| Chitosan oligosaccharides,especially the ones with degree of polymerization(DP)2-6,possess many bioactivities.Beacause of limitations of enzymolysis and solution properties of chitosan,so far the efficiency of chitosan oligosaccharides production was very low,and it is of great significance to explore how to produce chitosan oligosaccharides efficiently.In order to improve enzymolysis efficiency and yield of target chitosan oligosaccharides,enzymolysis of high concentration chitosan solution and its efficiency improvement were studied.Separation and purification process of hydrolysate obtained by hydrolysis of high concentration of chitosan solution was studied,and then enzymolysis and purification of high concentration of chitosan solution were combined in this study.Finally,microwave vacuum drying process of chitosan hydrolysate was studied.The results were as follows:1.Changes of content of reducing sugar and chitooligosacchairdes during enzymolysis of chitosan with various concentration were measured,it was found that the reducing sugar conversion rate decreased as concentration of chitosan increased.The conversion rate of reducing sugar in 8% chitosan solution was only 39.8%,while the yield of DP2-6 chitosan oligosaccharides was only about 50%.2.Effects of substrate concentration on enzymolysis of high concentration of chitosan were investigated,it was found that when concentration of the chitosan was low,the initial velocity of enzymolysis increased sharply with the increase of substrate concentration;when the substrate concentration increased,the initial velocity gradually tended to be stable,and then decreased with the substrate concentration.Final concentration of reducing sugar could be promoted by supplementing chitosan after hydrolysis for 4 h or 10 h,but the yield of target DP chitooligosacchrides decreased(45.58% and 46.68% respectively),and the production period became longer,which was not advantageous to the industrial production.3.Product inhibition during enzymolysis of high concentration of chitosan was investigated,it was found that degradation of chitosan can be further promoted effectively by procedural and repeated addition of chitosanase.The more the amount of enzyme was added,the higher the content of final reducing sugar in hydrolysate.The conversion rate of reducing sugar could be promoted greatly by supplymenting chitosanase 5 U/g chitosan after hydrolysis for 4 h and 8 h respectively,and average Mw of the hydrolysate was smaller.What is more,the yield of target chitooligosaccharides increased to 75.10%,and chitopentaose and chitohexaose with highest physiological activity also increased significantly.According to the results of supplementing chitosan and procedural supplementing chitosanase,it was reasoned that in the process of enzymolysis of the high concentration of chitosan solution,eyzmatic efficiency would declined as the formation of unproductive substrate-enzyme complexes.Subsrate inhibition was obvious,and supplementing chitosanase could improve enzymatic efficiency and the yield of target chitooligosaccharides.4.Ultrafiltration of the hydrolysate of high concentration of chitosan solution was carried out.Membrane flux increased gradually with pressure,and then remained stable.With the increase of pressure,contents of various chitosan oligosaccharides(DP 2~6)in permeate decreased in various degrees,and permeate rate of DP 2~6 chitooligosacchrides decreased gradually and then remained stable.Membrane flux increased gradually with the increase of temperature.In the range of 25 ~50 ?,temperature did not much affect permeate rate of DP 2~6 chitooligosaccharides and its proportion in permeate.2500 Da ultrafiltration membrane could not only penetrate the target oligosaccharides(DP 2~6),but also intercepted impurities with large molecular weight,which was suitable for this production.5.Hydrolysis of high concentration of chitosan was combined with ultrafiltration through inserting ultrafiltration operation in different hydrolysis time.Various schemes all could improve the the yield of DP 2~6 oligosaccharides.Enhancement in the yield of DP 2~6 oligosaccharides was reduced when less chitosanase was supplymented.6.Chitooligosaccharides were dried by microwave vacuum drying.Under the microwave power density of 2~4 W/g,there was obvious stage with constant drying rate during dehydration.while under the microwave power of 6~10 W/g,the drying rate reached its peak and then gradually decreased.Dissolution time of chitooligosachharides increased significantly(P<0.05)under too high microwave power density,and products became browning and coking.Clearance rate of hydroxyl radical OH also declined dramatically.When vaccum degree increased,dissolution time of chitooligosacchrides was significantly decreased(P<0.05),and the clearance rate of hydroxyl radical increased significantly(P<0.05).Brightness of products increased and browning degree decreased.In the range of material thickness of 0.4~1 cm,the products obtained by microwave vacuum drying showed no significant difference in solubility,oxidation resistance and color(P>0.05).In response surface optimization(index: solubility and antioxidation),the regression equations were as follows:Dissolution time =14+4.75A-2.88B-0.13C+0.5AC-0.75BC+5.13A2+2.37B2+0.88C2Clearance rate =84.99-21.42A+8.71B+1.1C+3.38AB+1.52AC+1.11BC-18.96A2-2.95B2-0.022C2A was microwave power density(W/g);B was vacuum degree(MPa);C was the material thickness(cm).All the factors in the regression equation had significant influence on the response value,and the optimal conditions were obtained: power density was 3 W/g,vacuum degree was 0.08 MPa and material thickness was 0.7~ 0.8cm.Except browning,the effect of microwave vacuum drying was similar to that of freezedrying,but cost was lower and drying time was shorter,which had a good applied prospect in the production of chitooligosaccharides. |
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