Enhanced antioxidant peptide extraction from zein using optimized ultrasound-assisted dual-enzyme methods hydrolysis

In this study, we developed an ultrasound-assisted dual-enzyme hydrolysis method for extracting low-molecular-weight peptides from maize alcohol-soluble proteins. Using a Box-Behnken design within response surface methodology (RSM), we optimized the hydrolysis process, achieving a maximum protein hydrolysis degree of 26.5 % under optimal conditions: substrate concentration 4 g/L, pH 8.3, enzyme addition 6 %, ultrasonic power 480 W/L, ultrasonic duration 2.3 h, and hydrolysis temperature 60 °C. Structural analysis revealed that ultrasound-treated samples exhibited looser structures and more wrinkled surfaces, indicative of increased exposure of hydrophobic and aromatic amino acids. In vitro antioxidant assays demonstrated significantly higher DPPH, hydroxyl radical, and superoxide anion radical scavenging activities in ultrasound-treated samples ( p  < 0.05), with the <3 kDa peptide fraction showing the highest activity. This study highlights the potential of ultrasound-assisted dual-enzyme hydrolysis for enhancing zein utilization and advancing sustainable food additive development.