Isolation, Purification, Structural Identification and Action Mechanism of Antioxidant Peptides Derived from Porcine Bones
CHEN Lulu, LEI Yang, HAO Yangxue, Lü Yangyong, WEI Shan, ZHAO Fengguang, HU Yuansen
1. School of Biological Engineering, Henan University of Technology, Zhengzhou 450001, China; 2. Henan Yuxin Sugar Alcohol Co. Ltd., Anyang 456171, China; 3. Food Laboratory of Zhongyuan, Luohe 462300, China
Abstract：The enzymatic preparation of antioxidant peptides from porcine bone meal was optimized by the combined use of single factor experiments and response surface methodology. The antioxidant peptides were separated and purified by successive ultrafiltration and Sephadex G-25 column chromatography, and the peptides with high antioxidant activity were selected and identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with PeptideRanker. The binding of the peptides to the protein Kelch-like ECH-associated protein 1 (Keap1) was analyzed by molecular docking technology. The results showed that the optimal process parameters were as follows: pH 6.8, 50 ℃, 6 000 U/g flavourzyme, 4 h hydrolysis, and 4 g/100 mL substrate concentration. Among three fractions (F-1, F-2, and F-3) obtained from successive ultrafiltration and Sephadex G-25 column chromatography, F-2 had the highest antioxidant activity, which scavenged (94.57 ± 1.08)% of 2,2’-azino-bis(3-ethylbenzothiazoliline-6-sulfonic acid) (ABTS) cationic radical and (79.08 ± 0.61)% of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. LC-MS/MS identified 232 peptides from F-2, and three peptides, GPVGAAGP, APGPVGP and QPGPAGPG, were predicted by PeptideRanker, hydrophobic amino acids accounting for more than 80% of their total amino acids, indicating that they may have high antioxidant activity. The three peptides bound to Keap1 protein by hydrogen bonding and hydrophobic interaction, causing the release of nuclear factor erythroid 2 related factor 2 (Nrf2) into the nucleus and the activation of the Keap1-Nrf2/antioxidant response element pathway to exert antioxidant effects. Therefore, GPVGAAGP, APGPVGP and QPGPAGPG can be used as new antioxidants, and the findings from this research provide a theoretical basis for the development and utilization of porcine bone-derived antioxidant peptides.
2025, Vol. 39 
