Preparation,Purification And Bioactivities Of Chitooligosaccharides

Chitosan,composed of β-(1→4)-linked N-acetyl-glucosamine(Glc NAc unit) and deacetylated glucosamine(Glc NH2 unit), is a derivative of chitin, mainly derived from shrimp and crab shell. The application of chitosan is limited due to its poor water-solubility. Chitooligosaccharides(COS), has small molecular weight, good water solubility and absorption, is a partially hydrolyzed product of chitosan. It has superior biological activity than chitosan. But all these bioactivities of COS are often determined using relatively poorly characterized oligomer mixtures, which is difficult to make further research on the mechanism. This condition was mainly confined to the complex process of COS monomers separation. Therefore, it’s clearly essential that well-characterized and highly purified oligosaccharides preparation so as to understand in-depth knowledge on the mode of the bioactivity of COS. In this study,we prepared COS by enzymic method and obtained a single degree of polymerization of COS by gel chromatography, meanwhile its in vitro and in vivo hypolipidemic and antioxidant activity was studied. The main results are as follows:(1) Preparation of COS: Single factor experiments and orthogonal experiments were used to optimize the process conditions for the preparation of COS by enzymatic hydrolysis. Meanwhile, its molecular weight, composition and content were detected. Results showed that the key points of technological parameter and the bests elected condition were: reaction temperature was 50℃, p H was 5.6, the dosage of chitosanase was 58 U?g-1, concentration of substrate was 3.0% and hydrolysis time was 4 h. The molecular weight, the composition and content of the sugar in hydrolysed chitosan were analysed by the method of acetyl acetone, TLC and HPLC,the results showed its molecular weight was 1457 Da, its content was 96.5%,while the sugars detected in the COS sample were Glc N,(Glc N)2,(Glc N)3,(Glc N)4, and(Glc N)5 with the content of 1.24%、40.42%、33.04%、12.83%和 12.46% respectively.(2) Separation of COS: Polyacrylamide gel was used for COS separation. The sample volume and the elution flow rate were investigated. The separating effect wasperfect in the conditions. Column volume: 340 m L; flow rate: 0.20 m L?min-1; loading amount: 0.10 g. Purity quotient of COS monomers was analysed by HPLC. Results indicated that the purity of(Glc N)2,(Glc N)3,(Glc N)4 and(Glc N)5 were 95.7%,97.1%,97.7% and 89.4%.(3) The antihyperlipidemic activity of COS in vivo was studied, which with molecular weight of 1457 Da and composition of Glc N to(Glc N)5. A rat model of hyperlipidemia was established by feeding high fat diet. The effect of high(375mg?kg-1?d-1), medium(187.5 mg?kg-1?d-1) and low(93.75 mg?kg-1?d-1) dose of COS on serum lipid levels, lipid levels, hepatic indicators and antioxidant activities in rats was studied after intragastric administration for 6 weeks. Results showed that COS significantly lowered the serum total cholesterol(TC), triglyceride(TG), low density lipoprotein cholesterol(LDL-C) concentrations and elevated the high density lipoprotein cholesterol(HDL-C) level. Meanwhile COS could also significantly increased the activity of LPL, HL, SOD and GSH-Px and decreased the content of ALT, AST, MDA and Liver coefficient. The results enlighted that COS has antihyperlipidemic and antioxidant activities.(4) To study the effects of different concentrations of COS monomers(DP of 2 to 5)on cholesterol and triglyceride levels in cells. Human hepatoma cell line Hep G2 was selected as the research subject to establish the hypercholesterolemia and hypertriglyceridemia model. The results revealed that with the degree of polymerization increasing, the effect of cholesterol and triglyceride decreased(chitobiose> chitotriose>chitotetraose>chitopentose). Antioxidantion in vitro showed that scavenging activities for DPPH and superoxide radical elevated with the increase of the polymerization degree. However, the effect for hydroxyl radicals was quite the contrary.In summary, the preparation, separation and in vitro and in vivo hypolipidemic and antioxidant activity of COS were studied, which laid the foundation for the development of functional products of COS, and also provide a scientific basis for and utilization of the further development of COS.