Study Of Enzymatic Hydrolysates Of Glucanase And Beta Galactosidase For The Curculigo Polysaccharides

Curculigo polysaccharides could enhance immune activity,antioxidant,anti fatigue and had other good biological activity,and showed high medicinal value and economic value.This experiment investigated the process of enzymatic extraction,separation and purification of the Curculigo polysaccharides,and obtained one pure polysaccharide called XMPS-1,determining purity and relative molecular weight of it.Using glucanase and beta galactosidase to modificate XMPS-1 respectively,and two kinds of enzymolysis technology were investigated.The enzymatic hydrolysate were separated by gel chromatography column and were analyzed by TLC.The activity of it was studied by cell experiments in vitro,to lay the foundation for finding activity center fragment of Curculigo polysaccharides that could activate macrophages.The main results were as follows:1.On the basis of single factor test,using Design-expert 8.0.5b software and the response surface method to optimize extraction process of Curculigo polysaccharides.The optimal conditions were obtained as enzyme dosage of 1.3 %,enzymolysis temperature of 58 ℃,enzymolysis time of 110 min and pH value of 5.7,while the extraction rate of Curculigo polysaccharide reached 20.52 %.Compared with ultrasonic extraction and hot water extraction,the extraction rate increased by129.8 % and 54.9 % respectively.Pure polysaccharide called XMPS-1 was separated by DEAE cellulose column and Sephadex G-50 gel column.The results of gel column chromatography and GPC showed that XMPS-1 had high purity,so XMPS-1 was a kind of holosaccharide.The molecular weight of XMPS-1 was 3031 Da.2.The effects of enzyme dosage,temperature,time and pH on enzymatic hydrolysate of XMPS-1 hydrolyzed by glucanase were investigated.On the basis of single factor test,the process was optimized by response surface methodology.With3.the model P < 0.0001,missing item P = 0.3433 > 0.05,indicating that the fitting degree of model was good,and the method had the design significance.The optimal conditions were obtained as enzyme dosage of 56 %,enzymolysis temperature of 52 ℃,enzymolysis time of 12 h and pH value of 6.0,while the content of reducing sugar reached 0.2019 mg/mL.The experimental results showed that the D-value between the measured value and the expected value was less than 5% with higher reliability.The enzymatic hydrolysis products were separated by Sephadex G-25 gel column and analyzed by TLC.While the enzymolysis time was 7~9 h,pure hydrolysates fragmentand were produced.4.Based on the yield of reducing sugar,the response surface methodology and central composite design method were used to construct the model of the effects of four factors on the enzymatic hydrolysis of XMPS-1 by beta galactosidase.Increasing the appropriate amount of time and enzyme dosage,with the suitable pH value and temperature,could promote the production of reducing sugar,and the impact strength of four factors was: time > enzyme dosage > temperature > pH.The optimal conditions were obtained as enzyme dosage of 10000 U/g,enzymolysis temperature of 46 ℃,enzymolysis time of 10 h and pH value of 6.6.The enzymatic hydrolysis products were separated by Sephadex G-25 gel column and analyzed by TLC.Pure hydrolysates fragmentand were obtained by dialysis in these four batches of products.5.The 12 batch of enzymatic hydrolysate of XMPS-1 was obtained by glucanase.The results of MTT assay showed that when the concentration of the sample was less than 500 g/mL,and the incubation time was less than 48 h;XMPS-1 had no obvious toxic effect on cells.The cell survival rate of most samples was higher than 100%,which indicated that most of the enzymatic hydrolysates were not only no toxic to cells,but also could promote the growth of cells.The contents of TNF-α,IL-1 and NO secreted by macrophages were detected by ELISA test kit and Griess method.It was concluded that the majority of enzymatic hydrolysates had a good effect on the secretion of three kinds of cytokines by synthesizing the effects of the enzymatic hydrolysates on the three cytokines.Especially the enzymatic hydrolysis obtained by5 h was the most prominent,and it had a better effect on the secretion of the three6.cytokines at different incubation time and different sample concentration,with in-depth research value.