Purification, Identification and Evaluation of Antioxidant Peptides from Pea Protein Hydrolysates

Food-derived antioxidant peptides can be explored as natural antioxidants due to their potential health benefits. In this study, antioxidant peptides were isolated and purified from pea protein hydrolysates (PPH). The DPPH and ABTS radical scavenging activities were used as indexes to purify the antioxidant peptides by a series of purification steps including ultrafiltration, ion exchange chromatography, G25 gel filtration chromatography, and reversed-phase chromatography. Three novel antioxidant peptides YLVN, EEHLCFR and TFY were identified, which all exhibited strong antioxidant activity in vitro. EEHLCFR showed stronger DPPH scavenging activity with an IC₅₀ value of 0.027 mg/mL. YLVN showed stronger ABTS scavenging activity with an IC₅₀ value of 0.002 mg/mL and higher ORAC values of 1.120 ± 0.231 μmol TE/μmol, which is even better than that of GSH. Three novel antioxidant peptides significantly elevated LO2 cells viability even at the concentration of 0.025 mg/mL, and cell viability enhanced to 53.42 ± 1.19%, 55.78 ± 1.03%, and 51.09 ± 1.06% respectively, compared to that of H₂O₂ injury group (48.35 ± 0.96%), and prevented the accumulation of ROS by enhancing the activities of antioxidant enzymes in H₂O₂-induced oxidative stress LO2 cells. The molecular docking results showed that the potential molecular mechanism of the three novel antioxidant peptides may be in high correlation with the activation of the Keap1-Nrf2 pathway by occupying the Keap1-Nrf2 binding site. These results demonstrate that the three novel antioxidant peptides are potential natural antioxidants that can be devoted to medicine or functional food ingredients.