Novel Antioxidant\nPeptides from Fermented Whey Protein\nby <i>Lactobacillus rhamnosus</i> B2-1: Separation and Identification\nby <i>in Vitro</i> and <i>in Silico</i> Approaches

Whey is a byproduct\nof the dairy industry and is rich\nin protein.\nTo enhance the significance of such byproducts and find efficacious\nantioxidants for combating oxidative stress, this study reported on\nthe preparation, purification, and identification of novel peptides\nwith antioxidant activities from whey protein metabolites following\nfermentation by <i>Lactobacillus rhamnosus</i> B2-1. The\nisolation and identification processes involved macroporous adsorption\nresin column chromatography, gel filtration column chromatography,\nand liquid chromatography-tandem mass spectrometry. Therein, three\nnovel antioxidant peptides (PKYPVEPF, LEASPEVI, and YPFPGPIHNS) were\nselected to be synthesized, and they demonstrated remarkable antioxidant\nactivities <i>in vitro</i> chemical assays. PKYPVEPF, LEASPEVI,\nand YPFPGPIHNS (100 μg/mL) displayed a notable cytoprotective\nimpact on HepG2 cells under oxidative stress induced by H<sub>2</sub>O<sub>2</sub>, increasing the cell viability from 49.02 ± 3.05%\nto 88.59 ± 10.49%, 82.38 ± 19.16%, and 85.15 ± 7.19%,\nrespectively. Moreover, the peptides boosted the activities of catalase\nand superoxide dismutase in damaged cells and reduced reactive oxygen\nspecies levels. The molecular docking studies highlighted that these\nantioxidant peptides efficiently bound to key amino acids in the Kelch\ndomain of Keap1, thereby preventing the interaction between Keap1\nand Nrf2. In conclusion, PKYPVEPF, LEASPEVI, and YPFPGPIHNS demonstrated\nsubstantial antioxidant activity, suggesting their potential for widespread\napplication as functional food additives and ingredients.