Process Optimization of Antioxidant Peptides Preparation from White Kidney Beans via Bacteria-Enzyme Synergy and Analysisof Their Antioxidant Activity

In this study, white kidney bean protein was utilized as a primary material to produce antioxidant peptides through a combined approach involving Bacillus subtilis fermentation and neutral protease treatment. The optimal processing conditions for peptide production were determined using single-factor experiments and response surface methodology, and the amino acid composition and the molecular weight distribution of the peptides were analyzed. Ultrafiltration was employed to isolate specific peptide fractions, and their antioxidant properties were assessed. The most promising antioxidant peptides were identified through liquid chromatography-tandem mass spectrometry (LC-MS/MS). Further investigation into the peptide's potential antioxidative properties was conducted through virtual screening, molecular docking, and molecular dynamics simulation. Results indicated that the ideal conditions for peptide formation were a fermentation temperature of 37.0 ℃, a fermentation period of 48.0 h, and an inoculum level of 5.0%. Under these conditions, the DPPH radical scavenging ability reached 76.12%±0.36%, and the peptide yield was 17.59%±0.16%. In WKBPs, the content of hydrophobic, basic, and acidic amino acids was relatively high, at 21.932%, 11.331%, and 20.583% respectively. The molecular weight distribution was predominantly below 3 kDa, and ultrafiltration showed that peptides with molecular weights under 3 kDa exhibited substantial antioxidant activity, as indicated by their DPPH, ABTS+, and hydroxyl radical scavenging rates, with IC50 values of 0.307, 0.310, and 0.361 mg/mL, respectively. The total antioxidant capacity was measured at 0.66±0.0018 mmol Fe2+/g at a concentration of 1 mg/mL. Among the 6364 peptide sequences analyzed, three active pentapeptides (FGWGP, FGHPEW and FGPYF) were identified, each forming a stable complex with Keap1 protein through hydrogen bonds and hydrophobic interactions. Molecular dynamics simulations indicated that these peptides could bind tightly to Keap1, forming stable conformations. This study provides both a technical guide and a theoretical foundation for further exploration and development of white kidney bean proteins and antioxidant peptide-based products.