Protease specificity governs antioxidant peptide release and activity from camel milk β-casein

Although camel milk β-casein is a promising source of bioactive peptides, the impact of protease specificity on antioxidant peptide release remains poorly understood. This study investigated how enzyme type shapes the hydrolysis profile and antioxidant capacity of β-casein in camel milk. Purified β-casein was obtained by anion-exchange chromatography and hydrolysed in vitro with pepsin, trypsin or chymotrypsin for 4 h. The degree of hydrolysis was quantified by the trinitrobenzenesulphonic acid assay, and structural changes and peptide patterns were characterised by urea-polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulphate-PAGE (SDS-PAGE) and reversed-phase high-performance liquid chromatography. Antioxidant activity was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical scavenging and ferric reducing antioxidant power (FRAP) assays. Protease specificity significantly influenced hydrolysis kinetics and peptide composition (P < 0.05). Pepsin yielded the highest degree of hydrolysis and the most diverse peptide profile, which correlated with superior antioxidant activity. All hydrolysates exhibited significantly enhanced radical scavenging and reducing power compared to intact β-casein (P < 0.05). Pepsin-derived peptides showed the lowest half-maximal inhibitory concentrations (IC50) values in DPPH (0.51 mg/ml) and ABTS (0.64 mg/ml) assays and the highest FRAP values, outperforming chymotrypsin and trypsin hydrolysates. ABTS•⁺ scavenging was consistently higher than DPPH• scavenging across all samples. These findings demonstrate that enzyme selection critically governs antioxidant peptide formation from camel β-casein via differential cleavage patterns. Pepsin hydrolysis represents an effective approach for generating potent antioxidant peptides from camel β-casein with potential application in dairy-based functional foods.