Model systems

The mechanism of denaturation and aggregation is complex, especially in whole systems such as milk or whey protein products. Therefore, individual whey proteins and mixtures of purified proteins have been studied as “model” systems to help elucidate the likely mechanisms that might govern whey protein interactions during heating. β- Lactoglobulin especially has been studied in model systems as it is the most abundant whey protein and dominates the heat-induced behaviour of whey protein systems.

Because of the heterogeneity of whey protein systems, and because individual proteins exhibit different behaviour under heating, it is difficult to relate the findings from model systems directly to those occurring in whey protein systems where other components such as lipids, lactose, minerals are also present and affect the protein thermal behaviour. The thermal denaturation and aggregation of the total whey proteins in such systems reflects the collective response of the component proteins (de Wit & Klarenbeek, 1984). Whey protein denaturation and aggregation have been extensively studied in a wide range of whey protein systems such as milk, skim milk, and cheese whey, but for the purpose of this study the following section will focus on the thermal denaturation and aggregation of whey proteins in model systems and in the complex whey protein systems, WPI and WPC.

2.4.2.1 β-Lactoglobulin

McKenzie (1971) concluded that β-lactoglobulin denaturation and aggregation at neutral pH involved several steps with a number of intermediate species. Mulvihill and Donovan (1987) reviewed earlier work and reported the mechanism of denaturation and

aggregation of β-lactoglobulin being as below:

- dimer - monomer dissociation N2 ֐ 2N

- reversible denaturation 2N ֐ 2D - irreversible aggregation 2D ௧௬௣௘ூሱۛۛሮ A1 ௧௬௣௘ூூ ሱۛۛۛሮ An or 2D ௧௬௣௘ூூூ ሱۛۛۛۛሮ Ax

At room temperature, β-lactoglobulin in solution exists in a dynamic equilibrium between its dimeric (N2) and monomeric (2N) forms. Under heating, there is

dissociation of the native dimer to native monomer, then conformational changes of the native monomer (2N ֐ 2D), which is the denaturation step (unfolding). This step involves the exposure of apolar residues to the solvent and disruption of hydrogen and hydrophobic bonding with loss of tertiary and secondary structures (Dupont, 1965b). Unmasking of thiol groups also occurs (Mulvihill & Donovan, 1987). The denaturation is reversible under mild conditions and on restoration of the original environmental conditions.

At higher temperatures, interactions occur via a series of irreversible associations to form a polydisperse set of aggregates. Two distinct aggregations have been discovered, Type I and Type II aggregations, resulting in a series of small (A1) and large aggregates

(An) (McKenzie, 1971). Type I aggregation is the tetramerization of four monomers

(Pantaloni, 1964b), involving intermolecular disulphide bond formation (Sawyer, 1968). Then, the Type II aggregation is the conversion of these small aggregates (A1) into

larger aggregates (An), involving non-covalent bonds. It is a “non-specific” aggregation

without involvement of thiol groups (Sawyer, 1968). When free thiol groups are alkylated with N-ethylmaleimide (NEM), a thiol group blocking agent, prior to heating, Type I aggregation cannot occur (and so neither can Type II aggregation), and a third

type of aggregation of denatured β-lactoglobulin molecules (2D) takes place without involving disulphide bonds (Ax) (Sawyer, 1968).

However, it would be wrong to assume that the thermal denaturation and aggregation phenomena are principally the result of any one of the following: disulphide interchange reactions, hydrophobic interactions or ionic effects.

2.4.2.2 α-Lactalbumin

α-Lactalbumin does not contain a free thiol group, and when heated under mild conditions (80qC, pH ~ 6.7) α-lactalbumin does not form aggregates (Hines & Foegeding, 1993; Matsudomi, Oshita, Kobayashi, & Kinsella, 1993; Matsudomi, Oshita, Sasaki, & Kobayashi, 1992; Schokker, Singh, & Creamer, 2000). However, when heated under severe conditions (100qC, 10 – 30 min), α-lactalbumin formed polymers linked by disulphide bonds and modified monomers (Chaplin & Lyster, 1986; Hong & Creamer, 2002). Kuwajima (1996) suggested that the latter were probably in the molten globule state or in other words that the disulphide bonds were not in their native state (Hong & Creamer, 2002). Ruegg, Moor, and Blanc (1977) demonstrated by DSC that α- lactalbumin undergoes a reversible transition at 64qC.

2.4.2.3 Bovine serum albumin

Little attention has been paid to the thermal behaviour of bovine serum albumin on its own from a food science point of view, but de Wit and Klarenbeek (1984) found that under a range of heating conditions at near neutral pH, bovine serum albumin is the most heat sensitive of the whey proteins. It has been suggested that bovine serum albumin thermal interactions take place in a similar way to those previously reported for

β-lactoglobulin (Gezimati, Singh, & Creamer, 1996a, 1996b). Similarly to β- lactoglobulin, bovine serum albumin contains one free thiol group, and, on this basis, similar behaviour can be expected. Electrophoretic studies showed that bovine serum

albumin aggregates were held together by disulphide bonds, and also by hydrophobic

interactions. The major difference with β-lactoglobulin is that bovine serum albumin aggregation began at a lower temperature.

2.4.2.4 Mixtures of β-lactoglobulin and α-lactalbumin

When heated together, β-lactoglobulin and α-lactalbumin aggregated mainly through disulphide-linkages and to a lesser extent by non-covalent interactions (Dalgleish, Senaratne, & Francois, 1997; Gezimati, Creamer, & Singh, 1997; Havea, Singh, & Creamer, 2001; Hines & Foegeding, 1993; Matsudomi, Oshita, Kobayashi, & Kinsella, 1993; Matsudomi, Oshita, Sasaki, & Kobayashi, 1992; Schokker, Singh, & Creamer, 2000). Havea, Singh, and Creamer (2001) observed the formation of intermediate disulphide-linked homopolymers (especially dimers and trimers) of β-lactoglobulin and

α-lactalbumin, and in addition Hong and Creamer (2002) found the presence of the heterodimer 1:1 α-lactalbumin – β-lactoglobulin. When heated in the presence of β- lactoglobulin, at β-lactoglobulin to α-lactalbumin ratios varying from 1:1 to 20:1, native

α-lactalbumin disappeared faster than when heated alone, whereas native β- lactoglobulin disappearance was not affected by the presence of α-lactalbumin (Hines & Foegeding, 1993; Hong & Creamer, 2002; Matsudomi, Oshita, Sasaki, & Kobayashi, 1992; Schokker, Singh, & Creamer, 2000). The fact that β-lactoglobulin disappearance

was not affected by α-lactalbumin indicated that the initial heat-induced intramolecular

reorganisation of the β-lactoglobulin disulphide bonds happened before α-lactalbumin

induced any modification to the aggregation mechanism. However, at a β-lactoglobulin

to α-lactalbumin ratio of 3:1, β-lactoglobulin aggregates were markedly reduced by the

presence of α-lactalbumin. Dalgleish, Senaratne, and Francois (1997) found that during the early stages of heating, the aggregates formed contained more β-lactoglobulin than

α-lactalbumin, whereas in the later stages, they contained equal amounts of both proteins.

2.4.2.5 Mixtures of β-lactoglobulin and bovine serum albumin

The intermediate aggregates formed in heated mixtures of bovine serum albumin and β- lactoglobulin were mostly homopolymers of each protein (Gezimati, Singh, & Creamer, 1996a; Havea, Singh, & Creamer, 2001) probably due to the lower thermal transition temperature of bovine serum albumin (Ruegg, Moor, & Blanc, 1977), allowing the majority of the bovine serum albumin molecules to denature and aggregate in the early stage of heating. Havea, Singh, and Creamer (2001) observed very small quantities of

bovine serum albumin and β-lactoglobulin complexes after heating at 75ºC for 10 min, fine and lightly stained bands were observed in 2D PAGE gels. Gezimati, Singh, and Creamer (1996b) suggested that bovine serum albumin formed polymers prior to the unfolding of β-lactoglobulin at moderate (75ºC) heat-treatment temperatures. Hines and Foegeding (1993) found that the rate of aggregation of bovine serum albumin was much

greater than that of β-lactoglobulin when the proteins were mixed in an equimolar ratio (22:78 w/w β-lactoglobulin: bovine serum albumin) in the presence of 100 mM NaCl. Under these conditions, the addition of bovine serum albumin increased the rate of

aggregation of β-lactoglobulin. Kehoe, Morris, and Brodkorb (2007) also found that

bovine serum albumin increased the rate of denaturation of β-lactoglobulin. 2.4.2.6 Mixtures of α-lactalbumin and bovine serum albumin

As observed with mixtures of β-lactoglobulin and bovine serum albumin, Gezimati, Singh, and Creamer (1996b) suggested that bovine serum albumin formed polymers prior to the unfolding of α-lactalbumin at moderate (75ºC) heat-treatment temperatures. In a mixed bovine serum albumin – α-lactalbumin system, they also suggested that α- lactalbumin then probably unfolded and formed some sort of adduct with the bovine serum albumin polymers. Havea, Singh, and Creamer (2000) studied bovine serum albumin – α-lactalbumin 1:1 mixtures (heated at 75ºC) and suggested a possible mechanism for the formation of α-lactalbumin polymers and hydrophobically associated

α-lactalbumin.

2.4.2.7 Mixtures of β-lactoglobulin, α-lactalbumin and bovine serum albumin Havea, Singh, and Creamer (2001) showed that when heated at 75ºC a mixture of the three major whey proteins formed various disulphide homopolymers of each protein as well as various adducts of the three proteins. Initial aggregates were formed predominantly by polymerisation of bovine serum albumin with itself while the

aggregates involving β-lactoglobulin and α-lactalbumin, homopolymers and mixed aggregates, were generated at a later stage.