Mining KEAP1-Targeted Antioxidant Peptides from Porphyra umbilicalis Phycobiliproteins: In Silico Prioritization, Radical Scavenging, and Cellular Cytoprotection

Phycobiliproteins from the red alga Porphyra umbilicalis are rich precursors of bioactive peptides, but targeted identification of high-potency sequences remains challenging. To address this, we combined an in silico screening pipeline with experimental validation to discover novel antioxidant peptides. Virtual enzymatic hydrolysis generated 908 candidate sequences, which were filtered down to 12 peptides based on molecular weight, toxicity, hydrophilicity, and bioactivity predictions. Molecular docking and molecular dynamics simulations further identified four candidate peptides (FK, FKP, FRS, and RRF) that were computationally predicted to bind stably within the KEAP1 Kelch domain. Chemical assays confirmed that all four synthesized peptides exhibited dose-dependent DPPH and ABTS radical-scavenging activities, along with Fe2+-chelating capacity and ferric reducing antioxidant power. In H2O2-stimulated Caco-2 cells, peptide pretreatment protected against cellular oxidative stress by restoring endogenous antioxidant enzyme activities, increasing total antioxidant capacity, and reducing lactate dehydrogenase leakage. Among these, RRF displayed the strongest protective efficacy. Overall, this study presents a practical workflow for mining marine-derived peptides and highlights four candidate peptides whose potential as functional food ingredients warrants further validation in actual P. umbilicalis hydrolysates.