The Enzymatic Preparation And Analysis Of Chitooligosaccharides And Its Preliminary Separation

Chitosan is rich in nature and it has been widely applied in food, medicine, environmental protection industry. But its applications become limited because of its high molecular weights and poor solubility. Chitooligosaccharides are the products by the degradation of chitosan.Chitooligosaccharides not only retain some functional properties of chitosan, but also has water-soluble and other more valuable characteristics. Enzymatic degradation of chitosan is a widely-used method, it can splitβ-1,4-glycosidic bonds specifically and selectively. The process is done under mild conditions,easy to control,it can obtain different molecular weight of chitooligosaccharides through the control of different conditions.The strain Mitsuaria sp.141-2 collected in the laboratory was employed in this study. The study focused on the enzymatic degradation of chitosan. The fermentation condition of enzyme production was optimized; The condition of enzymatic degradation was optimized; The degradation products were detected and analyzed; The products were preliminarily purified. The specific results were as follows:1,The study used two-step method to optimize the enzyme production which includes the optimization of biomass and induction. The optimized conditions of biomass enrichment phase were:2.5% glucose,0.9% peptone,0.14%K2HPO4,0.06% KH2PO4, 0.1%MgSO4,180 mL/1000 mL,30℃,180 rpm; The optimized induction conditions were 1.5% chitosan colloid for three days. Eventually, the biomass is 3 times the initial conditions, the enzyme production increased from the initial 3.5 U/mL to 6.1 U/mL.2,The paper studied the conditions of enzymatic degradation of chitosan by single-factor experiments. The optimum conditions were:substrate concentration 3%, pH 5.2～5.6, temperature 65℃, enzyme dosage 7 U/g chitosan. The most suitable degree of deacetylation may be the middle for about 85%.3,Hydrolysates were detected and analyzed.Thin layer chromatography was used to analyze the hydrolysate of chitosan. The main products were the mixture of chitosan hydrolysates with different degree. The study also explored a new method to prepare the required molecular weight chitosan by selecting different enzyme dosage and hydrolysis time. The products were preliminarily purified. The 75% ethanol could remove the chitosan whose degree of polymerization was above 9.The 87.5% ethanol could remove the chitosan whose degree of polymerization was above 7.