Identification of novel antioxidant peptides from cottonseed meal co-fermented with Lactobacillus mucosae LLK-XR1 and acid proteases: In silico screening, molecular simulation, and in vitro functional analysis

This study is the first to employ a microbe-enzyme synergistic fermentation process to produce bioactive peptides from cottonseed meal (CSM). The peptide profile of the fermented CSM was identified using LC-MS/MS analysis, and common bioactivities were predicted through computational models. Molecular docking, molecular dynamics simulations, and cell experiments were conducted to validate the antioxidant functions and identify the potential best antioxidant peptides (AOP), S-7-H and E-7-H. The top five peptides with the best docking results were F-6-P, D-5-Y, S-7-H, D-5-F, and E-7-H, with binding energies of −10.4, −10.2, −10.1, −10.0, and − 9.8 kcal mol⁻¹, respectively. Molecular dynamics simulations indicated that S-7-H and E-7-H demonstrated the most stable interactions with the Keap1 protein. All five AOPs effectively alleviated oxidative stress-induced damage in macrophages caused by hydrogen peroxide. These findings provide two novel and safe antioxidant peptides, S-7-H and E-7-H, and offer a more reliable screening approach for developing natural plant-derived AOPs. © 2025 Elsevier Ltd.