Structural evolution of soy protein amyloid fibrils induced by high-temperature pretreatment

The fibrillation of proteins has emerged as an effective approach to modify the functional properties of food-derived proteins. The conventional acidic-heating fibrillation process of soy protein requires an extended hydrolysis period of at least 20 h at 85 °C. While current research on the thermal effects of amyloid fibrils primarily focuses on the morphological changes induced by high temperature, whether high-temperature pretreatment accelerates the fibrillation process of soy protein remains unclear. In this study, we demonstrated that high temperatures (Pre 100 °C and Pre 120 °C) induced rapid unfolding and hydrolysis of protein particles into small peptides within the initial acidic-heating for 20 min. This provides extremely favorable prerequisites for the nucleation and elongation processes. The β-sheet content of fibrils reached 53.31% (at 85 °C for 12 h), 57.65% (at 100 °C for 1 h and 85 °C for 11 h) and 68.28% (at 120 °C for 1 h and 85 °C for 11 h), respectively. Thioflavin T (Th T) fluorescence analysis revealed peak intensities at 6 h (85 °C), 1 h (Pre 100 °C) and 10 min (Pre 120 °C). Atomic force microscopy (AFM) images confirmed that high temperature pretreatment accelerated the formation of long-straight fibrils (internal contour lengths, ICL ≥ 600 nm), reducing the required time from 12 to 3 h. These results elucidated the acceleration mechanism of soy protein fibrillation regulated by high-temperature pretreatment (Pre 100 °C and Pre 120 °C), offering a novel reference for optimization of preparing technology of amyloid fibrils derived from food proteins.