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physicochemical conditions: structures, interactions and
functionalities
Muhammad Gulzar
To cite this version:
Muhammad Gulzar. Dry heating of whey proteins under controlled physicochemical conditions: struc-tures, interactions and functionalities. Food engineering. Université de Bretagne Occidentale; AGRO-CAMPUS OUEST, 2011. English. �tel-02807314�
Ph.D. Thesis at Agrocampus Ouest
Under the seal of European University of Brittany To obtain the degree:
Docteur de L’Institut Supérieur Des Sciences Agronomiques, Agro-alimentaire,
Horticoles Du Paysage
Specialization: Food Science
Doctoral College: VAS (Life – Agriculture – Health)
Presented by:
Muhammad Gulzar
Public defense « December 12, 2011 » in front of Examination Committee Composition of jury:
Dr. Didier Marion, Director of Research, INRA Nantes, France. Reviewer
Professor Jack Legrand, UMR 6144 GEPEA, CNRS, Saint Nazaire, France. Reviewer
Dr. André Brodkorb, Senior Researcher, TFRC, Moorepark, Ireland. Member
Dr. Camille Loupiac, Equipe EMMA, Université de Bourgogne AgroSup Dijon, France Member
Professor Thomas Croguennec, Agrocampus-Ouest, Rennes, France Ph.D. Supervisor
Dr. Saïd Bouhallab, Director of Research, INRA-UMR STLO, Rennes, France Ph.D. Co-Supervisor
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N° ordre: 2011-31 N° Série: B-220Acknowledgements
Acknowledgements
Acknowledgements
Acknowledgements
It’s a reality that this life is not sufficient for saying thanks to The Almighty Allah (SWT) for the countless blessings that He (SWT) has conferred on me. However in an effort, I would like to pay my special gratitude to The Almighty Allah (SWT) who gave me the capacity, courage and consistency to accomplish this task successfully. I dedicate this work to my Lord Allah (SWT), to Whom all of my efforts and all of my life belong to. May peace and blessings of Allah (SWT) be upon The Prophet Muhammad (SAW), Who is The Mercy and the source of Mercy of Allah (SWT).
After that I would also like to pay my special thanks to my teachers Sheikh Abdul Qadir Jilani (May Allah (SWT) be pleased with him) and Hafiz Ehsan Elahi (May the mercy of Allah (SWT) be upon him), who are a source of guidance for me in my life. I would also like to say thanks to all of my family members, who were away from me but they always encouraged me to do this job.
I am highly grateful to Sylvie Lortel (Director INRA UMR-STLO) for welcoming me at UMR-STLO to start my scientific life. I am also thankful to Mr. Pierre Guy Marnet for guiding me towards this lab. Thomas Croguennec, my PhD Supervisor, I will not say thanks to him because I don’t have words to thank him for his countless efforts that he invested to train me to pay my part in scientific evolution of milk and milk products. I do remember very well that he strived a lot to help me start my scientific life and he taught me things as we communicate life skills to children. I would also like to pay my special thanks to Said Bouhallab (Co-Supervisor), who was really very helping person and criticizes positively. I do not have words to pay my gratitude to these two persons. I am also grateful to Dr. Didier Marion and Professor Jack Legrand for accepting to review my thesis as well as Dr. André Brodkorb and Dr. Camille Loupiac for accepting to be the part of my Jury.
My special thanks to Valerie Lechevalier who saved my life which was being deteriorated by ignorance of statistical knowledge. Thanks to her who helped me for learning and using statistics and interpretation of statistical results. I am grateful to Mr. Pierre Schuck and Romain Jeantet for participating in several meetings and for giving fruitful advices. In addition, I would also like to say thanks to all the lab fellows who helped me directly or indirectly for accomplishing this honorable task. Among the lab fellows I will say special thanks to Arlette Loubeyre (Secretariat) for her special help at every event of my PhD thesis. I am thankful to Christope Geneste and Radwan Jalem for their help in informatics problems. I am thankful to Claire Prioul and Maryvonne Pasco for their help and advices in several experiments. I am thankful to Michel Piot for analysis of ions for protein powders, Julien Jardin, Danial Mollé and Valerie Briard Bion for analyzing my samples by Mass Spectrometry. I am thankful to Stephan Pezzenec for forming me for experiments of FTIR, Elizbeth Le Reumeur for training in CD experiments, Florence Rousseau for help in experiments by Nano Sizer, Anne Dolivet for my formation of aw meter, Jean Jacques Dubois
for experiment of protein quantification and for my formation of atomic absorption spectrometry.
Finally I would say that I have mentioned the names of a very few people but I was benefited by a number of people at UMR-STLO but I will be unable to mention the countless efforts of all the people working at UMR-STLO. I would like to say thanks to all the members of UMR-STLO who participated directly or indirectly in the evolution of my life (scientific and general) at this lab. I am also thankful to Paul Robin (UMR-SAS) for his moral support and encouragement in my work. I am also highly grateful to the Higher Education Commission (HEC) of Pakistan and INRA UMR-STLO for the financial support of this project.
General Introduction ... 1
Part 1: Review of Literature ... 9
1: Whey Proteins ... 11
1.1: Beta-lactoglobulin ... 13
1.2: Alpha-Lactalbumin ... 14
1.3: Bovine Serum Albumin... 16
1.4: Minor whey proteins ... 17
2: Functional properties of whey proteins... 17
2.1: Solubility ... 18
2.2: Gelling properties... 19
2.3: Emulsifying properties ... 21
2.4: Foaming properties... 22
3: Pre-texturization of whey proteins ... 23
3.1: Pre-texturization in aqueous solution... 24
3.1.1: Enzymatic modifications... 24 3.1.1.1: Hydrolytic enzymes ... 24 3.1.1.2: Crosslinking enzymes ... 25 3.1.2: Physical Modifications... 26 3.1.2.1: Heat treatment ... 26 3.1.2.2: High pressure ... 28 3.1.3: Chemical modifications ... 29 3.1.3.1: Glycation ... 29 3.1.3.2: Acylation ... 30
3.2: Pre-texturization in powder state ... 31
3.2.1: Chemical modifications under dry heating ... 31
3.2.2: Structural and functional changes during dry heating... 33
4: Purpose of the Study ... 37
Part 2: Materials and Methods ... 39
1: Protein samples ... 41
2: Preparation of dry heated Powders: ... 41
3: Structural characterization of dry heated powders... 42
3.1: Structural analysis in powder form ... 42
3.1.1: Fourier transform infrared (FTIR) spectroscopy... 42
3.1.2: Differential scanning calorimetry ... 42
3.2: Structural analysis on reconstituted solutions ... 43
3.2.1: Reconstitution of whey protein powders in solution... 43
3.2.2: Physical analysis ... 44
3.2.2.1: Protein solubility ... 44
3.2.2.2: Turbidity measurement ... 44
3.2.2.3: Determination of aggregate size... 45
3.2.3: Chemical analysis... 45
3.2.3.1: Gel permeation chromatography (GPC) ... 45
3.2.3.2: Reversed phase – high pressure liquid chromatography (RP-HPLC):... 46
3.2.3.3: SDS-PAGE Analysis... 46
3.2.3.4: Sulfhydryl Quantification... 46
3.2.3.5: Surface hydrophobicity ... 47
3.2.3.6: Intrinsic hydrophobicity ... 47
3.2.3.8: Mass Spectrometry... 48
3.2.3.9: Protein identification by in gel trypsinolysis ... 49
4: Functional characterization of dry heat samples ... 50
4.1: Gel Hardness and Water Holding Capacity ... 50
4.2: Colorimetric Measurement... 51
Part 3: Results and Discussion ... 53
Chapter 1: Influence of pH on the dry heat-induced denaturation/aggregation of whey proteins ... 55
1: Introduction, objectives and strategy ... 57
2: Article 1... 59
2.1: Introduction ... 61
2.2: Material and Methods ... 62
2.2.1: Materials... 62 2.2.2: Preparation of powders ... 62 2.2.3: Preparation of samples ... 63 2.2.4: Physical analysis ... 63 2.2.4.1: Turbidity measurement ... 63 2.2.4.2: Protein solubility ... 63
2.2.4.3: Determination of aggregate size... 63
2.2.5: Chemical analysis... 64
2.2.5.1: Gel permeation chromatography... 64
2.2.5.2: SDS-PAGE analysis... 64
2.2.5.3: Sulfhydryl quantification ... 65
2.3: Results ... 65
2.3.1: Composition of dry heated WPI... 65
2.3.2: Characterization of soluble aggregates ... 66
2.4: Discussion ... 71
2.5: Conclusion... 74
3: Additional Results and discussion ... 76
4: Marked Results... 80
Chapter 2: Structural consequences of dry heating on purified Beta-lactoglobulin and Alpha-lactalbumin ... 81
1: Introduction, objectives and strategy ... 83
2: Article 2... 85
2.1: Introduction ... 87
2.2: Materials and Methods ... 88
2.2.1: Materials... 88
2.2.2: Preparation of powders and dry heating treatment ... 88
2.2.3: Samples preparation ... 88
2.2.3.1: Protein sample reconstitution ... 88
2.2.3.2: Recovery of non-aggregated proteins ... 88
2.2.4: Samples analysis ... 89
2.2.4.1: Gel permeation chromatography... 89
2.2.4.2: SDS-PAGE Analysis... 89
2.2.4.3: Sulfhydryl Quantification... 89
2.2.4.4: Surface hydrophobicity ... 90
2.2.4.6: Mass Spectrometry (LC-MS) ... 90 2.3: Results ... 91 2.3.1: Beta-lactoglobulin ... 91 2.3.2: Alpha-lactalbumin... 97 2.4: Discussion ... 99 2.5: Conclusion... 102
3: Additional Results and Discussion... 104
3.1: Further characterizations of non-native monomers... 104
3.2: Characterization of dry heated samples (solution 2)... 107
3.2.1: In powder form... 107
3.2.2: In solution form (after reconstitution at pH 7) ... 111
4: Marked Results... 117
Chapter 3: The effect of protein powder medium on the denaturation/aggregation of whey proteins ... 119
1: Introduction, objectives and strategy ... 121
2: Results and Discussion... 123
3: Article 3... 126
3.1: Introduction ... 128
3.2: Material and Methods ... 129
3.2.1: Materials... 129
3.2.2: Preparation of dry heated powders... 129
3.2.3: Color analysis of dry-heated powders... 130
3.2.4: Sample analysis ... 130
3.2.4.1: Sample preparation for analysis ... 130
3.2.4.2: Gel permeation chromatography... 130
3.2.4.3: SDS-PAGE Analysis... 131 3.2.4.4: Mass spectrometry ... 131 3.3: Results ... 132 3.4: Discussion ... 138 3.5: Conclusion... 140 4: Marked Results... 143
Chapter 4: The effect of physicochemical parameters on the composition and gelling properties of whey proteins ... 145
1: Introduction, objectives and strategy ... 147
2.1: Introduction ... 150
2.2: Materials and Methods ... 151
2.2.1: Preparation of dry heated powders... 151
2.2.2: Determination of protein fractions ... 151
2.2.3: Gelling properties quantification... 152
2.2.4: Statistical analysis ... 152
2.3: Results and Discussion... 154
2.3.1: Effect of pH... 164
2.3.2: Effect of water activity... 164
2.3.3: Effect of heat treatment ... 165
2.4: Conclusion... 166
3: Marked Results... 167
Milk is a liquid secreted by the female of all mammalian species and is used to feed the neonates. Due to the presence of a variety of nutrients, it is considered to be the complete diet to fulfil the physiological and nutritional needs of the new born. However, besides feeding of new born, milk is widely used in the food industry as a source of valuable nutrients (proteins, fat, lactose and minerals, etc.) having wide biological and techno functional properties.
France is the 7th largest producer of cow milk producing about 24.5 billion liters of milk per year and its production is higher than its needs implying the export of a part. However the price of milk in France is one of the highest in Europe and the rest of world (Fig. 1) leading to difficulties for the export. In this competitive condition, there is a need to develop new ways of valorisation of milk, either by improving the functionality of basic dairy ingredients (specificity and reproducibility of the functionality) or by transforming it to valuable dairy products like cheese and yoghurt. More than 50% of milk is already used for the production of cheese and yoghurt in France placing it as the 3rd largest cheese producer and first largest exporter of cheese.
Inherently, a large quantity of whey is obtained from cheese making but also from casein preparation industry (Foegeding et al., 2002; Smithers, 2008a). Whey has been poorly valorized and for long time it has been either disposed off or given to the animals for feeding purposes. However the advancement in separation techniques, knowledge in the physicochemical and nutritional properties of whey constituents helped to find new ways for valorization of whey proteins (Fig. 2) (Smithers, 2008a). Now the whey is extensively used to produce various ingredients for food and non-food industries (Smithers, 2008a).
Whey proteins are about 20% of the total milk proteins, β-lactoglobulin, α-lactalbumin, and bovine serum albumin being the major proteins of whey. Whey proteins are used in a number of food products due to their good nutritional properties and diverse functionalities (solubility, fat binding capacity, gelling properties, foaming and emulsifying properties). These properties could be further tailored by structural modifications of whey proteins by appropriate processes. These processes include enzymatic modifications (protein hydrolysis and cross-linking), chemical modifications (acylation, glycation, phosphorylation etc.), and physical modifications (denaturation and aggregation of the proteins mainly using heat treatment and pressure).
Figure 1: The price of milk in France and other European countries.
Among the different processes used to modify the functional properties of whey proteins, heat treatment has been extensively used. Apart from its main function that is to ensure viral and microbial safety of food ingredients, heat treatment is also able to improve certain functional properties of whey proteins by conferring heat-induced structural modifications of these proteins. Heat-induced denaturation/aggregation of proteins is a complex mechanism that is highly sensitive to the physicochemical conditions of the medium (pH, ionic force, temperature, time for heating, protein composition) and protein ingredient
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history. In spite of tremendous research on whey proteins, the denaturation and aggregation mechanism of its composite proteins mixtures is not completely understood. However, in order to ensure the desired quality of final whey ingredients (safety/functionalities) this mechanism should be controlled.
Figure 2: Schematic representation of evolution in the value of whey proteins taken from (Smithers, 2008b).
Heating is done either in solution or in dry state (powder form). Numerous studies have been conducted on whey proteins heated in solution but little is known about dry heating of whey proteins. In fact in dry state the water activity of proteins is low making the proteins more heat-stable and in addition, the mobility of protein molecules is reduced, which slows down the kinetic of aggregation of protein molecules (Zhou & Labuza, 2007). For these reasons, dry heating is widely used in the pharmaceutical and food industry to ensure the viral and microbial decontamination of thermo sensible ingredients. In addition, dry heating has been shown to improve the functional properties (gelling, foaming and, emulsifying properties) of food proteins and is now widely used at industrial scale especially for modifying egg white proteins functionality. However, in the industry, variations in the quality of final products (mainly a lack of reproducibility of the functionalities) are observed. In fact heat intensity (temperature and time for dry heating) is often the only parameter to vary the level of denaturation and aggregation of the proteins. The physicochemical parameters of the
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R e la ti v e V a lu e ( $ ) Disposal Whey powders ($1 kg-1) WPC-35 Demin. Whey powders $3 kg-1) WPC- 75/80 ($6 kg-1) WPI- 90 ($10 kg-1) WPI-90+ ($12 kg-1) fractions ($15-$600 kg-1) Science undermining Market and Technology Development and Sophistication
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1950s 1960s 1970s
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Gutter to Good Gutter to Gold
R e la ti v e V a lu e ( $ ) Disposal Whey powders ($1 kg-1) WPC-35 Demin. Whey powders $3 kg-1) WPC- 75/80 ($6 kg-1) WPI- 90 ($10 kg-1) WPI-90+ ($12 kg-1) fractions ($15-$600 kg-1) Science undermining Market and Technology Development and Sophistication
usually not controlled. However, they may have important effect on the dry heat induced denaturation and aggregation mechanism and ultimately an effect on the quality of final product.
This study has been dedicated to elucidate the impact of pH, water activity, protein composition, mineral and lactose contents of whey protein powders and heat intensity for dry heating on the structural (level and characteristics of aggregates) and functional modifications (solubility, gelling properties: gel strength and water holding capacity) of dry heated whey proteins. The aim of this study is to determine the impact of physicochemical conditions on denaturation/aggregation of whey proteins and their functional properties, as well as to find out the relationship between structural modifications and functional properties to develop a better understanding of structure/function relationship of these proteins.
Thesis outline
1. In the first part of thesis, we present the main whey proteins and their functionalities. In addition, an overview of the main processes used for protein pre-texturization is addressed. Special attention is paid on what we know about the chemical and physical changes that occur during dry heating of food proteins and consequences on their functional properties. For comparison, the well studied behaviour of proteins under heat treatment in solution and related properties were also overviewed. At the end of this part the purpose of this study has been briefly explained.
2. The part 2 describes the material and methods used in this thesis.
3. The 3rd part of thesis describes the results and discussion. It is composed of 4 chapters. a) The 1st chapter reports on the denaturation/aggregation process of whey proteins during dry heating. The denaturation/aggregation mechanism of whey proteins has been found to be highly pH dependent.
b) Chapter 2 gives more insights on the mechanism of heat induced denaturation aggregation using pure protein powders (α-La and β-Lg) at the same pH range. The intermolecular and intramolecular modifications were studied. We evidenced the occurrence on some chemical reactions (dehydration) concomitantly to covalent aggregation.
c) In chapter 3, we investigated how molecular composition (α-La/β-Lg ratio, lactose, calcium) of commercial WPI powder affects the dry heat induced denaturation/aggregation process. For this purpose, an artificial WPI supplemented in lactose and calcium was used. It was observed that the
denaturation/aggregation mechanism of whey proteins was highly sensible to even small quantity of lactose. The presence of trace of lactose was shown to be a key element that modulates the denaturation/aggregation of proteins in particular at pH 6.5.
d) In chapter 4, the impact of dry heating at different pH on the gelling properties of whey proteins was evaluated. In addition the heat treatment intensity and water activity of the powders were also changed in order to modulate the kinetic of dry heat induced denaturation/aggregation of whey proteins. The gel strength and WHC were improved for some of the conditions tested for dry heating. Optimal gel strength and water holding capacity were reached for different dry heating conditions.
Milk is a liquid, which is secreted by the female of all mammalian species. It is an opaque, white heterogeneous fluid in which different constituents have been held in multi-dispersed phases of emulsion, colloidal suspension, or solution (Chandan, 2006). Its main function is to meet the complete nutritional requirements and several physiological functions for the neonate. The composition of milk is complex (Chandan, 2006) and is variable in different mammalian species according to the physiological and nutritional needs of neonates. The milk proteins provide the essential amino acids to young mammals for the development of muscles and other tissues containing proteins. The concentration of proteins varies among mammalian species from 1% (humans) to 24% (white-tailed jack rabbit) (Fox & McSweeney, 1998), bovine milk having 3.4% of protein content. The concentration of protein changes according to cow breed, animal feed, stage of lactation period, etc. There are two types of proteins in milk, the one which precipitate at pH 4.6 are called caseins while the fraction that remain soluble at this pH is called whey proteins (Chandan, 2006). Depending upon the mammalian species the ratio of caseins and whey proteins vary in milk. In bovine milk, caseins represent about 80%, against 20% of whey proteins. The caseins are very heat stable proteins; the milk may be heated up to 24 hours at 100°C without coagulation (Fox & McSweeney, 1998), while whey proteins are less stable to heating and are denatured completely after 10 min heating at 100°C. Caseins are phosphoproteins containing high quantity of phosphoserine, proline and cysteine but contain small amount of sulfur containing amino acids (Fox & McSweeney, 1998). This work focuses on whey proteins, so we will discuss only whey proteins in detail.
1: Whey Proteins
Whey is a by product of cheese and casein production industry. It was historically disposed off or were given to the animals for feeding purposes. But in the last few decades, it has been shown that whey contain constituents like proteins, lactose, minerals, vitamins etc. (Siso, 1996), which have very good nutritional and functional properties. Whey proteins are compact globular proteins and are mainly divided into two groups: lactoglobulins and lactalbumins. These two forms may be fractioned by using saturated solution of MgSO4. The
former are precipitated by saturated MgSO4, while latter remain soluble (Fox & McSweeney,
1998). Whey proteins are rich in essential amino acids, sulfur containing amino acids, branched chain amino acids and have high biological value (Fig. 3). The lactalbumin fraction of the whey protein contains the major whey proteins β-lactoglobulin, α-lactalbumin, and
bovine serum albumin, which constitutes about 50, 20 and 10% of whey proteins, respectively. The lactalbumin fraction also contains some minor proteins, like lactoferrin, serotransferrin and several enzymes, etc., which are present in trace amounts. The lactoglobulin fraction consists of immunoglobulins mainly IgG1 and small amount of IgG2,
IgA and IgM.
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Figure 3: Nutritional value of whey proteins compared with some other common edible
proteins (Smithers, 2008b). In sulfur amino acid composition of whey, meat and casein, black bars represents total sulfur containing amino acids, grey bars represents methionine and black bars represents cysteine.
1.1: Beta-lactoglobulin
The beta-lactoglobulin (β-Lg) is the major whey protein of cow’s milk, constituting about 50-60% of the total whey proteins and is largely responsible for the functional properties of whey proteins; solubility, fat binding capacity, gelling properties, foaming and emulsifying properties. It consists of 162 amino acids with a molecular weight of 18.3 kDa and an iso-electric pH of about 5.2. Several variants of β-Lg have been identified i.e., A, B, C, D, and E. Bovine milk contains mainly A and B variants, which are different at position 66 (Asp/Gly) and 118 (Val/Ala). β-Lg consists of 10-15% α-helix, 43% β-sheet and 47% unordered structure including β-turn (Fox & McSweeney, 1998). In its native form, β-Lg consists of 2 anti parallel β-sheets formed by 9 β-strands labelled A to I and one α-helix as determined by X-ray crystallography (Papiz et al., 1986) (Fig. 4). The β-sheets face each other and form a central cavity, site for the fixation of hydrophobic ligands (Brownlow et al., 1997). The β-Lg contains two disulfide bridges (Cys66-Cys160 and Cys106-119) and a free sulfhydryl group located at position 121 on β-strand H. In the native form, the free sulfhydryl group is buried in the interior of protein structure and is inaccessible for chemical reactions. These disulfide bonds and the free sulfhydryl group play an important role for stabilizing the protein structure (Jayat et al., 2004). β-Lg exists as dimer in ruminants at physiological pH, while in all other species where β-Lg is present, it is in monomeric form. By varying pH, different oligomerization states of bovine β-Lg have been observed. At a pH below 3.5 and above 7.5, β-Lg is mainly monomeric, between pH 3.5-5.5, it is mainly associated as octamer and between pH 5.5-7.5 β-Lg dimer predominates (Fox & McSweeney, 1998). These pH ranges vary according to the ionic strength, temperature and the presence of hydrophobic ligands in the central cavity of the protein.
Figure 4: Ribbon diagram of a single subunit of β-Lactoglobulin with labeled β-strands and
joining loops (Brownlow et al., 1997).
The biological function of β-Lg is not clear till date however it shows a sequence homology with lipocalins and bind hydrophobic molecules like retinol (Considine et al., 2005; Yang et al., 2008), various fatty acids (Considine et al., 2007b; Jiang & Liu, 2010; Loch et al., 2011), phospholipids (Martins et al., 2008), vitamins (Yang et al., 2009; Liang & Subirade, 2010), aromatic compounds (Farrell et al., 1987), cholesterol (Hasni et al., 2011) etc. β-Lg protects the hydrophobic ligands against oxidation and during their transport through the stomach to the small intestine. Due to its ability to bind free fatty acids, β-Lg stimulates lipolysis, which may be a probable function also. It also transfers the passive immunity to the new born (Madureira et al., 2007).
1.2: Alpha-Lactalbumin
The Alpha-lactalbumin (α-La) is the 2nd major protein of whey proteins, which accounts for about 20% of the total whey proteins. It is the principle protein in human milk, and is fully synthesized in mammary glands. It consists of 123 amino acids and has a molecular weight of 14.2 kDa. Its iso-electric point is between 4.2-4.5. α-La has close homology in sequence with hen egg white lysozyme (Brew et al., 1967). Among the 123 amino acid residues, 54 are identical to corresponding residues in lysozyme and a further 23 residues are structurally similar (Fox & McSweeney, 1998). It consists of 26% of α-helix,
14% of β-structure and 60% unordered structure (Fig. 5). The α-La is a monomeric protein. The α-La contains 8 cystein residues, all engaged in disulfide bonds (Cys6-Cys120; Cys28-Cys111; Cys61-Cys77; Cys73-Cys91) which stabilize the tertiary structure of the protein. In addition, α-La binds one calcium ion per molecule (KD= (2.9 × 108 M-1) (Hendrix et al.,
2000) in a pocket containing four Asp residues (Hiraoka et al., 1980). When the pH is decreased below 4, the Asp residues are protonated and lose their ability to bind calcium. When a calcium ion is bounded in the protein structure it is called holo α-La, while in the absence of calcium ion, it is called apo α-La. The loss of the calcium ion reduces the heat stability of protein and it is denatured quite rapidly as compared to the holo form (Bernal & Jelen, 1984): the temperature of denaturation of holo α-lactalbumin is around 60°C (de Wit & Klarenbeek, 1984), while in the absence of calcium the denaturation temperature is around 30°C (Bernal & Jelen, 1984).
Figure 5: Structure of alpha-lactalbumin molecule and its functional regions showing the
location of metal ions (Chrysina et al., 2000). The secondary structure elements are marked (S, β-strand; H, α-helix; h, 310 helix).
The α-La plays an important role in the biosynthesis of lactose, which is an important source of energy for new born. It’s a part of the lactose synthetase which catalyzes the biosynthesis of lactose. In fact lactose synthetase consists of α-La and UDP-galactosyl transferase. The former is regulatory component, while latter is catalytic component. In the absence of α-La, UDP-galactosyl transferase acts as a non-specific galactosyl transferase and transfers galactose from UDP-galactose to a range of acceptors. In contrast, in the presence of
α-La, it becomes highly specific and transfers galactose only to glucose to form lactose (Fox & McSweeney, 1998). For this reason the quantity of α-La in milk reflects directly the concentration of lactose in milk. The α-La is absent in the milk of marine mammals, as a result the lactose is absent in their milk. In contrast, human milk containing the highest quantity of α-La contains the highest quantity (7%) of lactose. α-La is rich in tryptophan, which is a precursor of serotonin. Recently it was demonstrated that a complex formed with partially unfolded α-La (calcium depleted form) and oleic acid exhibit lethal action against tumor cells (Zhang et al., 2009; Liskova et al., 2010; Tolin et al., 2010; Brinkmann et al., 2011).
1.3: Bovine Serum Albumin
Bovine serum albumin (BSA) is quantitatively the 3rd protein of the whey proteins and constitutes about 10% of total whey proteins. It is not synthesized in the mammary gland and is added in the milk due to passive leakage from blood stream. It consists of 582 amino acids with molecular weight of 66.4 kDa. It’s mainly a helical protein (Fig. 6) having an iso-electric point of 4.7. It is a monomeric protein containing one sulfhydryl group and 17 disulfide bonds, which stabilize the structure of the protein. All the disulfide bonds are relatively close together in the polypeptide chain, which is therefore organized in a series of short loops.
BSA binds large amounts of hydrophobic molecules (aromatic compounds, free fatty acids, and other lipids). It gives protection to hydrophobic ligand and ensures the transport of insoluble fatty acids. By its ability to bind free fatty acids BSA stimulates lipolysis, which may be a probable function of BSA.
Figure 6: Bovine serum albumin showing its three α-helical domains (I, II, III), each having
two subdomains that share common structural motifs (Huang et al., 2004).
1.4: Minor whey proteins
Among the minor proteins, lactoferrin is a glycoprotein which belongs to the transferrin family. It has a molecular weight of 80 kDa and has a strong affinity to iron (Adlerova et al., 2008). It is involved in the transfer of iron to the body cells and controls the iron level in the blood. Lactoferrin has anti-microbial function and is a part of defensive system of body.
Immunoglobulins are present in very minute quantities. In fact they are mainly present in the blood and come to the milk by leakage. In mammals five different immunoglobulins are found e.g. IgA, IgD, IgE, IgG, and IgM. These are large protein molecules of heterogenous composition.
In addition to these proteins several enzymes like lipase, proteinase, phosphatase and lactoperoxidase etc. are also present in trace amounts in whey.
2: Functional properties of whey proteins
The functional properties are defined as the properties, which determine the overall behavior of proteins in food system during their preparation, processing, storage and
consumption. Whey proteins are well known for their biological and techno functional properties. As above we have already discussed the biological properties of whey proteins, so here we will discuss only their techno functional properties (solubility, gelling, foaming and emulsifying properties).
2.1: Solubility
Protein solubility may be defined as the nitrogen proportion in a protein product, which is soluble under a specified procedure. This is an important parameter to predict the functional properties of proteins. In fact the protein solubility is a prerequisite for most of the functional properties of proteins. Whey proteins are highly used as food ingredients due to their high solubility in a wide range of pH.
The protein solubility is mainly affected by the degree of denaturation and aggregation of protein. In addition the protein solubility is affected by several intrinsic factors like protein concentration, pH, ionic strength, temperature, etc. (Trevino et al., 2008). The proteins have usually lower solubility at their iso-electric point but the native whey proteins are soluble under a large concentration range even at their iso-electric point. In contrast, the solubility of denatured and aggregated whey proteins is very low at their iso-electric point. This property is basically used to quantify the fraction of denatured and aggregated proteins in whey protein samples. Apart from iso-electric point whey proteins are soluble even in the form of small aggregates. In fact the net charge and the charge distribution of proteins and aggregates affect the binding forces between them. Under physiological conditions (pH 6.7), major whey proteins have a net negative charge and these charges are sufficient to maintain whey proteins soluble even in the form of small aggregates. In contrast close to iso-electric point the net charge of proteins is minimal and repulsion between proteins is reduced leading to aggregation if proteins have attractive forces on their surface (denatured form). The ionic strength affects the charge distribution of proteins hence affects its solubility. Protonation of charged amino acids (mainly aspartic acid and glutamic acid) is sensitive to the presence of salts in the medium. At low ionic strength, salt addition increases the number of charges on protein surface increasing its solubility. This phenomenon is called salting in. In contrast at higher salt concentration, the salt molecules occupy all the parts on the protein and interact with a higher number of water molecules. As a result the number of water molecules available to interact with proteins is reduced, so the protein-protein interactions become more important than protein-solvent interactions. This leads to the precipitation of protein molecules and its
solubility is reduced. This phenomenon is called salting out (de Wit & Kessel, 1996). In contrast to other proteins, whey proteins are not really sensitive to variation in solubility according to salt quantity in the medium in the range covered by food products. However, under denatured form, pH close to iso-electric point or in concentrated systems the whey protein solubility is sensitive to salt addition. The temperature is an important factor to the solubility of whey proteins, as increase in temperature leads to the denaturation/aggregation of whey proteins and hence reduces its solubility.
2.2: Gelling properties
The name gel was 1st used by Thomas Graham in 1869 (Sullivane et al., 2008). It is defined as a continuous network of macroscopic dimensions immersed in a liquid medium exhibiting no steady-state flow. Gelation is defined as an aggregation process of proteins, in which polymer – polymer and polymer – solvent interactions are so balanced that a tertiary network or matrix is formed. It is proposed that gelation is a three step mechanism: in the 1st step the proteins are unfolded and in the 2nd step aggregation of protein molecules occurs to form soluble aggregates; if the heating is continued and concentration of protein is sufficient then a gel network is formed in 3rd step. The whey proteins usually exhibit very good gelling properties. Heating of concentrated solutions of whey proteins results in strong gels with high water holding capacity, this phenomenon is called heat set gelation.
The ability of whey proteins to form gels depends upon their structure, interactions with other components and processing conditions (heating temperature, protein concentration, pH, and ionic strength, etc.). Native whey proteins below the temperature of denaturation, have their most reactive groups for intermolecular interactions (hydrophobic amino acids, free sulfhydryl groups) buried in the interior of the protein structure. In contrast, when the proteins are heated above their denaturation temperature, the reactive groups are exposed and are readily available for chemical reactions. Then, upon denaturation, the proteins are readily available to form aggregates and if the protein concentration is sufficient, aggregates connect each other and form a three dimensional network: a gel is obtained. In fact the temperature affects both the rate of protein unfolding and aggregation of proteins. For β-Lg the protein unfolding is rate determining factor below 90°C, while protein aggregation is the rate determining factor above 90°C (Tolkach & Kulozik, 2007). The aggregation rate is also strongly dependent on the physicochemical conditions of the medium, mainly pH and ionic strength. If the rate of aggregation is very much faster than the rate of protein unfolding then
the structure of gel will be adversely affected and a coagulum, precipitate-like structure, will be formed. In fact, due to limited protein unfolding fixation of water molecules is reduced. For optimum gelling properties, a temperature should be selected that balances the rate of unfolding and aggregation of proteins.
The concentration of proteins has an important effect on protein gelation as a minimal protein concentration is required to form a gel. The minimum concentration for gelation depends upon several factors like temperature, pH and ionic strength (Sullivane et al., 2008). If the electrostatic repulsions among the protein molecules are reduced (increasing ionic strength, pH closer to iso-electric point) then the minimum concentration for gelation is reduced and vice versa.
Figure 7: The effect of pH and ionic strength on aggregation of whey proteins (van der Linden & Venema, 2007). Depending on protein concentration different supramolecular structures (below critical concentration for gelation) or gels (above critical concentration for gelation) are obtained.
Electrostatic repulsions also control the structure of the aggregates and the properties of the gel formed. If protein net charge is high (pH far from iso-electric point, low ionic strength), then there are strong repulsions among proteins and aggregation results in mainly linear aggregates (Fig. 7); if protein concentration is sufficient, transparent gels are obtained. In contrast when the protein net charge is low (pH near to iso-electric point, high ionic
strength), the repulsions among proteins are reduced and random aggregates are formed (Fig. 7) resulting in opaque gels if protein concentration is above the critical protein concentration for gelation. The net charge of the protein is directly related to pH and this latter also affect the type of interactions among proteins (Schokker et al., 2000b). The reactivity of sulfhydryl groups is reduced and formation of disulfide bonds is limited at acidic pH (Schokker et al., 2000b): weak gels with essentially hydrophobic interactions and hydrogen bonding are formed. Sullivane et al. (2008) observed that at pH 3 or below the whey protein gels have fine stranded structure. In fact at this pH there is strong repulsion among the protein molecules that reduces their excessive aggregation leading to fine stranded gels. The formation of disulfide bonds increases gel hardness during the gelation of whey proteins (Alting et al., 2003).
Heat induced denaturation/aggregation of native β-Lg increased by adding salt (Croguennec et al., 2004; Unterhaslberger et al., 2006; Nicolai et al., 2011). The rate of depletion of native β-Lg on heating is increased by adding a small quantity of salt but for higher salt concentration (≥ 1M NaCl) then the rate of depletion of native protein starts decreasing (Verheul et al., 1998; Nicolai et al., 2011). The type of salt affects significantly the rate of denaturation/aggregation of whey proteins. It has been observed that the calcium salt are more effective in increasing the protein denaturation/aggregation as compared to sodium salt (Croguennec et al., 2004).
The composition of whey proteins strongly influences the gelling behavior of whey proteins. The β-Lg was readily gelled when heated at 80°C at a protein concentration of 8%, while α-La did not gel under same heating conditions (Gezimati et al., 1997). However, the gelling properties of these proteins are improved, when they are heated in mixture (Matsudomi et al., 1992; Rojas et al., 1997) or in the presence of BSA (Kehoe et al., 2007).
2.3: Emulsifying properties
An emulsion is the thermodynamically unstable dispersion of at least two immiscible liquids (an aqueous phase and a lipidic phase). Emulsion destabilization occurs mainly through creaming (the migration of oil droplets towards the emulsion surface under the effect of gravity), flocculation (the aggregation of two or more oil droplets that keep their integrity) and coalescence (the irreversible fusion of two or more oil droplets to form a large droplet). The flocculation and coalescence accelerate creaming. The emulsion stability occurs from the presence of amphiphilic molecules called emulsifiers or emulsifying agent having affinity for
both phases. The emulsifying agent decreases the surface tension between the two phases favoring interface creation and stabilizes emulsion against flocculation and coalescence. Due to amphiphilic properties, proteins may serve these functions. Proteins are readily adsorbed at the oil water interface where they unfold to reduce their free energy. The whey proteins are commonly used due to their ability to stabilize oil droplets in oil in water emulsions.
The emulsifying properties of proteins are strongly affected by intrinsic factors, protein solubility, protein concentration, the presence of salt, pH of the medium, and temperature. The emulsifying properties of proteins depend on conformation and flexibility as well as hydrophilic and lipophilic balance of the polypeptide chains (Sullivane et al., 2008).
Whey proteins have better emulsifying properties at pH value away from iso-electric point. In fact at iso-electric point the aggregation and precipitation of denatured proteins reduces the effective concentration of proteins for interfacial film formation. In addition, protein aggregation reduces the flexibility of proteins, hence reduces the emulsifying ability of proteins. The presence of salts and pH play an important role in stabilizing whey protein emulsions. At low ionic strength and pH away from iso-electric point the electrostatic forces are greater than van der Waals forces, so the whey protein emulsions are stabilized by high energy barrier preventing droplets against flocculation. In contrast an increase in ionic strength (>0.15 M NaCl) and a reduction of pH close to iso-electric point weaken the electrostatic barrier so much that a strong flocculation is observed (Dickinson, 2010). An increase of temperature enhances protein surface hydrophobicity improving the rate of adsorption of whey proteins at oil/water interface. This also increases diffusion of proteins at the interface by reducing the viscosity of the medium (Fox, 1989). These elements explain the temperature dependence of emulsifying properties of whey proteins.
2.4: Foaming properties
The creation or stabilization of gas bubbles in a liquid is called foaming. Foaming requires a rapid diffusion of proteins to air/water interface reducing surface tension followed by partial unfolding of the protein. This partial unfolding favors the lateral interactions among the adsorbed proteins resulting in the formation of intermolecular cohesive film at the air/water interface, that encapsulate air bubbles (Foegeding et al., 2002). The principle mechanisms of foam destabilization include drainage (the movement of liquid downwards under the effect of gravity) that favors coalescence (the irreversible fusion of two or more air bubbles to form a larger one) and Ostwald ripening (diffusion of gas from smaller to larger
bubbles through the continuous phase). The viscoelasticity of proteins is directly related to stabilization against drainage and coalescence (Rouimi et al., 2005; Murray, 2007). Due to their ability to strongly adsorb at air-water interface and to form a thick cohesive and elastic film at air/water interface, whey proteins are good foaming agents (Murray, 2007).
Usually the factors that affect the emulsifying properties, also affect the foaming properties: protein structure and flexibility, protein solubility, protein net charge and charge distribution on protein surface. These factors also depend on extrinsic factors such as pH, ionic force and temperature. Protein aggregation in bulk reduces the diffusion of whey proteins at the air/water interface but when it occurs at interface of air bubbles, increases foam stability. Hence reduction of electrostatic repulsions between whey proteins to some extent increases the stability of foams but beyond this limit it has adverse effect due to protein aggregation in the bulk. The protein composition and salt contents in a solution of whey protein strongly affects the foaming properties (Luck et al., 2002). Foaming properties may be affected by even very slight changes in the protein structure. It has been shown that the variant B of β-Lg is adsorbed more rapidly at the air/water interface as compared to variant A (Mackie et al., 1999). It has been proposed that the increased hydrophobicity and reduced negative charge of variant B as compared to variant A enhanced the adsorption at the air water interface.
3: Pre-texturization of whey proteins
For food purpose, protein pre-texturization concerns the modification of the protein structure and/or state of aggregation using food grade processes in order to advantageously modify the functional properties of the proteins. As indicated above the whey proteins are intensively used in food products due to their exceptional functional properties, but these latter could further be tailored for specific applications by controlled structural modifications (pre-texturization) using appropriate processes. These processes include enzymatic modifications (hydrolysis, cross-linking, modification of the lateral chain of the amino acids), chemical modifications (hydrolysis, modification of the lateral chain of the amino acids), and physical modifications (denaturation and aggregation of the proteins mainly using heat treatment or high pressure). These modifications are done using proteins either dispersed in solution or in dry state.
3.1: Pre-texturization in aqueous solution
Among the different pre-texturization procedures used for improving the functional properties of whey proteins in solution, enzymatic modifications, chemical modifications and heat treatment have been largely used.
3.1.1: Enzymatic modifications
The enzymes are used to modify the protein structure, this structural modification leave a particular effect on protein functionality (Panyam & Kilara, 1996; Kim et al., 2007; Rabiey & Britten, 2009). One of the advantages of using enzymes for modifying protein structure is that the enzymatic reactions are very specific. Some enzymes hydrolyze the polypeptide chains of protein, while some others incorporate intermolecular or intramolecular crosslinks or attach specific group to the protein.
3.1.1.1: Hydrolytic enzymes
All the enzymes which cleave proteins are called proteases or proteinases. They play a very important role in digestion of food as they break down proteins in the food into peptides and amino acids, which are essential for body management. The principle aim of enzyme hydrolysis is to release the smaller polypeptide fragments, hence exposing the hydrophobic parts of the proteins buried in interior of native protein. Some of the released peptides also exhibit interesting biological properties (release of bioactive peptides, reduction of allergenicity). The cleavage of polypeptide chain by enzymes is highly specific; each enzyme can break a particular peptide bond.
The hydrolysis of proteins by proteases results in a loss of original molecular structure and conformation of proteins. The original hydrophobic and hydrophilic balance of protein molecule is changed which have an important impact on the functional properties (solubility, heat stability, gelling, foaming and emulsification etc.) of proteins. Controlled proteolysis of proteins by enzymes can improve the functional properties of food proteins over a wide range of pH but the choice of right enzyme, degree of hydrolysis and environmental conditions for hydrolysis are critical (Panyam & Kilara, 1996).
Whey protein hydrolysis improves the solubility of previously denatured and aggregated whey proteins (Mutilangi et al., 1996). Hydrolysis resulted in an increase of whey proteins surface hydrophobicity leading to improved foaming and emulsifying properties. In addition the hydrolyzed proteins due to their smaller size move to the interface more rapidly than native proteins. However, they do not form a cohesive film as compared to native
proteins. This reflects that the degree of hydrolysis should be optimized for improved surface properties. A 10-20% hydrolysis of whey proteins significantly enhanced their emulsifying properties, but when the degree of hydrolysis was higher the emulsifying properties were adversely affected due to the presence of large number of very small sized peptides(Singh & Dalgleish, 1998). Moreover, the gelling properties of whey proteins are also enhanced by a mild (1 – 2.5%) hydrolysis (Rocha et al., 2009).
3.1.1.2: Crosslinking enzymes
Intramolecular or intermolecular protein crosslinking modifies protein stability and some of their functional properties (heat stability, gelling properties). Enzymes such as protein disulfide isomerase, thiol oxidase and protein disulfide reductase modify the repartition of sulfhydryl groups and disulfide bonds in the protein structure, while enzymes like transglutaminase, tyrosinase, peroxidase and laccase (Buchert et al., 2010) induce covalent interaction involving lateral chain of amino acids other than cystein lateral chain. In this section, we will discuss only on transglutaminase activity, which have got a lot of attention in the recent years for crosslinking food proteins.
Figure 8: The mechanism of formation of covalent bond induced by transglutaminase (Singh, 1991).
In the presence of calcium, transglutaminases catalyze the acyl-transfer reaction between γ-carboxamide group of glutamine and various primary amines (Fig. 8). The ε-amino
CH2 C O NH2 Protein CH2 C O NH (CH2)4 Protein Protein N H2 (CH2)4 Protein Transglutaminase Ca+2 + + NH3
and intramolecular crosslinks (Singh, 1991). Transglutaminase crosslinks whey proteins very efficiently in a wide pH range from 6.5 to 8.0 (Aboumahmoud & Savello, 1990). One molecule of ammonia per crosslink is produced during reaction (Buchert et al., 2010). The transglutaminase induced crosslinks have high resistance to proteolytic enzymes.
Crosslinking of β-Lg by transglutaminase improves its dispersibility and heat stability. It has been shown that transglutaminase modified β-Lg can withstand a temperature of 100°C up to 30 min, while native β-Lg is almost completely aggregated, when heated at 90°C for 10 min. Under neutral pH conditions, when β-Lg (10% in protein concentration) is heated at 95°C for 30 min, it form strong gels, while under the same conditions the modified β-Lg formed loose and soft coagulum. It has been proposed that the crosslinking of β-Lg by transglutaminase restrict the unfolding of protein molecule, that hinder the formation of a gel network (Singh, 1991). The transglutaminase induced aggregates significantly improved the acid (glucono delta-lactone) induced gelation of whey proteins (Eissa et al., 2004; Eissa & Khan, 2005).
3.1.2: Physical Modifications
The use of physical methods to modify the structure of proteins is called physical modifications. The most intensively used physical methods for manipulating whey protein structures to modify their functionality are heat treatment and high pressure.
3.1.2.1: Heat treatment
The heat induced structural and conformational modifications in proteins have strong impact on their techno-functional properties. Heat treatment leads to the formation of various types of whey protein aggregates depending on physicochemical conditions. These different aggregates have wide techno-functional applications (gelling, foaming, emulsifying, encapsulating etc.).
Basically, the whey proteins are heat treated under low ionic strength and on pH well away from iso-electric point and on low proteins concentration (below critical concentration for gelation) to avoid gelation. The size, shape and properties of protein structures obtained depend on the heating conditions especially pH. The heat induced denaturation/aggregation mechanism of whey proteins (Shimada & Cheftel, 1988; Verheul et al., 1998; Schokker et al., 2000b) and shape of heat induced aggregates (Jung et al., 2008a) formed is changed by varying pH for heating (Fig. 9). The fibrillar, spherical, or curved aggregates with varying size depending upon the heating conditions may be obtained. The heating of whey proteins at
low pH may also result in some hydrolysis of proteins. The type of salt also affects the denaturation/aggregation mechanism of whey proteins. The calcium salts are more efficient than sodium salt for enhancing the denaturation/aggregation mechanism (Xiong et al., 1993; Hollar et al., 1995; Simons et al., 2002; Croguennec et al., 2004; O'Kennedy & Mounsey, 2009; Kuhn et al., 2010; Nicolai et al., 2011).
Figure 9: Negative-staining TEM micrographs of β-Lg aggregates formed at pH 2.5 (A), pH
5.8 (B) and at pH 7.0 (C). These micrographs are taken from (Jung et al., 2008b).
The ratio of proteins in the whey protein isolate may have a very important impact on the denaturation and aggregation mechanism of whey proteins. β-Lg and α-La have synergistic effects (Elfagm & Wheelock, 1978; Matsudomi et al., 1992; Rojas et al., 1997; Gezimati et al., 1997; Schokker et al., 2000a) for denaturation/aggregation mechanism as there was almost no aggregation when α-La was heated alone but a drastic loss in the quantity of α-La was seen in the presence of β-Lg. In the presence of β-Lg, the α-La molecules participated to the formation of the aggregates via hydrophobic interactions and disulfide bonds (Gezimati et al., 1997; Schokker et al., 2000a). In addition, the presence of α-La induced the formation of larger aggregates in the mixtures of β-Lg and α-La (Schokker et al., 2000a), while smaller aggregates of β-Lg were formed in the absence of α-La.
The denaturation/aggregation mechanism of whey proteins is also affected by the presence of other proteins like caseins. The caseins exhibit a chaperone effect on whey proteins during heat treatment (Mounsey & O'Kennedy, 2009; Mounsey & O'Kennedy, 2010). In fact during the course of heating, the caseins are associated to β-Lg molecules and the resulting particles have greater stability against heat treatment.
induced and acid induced), heat set gelation, encapsulation, stabilization of foams and emulsions, etc. (Foegeding et al., 2002; Alting et al., 2004; Kim et al., 2005; Ako et al., 2010; He et al., 2011; Nicolai et al., 2011).
In cold set gelation, the aggregates formed during the pre-texturization process are subsequently gelled by adding salt, or by acidification (Bryant & McClements, 1998; Altlng et al., 2003; Kuhn et al., 2010). The gel is constituted of a network of protein aggregates. In most of the cases the aggregates are previously formed by heating at pH 7 but the aggregates, which are formed at pH 2 have also been used for cold gelation (Veerman et al., 2003).
The pre-texturized whey proteins are also used for heat set gelation of whey proteins (Nicolai et al., 2011). The use of pre-texturized whey proteins increased transparency of gel, gel fracture stress, storage modulus and water holding capacity. The pre-texturized whey proteins formed fine stranded gels which were 6 times harder than the gels formed from native proteins. The effect of pre-texturized whey proteins on gelation behavior is very salt specific as strong gels are formed using calcium salts as compared to sodium salts. It has also been observed that the gelation temperature of pre-texturized proteins was low (48°C) as compared to native proteins (72°C) (Nicolai et al., 2011).
The presence of heat induced aggregates enhances the foaming (Wierenga & Gruppen, 2010; Nicolai et al., 2011) and emulsifying properties of whey proteins (Dissanayake & Vasiljevic, 2009). The presence of only 10% (heat induced) aggregates significantly improved the foaming properties of whey proteins (Nicorescu et al., 2008; Nicorescu et al., 2009). In fact a partial unfolding of whey proteins enhances their surface hydrophobicity, which leads to an improvement in foaming and emulsifying properties (Moro et al., 2001; Kim et al., 2005; Nicolai et al., 2011) of these proteins. Moreover the heat treatment of β-Lg at low pH (pH 2) resulted in the formation of fibrils, which improved the emulsifying properties; it formed a highly elastic interfacial layer with strong modulus (Jung et al., 2010).
3.1.2.2: High pressure
High pressure treatment is an alternative of heat treatment, which is mainly used to kill the microorganism in the food ingredients without impairing its nutritional quality (Lopez-Fandino, 2006). However, it may confer some structural modifications in food proteins that may lead to a modification in their techno-functional properties. It mainly affect the functional properties of food proteins by affecting the hydrogen bonding and hydrophobic interactions (Lopez-Fandino, 2006).
As compared to β-Lg, the α-La is more stable towards pressure induced denaturation/aggregation because it does not contain free sulfhydryl and contain more disulfide bonds which stabilize its structure (Lopez-Fandino, 2006). The pressure induced denaturation/aggregation of β-Lg is enhanced at alkaline pH due to higher reactivity of sulfhydryl groups (Arias et al., 2000).
It has been shown that the pressure induced structural modifications significantly improved the foaming and emulsifying properties of whey proteins (Greta et al., 2006; Lee et al., 2006). These authors linked the observed improvement in foaming and emulsifying properties to the partial denaturation and aggregation of whey proteins resulting in their increased surface hydrophobicity. In addition, the pressure application resulted in aggregation of whey proteins by disulfide bonds that enhanced their gelling properties (He et al., 2010). The formation of disulfide bonds was enhanced at alkaline pH resulting in more rigid gels than at acidic pH.
3.1.3: Chemical modifications
The use of chemical reagents to manipulate the protein structure is called chemical modification. Chemical modifications such as glycation and acylation of proteins are used to modify the structure of whey proteins (Kidwai et al., 1976; Morgan et al., 1999b) with aim to modify their functionality. This increases the suitability and applicability of whey proteins as food ingredients.
3.1.3.1: Glycation
Glycation is the association of proteins with reducing sugars (glucose, fructose or larger sugar moiety). A covalent bonding is formed between the reducing function (aldehyde or ketone) of the carbohydrate moiety and an amine function, predominantly the lateral chain of a lysine in protein. It is a process, which occurs naturally in food products. However, recently artificial glycation in food proteins got a lot of interest due to significant impact on the functional properties of these proteins. It results in the loss of positive charges of the protein hence the iso-electric point of the protein is moved towards more acidic pH (Chevalier et al., 2001b). Glycation of proteins is obtained when heating a mixture of protein and reducing sugar. Heat induced glycation of whey proteins while heating in solution has been intensively studied.
