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Whey protein concentrate (WPC) solutions containing 10, 30, 60 and 120 g dry powder/kg were heated at 75°C and whey protein aggregation was studied by following the changes in the distribution of β-lactoglobulin, α-lactalbumin and bovine serum albumin, using one dimensional and two dimensional PAGE. The one dimensional PAGE results showed that a minimal quantity of large aggregates was formed when 10 g WPC/kg solutions were heated at 75°C for up to 16 min whereas appreciable quantities were formed when 30, 60 and 120 g WPC/kg solutions were similarly treated. The two dimensional PAGE analysis showed that some disulphide-linked β-lactoglobulin dimers were present in heated 10 g WPC/kg solution, but very little was present in heated 120 g WPC/kg solution. By contrast, SDS was able to dissociate monomeric protein from high molecular mass aggregates in heated WPC solution of 120 g/kg but not in 10 g WPC/kg solution heated for 30 min. The rates of loss of native-like and SDS-monomeric β-lactoglobulin, α-lactalbumin and bovine serum albumin during heating increased as the WPC concentration was increased from 10 to 120 g/kg. In 120 g WPC/kg solution heated at 75°C, the amounts of SDS-monomeric β-lactoglobulin in each sample were greater than the quantities of native-like protein. However, in WPC solutions of 10, 30 and 60 g/kg, the differences between the amounts of native-like and SDS-monomeric proteins were slight. The loss of the native-like or SDS-monomeric proteins was consistent with a first or second order reaction. In each case, the apparent reaction rate constant appeared to be concentration-dependent, suggesting a change of aggregation mechanism in the more concentrated solutions. Overall, these results indicate that in addition to disulphide-linked aggregates, hydrophobic aggregates involving β-lactoglobulin, α-lactalbumin and bovine serum albumin were formed in heated WPC solution at high protein concentration, as suggested by model studies using binary mixtures of these proteins.

Bovine β-lactoglobulin (β-lg), α-lactalbumin (α-la) and bovine serum albumin (BSA), dispersed in ultrafiltration permeate, that had been prepared from whey protein concentrate solution (100 g/kg, pH 6·8), were heated at 75 °C. The consequent protein aggregation was studied by one-dimensional (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE). When 100 g β-lg/kg permeate solution was heated at 75 °C, cooled and examined, large aggregates were observed. These aggregates were partially dissociated in SDS solution to give monomers, disulphide-bonded dimers, trimers and larger aggregates. When mixtures of β-lg and α-la or BSA were heated, homopolymers of each protein as well as heteropolymers of these proteins were observed. These polymer species were also observed in a heated mixture of the three proteins. Two-dimensional PAGE of mixtures demonstrated that these polymers species contained disulphide-bonded dimers of β-lg, α-la and BSA, and 1:1 disulphide-bonded adducts of α-la and β-lg, or BSA. These results are consistent with a mechanism in which the free thiols of heat-treated β-lg or BSA catalyse the formation of a range of monomers, dimers and higher polymers of α-la. It is likely that when whey protein concentrate is heated under the present conditions, BSA forms disulphide-bonded strands ahead of β-lg and that α-la aggregation with β-lg and with itself is catalysed by the heat-induced unfolded BSA and β-lg.

Heat-induced gelation (80 °C, 30 min or 85 °C, 60 min) of whey protein concentrate (WPC) solutions was studied using transmission electron microscopy (TEM), dynamic rheology and polyacrylamide gel electrophoresis (PAGE). The WPC solutions (150 g/kg, pH 6·9) were prepared by dispersing WPC powder in water (control), 10 g/kg sodium dodecyl sulphate (SDS) solution or 10 mM-dithiothreitol (DTT) solution. The WPC gels containing SDS were more translucent than the control gels, which were slightly more translucent than the gels containing DTT. TEM analyses showed that the SDS-gels had finer aggregate structure (≅10 nm) than the control gels (≅100 nm), whereas the DTT-gels had a more particulate structure (≅200 to 300 nm). Dynamic rheology measurements showed that the control WPC gels had storage modulus (G′) values (≅13500 Pa) that were ≅25 times higher than those of the SDS-gels (≅550 Pa) and less than half those of the DTT-gels after cooling. Compression tests showed that the DTT-gels were more rigid and more brittle than the control gels, whereas the SDS-gels were softer and more rubbery than either the control gels or the DTT-gels. PAGE analyses of WPC gel samples revealed that the control WPC solutions heated at 85 °C for 10 min contained both disulphide bonds and non-covalent linkages. In both the SDS-solutions and the DTT-solutions, the denatured whey protein molecules were in the form of monomers or small aggregates. It is likely that, on more extended heating, more disulphide linkages were formed in the SDS-gels whereas more hydrophobic aggregates were formed in the DTT-gels. These results demonstrate that the properties of heat-induced WPC gels are strongly influenced by non-covalent bonding. Intermolecular disulphide bonds appeared to give the rubbery nature of heat-induced WPC gels whereas non-covalent bonds their rigidity and brittle texture.

Heat-induced aggregation of whey proteins in solutions made from two commercial whey protein concentrates (WPCs), one derived from mineral acid whey (acid WPC) and the other from cheese whey (cheese WPC), was studied using polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography (SEC), and transmission electron microscopy (TEM). Heat treatment (75 degrees C) of acid WPC solutions (12.0%, w/w, pH 6.9) resulted in formation of relatively small "soluble" aggregates that were predominantly disulfide-linked. By contrast, heat treatment of the cheese WPC solutions (under the same conditions) caused formation of relatively large aggregates, containing high proportions of aggregates linked by noncovalent associations. The rate of aggregation of both beta-lactoglobulin and alpha-lactalbumin at 75 degrees C, measured as the loss of native proteins by PAGE, was higher in the cheese WPC solution than in the acid WPC solution. Cross dialysis of the two WPC solutions resulted in alteration of the mineral composition of each WPC solution and reversing their heat-induced aggregation behavior. The results demonstrated that the mineral composition is very important in controlling the aggregation behavior of WPC products.

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Heat-induced gelation (80 degrees C for 30 min or 85 degrees C for 60 min) of whey protein concentrate (WPC) solutions was studied using small deformation dynamic rheology, small and large deformation compression, and polyacrylamide gel electrophoresis (PAGE). The WPC solutions (15% w/w, pH 6.9) were prepared by dispersing WPC powder in water (control), 1% (w/w) sodium dodecyl sulfate (SDS) solution, and N-ethylmaleimide (NEM) solution at a protein/NEM molar ratio of 1:1 or in 10 mM dithiothreitol (DTT) solution. PAGE analyses showed that the heat treatment of control solutions contained both disulfide and non-covalent linkages between denatured protein molecules. Only disulfide linkages were formed in heated SDS-WPC solutions, whereas only non-covalent linkages were formed in DTT-WPC and NEM-WPC solutions during heating. In heated NEM-WPC solutions, the pre-existing disulfide linkages remained unaltered. Small deformation rheology measurements showed that the storage modulus (G') values, compared with those of the control WPC gels (approximately 14000 Pa), were 3 times less for the SDS-WPC gels (approximately 4000 Pa), double for the NEM-WPC gels (approximately 24000 Pa), and even higher for the DTT-WPC gels (approximately 30000 Pa). Compression tests suggested that the rubberiness (fracture strain) of the WPC gels increased as the degree of disulfide linkages within the gels increased, whereas the stiffness (modulus) of the gels increased as the degree of non-covalent associations among the denatured protein molecules increased.

Whey protein concentrate solutions (12% w/v, pH 6.65 +/- 0.05) were pressure treated at 800 MPa for 20-120 min and then examined using size exclusion chromatography (SEC), small deformation rheology, transmission electron microscopy, and various types of one-dimensional (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE). The pressure-treated samples showed a time-dependent loss of native whey proteins by SEC and 1D PAGE and a corresponding increase in non-native proteins and protein aggregates of different sizes. These aggregates altered the viscosity and opacity of the samples and were shown to be cross-linked by intermolecular disulfide bonds and by noncovalent interactions using 1D PAGE [alkaline (or native), sodium dodecyl sulfate (SDS), and SDS of reduced samples (SDS(R))] and 2D PAGE (native:SDS and SDS:SDS(R)). The sensitivity of the major whey proteins to pressure was in the order beta-lactoglobulin B (beta-LG B) > beta-LG A > bovine serum albumin (BSA) > alpha-lactalbumin (alpha-LA), and the large internal hydrophobic cavity of beta-LG may have been partially responsible for its sensitivity to high-pressure treatments. It seemed likely that, at 800 MPa, the formation of a beta-LG disulfide-bonded network preceded the formation of disulfide bonds between alpha-LA or BSA and beta-LG to form multiprotein aggregates, possibly because the disulfide bonds of alpha-LA and BSA are less exposed than those of beta-LG either during or after pressure treatment. It may be possible that intermolecular disulfide bond formation occurred at high pressure and that hydrophobic association became important after the high-pressure treatment.