Abstract

Rainbow trout (Oncorhynchus mykiss) skin is an underutilized, protein-rich by-product with high potential for bioactive peptide production. The objective of this study was to optimize enzymatic hydrolysis of rainbow trout skin proteins to generate and identify novel bioactive peptides with antioxidant and anticancer potential. Enzymatic hydrolysis was optimized using a central composite design to maximize antioxidant activity, employing Alcalase (R) and Flavourzyme (R). Under optimal conditions, DPPH radical-scavenging capacity and ferric reducing antioxidant power (FRAP) were significantly enhanced, yielding peptides predominantly below 6 kDa. Purified peptide fractions exhibited improved antioxidant activity (lower IC50 values) and selective cytotoxicity against human breast (MCF-7) and gastric (AGS) cancer cell lines, with limited hemolytic activity at concentrations <= 5 mg/mL. Peptidomic analysis by nLC-MS/MS identified nine peptide sequences, of which seven are reported here for the first time. In silico analyses predicted high antioxidant and anticancer potential for most newly identified peptides. These results highlight rainbow trout skin as a valuable source of novel bioactive peptides for food and nutraceutical applications. The integrated approach provides new insights into the relationship between hydrolysis conditions, peptide profile, and multifunctional bioactivity.