Optimization of enzymatic hydrolysis process for collagen peptides from Alaska pollock bone and characterization of their antioxidant activity

To develop marine-derived antioxidant peptides, we used Alaska pollock (Gadus chalcogrammus) bones as raw material to extract bone protein. The enzymatic hydrolysis process was optimized via single-factor experiments and response surface methodology. Subsequently, ultrafiltration was employed to separate the enzymatic hydrolysate into four fractions, and the antioxidant activities of fractions with different molecular weights were systematically evaluated using in vitro antioxidant experiments and a cellular oxidative damage model. The results demonstrate that alkaline protease was the most effective enzyme, yielding a protein recovery rate of 97.73%, with the <3 kDa fraction accounting for 37.44% of peptide content. The optimal hydrolysis conditions were determined as follows: temperature 55.0 ℃, pH 11.0, and solid-to-liquid ratio 1:25.5 (g·mL⁻¹). Under these conditions, the DPPH radical scavenging rate of the enzymolysate reached 38.02% at a concentration of 50 mg·mL⁻¹. Among the isolated fractions, the <1 kDa peptides exhibited the strongest in vitro antioxidant capacity, with significantly greater DPPH scavenging ability and iron-reducing power than the >5 kDa fraction (p<0.05). Furthermore, molecular synergy among fractions of different molecular mass was observed in superoxide anion scavenging assays. In the cellular model, the <1 kDa fraction provides superior cytoprotection against H₂O₂-induced (1.61 mmol·L⁻¹) oxidative damage in C2C12 cells: it significantly increased cell viability (Measured by CCK-8 assay), reduced LDH release by 46.4%, decreased ROS accumulation by 8.5%, enhanced SOD activity by 59.4%, and attenuated mitochondrial apoptosis by stabilizing mitochondrial membrane potential-evidenced by a 143.4% increase in the JC-1 red/green fluorescence intensity ratio.