Synergistic Alcalase-papain hydrolysis of egg white protein: enhancing structural unfolding, antioxidant activity, and gastrointestinal stability.

Egg white protein (EWP) is a promising source of bioactive peptides, but its compact structure limits enzymatic accessibility. This study compared single-protease hydrolysis using alcalase (Al) or papain (Pa) with composite alcalase-papain hydrolysis (Al-Pa) in terms of peptide production, structural modification, antioxidant activity, and digestive stability. Al-Pa achieved the highest hydrolysate yield (72.85%) and the largest proportion of peptides below 10 kDa (63.71%), including 26.34% below 3 kDa. Structurally, Al-Pa caused more extensive unfolding, hydrophobic group exposure, and microstructural reorganization than Al or Pa alone. During simulated gastrointestinal digestion, Al-Pa hydrolysates exhibited greater digestive stability, retained stronger DPPH radical-scavenging activity and high ABTS activity. The digested Al-Pa products also showed increases of 13.92% in branched-chain amino acids and 4.90% in hydrophobic amino acids, together with enrichment of sulfur-containing amino acids. These findings highlight the advantages of composite protease hydrolysis for producing EWP hydrolysates with sustained antioxidant functionality during digestion.