Preparation, Activity Evaluation and Molecular Mechanism of Antioxidant Soybean Peptides by Ultrasound-Assisted Enzymatic Hydrolysis

Soybean protein isolate (SPI) was used as raw material to prepare antioxidant peptides through ultrasound-assisted enzymatic hydrolysis in this study. SPI hydrolysate was separated by ultrafiltration into peptides with different molecular masses, whose antioxidant activity was evaluated by measuring the scavenging capacity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) cation radicals and hydroxyl radicals, as well as using the ferric reducing antioxidant power (FRAP) assay. The amino acid composition was analyzed, and peptides with high antioxidant activity were identified. Molecular docking was used to elucidate their antioxidant mechanisms. The results showed that peptides with a molecular mass of < 3 kDa exhibited the strongest DPPH, ABTS cation and hydroxyl radical scavenging capacity, as well as the highest FRAP(P < 0.05). Amino acid composition analysis revealed that the peptides contained higher levels of hydrophobic and antioxidant amino acids, which were positively correlated with their antioxidant activity. By liquid chromatography-tandem mass spectrometry (LC-MS/MS), 1217 unique peptides were identified, and 10 potent antioxidant peptides (activity score > 0.90) were selected from them. Molecular docking indicated that these peptides had low binding energy (-9.7 to-7.2 kcal/mol) with kelch-like ECH-associated protein 1(Keap1), primarily exerting antioxidant activity through hydrogen bonds and hydrophobic interactions. This study provides theoretical and technical support for the high-value utilization of soybean protein resources and the development of natural antioxidant peptides.