Study on the Two-Enzyme Preparation and Antioxidant Activity of Walnut Oligopeptides
Abstract
To realize the high-value valorization of cold-pressed walnut meal, walnut oligopeptides were fabricated via synchronous dual-enzyme hydrolysis combined with activated carbon decolorization and membrane separation purification. We optimized the overall preparation process and systematically characterized the products, including their amino acid profile, in vitro antioxidant capacity, cytoprotective effects against H2O2-triggered oxidative injury in PC12 cells, and regulatory activity toward acetylcholinesterase (AChE). The optimal hydrolysis conditions were identified as pH 10.0, total enzyme dosage of 11,000 U/g, a trypsin-to-alkaline protease ratio of 2.1:1, solid–liquid ratio of 1:26, temperature of 51 °C and reaction duration of 4 h, which produced a hydrolysis degree of 33.02%. The optimized decolorization parameters were pH 5.2, activated carbon dosage of 2.3%, treatment at 58 °C for 43 min, with a peptide recovery rate reaching 83.35%. Cold-pressed walnut meal is rich in glutamic acid, arginine and aspartic acid, which lay the molecular foundation for the bioactive properties of the derived oligopeptides. In vitro tests demonstrated that the oligopeptides possessed strong scavenging ability against hydroxyl, DPPH and superoxide anion radicals (clearance rates of 90.18%, 81.72% and 85.80%, respectively), and maintained 73.21% of antioxidant activity after simulated gastrointestinal digestion. Moreover, walnut oligopeptides at 0.8 mg/mL showed no cytotoxicity and afforded a 79.88% protective effect against oxidative damage. The peptides significantly boosted SOD and GSH-Px activities, lowered MDA accumulation, and strongly suppressed AChE activity, performing better than donepezil hydrochloride. This efficient, eco-friendly technique achieves high-value utilization of walnut processing by-products. The obtained oligopeptides possess great potential as natural antioxidants and neuroprotective ingredients for functional food development.