Biochemical Changes And Antioxidative Oligopeptides In The Autolysis Of Sea Cucumber Stichopus Japonicus

Sea cucumber is the most common invertebrate in the ocean with high nutritive value. It is susceptive to the environmental changes caused by physical, chemical and physiological factors and easy to undergo autolysis. The subsequent phenomena of gut vomitting and body wall softening will happened, which lead to severe deterioration in sea cucumber quality and bring great difficulties to the transportation and processing of sea cucumber. It is significant and necessary for the development of sea cucumber deep-processing industries to study the autolysis of sea cucumber systematicly.In the present study, the biochemical changes including main chemical composition changes and freshness changes were investigated and the influencing factors of sea cucumber autolysis process were determined according to the autolysis characteristic of sea cucumber body wall and guts. Meanwhile, the autolysis method was adopted to prepare oligopeptides from sea cucumber guts. The antioxidant activities of oligopeptides were evaluated and their amino acid sequences were identified. The main conclusions were as followed:(1) Both protein and sugar were changed during the autolysis process of sea cucumber body wall and guts. pH and temperature were found to be the two main factors by investigating the effects of pH, temperature, NaCl concentration and liquid to sample ratio on the autolysis process of sea cucumber body wall and guts. TCA-soluble oligopeptide was determined to be the evaluation indicator for the autolysis process of sea cucumber body wall and guts. Meanwhile, the reducing sugar content could also be used as the evaluation indicator for the autolysis of sea cucumber guts.(2) The activities of cathepsin B and cathepsin L from sea cucumber guts were enhanced by 6%～17% after autolysis under different temperatures (25℃and 50℃). The inhibitors of cathepsin B and cathepsin L including E-64, antipain and iodoacetic acid significantly inhibited the autolysis of sea cucumber guts. Their suppressing rates of TCA-soluble oligopeptides contents reached to 48.97%,47.24% and 12.24%, respectively. Meanwhile, dithiothreitol as a activitor of cathepsin B and cathepsin L obviously promoted the autolysis process of sea cucumber guts. These results suggest that cathepsin B and cathepsin L may play an important role in the sea cucumber autolysis process.(3) In order to control the autolysis the temperature should be low and the pH should be controlled in the alkaline range in the transportation and processing of sea cucumber. In addition, sea cucumber should be sheltered from direct sunlight to avoid the inductive effects of ultraviolet irradiation on its autolysis. When the ready-to-eat sea cucumber was produced, the heating treatment should be performed at the temperature higher than 60℃in order to inhibit the activities of endogenous enzyme and maintain good appearance. Ultraviolet irradiation could be used in utilization of sea cucumber autolysis, and the temperature and pH should be in the range of 37～60℃and 4-7, respectively.(4) The combined interaction of different influencing factors on the sea cucumber autolysis was studied by response surface methodology. The results showed that the two models which were established on the basis of temperature, pH, NaCl concentration and temperature, pH and substrate concentration, respectively, could reflect the interaction accurately. The combined effect of pH and substrate concentration on the autolysis process of sea cucumber was significant. The TCA-soluble oligopeptides content decreased more quickly as the pH got more lower when the substrate concentration was relatively low, which was worse for the autolysis of sea cucumber body wall.(5) The freshness changes during the autolysis process of sea cucumber body wall were investigated at both 25℃and 46℃. The results showed that pH reached to minimum at 20 h and then increased gradually. The contents of TVBN were increased gradually with the autolysis period prolonged, and reached higher than 20mg/100g when the body wall was autolyzed for 44 h and 32 h at 25℃and 46℃, respectively. When the body wall was autolyzed for higher than 8h, the TVC were increased gradually and reached to 106 cfu/g at 32 h. These results suggest that microorganisms were not involved in the autolysis process of sea cucumber body wall under the experimental conditions in the present study.(6) The optimum autolytic conditions of sea cucumber guts were obtained by response surface methodology. Temperature at 48.30℃, pH at 4.43, sample to ratio 1:3 and time 4 h were found to be the optimal conditions to obtain autolysis hydrolysates from the guts of sea cucumber.(7) The autolysis hydrolysates were mainly composed of components below 3 kDa and it contained 17 kinds of amino acids. The autolysis hydrolysates had DPPH radical scavenging capacity, Fe2+-chelating ability and reducing power. The values of SC50, CC50 and AC0.5 were 4.04,5.91 and 8.06 mg/mL, respectively.(8) Four different fractions, namelyⅠ,Ⅱ,Ⅲand IV, were obtained after the autolysis hydrolysates were separated by Sephadex G-15. Fraction IV exhibited the highest DPPH radical scavenging capacity, Fe2+-chelating ability and protective effect against hydroxyl radicals-induced DNA damage, which may be related with the high content of Ile, Leu and Val.(9) The fraction IV was analyzed by ESI-MS for molecular mass determination and ESI-MS/MS for the characterization of peptides. Three kinds of antioxidative oligopeptides, including two tetrapeptides Val-Thr-Pro-Tyr (479 Da), Val-Leu-Leu-Tyr (507 Da) and a hexapeptide Val-Gly-Thr-Val-Glu-Met (635 Da), were identified. All of them showed obvious protective effects against hydroxyl radicals-induced DNA damage. protective effect against DNA damage. These results suggest that antioxidant oligopeptides derived from the guts of sea cucumber by autolysis method could be utilized for functional foods.