Preparation Of Pentamer To Heptamer Chitooligosaccharides With Specific Degree Of Polymerization By Hydrochloric Acidic Degradation To Chitosan

This study outlines a method to prepare chitooligosaccharides with a specificdegree of polymerization5-7. It was carried out by hydrochloric acid hydrolysis in achitosan-degraded system and a followed procedure by gel filtration chromatography tofraction chitosan oligomers. They were detected by high performance liquidchromatography.13C and1H nuclear magnetic resonance was used to identify anunknown complex. The main results were listed as follows.（1） Established the condition of thin layer chromatograph （TLC） which coulddistinguish chitooligosaccharides with a degree higher than5. Thin layerchromatography （TLC） was used to analyze the distribution of oligosaccharides. Weestablished the condition of TLC that could detect chito-oligomers（DP=1-7） and theirdistribution. The reaction mixtures were separated on a Silica gel HSGF254plate withisopropanol-water-ammonia water （15:1:7.5v/v/v）. After rinsing the plates withvanillin-sulfuric acid-water reagent （1:12:88, w/v/v） and baking them at175°C for10min.（2） Screened the reaction conditions of pentamer and heptamer chito-oligosaccharides, which are obtained from hydrolysis of chitosan by hydrochloric acidicdegradation. The results showed that chitosan was degraded most effectively in9MHCl at60°C and the yeild of chito-pentaose and chitohexose reached16.2%, higherthan previous study. We showed the relationships between every factor and the reactionprocess, as well as the product. Temperature has a relatively high influence on the degradation reaction, based on the empirical data. Chitooligosaccharides of differentpolymerization have different distributions in every reaction periods. Thus to ensure thestability and repeatability of the reaction we sampled all the chitooligosaccharides fromequilibrium system.（3） Isolation and identification of chitooligosaccharides of specific degree ofpolymerization （DP=5-7）. A procedure using Sephadex G-15onto size-exclusioncolumn was further developed to narrow the range of oligomers and to obtain expectedfraction containing mainly chitosan pentamer and hexamer detected by thin layerchromatography. Yield as high as55%could be obtained by controlling the sampleamount at0.3⁰.4g. In this study we sampled0.31g, eluted it with deionized water at aspeed of3.6ml/h, collected the product every0.9ml fraction, and then had achitooligosaccharides productivity of58%. This study gave a beneficial exploration forthe future study of chitooligosaccharides with different degrees of polymerization.Chitooligosaccharides fractions were isolated on Asahipak NH2P-504E column（4.6mm id×250mm length） and eluted at30°C with an acetonitrile-water （70:30, v/v）solution at a flow rate of1ml/min. The eluant was sampled20ml then detected byrefractive index detector at30°C. The results showed that the anticipatedchitooligosaccharides pentamer（retention time10.7min） and hexamer（retention time13.3min）, were detected, based on the comparison to the standards.This study put forward new ideas of application of chitosanase onchitooligosaccharides identification. The study combined the NMR and enzymolysisanalysis, identified the complex X through resolving13C and1H NMR graphs andstudying the specific chitosanase enzymolysis. The NMR was conducted in BrukerAvance400MHz, dissolved in D₂O. The enzymolysis was conducted by mixing thechitosanase （15U/mg, appr.1mg） and chitooligosaccharides （appr.1mg） in sodiumacetate buffer （50mM, pH6.0） at37°C, reacting for1h. The results showed the complexis chitosan oligosaccharides, with a degree of polymerization of7i.e. chitosan heptamer.This study identified chitosan heptamer for the first time, laying a good foundation for the identification and isolation of chitooligosaccharides with higher degree ofpolymerization.This study found the way of producing chitooligosaccharides with specific degreeof polymerization （DP=5-7）, providing beneficial references for the identification andisolation of chitooligosaccharides with higher degree of polymerization. Thechitooligosaccharides （DP=5-7） obtained is highly deacetyl chitooligosaccharides, witha percentage of97%. When combined with F-6and F-10the purity could be enhancedto more than90%. Compared with the chitooligosaccharides standards, thechitooligosaccharides product had a better appearance. The degradation rate of chitosanwith hydrochloric acid is over60%, with16.2%chitooligosaccharides （DP=5-6） in it.Yield of chitooligosaccharides standard under Sephadex isolation was58%, with over90%chitooligosaccharides （DP=5-7） in it.