Chitosanase Preparation Of Chitooligosaccharides And Chitosan Oligosaccharide Antibacterial Activity

Chitooligosaccharides are isotypic or heterotypic oligomers of N-acetyl glucosamine and D-glucosamine with polymerization degree of 2-10.It is the only functional oligosaccharide with amino and positive charge,giving it a wide range of applications.Chitosan has low molecular weight,good water solubility,and high biological activity,and has better application value and physiological function than chitosan.Skin trauma is a major problem in medical care.There are about 300 million chronic trauma patients in the world.The traditional wound treatment is dry healing after iodophor treatment,which is not conducive to the migration of fibroblasts,wound pain,and scarring.Studies have shown that chitosan oligosaccharides can enhance the fibroblasts’motility in vitro,promote cell proliferation and migration,enhance the function of inflammation and repair cells to accelerate wound healing;they can absorb protons produced by vasokinin and acetic acid and have good pain relief Role;can inhibit the formation of typeⅠcollagen and reduce scars.Chitosan oligostomy wound treatment can effectively inhibit wound infection and promote wound healing,and has good medical value in wound treatment.In this paper,after rejuvenating the laboratory-preserved strain Aspergillus fumigatus UC-233 strain,a high genetically stable chitosanase-producing strain was successfully screened,and the enzyme-producing activity was stable at 28U/m L.Through single factor and orthogonal experiments,the enzyme production conditions of the rejuvenated strain Aspergillus fumigatus UC-233 were studied.The optimal fermentation production conditions were 3.5%colloidal chitosan,3.2%corn steep powder,0.1%Mg SO₄·7H₂O,0.1%KH₂PO₄,0.4%anhydrous Ca Cl₂,0.1%（v/v）Tween-80,initial p H 5.5,filling volume 40m L（250m L shake flask）,fermentation culture temperature 30℃,inoculation volume 2.0%spore suspension The fermentation culture time is 54h,and the enzyme activity is 42.27（±0.21）U/m L,which is 1.51 times before optimization,which realizes the rejuvenation of the strain and the improvement of enzyme activity after optimization.Factors such as the concentration of chitosan substrate,the amount of enzyme added in the reaction system,the reaction p H,temperature and stirring speed can all affect the enzymatic hydrolysis of chitosan.Through single factor and orthogonal experiments,the enzymatic hydrolysis conditions of chitosan enzymatic hydrolysis of chitosan were optimized,and the optimal enzymatic hydrolysis process was determined to be colloidal chitosan 4.5%,the optimal enzyme addition amount was 1.26 U/m L,and the optimal enzymatic hydrolysis The p H is 5.5,the optimal enzymolysis temperature is 60℃,the optimal stirring speed is 100 r/min,the optimal enzymolysis time is 3.5 h,and the reducing sugar content reaches 66.03（±0.33）μmol·m L^-1 under the optimal enzymolysis conditions.Using chitosanase prepared by fermentation to decompose chitosan to prepare crude chitosan oligosaccharide liquid.After decolorization optimization by activated carbon,1000Da ultrafiltration membrane was separated and purified to obtain high-purity chitooligosaccharide,which was mainly concentrated in DP2～4.Based on the optimization of parameters in industrial production,the decolorization parameters were studied and determined:the optimum decolorization material was activated carbon for 15#sugar,the optimum amount of activated carbon for 15#sugar was 2.0%,the optimum decolorization time was30min,and the optimum decolorization temperature was 60℃.After decolorized chitooligosaccharide solution was separated by 1000Da ultrafiltration membrane,the freeze-dried yield was 52.8%and the average degree of polymerization was3.03.With homemade low polymerization degree oligosaccharides with different shell oligosaccharide concentration and p H on Escherichia coli antibacterial circle experiment and antibacterial performance of research,the results showed that the lower the p H,shell oligosaccharide concentration is higher,the better the performance of bacteriostasis,low concentrations of p H4～6 shell oligosaccharide antibacterial performance is as high as 90%above,p H,concentration of 6.0 4.0g/L shell bacteriostatic properties as high as 96%,sugar solution has good antibacterial properties,for shell oligosaccharide is applied in wound therapy to lay the foundation.