The instability of whey protein during heat treatment has always been a major problem limiting its use in food. On heating they denature and form aggregates, and can form gels at high concentration. The present study aimed to investigate ways of reducing formation of aggregates from heat-denatured whey proteins. Inhibition of aggregate formation has considerable potential for alleviating the problems that arise from the instability of whey proteins. The approach taken in this study to inhibition was intercepting the reactive sulfhydryl (-SH) group from the major whey protein, β-lactoglobulin (β-Lg), with -SH specific reagents and hence reducing its participation in the formation of aggregated protein complexes.
The study of the modification of major whey proteins (β-Lg, α-lactalbumin (α-La) and bovine serum albumin (BSA)) was carried out on both single β-Lg and mixed whey protein systems at near neutral pH (pH 6.8) and low concentration of protein (1%, w/w). The heating conditions were set at low-moderate temperatures (55-85oC) for 10-30 min. Such conditions were chosen to allow the heat-induced changes of whey protein to be readily followed; higher temperature, different pH and higher concentration of protein would make this more difficult or impossible. The results of the modification were analysed using a combination of size exclusion-high performance liquid chromatography (SE-HPLC), reversed phase HPLC (RP-HPLC) and polyacrylamide electrophoresis (PAGE) techniques. To confirm the presence of interactions between β-Lg and -SH reagents, electrospray ionization/time of flight-mass spectrometry (ESI/TOF-MS) analysis was conducted.
The results from the single β-Lg system showed that blocking the free -SH group with dithio(bis)-p-nitrobenzoate (DTNB), N-ethylmaleimide (NEM), dihydrolipoic acid (DHLA) or reduced glutathione (GSH) resulted in formation of a new type of monomer (beside the dissociated reactive monomers), referred as the modified monomers in this thesis, and significantly less of the larger β-Lg aggregates compared with heated β-Lg with no reagents. The modified monomers are monomers with reagents attached to them and were found to form via covalent interactions. DTNB, NEM, DHLA or GSH were able to trap the monomer containing a free -SH group, to varying degrees, and prevent -SH/disulfide (S-S) exchange phenomena that lead to formation of S-S-linked protein aggregates. Furthermore, as the presence of these reagents was able to reduce formation of covalently linked aggregates, mostly non-covalent aggregates were left in the system. This fact denotes the importance of non-covalent interactions during aggregation reactions and led to an investigation using biotin and lecithin that are able to bind to the exposed hydrophobic site of β-Lg. The heat stability of the denatured β-Lg was significantly improved (p<0.05) by the presence of biotin or lecithin upon heating to 70oC or 75oC. As expected, biotin and lecithin were able to bind with the exposed hydrophobic surfaces of β-Lg heated at 70oC; hence formation of aggregates was significantly reduced (p<0.05) as compared to the non-modified β-Lg system. Nevertheless, upon further heating to 75oC the ability of both reagents to reduce aggregation was gradually decreased and was not seen at 85oC. These results indicate the involvement of non-covalent interactions in aggregation of β-Lg during mild heating (at ~70oC) but further heating (<70oC) mainly involved covalent interactions. The non-covalent interactions are thought to facilitate -SH/S-S interchange reactions in the later stages of aggregation. Thus, covalent interactions appear to play an important role in the formation of high-Mw aggregates.
In contrast to its use in the single β-Lg system, DHLA enhanced polymerization reaction when added to a mixed whey protein system, whey protein isolate (WPI), in this study. Although intermediate aggregates were significantly reduced (p<0.05) compared with heating the mixture alone, DHLA was very reactive towards the S-S bond of α-La, thus, initiating the formation of high-Mw aggregates. Similarly, GSH was reactive towards both the -SH group of β-Lg and S-S bond of α-La, leading to aggregation in the heated WPI system. Nevertheless, the formation of high-Mw aggregates was significantly less (p<0.05) than in DHLA-modified system indicating that GSH is less reactive towards S-S bonds of α-La than is DHLA. The presence of DTNB or NEM, on the other hand, considerably improved the heat stability of major whey proteins. The low (or no) reactivity of DTNB and NEM towards the S-S bond of α-La was the main reason for the improved heat stability of whey proteins in the presence of these reagents.
It can be concluded that this study demonstrated the possibility of improving the thermal stability of whey proteins by adding -SH-specific reagents and the ability of these reagents was strongly influenced by the presence of all whey proteins and not determined by any single whey protein. A mechanism describing the pathways of inhibition by these reagents was established.