Homogeneous nuclei-induced, secondary nuclei-induced, and spontaneous whey protein concentrate nanofibril formation through different pathways.

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.