Preparation Of Peptides From Enzymic Hydrolysates Of Stichopus Japonicus And Its Antioxidant Properties In Vitro

Fast autolysis was firstly studied on the preparation of peptides from stichopus japonicus; in order to obtain higher degree of hydrolysis, complex enzyme hydrolysis was adopt on the basis of the fast autolysis. The hysicochemical properties and molecular weight distribution were determined by ultra-filtration and HPLC, the antioxidative activity of which was studied as well.The body was hydrolyzed by autolytic process, according to the rate of hydrolysis, the optimum enzymolysis technology was confirmed through uniform design. The result showed that optimum hydrolytic conditions were determined: temperature , 50℃; pH, 13; irradiation time, 25 min; the solid/liquid ratio, 1:4 (w:w); hydrolysis time, 4h. Under the above conditions, the DH (degree of hydrolysis) is 38.8%. The protein was first hydrolated by autoenzyme, then trypsin, whose optimum hydrolytic condition is follows: temperature , 50℃; pH, 7.5; protease dosage , 2%; the solid/liquid ratio, 1:4 (w:w); hydrolysis time, 2 h. The complex enzyme hydrolysis technology was developed , with the DH of 57.8%.The peptides from the above two technologies were purified with ultrafitration and dialysis respectively and then its hysicochemical properties and molecular weight distribution were determined. The results showed that the desalting ratio of 85% was obtained; the peptides had high solubility, low viscosity and good thermal stability, which provided basis for product development of the peptides. The molecular weight of protein hydrolysate from complex enzyme hydrolysis were almostly less than 5000 Da. The propotions whose molecular weight was less 1500 Da were 60% , so complex enzyme hydrolysis showed significant effects.Complex enzyme protein hydrolysates (CPHs) was fractionated according to the molecular basis into four major types of CPH I (above 10 kDa), CPH II (5 kDa～10 kDa), CPH III (3 kDa～5 kDa) and CPH IV (below 3 kDa) using an ultrafiltration (UF) membrane bioreactor system, respectively. The provention effect of oxidative DNA damage, lipid peroxidation inhibitory, radical scavenging activity of superoxide anion (O2-·) and hydroxy radical (·OH) were evaluated using chemiluminescence (CL). A natural antioxidant, ascorbic acid (Vc) was used as positive control. The results showed that all fractions showed higher antioxidant activities than Vc and a good correlation existed between the amount of peptides and antioxidant activity. The fraction whose molecular weight was below 3 kDa showed the strongest antioxidative activity, the antioxidative activity of the four fractions from CPHs decreased in the order: CPH IV >CPH III > CPH II >CPH I .Thus, the CPHs exhibited remarkable antioxidative activity, and the antioxidative activity of peptides increased as the molecular weight decreased. In order to explore the relationship between the composition and content of amino acids and the antioxidative activity of peptides, amino acids contained in CPH II, CPH III and CPH IV were determined. The results showed that some amino acids, such as Glu, Gly, Ala, Asp, Pro, Val and Arg were found closely related to the antioxidant properties of peptides. The fraction which showed stronger antioxidative had more amount of amino acids mentioned above. So the composition and content of amino acids and molecular weight of peptides jointly decided the antioxidant properties of peptides.