Preparation Of Chitooligosaccharides By Enzymatic Method And Their Anti-obesity Activity

Objective Obesity is a global health problem caused by the imbalance of energy metabolism,which greatly reduces the quality of life and life span of human beings.Chitooligosaccharides(COS)are oligosaccharides hydrolyzed from chitosan,which have the characteristics of good water solubility,better biocompatibility and nontoxicity.They have a broad application prospect in the field of weight loss.Chitosanase hydrolysis method is an environmentally friendly and efficient method for obtaining COS.However,COS are mainly hydrolyzed from chitosan by non-chitosanase hydrolysis method,which greatly limits their application at present.Therefore,it is very important to find new chitosanase with excellent properties and to prepare COS with anti-obesity activity.Methods The marine bacteria producing chitosanase were screened from the sediments of the Yellow Sea,and their 16 S r DNA were amplified for sequence alignment.The bacterium was cultured in the medium containing chitosan,and the produced chitosanase was purified to obtain chitosanase and and its enzymatic properties were characterized.The optimum conditions for the preparation of COS by enzymatic hydrolysis were studied and the preparation process of COS was established in a 5-L fermenter,and then the composition of COS was analyzed.Obese KM mice induced by a high-fat diet were fed with COS,and the serum was collected for lipid analysis.The liver and epididymal fat of the mice were taken for tissue section analysis to study the anti-obesity activity of COS.Results Sequence alignment showed that the strain had the highest identity sequence with Pseudoalteromonas sp.and was named Pseudoalteromonas sp.SY39.The purified chitosanase was named Csn M,and its relative molecular weight was about 28 k Da.Csn M is a cold-adapted enzyme,which shows highest activity at 40°C and exhibits 30.6% and 49.4% of its maximal activity at 10 and 15°C,respectively.Csn M is also a thermo-tolerant enzyme that recovers 95.2%,89.1% and 88.1% of its initial activity after boiling for 5,10 and 20 min,respectively.The optimum conditions for the preparation of COS from chitosan hydrolyzed by Csn M were: 40℃,hydrolysis for 10 hours,p H 6.0,18 U/(g chitosan)chitosanase.The COS are consisted of the oligosaccharides with polymerization degree of 2-5.COS significantly reduced the weight of mice,the weight of liver and epididymal fat and were capable of decreasing serum TC and TG levels of the mice fed with high-fat diet.COS reduced the size of fat cells and alleviated hepatic steatosis,and have the function of liver protection and anti-obesity.Conclusion In this study,a new chitosanase Csn M with good cold-adapted and themo-recovered ability was obtained by cultured and purifyied method from a chitosanase-producing marine bacterium.The degree of polymerization of COS prepared by hydrolysis of chitosan with Csn M was 2-5,and COS showed good anti-obesity activity in mice..