The effect of physicochemical parameters on the composition and gelling properties of whey proteins

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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.

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.

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).


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.

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 inter actions 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, wh ile 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.

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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.

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.

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.

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).

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.

Table of contents :

Part 1: Review of Literature
1: Whey Proteins
1.1: Beta-lactoglobulin
1.2: Alpha-Lactalbumin
1.3: Bovine Serum Albumin
1.4: Minor whey proteins
2: Functional properties of whey proteins
2.1: Solubility
2.2: Gelling properties
2.3: Emulsifying properties
2.4: Foaming properties
3: Pre-texturization of whey proteins
3.1: Pre-texturization in aqueous solution
3.1.1: Enzymatic modifications Hydrolytic enzymes Crosslinking enzymes
3.1.2: Physical Modifications Heat treatment High pressure
3.1.3: Chemical modifications Glycation Acylation
3.2: Pre-texturization in powder state
3.2.1: Chemical modifications under dry heating
3.2.2: Structural and functional changes during dry heating
4: Purpose of the Study
Part 2: Materials and Methods
1: Protein samples
2: Preparation of dry heated Powders:
3: Structural characterization of dry heated powders
3.1: Structural analysis in powder form
3.1.1: Fourier transform infrared (FTIR) spectroscopy
3.1.2: Differential scanning calorimetry
3.2: Structural analysis on reconstituted solutions
3.2.1: Reconstitution of whey protein powders in solution
3.2.2: Physical analysis Protein solubility Turbidity measurement Determination of aggregate size
3.2.3: Chemical analysis Gel permeation chromatography (GPC) Reversed phase – high pressure liquid chromatography (RP-HPLC) SDS-PAGE Analysis Sulfhydryl Quantification Surface hydrophobicity Intrinsic hydrophobicity Circular Dichroism Mass Spectrometry Protein identification by in gel trypsinolysis
4: Functional characterization of dry heat samples
4.1: Gel Hardness and Water Holding Capacity
4.2: Colorimetric Measurement
Part 3: Results and Discussion
Chapter 1: Influence of pH on the dry heat-induced denaturation/aggregation of whey proteins
1: Introduction, objectives and strategy
2: Article 1
2.1: Introduction
2.2: Material and Methods
2.2.1: Materials
2.2.2: Preparation of powders
2.2.3: Preparation of samples
2.2.4: Physical analysis Turbidity measurement Protein solubility Determination of aggregate size
2.2.5: Chemical analysis Gel permeation chromatography SDS-PAGE analysis Sulfhydryl quantification
2.3: Results
2.3.1: Composition of dry heated WPI
2.3.2: Characterization of soluble aggregates
2.4: Discussion
2.5: Conclusion
3: Additional Results and discussion
4: Marked Results
Chapter 2: Structural consequences of dry heating on purified Beta-lactoglobulin and Alpha-lactalbumin
1: Introduction, objectives and strategy
2: Article 2
2.1: Introduction
2.2: Materials and Methods
2.2.1: Materials
2.2.2: Preparation of powders and dry heating treatment
2.2.3: Samples preparation Protein sample reconstitution Recovery of non-aggregated proteins
2.2.4: Samples analysis Gel permeation chromatography SDS-PAGE Analysis Sulfhydryl Quantification Surface hydrophobicity Circular Dichroism Mass Spectrometry (LC-MS)
2.3: Results
2.3.1: Beta-lactoglobulin
2.3.2: Alpha-lactalbumin
2.4: Discussion
2.5: Conclusion
3: Additional Results and Discussion
3.1: Further characterizations of non-native monomers
3.2: Characterization of dry heated samples (solution 2)
3.2.1: In powder form
3.2.2: In solution form (after reconstitution at pH 7)
4: Marked Results
Chapter 3: The effect of protein powder medium on the denaturation/aggregation of whey proteins
1: Introduction, objectives and strategy
2: Results and Discussion
3: Article 3
3.1: Introduction
3.2: Material and Methods
3.2.1: Materials
3.2.2: Preparation of dry heated powders
3.2.3: Color analysis of dry-heated powders
3.2.4: Sample analysis Sample preparation for analysis Gel permeation chromatography SDS-PAGE Analysis Mass spectrometry
3.3: Results
3.4: Discussion
3.5: Conclusion
4: Marked Results
Chapter 4: The effect of physicochemical parameters on the composition and gelling properties of whey proteins
1: Introduction, objectives and strategy
2.1: Introduction
2.2: Materials and Methods
2.2.1: Preparation of dry heated powders
2.2.2: Determination of protein fractions
2.2.3: Gelling properties quantification
2.2.4: Statistical analysis
2.3: Results and Discussion
2.3.1: Effect of pH
2.3.2: Effect of water activity
2.3.3: Effect of heat treatment
2.4: Conclusion
3: Marked Results
Part 4: Conclusions and Perspectives


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