Discovery of novel tyrosinase-inhibitory peptides FW and LAW from bullfrog (Lithobates catesbeianus) muscle protein: In silico screening, inhibition kinetics, gastrointestinal stability, and anti-melanogenic efficacy in zebrafish.

Bullfrog (Lithobates catesbeianus) muscle protein remains an underexplored source of bioactive peptides. This study applied an integrated in silico-in vitro-in vivo workflow to identify tyrosinase (TYR)-inhibitory peptides from bullfrog muscle protein hydrolysates. Among five proteases evaluated, acid protease hydrolysis for 3 h produced the highest TYR-inhibitory activity. Following ultrafiltration and Sephadex G-15 gel filtration, the most active fraction (<1 kDa) was analyzed by LC-MS/MS, yielding 71 candidate peptide sequences. The dipeptide FW and tripeptide LAW were prioritized through PeptideRanker scoring, safety prediction, and molecular docking against mushroom TYR (PDB ID: 2Y9X), with predicted docking scores of -8.0 and - 7.9 kcal/mol, respectively. Synthetic FW and LAW exhibited concentration-dependent TYR inhibition, with IC₅₀ values of 0.21 mg/mL (approximately 0.60 mM) and 0.14 mg/mL (approximately 0.36 mM), respectively. Kinetic analysis indicated competitive inhibition by FW and non-competitive inhibition by LAW, while molecular docking suggested distinct interaction patterns broadly consistent with these kinetic profiles. Following simulated gastrointestinal digestion, the corresponding post-digestion samples retained approximately 90% of their initial inhibitory activity, although LAW showed chromatographic evidence of partial degradation or transformation. In zebrafish larvae, neither peptide significantly affected survival or caused apparent developmental abnormalities at 0.0125-0.05 mg/mL, and FW and LAW reduced whole-body melanin content by 30% and 27%, respectively, at 0.05 mg/mL. These findings identify bullfrog muscle protein as a potential source of TYR-inhibitory peptides and support further investigation of FW and LAW for anti-melanogenic applications.