Study On The Enzymatic Preparation Of Sea Cucumber Peptide And Its Antioxidant And Anti-fatigue Activities

Sea cucumber has been widely used in China for thousands of years as an invigorant and a functional food. The sea cucumber peptide had many physiological activitites and its biological activities were related to its molecular weight distribution. The thesis was about the enzymatic preparation of bioactive peptides from Stichopus japonicus, and then in vitro antioxidant activity and in vivo anti-fatigue activity of the oligopeptide and polypeptide were studied, both of which were produced using an ultrafiltration membrane bioreactor system, aiming at providing the basis for the development of sea cucumber peptide food.In this study, six kinds of proteases were screened, neutral protease was finally been choosed. The enzymatic hydrolysis were mainly on the effect of pH, temperature, concentration of substrate, dosage of enzyme and hydrolysis time on the yield of polypeptide and molecular weight distribution. Results showed that the temperature was 52.0 °C, the dosage of protease was 6950 U/g, the concentration of substrate was 1.0% and hydrolysis time was 3 h. Under these condition, the yield of polypeptide was 35.11%. The molecular weight of 130-2000 Da was 27.62±1.07% and the molecular weight of 2000-5000 Da was 63.69±1.20%.The oligopeptide and ploypeptide were produced using an ultrafiltration membrane bioreactor system. Results showed that the oligopeptide of 130-2000 Da was 72.6% and 2000-5000 Da was 26.8%. The ploypeptide of 130-2000 Da was 28.1% and 2000-5000 Da was 73.1%.Five different chemical models in vitro were used to evaluate the antioxidant activities of the oligopeptide and ploypeptide. The results showed that the oligopeptide could scavenge DPPH radicals(IC50=7.38 mg/mL), ABTS radicals(IC50=4.25 mg/mL), hydroxyl radicals(IC50=1.15 mg/mL) and superoxide anion(IC50=12.37 mg/mL) and inhibit the lipid peroxidation(IC50=3.68 mg/mL). The ploypeptide could scavenge DPPH radicals(IC50=8.39 mg/mL), ABTS radicals(IC50=6.19 mg/mL), hydroxyl radicals(IC50=2.95 mg/mL) and superoxide anion(IC50=15.07 mg/mL) and inhibit the lipid peroxidation(IC50=6.26 mg/mL). The antioxidant activity of oligopeptide was superior to polypeptide.The anti-fatigue effects of the oligopeptide and ploypeptide on mice were studied through different dose. The results showed that the exhaustive swimming times of the mice in the low-dose, medium-dose and high-dose oligopeptide groups were 34.42±13.33, 41.75±17.31 and 50.17±17.60 min, respectively. The oligopeptide could significantly prolong the time of loaded-swimming in comparison with that of the control group(14.5±8.46 min). The exhaustive swimming times of the mice in the low-dose, medium-dose and high-dose ploypeptide groups were 44.45±22.19, 46.17±24.52 and 48.27±23.23 min, respectively. The ploypeptide could significantly prolong the time of loaded-swimming in comparison with that of the control group(17.64±5.05 min). The oligopeptide did better in degrading the content of blood urea nitrogen and increasing the content of hepatic glycogen in comparison with ploypeptide. Furthermore, the content of MDA and the activities of CAT, SOD and GSH-Px were also determined. Results showed that both of the oligopeptide and polypeptide could decrease the content of MDA and increase the activities of CAT, SOD and GSH-Px, but the effect of oligopeptide was superior to polypeptide. In conclusion, the anti-fatigue activity of oligopeptide was superior to polypeptide.