Study On Preparations And Antioxidant Activities Of Polysaccharides And Polypeptides From Acaudina Molpadioides

Acaudina molpadioides are domestic low-value sea cucumbers.The polysaccharides and protein are the most important bioactive substances of Acaudina molpadioides.In this dissertation,dried body walls of low-value Acaudina molpadioides were selected as research materials.The enzymatic hydrolysis technologies were optimized.During preparing step,polysaccharides and polypeptides with different molecular weight(MW)range were firstly isolated by ultrafiltration.Then the best method to purify polysaccharides by using column chromatography is studied,as well as the chemical component and preliminary structure of polysaccharides from Acaudina molpadioides.Furthermore,a primary research was made to contrastive study the antioxidant activities in vitro of polysaccharides and polypeptides from Acaudina molpadioides.The main results are listed below.Different enzymes,order and modes of hydrolysis were selected to get the optimum combined preparation technology.Under the condition of material liquid ratio 1:40 trypsin is used with the enzyme concentration 2.4%,at 45 ? with pH value of 7.5,to hydrolysis for 8h,and polypeptides of MW blow lOKDa were obtained by ultrafiltration separation.Then the neutral protease and papain were used at ultrafiltration cut-off liquid and precipitation of previous hydrolysis.Firstly adding 7%neutral protease to hydrolysis for 4h at 45?.Then adding 8%papain at 60? to hydrolysis for 4h,the whole process of pH value was 7.0.In this condition,the extraction rate of polypeptides was(29.602±1.012)%,and the extraction rate of crude polysaccharides was up to(14.511±0.162)%.According to the study,the best ultrafiltration conditions were confirmed as follows:operating pressure is 0.20MPa,material liquid concentration is 6%,and the operating temperature is 35?.As the ultrafiltration separation sequence from high MW to low MW,three polypeptides with different MW range were obtained,48.47%between 5?10KDa named P1,18.46%between 1?5KDa named P2 and 33.07%below 1KDa named P3.As the ultrafiltration separation sequence from low MW to high MW,four crude polysaccharides with different MW range were obtained,63.09%blow lOKDa named G1,7.24%between 10?100KDa named G2,4.67%between 100?200KDa named G3 and 25.00%above 200KDa named G4.Three pure polysaccharides G4-1,G4-2 and G4-3 were obtained by using Q-Sepharose-F-F ion-exchange column chromatography to isolate and purify.The elution concentrations are Omol/L,0.5mol/L and 1.5 mol/L.According to chemical component analysis,with the decrease of the molecular mass,the content of total sugar and sulfate increased,and protein content decreased.The content of total sugar is up to 55.99%and protein is down to 6.33%in G4.The content of total sugar,protein,hexosamine,uronicacid,fucose,and sulfate was 67.35%,4.98%,3.21%,7.99%,43.98%,1.29%in G4-1,was 34.45%,21.98%,11.17%,9.88%,10.18%,16.16%in G4-2,was 54.81%,1.08%,9.30%,9.58%,23.48%,25.59%in G4-3.From the chemical component,it can be speculated that G4-1 might be a kind of fucoidans with abit of sulfate,G4-2 might be glycosaminoglycan,G4-3 might be a kind of sulfated fucoidans.The result of IR analysis further confirmed G4-2 was a compound of protein and polysaccharides,G4-3 contained the most sulfate,the types of glycosidic bond in G4-1 and G4-3 were all a-pyran glucose type,in G4-1 was a-furan glucose type.The detection results of antioxidant activities in vitro showed that,the order of scavenging activity of polypeptides with different MW range on-OH was P3>P2>P1,on DPPH· and O-2·was P2>P3>P1.The order of scavenging activity of crude polysaccharides with different MW range on ·OH,DPPH·,and O-2· were G4>G1>G3>G2,G4>G3>G1>G2,and G1>G4>G2>G3.The order of scavenging activity of pure polysaccharides on ·OH was:G4-1>G4-2>G4-3,on DPPH· and O2· was G4-2>G4-1>G4-3.