Study On Preparations Technology Of Active Polysaccharides And Polypeptides From Low-Value Sea Cucumber

In this paper,the body wall of sea cucumber(Acaudina molpadioides)was used as the experimental material to prepare the hydrolysate by ultrasonic pretreatment and mixed enzyme hydrolysis.Then the polysaccharides and polypeptides were separated by ultrafiltration and alcohol precipitation.After that,polypeptides with different molecular weight were prepared by ultrafiltration and the crude polysaccharides were purified by Q-Sepharose Fast Flow anion exchange column.On this basis,the antioxidant activity of polysaccharides and different molecular weight peptides were determined,the antitumor activity of purified polysaccharide and the effect of different molecular weight peptides on the secretion of collagen were explored,so as to provide a theoretical basis for improving the utilization rate of sea cucumber resources.The main results of this study were as follows:1.Trypsin,neutral protease and compound protease were selected to hydrolyse the body wall of the sea cucumber,and the optimum dosage of each enzyme was 8000 U/g,10000 U/g and 12000 U/g.Taking polysaccharides content and hydroxyl radical scavenging capacity as indexes,single factor experiment and orthogonal test were used to determine the best enzymolysis process.On the basis of the best enzymolysis process,we investigated the effect of ultrasonic pretreatment on the contents of polysaccharides and hydroxyl radical scavenging capacity.The final enzymatic hydrolysis process was as follows:the ultrasonic power was 100 W,the ultrasonic time was 30 min,the proportion of the mixed enzyme was trypsin(8000 U/g):neutral protease(10000 U/g):the compound protease(12000 U/g)was equal to 1:1:1,the ratio of material to liquid was 1:40(W/V),the temperature of enzymatic hydrolysis was 50℃,and the time of enzymatic hydrolysis was 7 h.2.The enzymatic hydrolysate was prepared according to the final process.Ultrafiltration and alcohol precipitation methods were used to separate the polysaccharides and peptides.Then we prepared sea cucumber polypeptides with different molecular weight by ultrafiltration.The effects of ultrafiltration membranes on the separation results were investigated,and then the 50 kDa membrane was selected for subsequent experiments.The influence of operation pressure on membrane flux and membrane efficiency was explored to determine the best operating pressure,the final determination was 0.2 MPa.The antioxidant activities of peptides with different molecular weights were studied in vitro.The results showed that the free radical scavenging ability of different molecular weight peptides was stronger,and the free radical scavenging ability of each polypeptide was higher than 50%in a certain range,and the free radical scavenging ability increased with the increase of concentration.3.The crude polysaccharides were purified by anion exchange column and the two methods obtained three and four polysaccharides,respectively.They were named as CF1,CF2,CF3 and SPF1,SPF2,SPF3,SPF4.The monosaccharide composition and chemical structure of purified polysaccharides were determined by PMP precolumn derivatization and infrared spectroscopy.The scavenging free radical ability of purified polysaccharides in vitro was investigated.The results showed that CF2 was mainly composed of glucose,galactose,fucose,arabia sugar and galactohydronic acid.CF3 was mainly fucose,followed by galactosidonic acid and galactose.SPF2 was mainly glucose,followed by galactose and fucose.SPF3 consisted of five monosaccharides,mainly fucose,followed by galactose,glucose,mannose and glucuronic acid.SPF4 was mainly composed of fucose.FTIR analysis of purified polysaccharides showed that they were all typical absorption bands of polysaccharide structure.Scanning electron microscopy showed that CF2 was aggregated from flakes and dots,and CF3 showed strip structure.SPF3 was a mixture of granular and strip objects,SPF4 was formed from a strip of polysaccharide molecules.The results showed that each purified polysaccharide had strong scavenging ability and had a dose effect relationship in a certain range.The activity of CF2 is higher than that of CF3.The antioxidant activity of SPF2 and SPF3 were slightly higher than that of SPF4.4.In this part,we constructed a hydrogen peroxide induced oxidative damage model of Human skin fibroblasts to study the protective effects of different molecular weight peptides and purified polysaccharides on oxidative damaged HSF cells and investigate the effect of different molecular weight peptides on the secretion of HSF collagen and the inhibitory effect of purified polysaccharides on human hepatoma cells(HepG2).The results showed that:(Molecular Weight,MW)MW<1 kDa polypeptide and SPF3 had strong protective effects on HSF.MW<1 kDa,1 kDa<MW<5 kDa,5 kDa<MW<10 kDa three kinds of peptides all significantly promoted the secretion of collagen from the HSF cells.SPF3 and SPF4 had significant inhibitory effects on HepG2 cells,and SPF4 inhibition effect was better than SPF3.