Evaluation of the impact of stirring on the formation, structural changes and rheological properties of ovalbumin fibrils

The tendency to self-assemble into amyloid fibrils with an ordered cross-β structure is a generic feature of proteins. The formation mechanism of ovalbumin (OVA) fibrils at 85 °C and pH 2.0 under static or stirring condition was investigated in this study. The results showed that the formation of OVA amyloid-like fibrils was confirmed by Thioflavin T fluorescence, and the formation rate was accelerated by stirring treatment. The building blocks of OVA fibrils were peptides with molecular weight between 10 and 30 kDa, whether stirred or not. With the extension of heating time, more and more worm-like and flexible fibrils were observed by atomic force microscopy under stirring condition. The zeta potential results showed that OVA was positively charged in an acidic environment, which favored the formation of the ordered aggregates. Based on the results of circular dichroism, surface hydrophobicity and free –SH content, it could be concluded that both non-covalent interactions (hydrogen bonds and hydrophobic interactions) and covalent interactions (disulfide bonds) were involved in the formation of OVA fibrils. OVA fibrils formed under stirring condition showed a much higher viscosity and storage modulus than those formed under static condition. Furthermore, OVA fibrils showed no in vitro cytotoxicity, which has the potential for future applications. This work can further deepen the understanding of the formation mechanism of food protein amyloid-like fibrils.