Identification and molecular mechanism of novel antioxidant peptides from milk tofu cheese: Peptidomics, molecular dynamics, and cell experiments

Abstract

This study investigated the antioxidative and cytoprotective activity of antioxidant peptides from acid-cured milk tofu cheese (MTFC) and preliminarily explored their potential antioxidant mechanism. Of the 312 peptides identified in MTFC by LC-MS/MS, a novel antioxidant peptide NQFLPYPYY (NY9) exhibited significant ABTS radical scavenging activity of IC₅₀ = 11.06 μmol/L, as well as excellent thermal and pH stability. Molecular docking analyses revealed that NY9 interacts with key active sites of Keap1, including Leu557, Leu365, Val465, Thr560, and Gly464, through hydrogen bonding and hydrophobic interactions. Furthermore, molecular dynamics simulations confirmed the stable binding of NY9 to Keap1. Cell experiments showed that NY9 exhibited notable cellular antioxidant activity, effectively protecting HepG2 cells from oxidative damage by decreasing intracellular reactive oxygen species and malondialdehyde levels, and increasing catalase and glutathione peroxidase activities. This study provides a theoretical basis for the high-value utilization of MTFC and the development of highly active antioxidant peptides.