Identification, in silico selection, and mechanism study of novel antioxidant peptides derived from the rice bran protein hydrolysates.

The work aimed to assess the antioxidant ability and obtain a new antioxidant peptide from rice bran protein. Rice bran protein was hydrolyzed by Alcalase, Neutral, Pepsin, Chymotrypsin, and Trypsin, separately. Trypsin hydrolysate (T-RBPH) showed high Fe2+ chelating activity (IC50, 2.271 ± 0.007 mg/mL), DPPH and hydroxyl radical scavenging ability (IC50, 0.191 ± 0.006 and 1.038 ± 0.034 mg/mL). Moreover, T-RBPH could alleviate the H2O2-induced oxidative damage in Caco-2. The T-RBPH was purified and identified by UF, GF, FPLC, and LC-MS/MS. Finally, 9-amino acid peptide-AFDEGPWPK with low molecular weight (1045.48 Da), high antioxidant activity, good safety, and solubility was screened by in silico method and chemical oxidation determination, and its interaction with Keap1 was also demonstrated. The ORAC and DPPH radical scavenging ability of AFDEGPWPK were 44.16 ± 0.79 and 28.38 ± 0.14 µmol TE/mM. Moreover, the Molecular docking and Western blot (WB) results showed that AFDEGPWPK could enter the binding pocket in the Kelch domain and activate Keap1/Nrf2/HO-1 pathway.

Identification and in silico analysis of novel antioxidant peptides in broken rice protein hydrolysate and its cytoprotective effect against H2O2-induced 2BS cell model.

Broken rice is an important by-product during milling process of rice, which is rich in protein. To increase the value of by-products and search for effective antioxidants, the antioxidant peptides from broken rice protein hydrolysate were separated and identified by ultrafiltration, gel filtration chromatography, fast protein liquid chromatography, and LC-MS/MS in this study. These identified peptides were further screened using a combined in silico and in vitro method and their antioxidant mechanism was explored by Western blot and molecular docking analysis. Ninety-eight peptides were obtained after antioxidant activity-oriented isolation and four novel peptides, SGDWSDIGGR, DFGSEILPR, GEPFPSDPKKQLQ, and GEKGGIPIGIGK, with excellent solubility, safety, and antioxidant activity were synthesized. Among these, SGDWSDIGGR showed good antioxidant activities in the extracellular assay (41.57 µmol TE/g and 29.41 % in ORAC and DPPH assay, respectively.), and it possessed a protective effect against H2O2-injured oxidative stress in 2BS cells in a dose-dependent manner. Furthermore, Western blot and molecular docking results showed that SGDWSDIGGR achieves antioxidant ability by occupying the Nrf2-binding site, activating the Keap1-Nrf2 signaling pathway, and upregulating the expression of antioxidant enzymes. This study extends the rice industry chain and provides insights into the selection and mechanisms research of antioxidant peptides.