Whey protein, as a major by-product from cheese manufacture, is a versatile and nutritive food ingredient. In food and ingredient processing, thermal treatment is often applied to achieve specific product property, e.g. to increase product stability. However, undesirable modifications may also be promoted by heating such as protein oxidation and generation of off-flavor, leading to the decrease of nutritional value and/or consumer acceptance. To optimize the conditions of thermal treatment for specific product functionalities customization with minimized side effects on the final food applications, it is important and necessary to understand the mechanism of potential heat-induced protein modifications. In the present PhD study, whey protein modifications (e.g. disulfide rearrangement, protein oxidation and desulfurization) and structural changes were investigated under different heating conditions (i.e. 60-90 °C for 10 min, 90 °C for 120 min and 160 °C for 160 s (as a UHT-like condition)) using multiple analytical tools (e.g. liquid chromatography-mass spectrometry (LC-MS), size-exclusion chromatography (SEC) and gas chromatography-flame photometric detector (GC-FPD)). These modifications and changes were compared in different whey protein systems, including the two major whey proteins, i.e. beta-lactoglobulin (β-LG) and alpha-lactalbumin (α-LA), a whey-model system (α-LA + β-LG) and whey protein isolate (WPI).
Heating of β-LG at 70 °C and above was found to induce disulfide rearrangement, which might lead to irreversible protein unfolding and protein aggregation. Although disulfide rearrangement was observed to start at both native disulfide bonds in β-LG, the surface-located Cys66-Cy160 was suggested to be more reactive towards rearrangement compared to Cys106-Cys119. At ≥80 °C, the disulfide-linked protein oligomers were increasing in size with increasing heat load, while Cys66 was proposed to be a key Cys residue participating in the rearranged inter-molecular disulfide bonds. Meanwhile, hydrogen sulfide (H2S) was also released (presumably via Cys β-elimination), which contributed to the sulfurous odor. Only minor level of heat-induced protein side-chain oxidation products was detected in β-LG after all employed heat treatments. Nevertheless, higher levels of heat-induced oxidation products, especially at Trp residues, were detected in α-LA as compared to β-LG after the severe heat treatment (i.e. 90 °C for 120 min). The presence of free Cys residue in β-LG was proposed to scavenge oxidants during heating, making it less susceptible towards oxidation than α-LA. Formation of non-native disulfide bond was only observed in α-LA once the temperature reached 90 °C, at which free Cys residue started to release from the native disulfide bonds. However, H2S was not detected in heated α-LA, which
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emphasized the major role of β-LG in H2S formation from heated whey. In mixed protein systems (i.e. whey-model and WPI), the higher level of total Cys and cystine residues facilitated both heat-induced disulfide rearrangement and H2S formation as compared to the single protein systems (i.e. pure α-LA and β-LG). The resulting larger disulfide-linked protein aggregates was suggested to provide further physical protection against protein oxidation, which is supported by the significantly lower oxidation occupancies found in the heated mixed protein systems compared to the single protein systems, especially in α-LA.
Collectively, the formation of disulfide-linked protein aggregates, via disulfide rearrangement, was suggested to be a key outcome in WPI upon thermal treatment (60-90 °C), and the resulting compact protein structure was proposed to reduce the susceptibility of amino acid residues towards heat-induced changes (e.g. oxidation and formation of sulfur volatiles) by lowering their solvent accessibility. The wide range of interconvertible oxoforms of Cys derivatives (e.g. disulfide) was speculated to provide an in vitro redox-regulation mechanism in the free Cys-containing systems (i.e. β-LG, whey-model and WPI).