Thermally induced self-assembly of zein into amyloid fibrils under alkaline-ethanol conditions: Kinetics, structural evolution, and molecular interactions.

The formation behavior of zein into amyloid fibrils (ZAFs) under alkaline-ethanol conditions remains insufficiently understood. This study adopts orthogonal experiments combined with multidimensional structural and physicochemical characterization to elucidate the formation mechanism of ZAFs under heat treatment in high-ethanol alkaline environments. Ethanol concentration was identified as the dominant factor governing ZAFs formation, with optimal conditions determined as 60% ethanol, pH 10, and 90 °C. Under these conditions, zein self-assembly followed a typical sigmoidal kinetic profile, exhibiting a shortened half-time (0.78 h) and a high apparent growth rate. Secondary structure analysis revealed a transition from disordered/α-helical conformations to β-sheet-rich structures. Atomic force microscopy (AFM) observations confirmed the evolution from flexible protofibrils to rigid mature fibril bundles. These findings clarify the correlations between environmental conditions, fibrillation kinetics, and structural evolution, providing a basis for regulating protein self-assembly and developing zein-based functional materials.