Enzymatic Prepaeation And Separation Of Chitooligosaccharides And Its Antioxidant Activity

Chitooligosaccharide(COS)is the hydrolysis products of chitin and chitosan,which has extensive biological activities such as anti-tumor,anti-oxidant,antimicrobial and so on.Biological activities of COS are closely related to its degree of polymerization(DP).So it is necessary to preparative separate chitooligosaccharide monomers with different DP for the further research of COS.Bacillus thermophilus HEC08 was isolated from the soil samples of extreme place,which can simultaneously produce high activated deacetylase and chitinase.The optimal conditions for enzyme production by the strain were obtained:chitin 1.5%,yeast extract 0.14%,culture temperature 45 ?,pH value 7.6,inoculum amount 10%,shaking cultivation at 168rpm for 72h.Enzymology test showed the optimum temperature and pH value of deacetylase and chitinase were 46 ? and 7.4,respectively.Chitin was hydrolyzed by fermentation liquor of Bacillus thermophilus HEC08,which had high practical value for transformation from chitin to COS directly.A simple and rapid HPLC-ELSD method for simultaneous determination of COS with DPI?6 was developed to analyze the hydrolysate of chitin,which had high specificity,sensiticity,repeatability and six compounds can be completely separated in 12min.The main hydrolysate of chitin were COS with DP2?6.Exchange and adsorption of cation exchange resin for COS with DPI?6 was researched.HD-8 cation exchange resin was screened out to separate COS monomers with DP 1?6 for the first time.Under the optimal condition of 1.0g sample/100mL resin,gradient elution with 0.5?1.5mol/L HCl and elution velocity of 2 BV/h,six highly purified glucosamine oligomers containing 99.67%glucosamine,97.85%dimer,98.22%trimer,95.34%tetramer,90.03%pentamer and 87.57%hexamer were obtained respectively.HD-8 resin had characteristics of high loading capacity,good separation,can be reusable and economical,which can provide favorable conditions for industrial preparation of COS monomers.The in vitro antioxidant activity of the separated COS with DPI?6 were studied,including DPPH and hydroxyl radical scavenging activity and reducing power.Five monomers with DP2-6 showed good radical scavenging activity and reducing power.Antioxidant activity enhanced with reducing of DP.Chitobiose had the best antioxidant activity which can be a potential antioxidant.