The Study On The Production Of Fungal Chitosan By Fermentation And The Enzymatic Degradation Of Chitosan

Chitosan(CTS) is the only alkaline polysaccharide polymer existed in nature which is the product of chitin deacetylation. CTS has a unique physiological activity and good film forming, moisture retention, adsorption and antibacterial activity which make it useful in a variety of industrial fields. Chitosan oligosaccharides(COS), the degradation products of chitosan, has greatly improved water-soluble property which make it has lots of special physiological activities and function compared with chitosan. In this paper, fungus were used for the production of chitosan by fermentation, and non-specific enzymes used for the degradation of chitosan.Firstly, several fungus were used for the production of chitosan. Actinomucor elegans was found to be the best strain for preparation of chitosan. The medium and fermentation conditions were optimized by single-factor design and response surface methodology. The results showed that the optimal proportion of medium were corn steep liquor6.8%, liquefied corn flour4.2%, KH2PO40.2%, MgSO40.2%, and the optimal fermentation conditions were time48h, original pH7.0, temperature30℃, shaker speed200r/min, amount of inoculation4%, amount of load50ml(in250ml flask).Secondly, diluted alkali and acid treatments were used to extract chitosan from the dry mycedia, and the extraction procedure and parameters were optimized. The results were as follows:socked with4%sodium hydroxide, proportion of solid to liquid1:30, at115℃for4h to remove the protein, and then extracted with4%acetic acid at60℃for3h, the yield ratio of chitosan achieved13.48%. Under the optimum fermentation and extraction conditions, the yield of chitosan was up to4.152g/L, the degree of deacetylation of chitosan was90.25%, and the average molecular weight was2.1×105.Thirdly, cellulase, pectinase, papain and pepsin were used for the degradation of chitosan, these four non-specific enzyme hydrolysis tests carried out by single-factor analysis based on the release dose of reducing sugar. The results showed that the activity of degradation of chitosan was:cellulase>pepsin>pectinase>papain. The hydrolysis conditions of mixed cellulase and pepsin were also studied by the orthogonal test. The optimal parameters of enzymolysis were pH5.5, reaction temperature65℃, adding pepsin3U/mg and cellulose3U/mg. Under the optimum conditions, the yield of reducing sugar achieved4.64mmol/L, which was much higher than cellulase and pepsin individually.Fourthly, hydrolysis products were characterized by SephadexG-25gel chromatography and thin-layer chromatography(TLC). Elution curves of gel chromatography showed that there were two-eluting peaks standing for COS and Glucosamine. The results of TLC showed that hydrolysis products contained two low- molecular weight COS. Furthermore, according to the thin-layer chromatography tapping, the recovery rate of COS and glucosamine were54%and22%.