Structural properties of Kudzu protein enzymatic hydrolysate and its repair effect on HepG2 cells damaged by H2O2 oxidation

Abstract

We investigated the structural properties, foaming capacity and foaming stability, antioxidant activity, and amino acid composition of Kudzu protein (KP) and Kudzu protein hydrolysate (KPH). The peptide sequence of KPH was analyzed using ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS), and the binding ability of the peptide sequence to Keap1 was predicted through molecular docking simulations. The electrophoresis and molecular weight distribution analysis results showed that the molecular weight of KPH was significantly lower than that of KP, with a mean molecular weight of approximately 2000–5000 Da. The structures and properties were characterized using Fourier transform infrared spectroscopy, relative fluorescence, and circular dichroism. The results showed that KP exposed a large number of hydrophobic groups after enzymatic hydrolysis, and its structure changed from α-helical to random coils. KPH has a higher foaming capacity (200%) and foaming stability (97.5%) than KP, which may be related to the change in structure. These results indicate that moderate hydrolysis can improve the functional properties of KP, providing a new opportunity for its application as a food ingredient. The antioxidant assay results showed that KP and KPH had a good hydroxyl radical, superoxide anion, 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) scavenging capacity and a high reducing capacity. KPH exerted better antioxidant effects than KP. The scavenging rates for DPPH, ABTS, hydroxyl radicals, and superoxide anions were 89.31%, 93.14%, 85.74%, and 58.29%, respectively, and its reducing capacity was 2.191, which may be related to the increase in amino acids with antioxidant activity after enzymolysis. In vitro, KP and KPH could significantly repair H₂O₂-induced oxidative damage in HepG2 cells, reduce the apoptosis rate, activate the Nrf2-Keap1 signaling pathway, reduce the accumulation of reactive oxygen species and malondialdehyde after oxidative damage, increase the activities of superoxide dismutase and glutathione (GSH) peroxidase, and increase the content of GSH and the total antioxidant capacity. Twenty-one peptide components were identified in KPH using UPLC-MS/MS, and the binding ability of 21 peptide components to Keap1 was analyzed through molecular docking technology. The results showed that all 21 peptides in KPH had good antioxidant activity, and real-time quantitative PCR (qRT-PCR) analysis was conducted to further explain the high antioxidant activity of KPH at the genetic level. These results show that KP and KPH are suitable for preparing antioxidant foods and related health foods to prevent oxidation-related diseases. KPH has more beneficial effects than KP.