrosalicylic acid (Sigma-Aldrich, St. Louis,

Missouri, USA), 1.06 M potassium-sodium-tartrate (Merck KG, Darmstadt, Germany) and 0.4 M

sodium hydroxide (Merck KGaA, Darmstadt, Germany). Dinitrosalicylic acid was solubilized in

deionized water under moderate warming before combining with tartrate and base. In an amber

bottle at room temperature, the reagent is stable for 6 months.

100 μL of sample and 100 μL of reagent were allowed to react for 15 min in a heater set to 98 °C

(MKR 13, HLC BioTech, Bovenden, Germany). 900 μL of deionized water were added and the

absorbance was measured in semi-micro cuvettes (VWR International Ltd., Darmstadt, Germany)

at 540 nm (Lambda 25, Perkin Elmer, Waltham, Massachusetts). Deionized water was used for

sample dilution and for the determination of the blank value. Calibration with glucose (Merck

KGaA, Darmstadt, Germany) did not follow a linear relationship. To be able to quantify very small

amounts of reducing sugar as well, the calibration curve was prepared with concentrations

ranging from 0.1 to 8 mM and fit with a polynomial trend line (see Figure A. 11).

O

O O

ON N

OH

O OH

O

O

N NH2

OH

O OH

deoxidation


243

Figure A. 11: Calibration curve prepared with glucose for quantification of reducing sugars.

Raffinose equivalent sugars

Oligosaccharides in treatment media and extracts were quantified using a commercially available

test kit, based on enzymatic reactions (Megazyme International Ireland Ltd, Wicklow, Ireland).

Raffinose equivalents are hydrolyzed to D-galactose that can be further oxidized to D-galactonic

acid under the consumption of NAD+. The amount of formed NADH can be determined

photometrically at a wavelength of 340 nm (Lambda 25, Perkin Elmer, Waltham, Massachusetts).

All regents were prepared according to the enclosed method description and stored under the

recommended conditions until use. Solution 4 was tempered to 30 °C before use. All solutions

were directly pipetted into UV suitable semi-micro cuvettes (VWR International Ltd., Darmstadt,

Germany) according to the procedure shown in Table A. 7.

Samples were diluted to adequate values with deionized water. Molar concentrations of D-

galactose were calculated based on Lambert-Beer’s law by using an extinction coefficient of

6300 L/(mol*cm). Concentrations for raffinose equivalents including free galactose were adjusted

for the galactose concentration and transferred to mass concentration assuming an average

molecular weight of 504.5 g/mol.

0

1

2

3

4

5

6

7

8

9

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

redu

cing

suga

r con

cent

ratio

n in

mM

difference in absorbance [-]

y=((2.785 )+0.607)²R²=0.995


244

Table A. 7: Instructions for the quantification of raffinose equivalent sugars (adapted from the manual of Megazyme)

Blank for

raffinose + free

D-galactose

Raffinose +

free D-

galactose

Blank for free

D-galactose

Free D-

galactose

Sample solution

Deionized water

Solution 4 (α-Gal)

-

0.25 mL

0.05 mL

0.25 mL

-

0.05 mL

-

0.3 mL

-

0.25 mL

0.05 mL

-

Mix with plastic spatula and incubate for 20 minutes. Then add:

Solution 1 (buffer)

Deionized water

Solution 2 (NAD+)

0.1 mL

0.85 mL

0.05 mL

0.1 mL

0.85 mL

0.05 mL

0.1 mL

0.85 mL

0.05 mL

0.1 mL

0.85 mL

0.05 mL

Mix with plastic spatula, after 3 min read the absorbance of the solutions (A1) and then start

the reactions by adding:

Suspension 3 (β-GalDH) 0.01 mL 0.01 mL 0.01 mL 0.01 mL

Mix with plastic spatula, after approx. 40 min determine again the absorbance (A2) until it

remains constant.

Protein characterization

Absorbance at 605 nm

Electromagnetic waves can interact with material either by absorption or by scattering. If the

intensity of a light beam is reduced due to these interactions, the material appears turbid.

Particles with an average diameter of at least 2 nm have to be present to scatter visible light, for

example macromolecules like proteins or starch or aggregates and droplets (Universität

Regensburg, 2013).

The sample appears more turbid, if the particle size increases due to an external influence, as light

passing through it is scattered to a larger extend. There is no clear linear relationship between the

degree of aggregation and the absorbance as light diffusion can be more affected by many small

particles than by few large aggregates.


245

Absorbance measurements were performed in semi-micro cuvettes (VWR International Ltd.,

Darmstadt, Germany) at a wavelength of 605 nm (Lasa 100, Dr. Bruno Lange GmbH & Co. KG,

Berlin, Germany). Samples were diluted in an adequate extent to give absorbances below 0.9. Tap

water was used as a blank.

Particle size distribution

The Brownian motion describes the random movement of particles due to collision with the

molecules of the surrounding solvent. The larger the particles are, the slower is their movement.

Dynamic light scattering enables the observation of particle movements and the calculation of

their diffusion coefficient and their equivalent diameter.

Samples were characterized using the Zetasizer Nano ZS (Malvern Instruments GmbH,

Herrenberg, Germany) and the corresponding software. Laser light met the sample with a

wavelength of 633 nm and an intensity of 4 mW. The detection of backward scattering occurred

at an angle of 173 ° in relation to the incident light beam. Setting of the attenuation index and

focus position was automatically adjusted by the program.

Samples were centrifuged for 5 minutes at 500 g to remove large particles that undergo

sedimentation during measurement. The supernatant was analyzed in semi-micro cuvettes (VWR

International Ltd., Darmstadt, Germany) at a temperature of 25 °C.

Size distributions based on intensity, volume and number of analyzed particles can be obtained by

the software. Distributions according to particle number are the least accurate as measuring

errors multiply with data conversion. Nevertheless, results are presented in this way due to its

good comprehensibility.

Surface hydrophobicity

Fluorescence spectroscopy is based on the ability of molecules to absorb photons from an

excitation light beam and emitting photons of different energy levels and frequencies. Detecting

the intensities and frequencies of the emitted light allows a characterization of the analyzed

sample. Reagents, whose emitted light intensity depends on the polarity of their environment,

can be used to characterize hydrophobic and non-polar regions on the protein surface (Nakai et

al., 1996). As aliphatic and aromatic amino acids preferentially interact with aliphatic and

aromatic binding partners, respectively, a differentiation between aliphatic and aromatic

hydrophobicity is reasonable (Hayakawa & Nakai, 1985). Cis-parinaric acid (CPA), a conjugated

polyunsaturated fatty acid, and 1-anilino-8-naphthalene sulfonate (ANS), containing three

aromatic rings, are common representatives for each group and were chosen as fluorescence

probes for the conducted experiments. Their molecular structure is shown in Figure A. 12.


246

Figure A. 12: Molecular structure of ANS (top) and CPA (bottom).

To get detailed information on the interaction of fluorescence dye and protein sample, ANS and

CPA were added in increasing concentrations ranging from 30 to 150 and 20 to 100 μmol/g

protein, respectively. Figure A. 13 shows the progress of the fluorescence signal in dependence on

the dye concentration. Saturation of the fluorescence intensity with increasing dye concentration

can be observed. Both samples B and C showed a higher hydrophobicity in comparison to sample

A, but different curve progressions. To differentiate between both types of dye interaction, the

ratio between maximum fluorescence intensity and the apparent dissociation constant of the dye-

protein complex was used as a value for the protein surface hydrophobicity index (according to

Moro et al. (2001)).

Samples were analyzed in quartz cuvettes with a light path of 10 mm (101-QS, Hellma Analytics,

Müllheim, Germany) in a fluorescence photometer connected to a corresponding power supply

(650-10S and 150 Xenon Power Supply, Perkin Elmer, Waltham, Massachusetts, USA). A slit width

of 2 nm was used in all experiments. Fluorescence signals were recorded by a measurement

board (OMB-DAQ 3000, Omega Engineering Inc., Stamford, Connecticut, USA) and transmitted to

an evaluation software (LabView Version 8.5.1, National Instruments, Austin, TX, USA), which

averaged the data recorded within a 5 s measurement period.

For aromatic hydrophobicity, a 1 mM stock solution of ANS (ammonium salt, Sigma LifeScience,

St. Louis, Missouri, USA) was prepared in 10 mM phosphate buffer (pH 7) and diluted with the

same buffer to concentrations of 60, 120, 180, 240 and 300 μM. Mixing with the protein sample in

a ratio of 1:1 (v/v) finally led to the desired concentrations in the test sample. 390 nm and 470 nm

were used as excitation and emission wavelengths, respectively.

To determine aliphatic hydrophobicity, 1 mg of CPA (Biomol GmbH, Hamburg, Germany) was

diluted in 96 % undenatured ethanol (Carl Roth GmbH & Co KG, Karlsruhe, Germany), giving a

362 μM CPA solution. An equimolar concentration of butylated hydroxytoluol (SAFC, Sigma-

Aldrich, St. Louis, Missouri, USA) was added as antioxidant. The solution was purged with nitrogen

NH S OO

O-

OH

O

CH3


247

and stored in UV impermeable reaction tubes at -20 °C until usage. Due to the high instability of

CPA in the presence of oxygen no stable dilutions in phosphate buffer could be prepared before

analyses. Therefore, 2 mL of protein sample were filled directly in the fluorescence cuvette and

11.3 μL of CPA solution were added stepwise and mixed in with a plastic spatula. After each

addition, the sample was excited at 325 nm and the emission recorded at 410 nm.

Samples were diluted with deionized water to adequate concentrations before analyses. Protein

concentrations of 1 g/L and 0.1 g/L gave good results for the aromatic and aliphatic

hydrophobicity protocols applied. Fluorescence signals obtained were corrected by the signals of

the protein solution and the fluorescence dye in correspondent concentration.

Figure A. 13: Data analysing of surface hydrophobicity determination.

Quantification of accessible thiol groups

The number of reactive thiol groups plays an important role in protein aggregation and strongly

influences their sensitivity towards technological treatments (Beveridge et al., 1974). Reaction

with the aromatic disulphide 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB), known as Ellman’s

reagent, leads to colour formation, detectable with spectrophotometric measurement (Ellman,

1959). The reaction pathway is shown in Figure A. 14.

A 10 mM DTNB2- solution (Merck KGaA, Darmstadt, Germany) was prepared in 96 % undenatured

ethanol (Carl Roth GmbH & Co KG, Karlsruhe, Germany) and stored under light exclusion at 4 °C

until usage. 0.2 M TRIS-HCl buffer pH 8 was used as sample buffer. To differentiate between fast

0

20

40

60

80

100

120

140

0 20 40 60 80 100 120 140 160

fluor

esce

nce

signa

l [m

V]

ANS concentration in μmol / g protein

A B C

Fmax

kd

Fmax/2


248

accessible thiol groups and slow accessible groups that are buried in the interior of the protein

molecule, the same buffer containing 1 % (w/w) SDS was used in addition.

Figure A. 14: Reaction scheme of thiol groups with Ellman’s reagent according to Sedlak and Lindsay (1968).

200 μL of protein solution were given in semi-micro cuvettes (VWR International Ltd., Darmstadt,

Germany) and 1000 μL of the appropriate buffer were stirred in with a plastic spatula. Samples

containing SDS were incubated for at least 10 minutes for protein unfolding. The absorbance was

measured at a wavelength of 412 nm (Lambda 25, Perkin Elmer, Waltham, Massachusetts).

Afterwards, 20 μL of DTNB solution were mixed in with the plastic spatula. After 20 min of

reaction time, the absorbance was determined again. Deionized water was used for the

determination of blank values. The change in absorbance caused by cleavage of thiol groups was

calculated as follows:

where A+ and A- are the absorbances measured with and without DTNB.

A calibration curve with glutathione was prepared to calculate the molar concentration of thiol

groups (see Figure A. 15). Protein samples were diluted with deionized water to be in the range of

the calibration curve.

SS

N+ O-O

N+

O- O

O

OH

O

OH

R SH +

N+ O

-O

O

OH

SH

N+

O- OO

OH

SR

+


249

Figure A. 15: Calibration curve for the determination of fast and slow accessible thiol groups made with gluthathione in TRIS-HCl buffer pH 8.

Analysis of formed aggregates

The chemical stability of the aggregates towards different denaturing reagents was analyzed to

identify the interactions that led to precipitation of protein during treatments. Changes in sample

turbidity and protein solubility after addition of specific reagents indicate a decrease in cross-

linking and resolubilization of protein aggregates. Buffer systems were chosen according to

solubility tests for whey protein gels (Schmitt et al., 2010) and extruded meat analogues (Liu &

Hsieh, 2008). Urea, dithiothreitol (DTT), sodium dodecyl sulphate (SDS) and sodium chloride were

chosen to affect hydrogen bonds, disulphide bonds, hydrophobic and electrostatic interactions,

respectively.

100 mM phosphate citrate buffer pH 7 was supplemented with 8 M urea (Merck KGaA,

Darmstadt, Germany), 3 % SDS (w/v, Carl Roth GmbH & Co KG, Karlsruhe, Germany), 3 % sodium

chloride (w/v, Merck KGaA, Darmstadt, Germany), 0.1 M DTT (Sigma-Aldrich, St. Louis, Missouri,

USA) or all four reagents combined. Treated protein samples were well homogenized with a

vortex shaker and mixed with each buffer in a ratio of 1: 1 (v/v). After 30 minutes of residence

time at 25 °C and continuous shaking (MKR 13, HLC BioTech, Bovenden, Germany), the sample’s

absorbance was measured. Insolubilities were removed by centrifugation at 10 000 g for 5 min

(Biofuge pico, Heraeus, Osterode, Germany) and the protein content in the supernatant analyzed

y = 0.0115x + 0.0136R² = 0.9996

y = 0.0111x + 0.0127R² = 1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 10 20 30 40 50 60 70

diffe

renc

e in

abs

orba

nce

[-]

thiol concentration in μmol / L

fast accessible slow accesible Linear (fast accessible) Linear (slow accesible)


250

with the Biuret test. Soluble protein could not be analyzed in samples containing DTT due to

incompatibility of with the Biuret test.

Non-reducing SDS PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis)

Proteins move within an electric field due to their intrinsic charge according to the principles of

electrophoresis. In a gel matrix, the motion of proteins is retarded to a certain extent depending

on the protein’s molecular radius and the density of the gel structure. These properties can be

used for protein separation and identification in samples of unknown composition.

Presence of SDS leads to unfolding of the secondary, tertiary and quaternary protein structure,

resulting in a linear molecule with only a few Angstroms width and a length depending on the

protein’s molecular size. SDS donates a negative net charge to the protein, making all protein

molecules move towards the cathode at rates dependent on their molecular mass. Comparison of

a standard with known protein weights allows the identification of single protein fractions in the

sample.

SDS PAGE was run using an electrophorese unit (Mini PROTEAN® Tetra Cell, BioRad Laboratories

GmbH, Munich, Germany) enabling the mount of 4 gel cassettes in parallel. Commercially

available gels (Roti-Page, Carl Roth GmbH & Co KG, Karlsruhe, Germany) based on a TRIS/glycine

buffer system were used. Polyamide concentrations of the stacking and running gel amounted

4 % and 12 %, respectively. Gels were prepared according to the enclosed manual. Running buffer

was diluted 1:10 with deionized water and filled into the cell as labelled.

A commercial protein marker with molecular weights ranging from 10 - 150 kDa was used for

protein identification (Roti®-Mark 10-150 Plus, Carl Roth GmbH & Co KG, Karlsruhe, Germany).

Samples were diluted in a ratio of 1:1 (v/v) with sample buffer, the marker was used as delivered.

10 μL of marker or diluted sample were load into each well. Electrophoresis was started by

connecting the unit to a voltage generator (Electrophoresis power supply E835, Consort BVBA;

Turnout, Belgium) and applying a voltage of 250 V. The completion of the run was defined visually

when the dye front reached the end of the gel cassettes.

Gels were removed gently from the cassettes, rinsed carefully with deionized water and fixed by

inlaying in the fixative for 5 min. Protein bands were coloured in warmed staining solution at

approximately 60 °C for a residence time of 15 min under gently stirring. Excessive dye was

removed in several destaining steps with increasing residence times (5, 10, 30, 60 min and

overnight). The profile of the protein bands was conserved by scanning the gels between two

layers of overhead foil.


251

Techno-functionality tests

Solubility

Solubility was determined in dependence of the pH-value using 0.05 M phosphate-citrate buffer.

0.1 g of sample was weighed in small glass beakers of 15 mL volume. 10 mL of buffer were added

and the mixture stirred on a magnetic stirrer for 30 minutes at 700 rpm. Insoluble compounds

were removed via centrifugation at 10 000 g for 5 minutes (Biofuge pico, Heraeus, Osterode,

Germany) and the protein content of the supernatant analyzed via the Biuret method.

Fat binding capacity

The ability to bind lipids against centrifugal forces was determined according to Schwenke et al.

(1981) with some modifications.

1 ± 0.009 g of sample was weighed (Sartorius AG, Göttingen Germany) into a 30 ml centrifuge

beaker. 5 mL of commercially available sunflower oil were added and mixed with a stageless

agitator (Janke & Kunkel AG, IKA Werk, Staufen, Germany) equipped with a self-made screw

stirrer. One minute of homogenization was followed by a five minute resting period. This

procedure was conducted twice. Residual material was thrown off the stirrer within five seconds

of stirring. The sample was centrifuged for 15 min at 10 000 g (F21-8x50y, Sorvall RC 6+, Thermo

Fisher Scientific, Waltham, Massachusetts, USA), the supernatant was decanted and the beaker

put upside down on a tissue for 10 min to remove excessive oil before weighing. The amount of

bound oil was either referred to the dry matter or protein content.

Water binding capacity

To determine the water binding capacity of the samples, the method of Smith (1972) modified by

Quinn and Paton (1979) was applied.

1 ± 0.009 g of material was weighed into a 30 ml centrifuge beaker and mixed with tap water for

one minute using the aforementioned stirring unit (Janke & Kunkel AG, IKA Werk, Staufen,

Germany). The approximate amount of water needed for analysis was determined in preliminary

experiments according to Smith (1972) and Schwenke et al. (1981) and resulted in 2.5 mL of

added tap water for pea flours and 2 mL of water for protein concentrates. Centrifugation and

weighing was performed analogously to fat binding analyses (see above).

Gelation ability

Gels were prepared from dry pea flour by heat induced gelation. 100 mL of a 20 % (w/v)

suspension of flour were prepared in tap water. The suspension was heated in a 250 mL glass


252

beaker in a water bath set to 100 °C (GFL, Burgwedel Germany) for 5 min under continuous

stirring. The heated suspension was given into small petri dishes and cooled down at ambient

temperature for 3.5 h. Firmness of the gels formed was determined with a texture analyser

equipped with a cylindrical measurement body of 35 mm diameter (TA-XT2, Stable Micro

Systems, Godalming, UK). Analyses were performed with a forward speed of 2 mm/s, a test speed

of 0.1 mm/s and a displacement of 5 mm. Force-displacement curves were recorded and the gel

firmness calculated as:

Foaming properties

Foams were characterized using the Dynamic Foam Analyser DFA100 (Krüss GmbH, Hamburg,

Germany), an analytical device combining foam formation and stability control via an optical

measurement system. The sample is filled into a glass cylinder between a LED bar and a line

sensor. Differences in the optical density between liquid, foam and air enable a determination of

drainage and total height at the respective measurement time and therefore a calculation of the

current foam volume.

Protein samples were diluted with deionized water. Suitable protein concentrations were

determined empirically on the basis of foam decay within 30 minutes. 50 mL of the sample were

put into the glass cylinder CY 4501 with a diameter of 40 mm and a height of 250 mm. Settings of

the unit were altered during this thesis with the aim of optimizing the analytical procedure.

Parameters of the first experimental series are given in brackets. Ambient air was pumped

through the glass frit FL 4504 with a pore diameter of 10-16 μm on the bottom of the device with

a gas flow rate of 150 mL/min (500 mL/min) until a foam height of 160 mm (total height of

180 mm) was reached. Detection frequency amounted 10 Hz in the first 2 minutes and 1 Hz during

the rest of the measurement. Sensitivity of the grey scale was adapted depending on the results

obtained and curve progressions were recalculated accordingly.

Foams prepared from self-made potato protein were foamed using a commercially available

handheld electric mixer with wire whisk. 20 mL of protein solution with a concentration of 5 g/L

were filled in a 250 mL graduated plastic beaker and whipped for 2 minutes at maximum

intensity. The sample was weighted and the volume of foam and drainage read off directly after

whipping. Changes in the volume of foam and liquid were again determined 15, 30, 45 and

60 minutes after foam formation.


253

Foam expansion is calculated as foam volume divided by sample volume multiplied by 100 %.

Foam stability is given as the current foam volume divided by the maximum foam volume

multiplied by 100 %. Half-life of the foam corresponds to the time when 50 % foam stability was

reached.

Evaluating impacts on sample shelf-life

Microbial inactivation

As the endogenous microbial load of the centrifuged protein solution was low and affected by

strong variations, microbial strains were purposefully added to evaluate the inactivation effect of

selected treatment intensities. In pea and potato processing inactivation of Listeria

monocytogenes as well as inactivation of coliform bacteria is of high importance (personal

communication with Christoph Pieper, Emsland-Stärke GmbH, Emlichheim, Germany). Escherichia

coli (6897, DSMZ GmbH, Braunschweig, Germany) and Listeria innocua (35/32, former BGA,

Berlin, Germany) were chosen as nonpathogenic substitutes for the recommended strains.

The two strains were obtained as freeze-dried cultures, transferred into cryo-cultures and stored

at -80 °C until usage. The cryo-cultures were recultivated in standard bouillon (Merck KGaA,

Darmstadt, Deutschland) in two stages to reduce the amount of dead cells in the suspension. The

standard bouillon was removed via centrifugation for 10 min at 10 000 g (Heraeus Megafuge 1.0R,

Thermo Fisher Scientific, Waltham, Massachusetts, USA) and resuspension of the pellet in ringer

solution (Merck KGaA, Darmstadt, Deutschland). After a second washing step, the pellet was

suspended either in diluted ringer (1/8) or protein solutions resulting in bacterial counts of

approximately 109 cfu/mL.

The microbial samples were transferred either to glass capillaries, nunc tubes or electroporation

cuvettes depending on the following treatment. Treatments were performed according to page

225 ff. Addition and removal of the sample had to occur under sterile conditions to avoid foreign

contamination. A decadic series of dilution up to 10-7 was made in microtiter plates (Carl Roth

GmbH, Karlsruhe, Germany) and 50 μL of diluted sample were plated on nutrient agar (Oxoid Ltd.,

Basingstoke, England). Samples were incubated for at least 24 h at 37 °C in an incubator (INE 600,

Memmert GmbH & Co. KG, Schwabach, Germany) and the number of colony forming units

counted manually. Plate counts varying between 10 and 200 were considered for evaluating the

inactivation effect expressed as reduction of log cycles in cfu/mL.


254

Enzyme activity

The effect of heat or pressure on enzymatic activity was both determined in pure enzyme

solutions and in extracts from peas that were subjected to the respective technologies. Solutions

and peas were processed according to page 225 ff. Extraction of enzymes was performed as

described on page 234. The activity of treated enzyme solutions was related to solutions

permanently stored on ice. Activity in the extracts was related to that from unswollen,

unprocessed peas under consideration of the protein content in the extracts determined with

biuret complexation.

Lipoxidase:

Lipoxidase catalyzes the peroxidation of various fatty acids. The increase in oxidized linoleic acid

can be determined by measuring the absorbance at 234 nm.

Purified lipoxidase from soybean (EC 1.13.11.12, type I-B, Sigma-Aldrich, St. Louis, Missouri, USA)

was dissolved in 50 mL of 200 mM borate buffer pH 9 (Merck KGaA, Darmstadt, Germany), frozen

in liquid nitrogen and stored at -20 °C until use. The thawed sample was further diluted with

deionized water in a ratio of 1:20 before heat and pressure treatments.

500 μL linoleic acid (Sigma-Aldrich, St. Louis, Missouri, USA) were mixed with the same amount of

ethanol (95 %, Carl Roth GmbH & Co KG, Karlsruhe, Germany). The mixture was aerated with

nitrogen to prevent oxidation during storage at -20 °C. 100 μL of this mixture were diluted to

50 mL with borate buffer, whereof 5 mL were further diluted with 20 mL of borate buffer and

5 mL deionized water to a final stock solution. Appearing turbidities were removed in an

ultrasonic bath. The substrate solution was stored in light-proof plastic tubes on ice until analysis.

Buffer and stock solution were tempered to 25 °C directly before performing the assay. The

photometer was equipped with a tempering unit and a water bath set to 25 °C (Ministat, Peter

Huber Kältemaschinen GmbH, Offenburg, Germany). To determine the activity of the pure

enzymes, 1000 μL of borate buffer pH 9 and 2000 μL of substrate solution were mixed in quartz

glass cuvettes (100-QS, Hellma Analytics, Müllheim, Germany). 100 μL of enzyme solution were

added, carefully homogenized with a cuvette stirrer (neoLab Migge Laborbedarf-Vertriebs GmbH,

Heidelberg, Germany) and incubated for one minute in the sample holder of the photometer. The

increase in absorbance was recorded from 60-180 s at a wavelength of 234 nm (Lambda 25 Perkin

Elmer, Waltham, Massachusetts). The blank was recorded using 100 μL buffer instead of enzyme

solution.

The assay was not suitable for extracts from pea flour due to a different pH optimum of the

enzymes. The extracts were analyzed according to Gökmen et al. (2005). A substrate solution of


255

157 μL linoleic acid (Sigma-Aldrich, St. Louis, Missouri, USA), 157 μL Tween 20 (Carl Roth GmbH &

Co KG, Karlsruhe, Germany) and 10 mL deionized water was clarified by adding 1 mL of 1 N

sodium hydroxide solution (Merck KGaA, Darmstadt, Germany) and further diluted to 200 mL with

100 mM phosphate buffer pH 6. 7.25 mL of substrate solution were tempered in a water bath to

25 °C (Ministat, Peter Huber Kältemaschinen GmbH, Offenburg, Germany) and 100μL mL of

extract and 150 μL tap water were added and mixed in thoroughly. In constant time intervals,

500 μL of sample were taken from the mixture and added to 2000 μL of 0.1 N sodium hydroxide

solution to stop the enzymatic reaction. Time intervals of 0, 15, 30, 45 and 60 s or 0, 30, 60, 90,

120, 150 and 180 s were chosen according to the activity in the extracts. The absorbance of the

samples was measured in quartz glass cuvettes (100-QS, Hellma Analytics, Müllheim, Germany) at

a wavelength of 234 nm (Lambda 25 Perkin Elmer, Waltham, Massachusetts). The blank was

prepared by mixing 500 μL of substrate solution with 2000 μL of 0.1 N sodium hydroxide

solutions.

Peroxidase:

Peroxidases catalyze the reduction of hydrogen peroxide to water under consumption of different

substrates. Their activity can be determined photometrically via the dehydrogenation of

pyrogallol to pyrogallin.

A 5 % pyrogallol solution (Sigma-Aldrich, St. Louis, Missouri, USA) and a 0.5 % peroxide solution

(Merck KGaA, Darmstadt, Germany) were freshly prepared in deionized water on each day of

analysis. Both solutions were stored on ice under exclusion from light until use. 160 μL of peroxide

solution, 320 μL of pyrogallol solution, 320 μL of 100 mM potassium phosphate buffer (pH 6) and

2100 μL of deionized water were mixed in disposable macro cuvettes (VWR International Ltd.,

Darmstadt, Germany) and tempered to room temperature. 100 μL of enzyme solution or extract

were added, mixed in immediately with a cuvette stirrer (neoLab Migge Laborbedarf-Vertriebs

GmbH, Heidelberg, Germany) and the increase in absorbance was recorded at a wavelength of

420 nm for 120 s (Lambda 25, Perkin Elmer, Waltham, Massachusetts). A blank value was

recorded with deionized water instead of sample.

Peroxidase from horseradish (EC 1.11.1.7, type I, Sigma-Aldrich, St. Louis, Missouri, USA) was

taken as reference for the heat and pressure sensitivity of purified enzymes. The whole sample

with a total activity of 5 kU was diluted in 50 mL of 0.1 % bovine serum albumin solution, frozen in

liquid nitrogen and stored at -20 °C until experiments. The thawed sample was further diluted

1:100 with phosphate buffer before treatments.


256

Enzymatic digestibility

Pea flours were subjected to a simulated digestion to investigate the accessibility of protein and

starch to enzymatic degradation. All enzymes were purchased in pure form and solved separately

directly before usage (see Table A. 8). Oligo-α-1,6-glucosidase and Sucrose-α-glucosidase came

from Coring System Diagnostix GmbH (Gernsheim, Germany). All other enzymes were purchased

from Sigma-Aldrich (St. Louis, Missouri, USA). Enzyme activities in the digested samples were

chosen according to information in literature (Alvarez et al., 1935; Dunaif & Schneeman, 1981;

Nater et al., 2006; Rohleder et al., 2006) or arose from the specifications of the enzyme

preparation available.

Table A. 8: Enzyme solutions used for digestibility tests

Enzyme Activity / Concentration Solvent

Mouth α-amylase (EC 3.2.1.1)

from human saliva 100 Units / mL deionized water

Stomach Pepsin

(EC 3.4.23.1) from porcine gastric mucosa

250 mg / L deionized water

Duodenum

Trypsin (EC 3.4.21.4)

from porcine pancreas 100 Units / mL 1 mM HCl

Chymotrypsin (EC 4.4.21.1)

from bovine pancreas 800 Units / mL 1 mM HCl

α-amylase (EC 3.2.1.1)

from porcine pancreas 200 Units / mL deionized water

Small intestine

Dipeptidyl peptidase IV (EC 3.4.14.5)

from porcine kidney 1 Unit / L 0.1 M TRIS-HCL buffer

pH 8

α-glucosidase (EC 3.2.1.20)

from S.cerevisiae 100 Units / mL deionized water

Oligo-α-1,6-glucosidase (EC 3.2.1.10)

microbial origin 100 Units / mL deionized water

Sucrose-α-glucosidase (EC 3.2.1.48)

microbial origin 10 Units / mL deionized water


257

5 ± 0.05 g of dry pea flour were weighed in plastic tubes. 15 mL of 20 mM phosphate buffer pH 7

were added and the sample was homogenized for 30 s with a static laboratory mixer (Vortex

Genie 2, Scientific Industries, Bohemia, New York, USA). A sample of 2 mL was taken and 2 mL of

amylase solution was added to the flour in the plastic tube. Mixing was further continued for

another 30 s. The suspension was transferred into a 250 mL glass beaker and remainders in the

plastic tube were also washed into the beaker with 20 mL of 0.5 % hydrochloric acid (Merck KGaA,

Darmstadt, Germany). Another 2 mL of sample were taken and 2 mL of pepsin solution were

added to the glass beaker. The beaker was put on a shaker plate (Certomat U, B. Braun Biotech

Inc., Allentown, Pennsylvania, USA), warmed to 37 °C with a heating jacket (Certomat HK, B.

Braun Biotech Inc., Allentown, Pennsylvania, USA). After 60 min, addition of 40 mL of 150 mM

sodium hydrogen carbonate solution (Merck KGaA, Darmstadt, Germany) raised the pH to neutral

values. A sample was taken and 1 mL of trypsin and chymotrypsin and 2 mL of amylase were

added. After 10 min, a sample was taken again and 1 mL of each enzyme occurring in the small

intestine was added. Henceforward, samples of 2 mL were taken in regular intervals – every

15 min in the first hour, every 30 min in the two following hours.

All samples taken were heated for 10 min in boiling water to stop enzymatic activity. Heated

samples were centrifuged for 5 min at 10 000 g (Biofuge pico, Heraeus, Osterode, Germany) and

the supernatants were frozen in liquid nitrogen and stored at -20 °C until analysis. The increases

in accessible amino groups and reducing sugars were used as indicators for enzymatic

degradation of proteins and starch (see page 241 and 242). The digestion was as well performed

using deionized water instead of pea flour to subtract the number of accessible amino groups

originating from enzyme molecules.

Data evaluation and statistical analysis

All treatments were performed at least in duplicate, with the exception of the PEF treatment of

10 kV/cm, which was not repeated due to a damaged treatment chamber. All extractions,

digestions, dilutions, photometric and techno-functional analyses were done in triplicate. Results

are given as mean and standard deviations between the multiple treatments.

Statistical significance between sample means was analyzed with Tukey’s range test and a

significance level of 0.05. Analysis of variance was conducted with the ANOVA function of

originpro 7 (OriginLab Corporation, Northampton, MA, USA). Different letters next to the results

indicate significant differences between mean values.


258

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Bernfeld, P. (1955). Amylases, alpha and beta. Methods in Enzymology, 1, 149-158.

Beveridge, T., Toma, S. J. & Nakai, S. (1974). Determination of SH-groups and SS-groups in some food proteins using Ellmans reagent. Journal of Food Science, 39(1), 49-51.

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Schmitt, C., Moitzi, C., Bovay, C., et al. (2010). Internal structure and colloidal behaviour of covalent whey protein microgels obtained by heat treatment. Soft Matter, 6(19), 4876-4884.

Schwenke, K. D., Prahl, L., Rauschal, E., et al. (1981). Functional properties of plant proteins. 2. Selected physicochemical properties of native and denatured protein isolates from faba beans, soybeans, and sunflower seed. Nahrung-Food, 25(1), 59-69.

Sedlak, J. & Lindsay, R. H. (1968). Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Analytical Biochemistry, 25(1-3), 192-205.

Sigel, H. & Martin, R. B. (1982). Coordinating properties of the amide bond - Stability and structure of metal-ion complexes of peptides and related ligands. Chemical Reviews, 82(4), 385-426.

Street, C. A. (1991). Flour confectionary manufacture. Blackie and Son Ltd., Glasgow, UK.

Universität Regensburg (2013). Dynamische Lichtstreuung an kolloidalen und makromolekularen Systemen - Bestimmung von Partikelgrößen. Source: http://www.chemie.uni-regensburg.de/ Physikalische_Chemie/Schmeer/PDF_Files/Lichtstreuung_n.pdf. Accessed on: 18.06.2013.

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262

List of figures

Figure I.1: Schematic representation of the metabolic demands for amino acids (according to

WHO et al., 2007). .............................................................................................................................. 6

Figure I.2: Dietary protein consumption and percentage coming from livestock products

(according to FAO, 2013). ................................................................................................................... 8

Figure I.3: Trends in the consumption of livestock products: a) development of the global

production and per capita consumption of livestock goods and fish (FAO, 2013); b) meat

consumption in the EU 25 per weak (European Commission, 2005). ................................................ 9

Figure I.4: Reasons cited for being a vegetarian, agreement in % (Statistic Brain, 2012). .............. 13

Figure I.5: Contribution of noncommunicable diseases to the overall mortality in Germany (% of

total deaths, all ages, WHO, 2011b). ................................................................................................ 14

Figure I.6: Composition of the vegetal protein supply calculated from the daily protein

consumption in 2009 (FAO, 2013). ................................................................................................... 15

Figure I.7: Simplified flow chart of potato starch production according to Witt (1996) and

Bergthaller et al. (1999).................................................................................................................... 17

Figure I.8: Production of oil and defatted meal from different oilseeds in the EU 27 (FEDIOL, 2013).

.......................................................................................................................................................... 20

Figure I.9: Functional properties of selected domestic protein alternatives in comparison to

soybean (=1). Data obtained from Krause et al. (2007), Holm and Eriksen (1980), Fernandez-

Quintela et al. (1997), Aluko et al. (2009). ....................................................................................... 24

Figure II.1: Summer menu of star restaurant VAU in Berlin downloaded on 26th June 2013. ....... 34

Figure II.2: Sequence of protein-water interaction for dry protein according to Chou and Morr

(1976). .............................................................................................................................................. 37

Figure II.3: Destabilisation mechanisms of foams and emulsions. .................................................. 41

Figure II.4: The four sublevels of the protein structure (redrawn according to common illustrations

in literature). .................................................................................................................................... 44

Figure II.5: General scheme of pressure-temperature phase diagrams of proteins and pressure

effects on protein structure redrawn according to Messens et al. (1997). ..................................... 50

Figure II.6: Solubility profiles of pea protein concentrates won by isoelectric precipitation and

freeze drying or ultrafiltration with a 10 kDa cut off and subsequent freeze or spray drying. ....... 52

Annex B – Indices

263

Figure III.1: Flow chart of the experiments and analyses performed with protein in solution. ...... 62

Figure III.2: Influence of thermal treatments at ambient pressure on solubility and absorbance at

605 nm of potato protein solutions pH 7; sample: self-made protein concentrate; initial protein

concentration: 10 g/L; treatment time: 10 min protein quantification via Biuret method. ............ 64

Figure III.3: Particle size distributions in potato protein solutions pH 7 treated at different

temperatures; sample: self-made protein concentrate; initial protein concentration: 10 g/L;

treatment time: 10 min; centrifugation for 5 min at 500 g. ............................................................ 65

Figure III.4: Influence of high isostatic pressure on protein solubility and absorbance at 605 nm of

potato protein solutions pH 7; sample: self-made protein concentrate; initial protein

concentration: 10 g/L; treatment time: 10 min; protein quantification via Biuret method. ........... 66

Figure III.5: Particle size distributions in potato protein solutions pH 7 exposed to different

pressure-temperature combinations; sample: self-made protein concentrate; initial protein

concentration: 10 g/L; treatment time: 10 min; centrifugation for 5 min at 500 g. ........................ 67

Figure III.6: Surface hydrophobicity of the soluble protein fraction of potato protein solutions pH 7

subjected to following treatments: top: thermal treatments at atmospheric pressure; bottom:

pressure-temperature-combinations; sample: self-made protein concentrate; initial protein

concentration: 10 g/L; treatment time: 10 min; protein concentration during fluorescence

analysis: 1 g/L for aromatic hydrophobicity, 0.1 g/L for aliphatic hydrophobicity; protein

quantification via Biuret method. .................................................................................................... 68

Figure III.7: Non-reducing SDS PAGE of the soluble protein fraction of potato protein solutions

pH 7 subjected to following treatments: top: thermal treatments at atmospheric pressure;

bottom: pressure-temperature-combinations; sample: self-made protein concentrate; initial

protein concentration: 10 g/L; treatment time: 10 min; A: Lipoxidase (85 kDa); B: Patatin

monomer (41 kDa); C: Protease inhibitors (19-25 kDa). .................................................................. 70

Figure III.8: Protein solubility of potato protein solutions pH 7 and pH 6 subjected to following

treatments: top: thermal treatments at atmospheric pressure; bottom: pressure-temperature-

combinations; sample: commercial protein isolate; initial protein concentration: 10 g/L; treatment

time: 10 min; protein quantification via Biuret method. ................................................................. 71

Figure III.9: Aromatic surface hydrophobicity of the soluble protein fraction of potato protein

solutions pH 7 and pH 6 subjected to following treatments: top: thermal treatments at

atmospheric pressure; bottom: pressure-temperature-combinations; sample: commercial protein

isolate; initial protein concentration: 10 g/L; treatment time: 10 min; protein concentration during

fluorescence analysis: 1 g/L; protein quantification via Biuret method. ......................................... 73

Annex B – Indices

264

Figure III.10: Non-reducing SDS PAGE of the soluble protein fractions of potato protein solutions

pH 7 and pH 6 exposed to following treatments: 60 °C, 80 °C at ambient pressure and 600 MPa at

40 °C; sample: commercial protein isolate; initial protein concentration: 10 g/L; treatment time:

10 min; A: Lipoxidase (85 kDa); B: Patatin monomer (41 kDa); C: Protease inhibitors (19-25 kDa).

.......................................................................................................................................................... 75

Figure III.11: Stability of aggregates formed in potato protein solutions during treatments at 80 °C

and 0.1 MPa or 40 °C and 600 MPa towards addition of different reagents: top: absorbance of the

samples in respective reagents at 605 nm; bottom: proportion of precipitated protein in the

samples mixed with respective reagents; not precipitated protein was quantified via Biuret

method; sample: commercial protein isolate and self-made protein concentrate; initial protein

concentration: 10 g/L; treatment time: 10 min. .............................................................................. 77

Figure III.12: Influence of post-process medium changes on the solubility of protein solutions

subjected to following treatments: top: thermal treatments at atmospheric pressure; bottom:

pressure-temperature-combinations; sample: commercial protein isolate; initial protein

concentration: 10 g/L; treatment time: 10 min; HCl: addition of HCl to decrease pH value from 7 to

6; NaCl: adjustment of equimolar NaCl concentration at pH 7; protein quantification via Biuret

method. ............................................................................................................................................ 79

Figure III.13: Influence of thermal treatments at ambient pressure on protein solubility and

absorbance at 605 nm of pea protein solutions pH 7; sample: self-made protein concentrate;

initial protein concentration: 10 g/L; treatment time: 10 min; protein quantification via Biuret

method. ............................................................................................................................................ 80

Figure III.14: Influence of high isostatic pressure on protein solubility and absorbance at 605 nm of

pea protein solutions pH 7; sample: self-made protein concentrate; initial protein concentration:

10 g/L; treatment time: 10 min; protein quantification via Biuret method. ................................... 81

Figure III.15: Particle size distributions in heat treated pea protein solutions pH 7; sample: self-

made protein concentrate; initial protein concentration: 10 g/L; treatment time: 10 min;

centrifugation for 5 min at 500 g. .................................................................................................... 81

Figure III.16: Particle size distributions in pea protein solutions pH 7 exposed to different pressure-

temperature combinations; sample: self-made protein concentrate; initial protein concentration:

10 g/L; treatment time: 10 min; centrifugation for 5 min at 500 g. ................................................ 82

Figure III.17: Protein solubility and absorbance at 605 nm of pea protein solutions pH 7 subjected

to following treatments: top: thermal treatments at atmospheric pressure; bottom: pressure-

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265

temperature-combinations; sample: air-classified pea flour batch two; initial protein


Figure III.18: Particle size distribution of pea protein solutions pH 7 subjected to following

treatments: top: thermal treatments at atmospheric pressure; bottom: pressure-temperature-

combinations; sample: air-classified pea flour batch two; initial protein concentration: 10 g/L;

treatment time: 10 min; centrifugation for 5 min at 500 g. ............................................................ 84

Figure III.19: Influence of thermal treatments at ambient pressure (top) and different pressure-

temperature-combinations (bottom) on the surface hydrophobicity of the soluble fraction of pea

protein solutions pH 7; aromatic and aliphatic hydrophobicity of samples made of self-made

protein concentrate, aromatic hydrophobicity of samples prepared from air-classified pea flour

batch one; initial protein concentration: 10 g/L; treatment time: 10 min; protein concentration

during fluorescence analysis: 1 g/L for aromatic hydrophobicity, 0.1 g/L for aliphatic

hydrophobicity; protein quantification via Biuret method. ............................................................. 87

Figure III.20: Stability of aggregates formed in pea protein solutions during treatments at 80 °C

and 0.1 MPa or 40 °C and 600 MPa towards addition of different reagents: top: absorbance of the

samples in respective reagents at 605 nm; bottom: proportion of precipitated protein in the

samples mixed with respective reagents; not precipitated protein was quantified via Biuret

method; samples: self-made protein concentrate and protein-enriched flour batch two; initial

protein concentration: 10 g/L; treatment time: 10 min. ................................................................. 88

Figure III.21: Non-reducing SDS PAGE of the soluble protein fraction in solutions pH 7 prepared

from pea flour batch one after thermal treatments at ambient pressure and pressure treatments

at 40 °C; initial protein concentration: 10 g/L; treatment time: 10 min; A: Lipoxidase (90 kDa); B:

Convicilin (71 kDa); C: Legumin (60 kDa); D: Vicilin (17-50 kDa); E: PA2 (26 kDa). .......................... 90

Figure III.22: Non-reducing SDS PAGE of the soluble protein fraction in solutions pH 7 prepared

from pea concentrates after exposure to different treatments; top: thermal treatments at

ambient pressure; bottom: pressure treatments at 20 and 40 °C; initial protein concentration:

10 g/L; treatment time: 10 min; A: Lipoxidase (90 kDa); B: Convicilin (71 kDa); D: Vicilin (17-

50 kDa); E: PA2 (26 kDa). .................................................................................................................. 92

Figure III.23: Influence of thermal treatments at ambient pressure on total and fast accessible

thiol groups of pea protein solutions pH 7; top: samples prepared of self-made protein

concentrate; bottom: samples made of air-classified pea flour batch two; initial protein

concentration: 10 g/L; treatment time: 10 min protein quantification via Biuret method. ............ 93

Annex B – Indices

266

Figure III.24: Influence of several pressure-temperature-treatments on total and fast accessible

thiol groups of pea protein solutions pH 7; top: samples prepared of self-made protein

concentrate; bottom: samples made of air-classified pea flour batch two; initial protein


Figure III.25: Quantification of soluble protein via different photometric methods in potato

protein solutions pH 7 subjected to pulsed electric fields; sample: self-made protein concentrate;

initial protein concentration: 10 g/L. ............................................................................................... 96

Figure III.26: Quantification of soluble protein via different photometric methods and aromatic

surface hydrophobicity of the soluble protein fraction of whey protein solutions pH 7 subjected to

pulsed electric fields; sample: commercial whey protein isolate; initial protein concentration:

10 g/L; protein concentration during fluorescence analysis: 2 g/L; protein quantification via Biuret

method. ............................................................................................................................................ 97

Figure III.27: Quantification of soluble protein via different photometric methods and aromatic

surface hydrophobicity of the soluble protein fraction of pea protein solutions pH 7 subjected to

pulsed electric fields; sample: air-classified pea flour batch one; initial protein concentration:

10 g/L; protein concentration during fluorescence analysis: 1 g/L; protein quantification via Biuret

method. ............................................................................................................................................ 98

Figure III.28: Non-reducing SDS PAGE of protein solutions pH 7 prepared from pea flour and

treated with pulsed electric fields of varying intensity; initial protein concentration: 10 g/L;

treatment time: 10 min; A: Lipoxidase (90 kDA); B: Convicilin (71 kDa); C: Legumin (60 kDa); D:

Vicilin (17-50 kDa); E: PA2 (26 kDa). ................................................................................................. 99

Figure IV.1: Half-life of foams prepared from heat treated pea flour suspensions pH 7; treatment

time: 10 min; protein concentration during treatment: 10 g/L; protein concentration during foam

analyses: 0.4 g/L; dilution medium: deionized water. ................................................................... 107

Figure IV.2: Half-life of foams prepared from pressurized pea flour suspensions pH 7; treatment

time: 10 min; protein concentration during treatment: 10 g/L; protein concentration during foam

analyses: 0.4 g/L; dilution medium: deionized water. ................................................................... 108

Figure IV.3: Foam stability of the supernatants obtained from pea flour suspensions pH 7 after

heating at atmospheric pressure and pressure treatments at 40°C; treatment time: 10 min;

protein concentration during treatment: 10 g/L; protein concentration during foam analyses:

0.33 g/L; dilution medium: deionized water; detection time: 10 min. .......................................... 109

Figure IV.4: Mechanistic proposal for the structural alterations after application of heat (60 °C,

80 °C) or pressure (p), and after centrifugation (RCF) and adjustment of remaining protein

Annex B – Indices

267

concentration, effects on foam stability (FS) and volume expansion during foam formation (FE).

........................................................................................................................................................ 112

Figure IV.5: Half-life of foams prepared from solutions of commercial potato protein pH 7

subjected to different temperatures; treatment time: 10 min; protein concentration during

treatment: 10 g/L; protein concentration during foam analyses: 0.6 g/L; dilution medium:

deionized water. ............................................................................................................................. 113

Figure IV.6: Half-life of foams prepared from solutions of commercial potato protein pH 7

subjected to different pressure-temperature combinations; treatment time: 10 min; protein

concentration during treatment: 10 g/L; protein concentration during foam analyses: 0.6 g/L;

dilution medium: deionized water. ................................................................................................ 114

Figure IV.7: Stability of foams prepared from PEF treated pea flour (batch one) suspensions pH 7;

protein concentration during treatment: 10 g/L; protein concentration during foam analyses:

0.33 g/L; dilution medium: deionized water. ................................................................................. 115

Figure IV.8: Stability of foams prepared from PEF treated solutions pH 7 prepared from self-made

protein concentrate; protein concentration during treatment: 10 g/L; protein concentration

during foam analyses: 0.6 g/L; dilution medium: deionized water. .............................................. 117

Figure IV.9: Inactivation of vegetative microbial cells by heat treatment at 60 °C in 1/8 ringer

solution or solutions made of pea and potato protein concentrate with a protein concentration of

10 g/L; top: E. coli; bottom: L. innocua. ......................................................................................... 120

Figure IV.10: Inactivation of vegetative microbial cells by pressure treatment at 400 MPa and

40 °C in 1/8 ringer solution or solutions made of pea and potato protein concentrate with a

protein concentration of 10 g/L; top: E. coli; bottom: L. innocua. ................................................. 122

Figure IV.11: Inactivation of vegetative microbial cells in 1/8 ringer solution by pulsed electric field

treatment of various intensities at different starting temperatures; top: E. coli; bottom: L. innocua.

........................................................................................................................................................ 124

Figure V.1: Cell disintegration indices (CDI) of potato tissues subjected to PEF treatments of

different intensities in relation to untreated tissue calculated via electrical conductivity at

frequencies of 5.5 kHz and 2.8 MHz. Columns with different letters deviate significantly (α = 0.05).

........................................................................................................................................................ 133

Figure V.2: Microscopic pictures of tissue sections taken from peas exposed to different physical

treatments in a swollen state and re-dried in warm air at 50 °C. Top left: 20°C, top right: PEF

treatment of 5 kV/cm and 125 kJ/kg; middle: PEF treatment of 10 kV/cm and 125 kJ/kg, bottom

Annex B – Indices

268

left: thermal treatment at 80 °C, bottom right: pressure treatment at 400 MPa and 40 °C; staining

with toluidine blue. ........................................................................................................................ 134

Figure V.3: Photographs of agar plates used for germination tests of swollen peas subjected to

different treatments. From left to right: ungerminated and germinated samples after 24 h, root

development after 48 h; microbial growth on ungerminated samples after 48 h. ....................... 135

Figure V.4: Protein release into treatment medium in dependence of cutting geometry; pulsed

electric field treatment with 1 kV/cm and 5 kJ/kg; calculation based on a protein content of

20 g/kg fresh potato; columns with different letters deviate significantly (α = 0.05). .................. 137

Figure V.5: Release of protein and oligosaccharides from whole peas into treatment medium in

dependence of the electric field strength applied; energy input: 125 kJ/kg.; time into treatment

medium: 10 min; determination of protein content with Bradford reagent; quantification of

oligosaccharides with an enzymatic test kit; columns with different letters deviate significantly (α

= 0.05). ............................................................................................................................................ 139

Figure V.6: Influence of heat and pressure on the release of protein and oligosaccharides from

whole peas into the surrounding medium; top: thermal treatments at ambient pressure; bottom:

high pressure treatments at 40 °C; treatment time: 10 min; release time: 15 min; quantification of

protein with Bradford reagent; quantification of oligosaccharides with an enzymatic test kit;

columns with different letters deviate significantly (α = 0.05). ..................................................... 140

Figure V.7: Influence of pulsed electric fields on the solid-liquid separation of potato tissue in a

manual press. The moisture content in the press residue was determined using a moisture

analyzer (Sartorius MA 35). ............................................................................................................ 141

Figure V.8: Drying rates of potato press cakes in relation to its moisture content; press cakes were

obtained from manual pressing of grinded potatoes previously treated with pulsed electric fields

of different intensities; drying was performed for 300 min at 50 °C in a drying cabinet. ............. 142

Figure V.9: Drying rates of whole peas influenced by different treatments in relation to their

moisture content; top: heat treatment; middle: pressure treatment at 40 °C, bottom: PEF

treatment: treatment times amounted 10 min for heat and pressure and were dependent on the

pulse number for PEF treatments. Peas were dried for 180 min at 50 °C in a moisture analyzer. 143

Figure V.10: Visual appearance of peas re-dried after exposure to different treatments: Top:

pressure treatment; bottom left: heat treatment; bottom right: pulsed electric fields; treatment

times amounted 10 min for heat and pressure and were dependent on the pulse number for PEF

treatments; peas were dried for 6 h at 50 °C. ................................................................................ 144

Annex B – Indices

269

Figure V.11: Microscopic pictures of tissue sections taken from peas exposed to pressure

treatments at 40 °C in a swollen state; left: 600 MPa; right: 400 MPa; treatment time: 10 min;

drying for 6 h at 50 °C..................................................................................................................... 145

Figure V.12: Rehydration of whole peas influenced by different technologies; top: heat treatment;

middle: pressure treatment at 40 °C, bottom: PEF treatment; treatment times amounted 10 min

for heat and pressure and were dependent on the pulse number for PEF treatments; peas were

dried for 6 h at 50 °C; water uptake occurred for 24 h at room temperature. ............................. 147

Figure V.13: Rehydration of whole peas influenced by different technologies; top: heat treatment;

middle: pressure treatment at 40 °C, bottom: PEF treatment; treatment times amounted 10 min

for heat and pressure and were dependent on pulse number for PEF treatments; peas were dried

for 6 h at 50 °C; water uptake occurred for 2 h in boiling water. .................................................. 148

Figure VI.1: Protein content in potato fruit juice won by a manual pressing after pulsed electric

field treatment of whole potato tubers and calculated protein yields in relation to tuber dry

matter; protein quantification via Bradford analysis; columns with different letters deviate

significantly (α = 0.05). ................................................................................................................... 157

Figure VI.2: Protein yield determined via Bradford analysis obtained by extraction of freeze-dried

potato cubes or solid-liquid separation and subsequent press cake extraction; PEF treatment: 1

kV/cm, 5 kJ/kg, 666 pulses, 2 Hz; percentages imply the changes due to PEF in relation to the

control sample................................................................................................................................ 161

Figure VI.3: Changes in protein solubility determined with Bradford assay during pH-adjustment

and re-setting to the initial value in potato fruit juice. .................................................................. 162

Figure VI.4: Foam stability in 1 % protein solutions pH 7 prepared from self-made potato protein

concentrate whipped with a handheld electric mixer; PEF treatment: 1 kV/cm, 5 kJ/kg, 666 pulses,

2 Hz; acid treatment: setting to pH 4.5 by HCL addition and resetting to pH 6 with NaOH. ......... 164

Figure VI.5: Buffer and water extractability of protein from wet-grinded pea flour subjected to PEF

treatments of 125 kJ/kg at room temperature; extraction time 30 min; columns with different

letters deviate significantly (α = 0.05). ........................................................................................... 165

Figure VI.6: Extractability of protein from wet-grinded pea flour subjected to thermal and high

pressure treatments with 10 min dwell time; protein extraction in buffer at pH 9 within 30 min

under continuous stirring; columns with different letters deviate significantly (α = 0.05). .......... 167

Figure VI.7: Protein extractability from wet-grinded pea flour subjected to thermal and high

pressure treatments with 10 min dwell time; protein extraction in tap water within 30 min under

continuous stirring; columns with different letters deviate significantly (α = 0.05). ..................... 168

Annex B – Indices

270

Figure VI.8: Non-reducing SDS PAGE of protein extracts from wet-grinded peas after pulsed

electric field, thermal and high pressure treatments; extraction medium: tap water; extraction

time: 30 min; A: Lipoxidase (90 kDa); B: Convicilin (71 kDa); C: Legumin (60 kDa); D: Vicilin (17-

50 kDa); E: PA2 (26 kDa). ................................................................................................................ 169

Figure VI.9: Solubility of pea protein concentrates in phosphate-citrate buffer of different pH after

30 min of stirring; protein concentrates won from swollen peas subjected to 20 °C (untreated), 80

°C (heat treated) or 600 MPa (pressure treated); initial powder concentration 10 g/L; protein

quantification via Biuret method. .................................................................................................. 171

Figure VI.10: Foam stability detected for solutions prepared from pea protein concentrates in tap

water at pH 7; protein concentrates won from swollen peas subjected to 20 °C (untreated), 80 °C

(heat treated) or 600 MPa at 40 °C (pressure treated); protein quantification in clear solutions via

Biuret assay; protein concentration for foam analyses: 0.35 g/L; dilution medium: deionized

water; testing unit: DFA 100. ......................................................................................................... 172

Figure VII.1: Water binding capacity of flours prepared from peas treated at different pressure-

temperature combinations; peas were swollen for 20 h in tap water before treatments; treatment

time: 10 min; drying of peas for 6 h at 50 °C; binding capacity was determined at ambient

temperature against a centrifugation at 10 000 g for 15 min. ...................................................... 179

Figure VII.2: Fat binding capacity of flours prepared from peas treated at different pressure-

temperature combinations; peas were swollen for 20 h in tap water before treatments; treatment

time: 10 min; drying of peas for 6 h at 50 °C; binding capacity was determined at ambient

temperature against a centrifugation at 10 000 g for 15 min. ...................................................... 180

Figure VII.3: Tap water and fat binding of protein concentrates prepared from peas treated at

20 °C or 80 °C at ambient pressure or at 40 °C and 600 MPa; peas were treated after 20 h of

swelling in tap water; treatment time: 10 min, binding capacity was determined against

centrifugation for 15 min at 10 000 g. ........................................................................................... 181

Figure VII.4: Flour suspensions in a transmitted light microscope prepared from peas subjected to

the following treatment conditions: Top: 20 °C (left), 60 °C (middle) and 80 °C (right) at

atmospheric pressure; bottom: 200 MPa (left), 400 MPa (middle) and 600 MPa (right) at 40 °C;

peas were swollen in tap water for 20 h previous to treatments; treatment time: 10 min. ......... 182

Figure VII.5: Microscopic pictures of tissue sections taken from peas exposed to 10-minute

treatments in a swollen state and re-dried in warm air at 50 °C. Left: 80 °C; right: 600 MPa at

40 °C. .............................................................................................................................................. 183

Annex B – Indices

271

Figure VII.6: Firmness of gels made of 20 % (w/v) pea flour suspensions heated to 100°C for 5 min

and cooled down to ambient temperature for 3.5 h; flours were made from peas treated at

different temperature-pressure combinations for 10 minutes in a swollen state and re-dried for

6 h at 50 °C; gel firmness was calculated via force-displacement curves of the texture analyzer. 184

Figure VII.7: Activity in pure enzyme solutions after exposure to different temperatures at

ambient pressure in relation to samples treated at 20°C; top: lipoxidase from soybeans; bottom:

peroxidase from horseradish. ........................................................................................................ 187

Figure VII.8: Activity in pure enzyme solutions after pressure treatments at 40°C in relation to

untreated control; top: lipoxidase from soybeans; bottom: peroxidase from horseradish. ......... 189

Figure VII.9: Lipoxidase activity in extracts from peas treated at different pressure-temperature

combinations in relation to activity in untreated, unswollen peas; peas were treated after 20 h

swelling in tap water; treatment time: 10 min; drying for 6 h at 50 °C. ........................................ 190

Figure VII.10: Peroxidase activity in extracts from peas treated at different pressure-temperature

combinations in relation to activity in untreated, unswollen peas; peas were treated after 20 h

swelling in tap water; treatment time: 10 min; drying for 6 h at 50 °C. ........................................ 192

Figure VII.11: Buffers, solutions and enzymes added during the different steps of the simulated

digestion. ........................................................................................................................................ 194

Figure VII.12: Influence of thermal and pressure treatments of whole peas on the protein

digestibility in the respective dry pea flour. Peas were swollen for 20 h in tap water and subjected

to 80 °C for 10 min (heat treated), 600 MPa at 40 °C for 10 min (pressure treated) or not further

treated (untreated); drying of whole peas for 6 h at 50 °C; digestion according to Figure VII.11 and

annex page 253. ............................................................................................................................. 196

Figure VII.13: Protein digestibility in pea flours made of heat and pressure treated whole peas.

Peas were swollen for 20 h in tap water and subjected to 80 °C for 10 min (heat treated), 600 MPa

at 40 °C for 10 min (pressure treated) or not further treated (untreated); drying of whole peas for

6 h at 50 °C; digestion according to Figure VII.11 and annex page 253. ........................................ 197

Figure VII.14: Starch digestibility in pea flours exposed to heat and pressure treatments. Peas

were swollen for 20 h in tap water and subjected to 80 °C for 10 min (heat treated), 600 MPa at

40 °C for 10 min (pressure treated) or not further treated (untreated); drying of whole peas for

6 h at 50 °C; digestion according to Figure VII.11 and annex page 253. ........................................ 199

Figure VII.15: Influence of thermal and pressure treatments of whole peas on the starch

digestibility in dry pea flour. Peas were swollen for 20 h in tap water and subjected to 80 °C for

10 min (heat treated), 600 MPa at 40 °C for 10 min (pressure treated) or not further treated

Annex B – Indices

272

(untreated); drying of whole peas for 6 h at 50 °C; digestion according to Figure VII.11 and annex

page 253. ........................................................................................................................................ 201

Figure VIII.1: Possible ways to implement pulsed electric fields and high pressure into existent

potato processing strategies. ......................................................................................................... 214

Figure VIII.2: Possibilities to implement pulsed electric fields and high pressure into different pea

process options. ............................................................................................................................. 215

Annex B – Indices

273

List of tables

Table I.1: Environmental evaluation of animal and vegetable protein production ......................... 10

Table I.2: Survey of the amino acid content of different food protein sources (Young & Pellett,

1994) ................................................................................................................................................ 12

Table I.3: Lipid and protein contents of selected oilseeds of interest for the food industry .......... 19

Table I.4: Biochemical composition of selected legume seeds compiled by Gueguen (1983) from

several references ............................................................................................................................ 21

Table I.5: Nutritional comparison of selected plant proteins .......................................................... 23

Table II.1: Major functional proteins (Zayas, 1997) ......................................................................... 35

Table II.2: Functional protein properties and their relation to structural attributes ...................... 36

Table II.3: Molecular protein characteristics affecting its functional properties (Kinsella, 1982) ... 43

Table III.1: Characterization of the protein rich basic material and the 1 % protein solutions made

of them ............................................................................................................................................. 63

Table III.2: Average particle diameter of thermal and high pressure treated protein solutions

made of commercial isolate at pH 7 and pH 6; initial protein concentration: 10 g/L; treatment

time: 10 min; solutions were centrifuged for 5 min at 500 g .......................................................... 72

Table III.3: Absorbance read at 540 nm of the centrifuged pea flour (batch two) solutions pH 7

subjected to heat or high pressure diluted 1:10 with deionized water (analogous to conduct of the

Biuret analysis); initial protein concentration: 10 g/L; treatment time: 10 min; centrifugation for

10 min at 5 min at 10 000 g .............................................................................................................. 85

Table IV.1: Time to reach maximum foam height of 160 mm; sample: protein-enriched pea flour;

treatment time: 10 min; protein concentration during treatment: 10 g/L; protein concentration

during foam analyses: 0.4 g/L for whole samples, 0.33 g/L for supernatants; dilution medium:

deionized water .............................................................................................................................. 111

Table IV.2: Time to reach maximum foam height of 160 mm; sample: protein-enriched pea flour

and self-made potato protein concentrate; protein concentration during treatment: 10 g/L;

protein concentration during foam analyses: 0.33 g/L for pea protein; 0.6 g/L for potato protein;

dilution medium: deionized water ................................................................................................. 116

Table V.1: Germination ability of differently treated whole peas – (x) germination occurred within

48 h after treatment; (/) no germination detected within this time period; pressure treatments

were performed at 40 °C, pulsed electric field treatments were conducted at ambient

Annex B – Indices

274

temperature; dwell time for thermal and high pressure treatments amounted 10 min, duration of

the pulsed electric field treatment was determined by the pulse number ................................... 136

Table V.2: Density of the peas after different treatments and warm air drying at 50 °C in relation

to control (peas treated at 20 °C and atmospheric pressure without application of an electric

field); dwell time for thermal and high pressure treatments amounted 10 min, duration of the

pulsed electric field treatment was determined by the pulse number ......................................... 145

Table V.3: Characterization of the rehydration medium used for differently pre-treated peas: total

soluble solids in °Bx and light absorbance and at 412 nm; treatment time: 10 min; drying for 6 h at

50 °C, rehydration in tap water for 24 h ........................................................................................ 149

Table V.4: Specific density of the peas after rehydration in relation to peas treated at 20 °C and

atmospheric pressure; treatment time: 10 min; drying for 6 h at 50 °C, rehydration in tap water

for 24 h ........................................................................................................................................... 149

Table VI.1: Water extractable protein in mg/g dry matter determined via different photometric

methods. PEF treatment: 1 kV/cm, 5 kJ/kg, 666 pulses, 2 Hz ........................................................ 158

Table VI.2: Foam formation characteristics in 1 % protein solutions pH 7 prepared from self-made

potato protein concentrate whipped with a handheld electric mixer; PEF treatment: 1 kV/cm,

5 kJ/kg, 666 pulses, 2 Hz; acid treatment: setting to pH 4.5 by HCL addition and reset to pH 6 with

NaOH .............................................................................................................................................. 163

Table VI.3: Composition of protein concentrates won from swollen peas subjected to 20 °C

(untreated), 80 °C (heat treated) or 600 MPa at 40 °C (pressure treated) .................................... 170

Table VII.1: Protein content and specific volume of protein concentrates prepared from peas

treated at 20 °C or 80 °C at ambient pressure or at 600 MPa at 40 °C; peas were treated after 2 h

of swelling in tap water; treatment time: 10 min .......................................................................... 182

Table VIII.1: Estimated costs for pulsed electric field treatment on industrial scale ..................... 217

Table VIII.2: Estimated processing costs for a high pressure treatment in an industrial unit of the

type wave 6000 model 300 (Hiperbaric, Burgos, Spain) ................................................................ 219

Annex B – Indices

275

Abbreviations

ANOVA Analysis of variance

ANS 8-Anilinonaphthalene-1-sulfonic acid

BMI Body mass index

BSE Bovine spongiforme Enzephalopathie

CDI Cell disintegration index

Cfu Colony forming units

CPA cis-Parinaric acid

DTT Dithiothreitol

EC Enzyme Commission numbers

EU European Union

EU 25

25 member states of the European Union

(accession until December 2006)

EU 27

27 member states of the European Union

(accession until June 2013)

HP High pressure

LOX Lipoxidase

n.s. Not specified

OPA ortho-Phthaldialdehyd

PAGE Polyacrylamide gel electrophoresis

PEF Pulsed electric fields

Annex B – Indices

276

POD Peroxidase

SDS Sodium dodecyl sulfate

UK United Kingdom

USA United States of America

Annex C – Personal information

277

Annex C Personal information


278

Curriculum vitae

Personal details:

Name:

Date of birth:

Place of birth:

Family status:

Children:

Nationality:

Anne Kathrin Baier née Heckelmann

25 March, 1983

Heppenheim an der Bergstraße

Married

Emilie Luise (* 03 May 2014)

German

Work Experience:

since Oct. 2007: Research associate and PhD candidate at the Department of Food

Biotechnology and Food Process Engineering, Technische Universität

Berlin

Dec. 2006 – Sep. 2007:

Student assistant at the Department of Food Biotechnology and Food

Process Engineering, Technische Universität Berlin, in the EU project

Novel processing methods for the production and distribution of high

quality and safe foods (NovelQ)

Jul. 2004 – Oct. 2004: Internship at August Storck KG, Berlin

Research projects:

Entwicklung eines Hochspannungsimpulsunterstützten Verfahrens zur Verdrängungsextraktion von

Ölen und funktionellen Proteinen aus Ölsaaten am Beispiel von Raps

LeguAN - Innovative und ganzheitliche Wertschöpfungskonzepte für funktionelle Lebens- und

Futtermittel aus heimischen Körnerleguminosen vom Anbau bis zur Nutzung


279

Education:

Oct. 2002 – Sep. 2007: Studies in Food Technology at Technische Universität Berlin

Degree: M.Eng (Dipl.-Ing.) Food Technology

Feb. 1994 – Jun. 2002: Evangelische Schule Frohnau, Berlin (Primary and grammar school)

Degree: Abitur (A-Levels)

Aug. 1993 – Feb. 1994: Albertus-Magnus-Schule, Viernheim (Grammar school)

Aug. 1989 – Jun. 1993: Nibelungenschule, Viernheim (Primary school)

Diploma thesis:

Title: “Influence of low intensity pulsed electric fields on the activity of Polyphenoloxidase and

Peroxidase”

Supervision: Prof. Dr. Dipl.-Ing. Dietrich Knorr, Dipl.-Ing. Ana Balasa

Language skills:

German:

English:

French:

Native

Fluent

Basic skills

Berlin, 30.06.2015


280

Publications / Presentations

Peer reviewed journal articles:

Baier, A.K., Bußler, S., Knorr, D. Potential of high isostatic pressure and pulsed electric fields to

improve mass transport in pea tissue. Food Research International (2014),

http://dx.doi.org/10.1016/j.foodres.2014.11.043 (article in press).

Baier, A.K., Knorr, D. Influence of high isostatic pressure on structural and functional

characteristics of potato protein. Food Research International (2015),

http://dx.doi.org/10.1016/j.foodres.2015.05.053 (article in press).

Oral and poster presentations:

Baier, A.K., Rauh, C., Knorr, D. High pressure processing of starchy foods. Poster presentation at

“20th international IGV conference – healthy grain for a healthy diet”. April 2015 in Nuthetal,

Germany.

Baier, A.K., Baier, D., Rauh, C., Knorr, D. Potenzial hoher isostatischer Drücke und gepulster

elektrischer Felder für die Leguminosenverarbeitung. Oral presentation at „GDL Kongress

Lebensmitteltechnologie 2014“. Oktober 2014 in Frankfurt, Germany.

Heckelmann, A.K., Baier, D., Knorr, D. Einsatz innovativer Technologien in der

Proteinprozessierung. Oral presentation at “Jahrestreffen der ProcessNet-Fachgruppen

Lebensmittelverfahrenstechnik und Phytoextrakte”. Februar 2014 in Freising, Germany.

Heckelmann, A.K., Knorr, D. Potential of non-thermal technologies in protein processing. Oral

presentation at “19th international IGV conference - capabilities of vegetable proteins”. April

2013 in Nuthetal, Germany.

Purschke, B., Heckelmann, A.K., Knorr, D. Influence of thermal, high pressure and pulsed electric

field treatments on structure and foaming properties of soluble proteins of pea cultivar

Salamanca. Poster presentation at “19th international IGV conference - capabilities of vegetable

proteins”. April 2013 in Nuthetal, Germany.

Heckelmann, A.K., Purschke, B., Bußler, S., Rohn, S., Knorr, D. Einfluss isostatischen Hochdrucks

auf die Gewinnung, Konservierung und funktionellen Eigenschaften pflanzlicher Proteine. Invited


281

talk at „Regionalverbandstagung Nordost der Lebensmittelchemischen Fachgruppe in der

Gesellschaft Deutscher Chemiker“. March 2013 in Berlin, Germany.

Bußler, S., Heckelmann, A.K., Knorr, D. Influence of High Isostatic Pressure on the Functional

Properties of Pea and Pea Flour. Poster presentation at “EFFoST annual meeting 2012”. November

2012 in Montpellier, France.

Heckelmann, A.K., Purschke, B., Knorr, D. High isostatic pressure as a tool for pea protein

modification. Poster presentation at “European High Pressure Research Group meeting 2012”.

September 2012 in Thessaloniki, Greece.

Heckelmann, A.K., Knorr, D. Influence of pulsed electric fields on potato protein extractability.

Poster presentation at “EFFoST annual meeting 2011”. November 2011 in Berlin, Germany.

Bußler, S., Heckelmann, A.K., Knorr, D. Effect of Pulsed Electric Fields on the Yield and Properties

of Legume Proteins. Poster presentation at “EFFoST annual meeting 2011”. November 2011 in

Berlin, Germany.

Heckelmann, A.K., Knorr, D. Pulsed electric field assisted extraction of potato protein – effects on

protein recovery. Poster presentation at “iFood Conference 2011”. October 2011 in Osnabrück,

Germany.

Heckelmann, A.K., Weiler, A., Knorr, D. Influence of pulsed electric fields on potato processing –

recovery of potato fruit juice and potato protein. Poster presentation at “BerlinFood 2010 –

European PhD Conference on Food Science and Technology”. September 2010 in Berlin, Germany.

Heckelmann, A.K., Weiler, A., Knorr, D. Improvement of potato protein extraction by pulsed

electric field treatment. Poster presentation at “EFFoST Conference 2009”. November 2009 in

Budapest, Hungary.

Heckelmann, A.K., Krause, J.-P., Knorr, D. Recovery of functional rape seed protein. Poster

presentation at “EFFoST First European Food Congress 2008”. November 2008 in Ljubljana,

Slovenia.

282

Eidesstattliche Erklärung

Hiermit versichere ich an Eides statt, dass ich die Dissertation selbständig verfasst habe. Alle

benutzten Hilfsmittel und Quellen sind aufgeführt.

Weiter erkläre ich, dass ich nicht schon anderweitig einmal die Promotionsabsicht angemeldet

oder ein Promotionseröffnungsverfahren beantragt habe.

Veröffentlichungen von irgendwelchen Teilen der vorliegenden Dissertation sind von mir, wie in

der vorstehenden Publikationsliste aufgeführt, vorgenommen worden.

Berlin, 30.06.2015

Anne Kathrin Baier

potential of high isostatic pressure and pulsed electric ... · different fields of work. i would...

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