Study on the Two-Enzyme Preparation and Antioxidant Activity of Walnut Oligopeptides

To achieve high-value valorization of cold-pressed walnut meal, walnut oligopeptides were prepared using synchronous dual-enzyme hydrolysis coupled with activated carbon decolorization and membrane separation purification. Process optimization was conducted, followed by systematic evaluation of amino acid profile, in vitro antioxidant activity, protective effects against H2O2-induced oxidative injury in PC12 cells, and acetylcholinesterase (AChE) regulatory function. The optimal hydrolysis conditions were determined as pH 10.0, total enzyme dosage 11000 U/g, trypsin/alkaline protease ratio 2.1:1, solid-liquid ratio 1:26, 51℃ for 4 h, yielding a degree of hydrolysis of 33.02%. The optimized decolorization parameters were pH 5.2, activated carbon dosage 2.3%, 58℃ for 43 min, achieving a peptide recovery rate of 83.35%. Walnut meal was abundant in glutamic acid, arginine, and aspartic acid, which served as the molecular basis for its bioactivities. In vitro assays revealed that the oligopeptides exhibited strong scavenging capacities against hydroxyl, DPPH, and superoxide anion radicals (90.18%, 81.72%, and 85.80%, respectively), while retaining 73.21% activity after simulated gastrointestinal digestion. Furthermore, 0.8 mg/mL walnut oligopeptides displayed no cytotoxicity and conferred a 79.88% protection rate against oxidative damage. They significantly elevated SOD and GSH-Px activities, reduced MDA accumulation, and markedly inhibited AChE activity, outperforming donepezil hydrochloride. This efficient and green approach enables the valorization of walnut processing by-products. The resulting oligopeptides show promising potential as natural antioxidant and neuroprotective agents for functional food applications.