Abstract
Whey is a byproduct of the dairy industry and is rich in protein. To enhance the significance of such byproducts and find efficacious antioxidants for combating oxidative stress, this study reported on the preparation, purification, and identification of novel peptides with antioxidant activities from whey protein metabolites following fermentation by Lactobacillus rhamnosus B2-1. The isolation and identification processes involved macroporous adsorption resin column chromatography, gel filtration column chromatography, and liquid chromatography-tandem mass spectrometry. Therein, three novel antioxidant peptides (PKYPVEPF, LEASPEVI, and YPFPGPIHNS) were selected to be synthesized, and they demonstrated remarkable antioxidant activities in vitro chemical assays. PKYPVEPF, LEASPEVI, and YPFPGPIHNS (100 μg/mL) displayed a notable cytoprotective impact on HepG2 cells under oxidative stress induced by H(2)O(2), increasing the cell viability from 49.02 ± 3.05% to 88.59 ± 10.49%, 82.38 ± 19.16%, and 85.15 ± 7.19%, respectively. Moreover, the peptides boosted the activities of catalase and superoxide dismutase in damaged cells and reduced reactive oxygen species levels. The molecular docking studies highlighted that these antioxidant peptides efficiently bound to key amino acids in the Kelch domain of Keap1, thereby preventing the interaction between Keap1 and Nrf2. In conclusion, PKYPVEPF, LEASPEVI, and YPFPGPIHNS demonstrated substantial antioxidant activity, suggesting their potential for widespread application as functional food additives and ingredients.