Studies On The Optimization Of Enzymatic Preparation Of Chito-oligosaccharides And Its Physiological Activities

Chito-oligosaccharides is the degradation product of Chitosan. The molecular structure of Chito-oligosaccharides is similar with Chitosan. Because of low polymerization degree, small molecular weight and good water-soluble, Chito-oligosaccharides have more extensive application space than Chitosan.In our country there are abundant sea foods. Shrimp shell and crab shell which are regarded as residual leftover after aquatic product processing, not only produce lots of waste but also pollute environment. This experiment explores optimum technological conditions of preparing Chito-oligosaccharides, and studies on physiological activities of MW<10kDa(molecular weight is less than 10000 Dalton) Chito-oligosaccharides such as antioxidant, hypoglycemia, reducing blood lipid and biosorption of heavy metal. The research results as follows:(1) This experiment selects cellulose which is low cost and has good degradation effect degrading Chitosan, and determines degradation effect in different enzymolysis time, enzyme concentration,pH and temperature. Through single factor experiment and orthogonal experiment, we obtain the best technological combination for producing Chito-oligosaccharides which the enzyme concentration is 1600U/g,enzymolysis time 7h, pH 5.4 and temperature 55℃. In four factors of influencing Chito-oligosaccharides production, pH has the most significant influence (P<0.05), and temperature has the least significant influence (P>0.05). On this technological combination of preparing MW<10kDa, the production rate of Chito-oligosaccharides is 69.51%.(2) Through the experiment of Chito-oligosaccharides scavenging(·OH)and(O2?·)we can know when the Chito-oligosaccharides concentration is 10 mg/ml, the clearance to(·OH)and(O2 ?·)can reach about 80%, Chito-oligosaccharides concentration is 30 mg/ml, the clearance to(·OH)and(O2 ?·)can reach close to 100%.After the mice given Chito-oligosaccharides by gavage 30d I determine the MDA, SOD and T-AOC indexs of the mice. The results showed that the SOD, T-AOC activity in liver and serum of middle dose group (400 mg/kg) and high dose group (800 mg/kg)mice is higher than high fat model group mice significantly (P<0.05), MDA content is lower than high fat model group significantly (P<0.05).(3) Chito-oligosaccharides have hypoglycemic function to diabetic mice induced by 60mg/kg alloxan. The results showed that the blood glucose value of mice given Chito-oligosaccharides by gavage have significant difference with the model control group (P<0.05). So the experiment illustrates Chito-oligosaccharides can decrease blood glucose value of diabetic mice obviously, but it can't reduced to the normal blood glucose value completely.(4) We choose the Chito-oligosaccharides of different molecular weigh to do descending blood lipid experiment in vitro. The result showed that the MW>2kDa Chito-oligosaccharides have good absorbability to oil, bile salts and cholesterol. Animal experiment also showed that MW>2kDa Chito-oligosaccharides have the function of reducing weight, decreasing blood lipid and protecting liver. Among which the high dose group has the most significant effect.(5) In the experiment of adsorbing heavy metal in shellfish, I feed clam in the seawater rich with heavy metal to make shellfish model rich with heavy metal. Then I took the clam rich with heavy metal into seawater containing Chito-oligosaccharides. After 5d I detect the difference of heavy metal residue in shellfish by atomic absorption. The result showed that Chito-oligosaccharides can promote the clam's metabolism of heavy metal. The effect of metabolism of Pb and Cu is better, when Chito-oligosaccharides concentration in seawater is 0.4g/L, the metabolic rate of Pb and Cu can reach to 26.00% and 21.47%, higher than model group which is 10.64% and 11.83%; the metabolic rate of Zn is 8.40%, in the second place, higher than model group which is 7.41% and to Cd it has not significant effect.