Novel ACE-inhibitory peptides from egg yolk protein by stepwise enzymatic hydrolysis: Optimization, structural-activity characterization and molecular dynamics simulation.

To overcome the limited functionality of egg yolk protein from single-protease hydrolysis, this study developed a stepwise enzymatic strategy using alkaline (AH) and flavor (FH) proteases to concurrently enhance physicochemical properties and generate antihypertensive peptides. The optimal AH + FH treatment significantly improved powder solubility (78.88%, P < 0.05) and protein solubility (67.31%, P < 0.05). These improvements were associated with a reduction in β-sheet content and enhanced molecular flexibility. The hydrolysis also modified surface properties by increasing net negative charge and reducing surface hydrophobicity. The <1 kDa hydrolysate fraction exhibited potent ACE-inhibitory activity (70.51%, P < 0.05). Among identified peptides, KLPDMILY was validated as the most potent inhibitor (94.87%, P < 0.05), with docking studies attributing its high activity to extensive hydrogen bonding and Zn2+ coordination with ACE. This work demonstrates an effective strategy to produce dual-purpose functional ingredients from egg yolk, enhancing both its food processing characteristics and health-promoting value.