Antioxidant peptides from silver carp steak by alkaline protease and flavor enzyme hydrolysis: Characterization of their structure and cytoprotective effects against H₂O₂-induced oxidative stress

Silver carp steak is a rarely utilized silver carp processing byproduct. This study aimed to optimize a dual enzymatic method to extract antioxidant peptide components from silver carp steak and characterize their structure and in vitro antioxidant activity through ultrafiltration purification, response surface methodology, molecular docking, and radical scavenging activity analysis. The optimal extraction conditions for silver carp steak antioxidant peptides (SCSAP) were determined as 1:6 solid-liquid ratio, 1500 U/g alkaline protease addition, 4 h alkaline protease hydrolysis time, 1946 U/g flavor enzyme addition, and 2.5 h flavor enzyme hydrolysis time. The <3 kDa SCSAP component (SCSAP-3kDa) showed the strongest antioxidant activity, with its 1,1-diphenyl-2-trinitrophenyl hydrazine (DPPH) radical scavenging rate, ABTS radical scavenging rate, hydroxyl radical scavenging rate, metal ion chelating rate, and reducing capacity reaching 88.75%, 91.21%, 67.02%, 69.07%, and 0.985, respectively. Moreover, the three peptides (PF-7, GP-8, and YF-10) of 100 µg/mL could protect HepG2 cells from oxidative stress damage by reducing the oxidative damage level and activating Keap1-Nrf2-ARE pathways, enabling an increase of superoxide dismutases (SOD) activity, and a decrease of malondialdehyde (MDA) content and reactive oxygen species (ROS) level. The integrated results indicate the enormous potential of SCSAP-3kDa as a functional food ingredient in the food industry. PRACTICAL APPLICATION: This study selected the antioxidant capacity of silver carp steak peptides as the index and developed a facile dual enzymatic hydrolysis method to obtain three antioxidant peptides (PF-7, GP-8, and YF-10) with biological activity, providing a theoretical basis for bioavailability of antioxidant peptides from silver carp steak and contributing to their application in new functional foods.