Noncovalent Interaction-Driven Assembly Endows Casein Amyloid Fibrils with Inhibitory Activities against Starch-Hydrolyzing Enzymes

Casein, owing to its amphiphilic and self-assembling properties, can transform into amyloid fibrils (CNAF), which exhibit distinct bioactivity. In this study, it was innovatively found that the fibrillation of casein significantly enhances its inhibitory activity against starch-hydrolyzing enzymes. Specifically, the process followed an S-shaped thioflavin T fluorescence kinetic curve and involved conformational transition from its native disordered state to β-sheet-rich structures driven by hydrogen bonding, π-π stacking, and electrostatic interactions. Furthermore, the elongated fibrillar morphology of CNAF, with a high aspect ratio and large specific surface area, facilitated multivalent binding to enzyme surfaces. This interaction resembled a "grape-on-a-vine" arrangement and contributed to enhanced inhibition. CNAF exhibited mixed-type inhibition toward α-amylase and competitive inhibition toward α-glucosidase, indicating structure-specific enzyme inhibition mechanisms that collectively reduced starch hydrolysis. Consequently, CNAF effectively delayed in vitro starch digestion by modulating enzyme activity, indicating its potential as a functional ingredient for postprandial glycemic regulation.