To explore the two-step enzymatic hydrolysis process for preparing walnut meal enzymatic peptides, and study their in vitro antioxidant and anti-fatigue activities.

Single-factor experiments and response surface experiments were used to determine the optimal two-step enzymatic hydrolysis method for preparing walnut meal enzymatic peptides. The antioxidant capacity was evaluated by determining the scavenging effects on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, hydroxyl radicals, and superoxide anion radicals, as well as the total reducing power. The anti-fatigue effect was assessed using a mouse swimming exhaustion test.

The optimal combination for preparing walnut meal enzymatic peptides through two-step enzymatic hydrolysis was as follows: Alkaline protease at 2000 U/g, pH of 9.0 for alkaline protease, and flavor protease at 1000 U/g. Compared with ascorbic acid, the half-maximal inhibitory concentration for DPPH radical scavenging was (0.25±0.01) mg/mL, the half-maximal inhibitory concentration for hydroxyl radical scavenging was(0.96±0.01) mg/mL, and the half-maximal inhibitory concentration for superoxide anion radicals scavenging was(0.25±0.01) mg/mL, indicating that walnut meal enzymatic peptides had certain in vitro antioxidant activity. The mouse experiments revealed that walnut meal enzymatic peptides had no negative effects on mice and could effectively prolong their swimming time, maintain the levels of glycogen and antioxidant enzymes in the body, and reduce the accumulation of serum metabolic waste and peroxides, demonstrating a certain anti-fatigue effect.

The two-step enzymatic hydrolysis method is successfully used to prepare walnut meal enzymatic peptides with in vitro antioxidant and in vivo anti-fatigue activities, which have great application potential in the development of health foods or functional food ingredients with antioxidant and anti-fatigue functions.