Four proteases: trypsin, protease A, pepsin, and protease M were selected to modify whey protein concentrate (WPC) at a low degree of hydrolysis (0.1, 0.2, and 0.3%) before adjusting to pH 2.0 and heating at 90°C to gain insight into the influence of proteolysis on fibril formation. The kinetics of fibril formation were performed on native and modified WPC using the fluorescent dye thioflavin T in conjunction with transmission electron microscopy and far-UV circular dichroism spectroscopy for the morphological and secondary structural analyses. The change in surface hydrophobicity and content of free sulfhydryl groups were also observed during the formation of fibrils for the native and modified WPC. The content of aggregation and thioflavin T kinetic data indicated that the ability of fibril formation was apparently different for WPC modified by the 4 proteases. Whey protein concentrate modified by trypsin aggregated more during heating and the fibril formation rate was faster than that of the native WPC. Whey protein concentrate modified by the other proteases showed slower aggregation with worse amyloid fibril morphology. Compared with the native WPC, the structure of WPC changed differently after being modified by proteases. The state of α-helix structure for modified WPC played the most important role in the formation of fibrils. Under the mild conditions used in this work, the α-helix structure of WPC modified by trypsin caused little destruction and resulted in fibrils with good morphology; the content of α-helices for WPC modified by other proteases decreased to 36.19 to 50.94%; thus, fibril formation was inhibited. In addition, it was beneficial for the modified WPC to form fibrils such that the surface hydrophobicity increased and the content of free sulfhydryl groups slightly decreased during heating.

The addition of homogeneous nuclei (HN) or secondary nuclei (SN) could lead to different kinetics and thermodynamics as the nucleation energy barrier decreases and the lag time is shortened to different degrees compared with spontaneous fibrillation. To explain these differences, we monitored the formation and depletion of HN during fibril formation and found that both SN-induced fibrils and HN-induced fibrils follow the same nucleated growth pathway as spontaneously formed WPC fibrils. Moreover, there were also other paths, which were confirmed by X-ray diffraction, transmission electron microscopy, and atomic force microscopy. The surfaces of the SN could recruit monomers and resulted in stronger intersheet stacking and a larger fibril height and periodicity. The HN incorporation led to a propensity for hydrogen-bonding interactions and a longer fibril. Fibrillation by the addition HN and SN followed both common and distinct pathways, as spontaneous fibrillation and led to different capacities to induce fibrillation.