Low Temperautre Bacteria Proteasa And Its Preliminary Study On Extraction Of Sea Cucumber Active Substances

Sea cucumber is one of the most important marine foods and medicinalresources, which contains a large number of proteins, amino acids, carbohydrates,complex vitamins and mineral elements. However, the traditional processing ofcooking sea cucumber seriously limits the nutrients of sea cucumber which will beabsorbed by human bodies, causing the waste of nutrition. The purpose of the paperis to research and find out an appropriate method to improve the digestibility so thatthe extract active substances of sea cucumber can be absorbed much better by humanbodies. The method in this paper is to use alkaline protease,which is isolated fromthe Antarcticstrains with hydrolysis, neutral flavourzyme, providing withhydrolysisand, and water treatment group to process the sea cucumbers separately. Atthe same time, optimize the best technological parameter of these two enzymes onthe basis of crude ratio of polysaccharides and polypeptide.According to theconsequences, the relatively higher crude rate of polysaccharides and polypeptide isacquired in the group treated by alkaline protease and flavourzyme. This paper alsoexplores the inoxidizability and anti-tumor efficacy of the active substances. Theprimary research results are listed as followed:First, a series of strains screened in the Antarctic are inoculated; then the strainswith high protease activity were preliminary screened by the method of CoomassieBrilliant Blue G-250, finally, the strain with the highest protease activity defined asI64which would be high-producing strain were screened by FLION phenol.Hereafter,the fermentation enzyme-producing conditions were tentatively optimized based onthe two indicators of cell concentration and enzyme activity of fermented liquid andthe most appropriate pH is confirmed as7.0, best developing temperature is10℃,2216E couLd be confirmed as the appropriate medium; The appropriate pH foralkaline protease is9.0, the most suitable temperature is30℃.Using the flavourzyme, alkaline protease that extract from the Antarcticmicroorganisms, and water group to process sea cucumber, making use of CoomassieBrilliant Blue to measure the content and degree of hydrolysis of soluble protein,taking advantage of sulfuric acid-phenol method to measure the content ofpolysaccharide,sea cucumber crude polysaccharide and sea cucumber polypeptidewould be used as two indicators to evaluate the optimize hydrolysis conditions ofthese three processing methods above. The best optimizational conditions forflavourzyme treatment group are: the amount of water is six fold, the dosage ofenzyme1.5%, temperature is40℃, working pH is7.0, enzymatic hydrolysis time is6hours; The best optimization conditions for low temperature proteolytic enzymes that extract from the Antarctic microorganisms are: the amount of water is six fold,the dosage of enzyme is1.5%, enzyme appropriate temperature is30℃, pH is9.0,3hours’enzymolysis time.The best optimization conditions for water treatment groupare:45℃,3and9hours.The amount of active material acquired from enzymolysisof low temperature alkaline protease is3%to50%higher than that of neutralproteinase,85%to95%higher than that of water treatment group, which could beconfirmed as the best enzyme preparation.The material acquired in the experiments, such as sea cucumber enzymatichydrolysate and crude polysaccharide, is applied to neuroblastoma cells andpancreatic cancer cells so preliminary study can be conducted about the oxidationresistance and anti-tumor.The protective effect of enzymatic hydrolysate of sea cucumber and crudepolysaccharide for neuroblastoma cell treated by H2O2has been proved by the cellexperiment. Micro plate reader and Antioxidants kit are used to determine theinoxidizability of enzymatic hydrolysate of sea cucumber and crude polysaccharide.The consequences show that all the polysaccharide and enzymatic hydrolysateacquired by the three groups present certain inoxidizability and dependence onconcentration. The ability to eliminate hydroxyl radical of enzymatic hydrolysate andcrude polysaccharide in group3is much stronger than the ability to eliminatesuperoxide anion free radical; the ability to eliminate hydroxyl radical, superoxideanion free radical of enzymatic hydrolysate and crude polysaccharide in the sameconcentration shows as follow: the low temperature proteolytic enzyme treatmentgroup is higher than the low temperature enzyme treatment group, followed by thewater treatment group.And hydroxyl radical scavenging rate range of polysaccharidewhich is in2.5mg/mL concentration reaches up to50.6％~68.2％in the lowtemperature proteolytic enzymes treatment group.MTT method was used to detectthe proliferation activity of cultured human pancreatic cancer cell line, which isinfluent in the sea cucumber polysaccharide and the sea cucumber enzymatichydrolysate.The sea cucumber polysaccharide and enzymatic hydrolysate cansignificantly inhibit the proliferation of PANC-1activity, while the peptide had nosignificant effect on the proliferation activity of tumor cells.