The Oxidative Degradation Of Chitosan And The Purification Of Oligochitosan

Chitosan, the deacetylated derivative of chitin, consists of β-1,4linkedD-glucosamine （GlcN） and partially N-acetyl-D-glucosamine （GlcNAc） in varyingproportions. The application of chitosan is limited due to its poor water-solubility.Oliogochitosan, the degradation product of chitosan, has the same structure withchitosan but superior biological activity, thus more and more importance is attached tooligochitosan according to its special functions in medicine, health food, biologicalmaterial, etc. At present, oligochitosan with specific degrees of polymerization （DP）were prepared by enzymatic or acid degradation methods, however, oligochitosanwith DPs up to6prepared by oxidative degradation hasn’t been reported. In this paper,chitosan was degraded by H₂O₂and the oligochitosan products were purified andseparated effectively, oligochitosan with DP from1to5were obtained in the end. Themain points in this paper are as follows:（1） The degradation process and experimental rules of chitoson by H₂O₂werestudied systematicly. Viscometric and the end group reduction method were used todetermine the degree of degradation. According to single factor experiments andorthogonal experiments, the key points of technological parameter and the bestselected condition were: degradation temperature of60℃, degradation time of6h,concentration of4.0%H₂O₂and4.0%acetic acid. The distribution of chitosan and thedegraded product were analyzed by gel permeation chromatography, the resultsshowed chitosan had been completely degraded and the molecular weight （Mw） wasbelow2000, while the polymerization degree of degraded chitosan was below10afterbeen detected and analysed by electro-spray ionization mass spectrometry （ESI-MS）.The Mw of structure unit was161, it’s the same with glucosamine.（2） Oligochitosan with DPs up to6has a high biological activity. In order toobtain the oligochitosan with DP from1-6, ion chromatography （IC） was used forquality and quantity dertermination and response surface methodology （RSM） wasapplied to optimize the technological parameter.According to the results ofBox-Benhnken Design, the optimum condition was prepared at56℃for6h withconcentration of4.5%H₂O₂.（3） The purification and separation of degradation product were studiedsystematicly. D301weak anion resin and P-2polyacrylamide gel were used foroligochitosan purification and separation, oligochitosan of DP1-5were obtained with the purity of75.81%,62.47%,60.01%,63.27%and54.49%respectively. Through theprocess of degradation and separation, different DP fractions were obtained fromchitosan, the final yield of glucosamine, dimei,trimer, tetramer, pentamer and DPhigher than hexamer were, in order,7.24%、5.18%、8.30%、3.41%、2.38%、10.09%.（4） The mechanism of oxidative degradation was discussed. From thecomprehensive results of IR, X-RAD and1H-NMR spectrums, it can be concludedthat the constitutional unit didn’t change from raw material to degradation product,however, few peroxide by-products with C=O groups were formed. In the process ofdegradation, it can be conjectured that HO, generated by H2O2, may attack β-1,4glucosidic bond in the structure of chitosan macromolecular and break O-O etherlinkage through dehydrogenation reaction.（5） The antimicrobial activity experiment of seperated oligochitosan was studied.Oxford-cup method had been used to measure the size of inhibition zone. Resultsshowed that oligochitosan had a siginificant effect on Escherichia coli, but a slighteffect on StapHylococcusaureus and Listeria monocytogenes. Oligochitosan of DP5had a visible effect on Escherichia coli when its concentration was up to1mg/mL.