The Extraction Of The Bioactive Peptides From Oyster And Preliminary Study On Its Activity

Part1 Preliminary purification of Bioactive Peptides from Oyster(BPO)Objective To study the isolation and purification of BPO.Methods The sample pretreatment steps play a key role in gel-filtration chromatography.Four methods for sample pretreatment were used to clarify enzymatic hydrolysate from oyster,including filtration with filter paper,micromembrane filtration,low-temperature high-speed centrifugation and chitosan flocculation method.BPO was purified by gel-filtration chromatography of sephadex G- 10,G- 15,G-25,G-50 and G-75 respectively.The molecule weight distribution of peptide fractions was analyzed by SDS-PAGE and tricine-SDS-PAGE.Results 1.Paper filter filtration for enzymatic hydrolysate from oyster was difficult,and there was turbidity in filtrate after stored at 4℃for 24h.The clarification effects of micromembrane filtration,low-temperature high-speed centrifugation and chitosan flocculation method to enzymatic hydrolysate from oyster were significant.They were easy except micromembrane filtration.2. After isolation and purification,it revealed that the separation effects of sephadex G-15 to enzymatic hydrolysate from oyster was the best.The eight peaks were obtained from enzymatic hydrolysate of oyster separated by chromatography column of sephadex G-15.Peak shape was symmetrical,and peak width was narrow.3.The molecule weight distribution(MWD)of G-25â… ,G-25â…¡and G-25â…£fractions were found to be about 66200u,8159～66200u and under 2512u respectively.In addition,freeze-dried products of G-25â…¢fraction were insoluble in eluant and electrophoresis sample solution,and its results could not be judged.Conclusion As a pre-treatment method of enzymatic hydrolysate from oyster,low-temperature high-speed centrifugation was simple and fast,suitable for experimental study.The separation effects of sephadex G-15 to enzymatic hydrolysate from oyster was the best.Experiment result indicated that the molecular weight distribution of BPO was extensive,and the composition of BPO was complex.Part2 The preliminary study on inhibitory effects of bioactive peptides from oyster on human tongue squamous cell carcinoma Tca8113 cellsObjective To study on inhibitory effects of bioactive peptides from oyster BPO G50-â…¡on human tongue squamous cell carcinoma Tca8113 cells.Methods The inhibitory effects of BPO G50-â…¡was studied in different concentrations and different time on human tongue squamous cell carcinoma Tca8113 cells by resisted-colour trypan blue.The morphological changes were determined by means of H.E dyeing and electron microscopy respectively.Cells apoptosis induced of BPO G50-â…¡was observed by acridine orange and ethidium bromide flurescence staining methods(AO-EB).Results The median inhibitory concentration(IC₅₀)of BPO G50-â…¡to Tca8113 in 24h,48h and 72h were 1.50mg/ml,0.57mg/ml and 0.20mg/ml respectively.BPO G50-â…¡could significantly inhibit Tca8113 cells growth and proliferation,it was relied on concentrations and time.The morphological changes of the tca8113 cells treated with BPO G50-â…¡were observed under light and electronic microscope respectively.The morphological changes were characterized by some typical features of apoptosis,such as cells shrinkage, vacuolus in cytoplasm,pyknosis,aniso chromatin,chromatin margination and etc.The result of AO/EB flurescence staining showed that the nuclear chromatins of living cells were green and those of dead cells were orange.The nuclear chromatins of the early apoptotic cells were green which showed condensation or fragment.And that of lately apoptotic cells were orange which showed condensation or debris.Conclusion BPO G50-â…¡could inhibit cell proliferation,change cell morphology and induce cell apoptosis.Part3 Study on the antioxidant activities of Enzymatic Hydrolysate from OysterObjective To study the antioxidant activities of enzymatic hydrolysate from oyster.Methods Its reducing power was detemined by the Pursian blue reaction.Scavenging effects on DPPH radical,hydroxyl free radical(·OH) produced from Fenton system and superoxide anion free radical(O^2-·) produced from the self oxidation of pyrogallol were measured.In addition,the antioxidant effect on peroxidation of polyunsaturated fatty acid from lipoprotein induced by iron was studied.Results The concentration of scavenging 50%(EC₅₀)DPPH·and·OH of enzymatic hydrolysate from oyster were 7.313 mg/ml and 1.330 mg/ml respectively.The inhibition rate of enzymatic hydrolysate(17.800mg/ml)on the pyrogallol autoxidation and lipid peroxidation reached 19.86%and 82.97% respectively.Conclusion Enzymatic hydrolysate from oyster had the evident effects of scavenging DPPH·and·OH.The results showed the effect of enzymatic hydrolysate was related to the concentration.In addition,enzymatic hydrolysate also had the effect of inhibition on the peroxidation of PUFA.