Preparation, Separation And Analysis, Antibacterial Activity Of Chitosan Oligosaccharide

Chitooligosaccharide (COS) is the hydrolysis products of chitosan. With the advanage of high water solubility and bioavailability, it has been widely used in the fields of anti-microbial, anti-oxidant, anti-inflammatory, regulation of blood lipids and blood glucose, anti-tumor, enhance immunity, activation of the intestinal flora, etc. Therefore the research and development of COS has become the hot spot in both domestic and foreign.Water-soluble COS was prepared using the microwave-assisted enzymatic hydrolysis method, and water soluble COS with low-degree polymerization was prepared by ethanol precipitation. TLC、HPLC、UPLC-MS were used to analyze COS. COS with different polymerization degree can be clearly separated with TLC when the volume raito of ethyl Isopropanol/Water/Ammonia was4/2/3. HPLC was used to analyze COS accurately and quantitatively. UPLC-MS was used to identify the N-acetylated constituents in COS. The chromatographic behavior of COS was also studied, the results illustrated that the reason of the chromatographic separation of COS was not in accordance with its polymerization degree was mainly caused by the N-acetylated constituent. It could provide refernce for analyzing and separating the constituents of COS.Polyacrylamide gel (Bio-Gel P-2) was used to separate COS monomers and the purity of COS monomers was analysed by TLC and UPLC-MS. Results indicated that the components were chitobiose, chitotriose, chitotetraose, chitopentaose and chitohexaose. The purity of chitobiose and chitotriose were both up to96%, while the purity of chitotetraose, chitopentose and chitohexaose were lower, and need to be further purified.The antibacterial activity of COS with different deacetylation degree (DD) was studied. It showed that when pH was5.0, the IC50of COS with the DD95%,65%,45%was2.5,5,40g/L respectively, which illustrated that the lower DD of COS, the better antibacterial activity to Escherichia coli under the same pH. For the COS with DD95%, the IC50was2.5,2.5,10g/L when pH is5,6,7respectively, which illustrated that the lower pH of the solution, the better antibacterial activity to E.coli under the same DD of COS. Besides, the antibacterial activity had an obvious difference to E.coli, Staphyloccocus aureus, Fusarium oxysporum between the COS monomers, the inhibition rate of chitohexaose is the highest and the chitobiose’s is the lowest, while glucosamine showed no antibacterial activity. For example, the inhibition rate of chitohexaose to F.oxysporum was70.5%, while chitobiose was only38.8%.