Seperation And Antioxidant Activities Of Maillard Reaction Products Of Hydrolysates From Sea Cucumber (Stichopus Japonicus)Gut

Sea cucumber（Stichopus japonicus）gut is a by-product during process of sea cucumber containing high level of protein.It is a good source to develop active peptides.In the present study,S.japonicus guts as raw materials were hydrolyzed by neutrase.The Maillard reaction products（MRPs）were prepared from lyophilized powder of sea cucumber gut hydrolysates and D-ribose system.MRPs were further isolated by ultrafiltration with 5,3 and 1 kDa membrane,respectively.Four fractions including fraction Ⅰ（>5 kDa）,fraction Ⅱ（3～5 kDa）,fraction Ⅲ（1～3 kDa）and fraction Ⅳ（<1 kDa）were obtained.The characteristics of MRPs-C were compared with each fraction by FT-IR analysis,color measurement,spectrophotometric analysis,browning degree analysis,ultraviolet absorption,and antioxidant activities.The antioxidant activities were evaluated by detecting TEAC ability,Fe²⁺ chelation ability,reduction ability,lipid peroxidation inhibition ability and DPPH-scavenging ability.Firstly,the results showed that the Maillard reaction had generated substances,such as aldehydes,ketones and so on by FT-IR analysis.The degree of browning and fluorescence intensity decreased with the decreasing of fraction molecular weight.Fraction I showed the highest level of browning degree,ultraviolet absorption value and fluorescence intensity.It is suggested that the fraction I（>5 kDa）has a great contribution to the color of the MRPs.The degree of antioxidant activity increased with the decreasing of fraction molecular weight.Fraction IV had the stronger antioxidant activity than the other three fractions.Therefore,the antioxidant activity of the ultrafiltration components of Maillard reaction products of hydrolysates from sea cucumber S.japonicus gut was mainly determined by the low molecular weight component（<1 kDa）of MRPs.Secondly,the crude fraction and the fraction IV were extracted by methanol and ethanol respectively.Then the antioxidant capacity of ABTS free radical,DPPH radical scavenging ability and linoleic acid anti-lipid peroxidation ability were evaluated.The results showed that the antioxidant semi inhibitory concentration of the ultrafiltration components of Maillard reaction products of hydrolysates from sea cucumber S.japonicus gut were all decreased significantly（p<0.05）after extraction with organic reagent.The antioxidant properties of the methanol extraction of the ultrafiltration components was significantly higher than that of the ethanol extraction,which antioxidant semi inhibitory concentrations of ABTS free radical,DPPH radical scavenging ability and linoleic acid anti-lipid peroxidation ability were 2.63±0.06 mg/mL,0.22 ± 0.01 mg/mL and 18.66±0.22mg/mL respectively.Finally,the protective effects of Maillard reaction products of hydrolysates from sea cucumber S.japonicus gut on oxidative stress injury in HepG2 cells was detected with the hydrolysates,Maillard reaction products and the methanol extraction of Maillard reaction products of hydrolysates from sea cucumber S.japonicus gut.The results showed that there was no significant effect on the survival rate of HepG2 cells induced by H₂O₂ of the hydrolysates in the concentration range of 20 μg/mL～200 μg/mL（p>0.05）,while the Maillard reaction products and the methanol extraction of Maillard reaction products were significantly protected the cells and the effect of the methanol extraction on cell protection was stronger than the ethanol extraction of Maillard reaction products.The survival rate of HepG2 cells induced by H₂O₂ of the methanol extraction of the crude MRPs was 1.74-fold of the crude MRPs,3.97-fold of the H₂O₂ processing group.It is suggested that the methanol extraction of Maillard reaction products of hydrolysates from sea cucumber S.japonicus gut could inhibit the oxidative stress injury of HepG2 cells induced by H₂O₂.