Screening, Fermentation And Preliminary Purification Technology For Absidia Glauca CGMCC No.4316Producing Glycoside Hydrolase Of Ginsenoside

The patent strains of Absidia glauca CGMCC No.4316and Zygorhynchus moelleriCGMCC No.4315have the unique ability transform ginsenoside Rb1into Rd, which weremainly separated from ginseng root. However their efficiencies in fermentation and enzymeproduction capacity have not been clarified. This thesis is aimed at screening highly activefungal strains for ginsenoside transformation, determinating optimizing fermentationcondition and purification of ginsenoside hydrolase. The test of separation and purification ofginsenosides in leaves powder was conducted. The research work confirmed the commercialpotential of these fungal strains to biologically transform lower active ginsenosides intohigher active rare ones.By means of agar block combined with TLC spot detection for colorimetric qualitativescreening, and liquid fermentation associated with HPLC quantitation for repeated screening,a rapid detection method was set up for accurate analysis of ginsenosides. The fungal strain ofCCGMCC No.4316was confirmed to be an optimum isolate for higher production ofginsenoside hydrolase and fermentation and purification of rare ginsenosides.The enzymatic reaction was analyzed by means of TLC. The key factors affectingfermentation to produce enzyme were investigated by single factor experiment. The methodof response surface analysis was applied, in association with glycosidase ginsenosides activityas response value, to establish3D regression model in order to optimize the variousinfluencing factors of fermentation medium. The results showed that the enzyme activityreached92.78U/mL, under the optimal conditions of wheat bran20.47g/L as carbon source,peptone9.74g/L as nitrogen source, initial pH6.0, culture medium volume45mL/250mL,shake flask revolution of180r/min, inventory10.00%, culture temperature30℃for72hfermentation.After fermentation to produce enough enzymes, the methods of ammonium sulfateranking precipitation and ethanol precipitation were tested to separate and purify theginsenoside hydrolase. It showed that the ammonium sulfate salting method was effective forpreliminary purification of the enzyme. Through product extracting plus silica gelchromatography for accurate separation and purification of ginsenosides, the highly purifiedRd was finally obtained.In addition, by using ethanol heating reflux extraction combined with macroporousadsorption resin purification technology as auxiliary method, the ginsenosides were separatedand purified from ginseng leaf powder. A precise mensuration was successfully establishedfor ginsenoside content in product.