Antioxidant peptides derived from mulberry seed protein by ionic liquid-enhanced microfluidic hydrolysis with immobilized protease

To acquire a further utilization of mulberry seed, antioxidant peptides derived from defatted mulberry seed protein was prepared using a continue-flow microreactor with the synergistic effects of immobilized protease and nontoxic ionic liquid. The mulberry seed meal was pretreated with the combined simultaneous ultrasound treatment at a frequency combination of 20/28/40 kHz, solid–liquid ratio of 1:30, output power of 400 W, and temperature of 40 °C; the extraction rate of mulberry seed protein was up to 70.1%, 21.54% higher than that obtained with the single-frequency ultrasonic treatment (p < 0.05). The hydrolysis process of mulberry seed protein in the continue-flow microreactor was catalyzed by the immobilized protamex combined with choline chloride, the flow rate was 2.5 mL/min, the temperature was 50 °C, and the hydrolysis rate was up to 83.33%, which was 1.14 times higher than that without ionic liquid. Moreover, the immobilized protease can be used 12 times under continue-flow condition, and the enzyme activity was still more than 80% of the original enzyme. Four antioxidant peptides including AMDGK (896.24 Da), KFRAKDG (1109.47 Da), FPSNEPLLG (1287.15 Da), and SNVPNKEL (1005.14 Da) were obtained by ionic liquid-enhanced microfluidic hydrolysis with immobilized protease from mulberry seed protein, and AMDGK had not been reported before. These peptides exhibited strong scavenging activities on 2,2-diphenyl-1-picrylhydrazyl radical (DPPH˙) (EC₅₀ were 2.64, 1.89, 2.16, and 1.34 mg/mL, respectively) and superoxide anion radicals (·O₂⁻) (EC₅₀ were 3.36, 2.78, 2.56, and 1.77 mg/mL, respectively). Thus, the defatted mulberry seed protein possesses good edible development potential and economic value in the production of antioxidant peptides.