Enzymatic modification of millet prolamin by alcalase: structural characterization and functional enhancements

This study investigated the impact of alcalase hydrolysis (10–180 min) on the structural and functional properties of millet prolamin (MP). Alcalase exhibited exceptional proteolytic efficiency, achieving a hydrolysis degree of 27.69% at 180 min, outperforming trypsin, α-chymotrypsin, and papain. Tricine-SDS-PAGE and SEM confirmed that alcalase hydrolyzed MP aggregates into small peptides (≤14 kDa) and disrupted its spherical structure. The millet prolamin hydrolysates (MPHs) showed markedly improved solubility (96.82% at 180 min), with surface hydrophobicity peaking at 30 min. MPHs also exhibited enhanced emulsifying and foaming properties, with emulsifying indices peaking at 120 min (30.14 m2/g, 98.06 min) and foaming capacity and stability peaking at 30 min (147.50%) and 120 min (86.23%), respectively. Antioxidant activity was significantly elevated, with DPPH scavenging activity nearly doubled within 10 min, ABTS scavenging activity peaking at 76.43% at 60 min, and reducing power increased. Intrinsic fluorescence, UV–Vis, and FTIR spectroscopy confirmed protein conformational transitions through increased exposure of hydrophobic and aromatic residues and changes in amide bands. These findings demonstrate alcalase hydrolysis is an efficient strategy for producing bioactive peptides from MP, supporting its potential as a versatile, high-solubility functional ingredient in plant-based formulations, particularly in the development of food emulsions and antioxidant-rich dietary supplements.