| Many studies have shown that chitooligosaccharides have several bioactivities,including anti-microbial,anti-oxidation,reducing blood sugar and so on.This indicates that it is very meaningful for chitooligosaccharides production,in which it is better to degrade chitosan by chitosanase.But so far,only a small number of studies about chitosan degradation at a concentration of more than 6%have been seen,which leads to lower efficiency for chitooligosaccharides production.In order to improve the chitosan concentration in the enzymatic hydrolysis,and promote the hydrolysis process of chitosan solution with high concentration,and achieve high efficient enzymatic hydrolysis of chitosan solution with high concentration,the dissolution behaviors and solution properties of chitosan were investigated.Then the stability of pH and temperature of chitosanase were studied and the composition of enzymatic hydrolysates was analyzed,which laied the foundation for improving the conditions of enzymatic hydrolysis.Finally,based on the dissolution behavior of chitosan,the substrate reaction concentration was up to 10%?12%,the feasibility of adding enzymes in the process of dissolving was analyzed,and the effects of different enzymatic methods on the viscosity of solution and the concentration of reducing sugar in the hydrolysate were compared.The results of the study were as follows:l.In the process of dissolution,with the addition of acid,until the the HCl concentration reached 0.44 mol/L,the transmittance of chitosan solution at different concentration was initially low,and then rose sharply,until the rise trend was flat.The higher the chitosan concentration and degree of acetylation,the higher the HCl concentration at which transmittance began to increase sharply.When the chitosan concentration was 8%,1#and 2#solution transmittance increased significantly(p<0.05)at the HCl concentration of 0.23?0.34 mol/L,3#and 4#solution transmittance began to increased significantly(p<0.05)at the HCl concentration of greater than 0.29 mol/L.After finishing adding HCl,the lower the molecular weight of 4#chitosan,the higher the final transmittance of chitosan solution.2.When HCl concentration increased at ratio of about 0.06 mol/L,the conductivity of chitosan solution with a concentration of 8%initially increased at a small and even ratio.When HCl concentration was higher enough,the conductivity of solution rose at a larger ratio.The concentration range of HCl,at which the conductivity of 1#-4#solution began to increase dramatically,were 0.29?0.34,0.34?0.39,0.39?0.44,0.39?0.44 mol/L,respectively.It could conclude that in the range of the concentration of HCl,the chitosan molecules were almost completely protonated and fully dissolved,so the transmittance was higher.And the higher the degree of deacetylation of chitosan,the higher the HCl concentration at which conductivity of solution began to increase dramatically.This was corresponding to the change of the transmittance of the solution.3.When the chitosan concentration was 8%,1#and 2#,3#and 4#chitosan solution reached the maximum viscosity at concentration of HCl of 0.29 mol/L and 0.34 mol/L,respectively,and chitosan molecules were stretched.After that,chitosan molecules were curled into spherality and the solution viscosity decreased significantly(p<0.05),.Temperature increasing led to the decrease of solution viscosity,but the change trends of the solution viscosity with the concentration of HCl were the same.4.Microscopy observation showed that the number of chitosan particles decreased obviously with addition of HCl.Chitosan with a higher degree of deacetylation fully disintegrated at a higher HCl concentration.The transmittance and conductivity of solution increased obviously at the stage at which of chitosan particles disintegrated fully.5.The chitosanase was stable in the pH range of 4.5?5.5 and under 55?.According to the stability range of pH,it could lay the foundation for the subsequent addition of enzyme to reduce viscosity.With reference to its temperature stability range,the dissolution temperature could be increased to 55?.If the temperature was increased to the 60?,the enzyme activity would be seriously damaged.6.Under 55?,pH5.5,and enzyme content of 5 U/g chitosan,4#chitosan was hydrolyzed at the concentration of 8%for 12 h,and content of reducing sugar produced was about 18 mg/mL.Analysis of enzymatic hydrolysates by TLC and HPLC showed that the chitosanase was an endo-type cleavage enzyme,the hydrolyzed products consisted of only chitooligosaccharides,which varied in the composition with reaction time,and the chitooligosaccharides concentration of DP 2?6 produced for 12 h reached 14.77 mg/mL.7.According to the results of the study on the dissolution behavior of chitosan,it was known that HCl played a key role in the dissolution of chitosan molecules.In order to increase the concentration of chitosan in the solution,the amount of HC1 should be increased accordingly.When chitosan molecules were stretched,more glycosidic bond sites were exposed,and a certain amount of enzyme was added,which was beneficial for the degradation reaction,and could restrain the tendency of the viscosity of the high concentration chitosan solution to increase greatly.Thus,the dissolution process was combined with the enzymatic hydrolysis process.According to the results of pH stability of the enzyme,the amount of HCl should be controlled to prevent the pH of the solution beyond the scope of the appropriate pH value of the enzyme.Based on the above results,4#chitosan could be fully dissolved by adding 3/4 mL 6mol/L HCl per gram of chitosan.In this process,the chitosan molecular chains were more and more extended due to electrostatic repulsion with the addition of HCl,the final pH of the solution was about 5.00,and the pH had no significant effect on the enzymatic hydrolysis.8.The promotion of dissolution and hydrolysis process of 4#chitosan at the concentration of 10%was as follows:When the concentration of HCl was 0.32 mol/L,the viscosity of chitosan solution with 10%concentration was very large.So some enzyme needed be added to reduce viscosity when the concentration of HCl was less than 0.32 mol/L.The pH of the solution changed constantly with adding acid,and it also caused the local acid of the solution,which affected the enzyme activity.Therefore,different enzyme addition times were investigated,and according to 5 U/g chitosan enzyme,adding a part of the enzyme was appropriate in the process of dissolving,and after the dissolution,the remaining enzyme was added.The results showed that:At 55?,when the HCl concentration reached about 0.17 mol/L,chitosanase content of 1.7 U/g chitosan was added.The viscosity of the solution decreased about 53.7%compared with the control group without enzyme.After hydrolyzed for 12 h,content of reducing sugar produced was about 30 mg/mL.If enzyme content of 2.5 U/g chitosan was added again when enzymolysis was for 240 min,content of reducing sugar produced for 12 h could reach about 43 mg/mL,and analysis of enzymatic hydrolysates by TLC and HPLC showed that the the hydrolyzed products consisted of only chitooligosaccharides,and the concentration of chitooligosaccharides with DP 2?6 reached 25.43 mg/mL.9.The promotion of dissolution and hydrolysis process of 4#chitosan at the concentration of 12%was as follows:At 55?,when the HCl concentration reached about 0.21,0.41 mol/L,respectively,chitosanase content of 7.5,2.5 U/g chitosan were added,respectively.The viscosity of the solution decreased more than 70%compared with the control group without enzyme.After the dissolution,chitosanase content of 5 U/g chitosan were added.When enzymolysis was for 240 min,chitosanase content of 1.7 U/g chitosan was added again.The viscosity of enzyme hydrolysis solution finally dropped to about 400 cp.The degradation of chitosan with the concentration of 12%was achieved. |
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