Improvement Of Extraction Process Of Diosgenin And Screening Saponin Transformed Strains

Dioscorea zingiberensis C.H.Wright is a dioscorea plant,belonging to the genus Dioscorea.It contains a large number of diosgenin with pharmacological activity in itsrhizome.Diosgenin,as an important intermediate of steroid hormone drugs,plays an significant role in anti-tumor,anti-aging,anti-allergy,hypolipidemic and other aspects.At present,the production of diosgenin uses mainly direct acid hydrolysis.The method existed so many problems such as low extraction rate,high cost,low resource utilization,and serious environmental pollution.In this paper,the biological extraction process of diosgenin were optimized to improve the yield of diosgenin,to achieve efficient use of resources,and to ruduce environmental pollution.The details are as follows:(1)The pre-fermentation conditions(temperature and time)in the extraction process of diosgenin were optimized and the optimal conditions were obtained.The optimal conditions of natural spontaneous fermentation were as follows:45 ?for 24 hours firstly,52 ?for 12 hours secondly,62 ?for 36 hours finally.The effects of cellulase,xylanase and pectinase on the yield of diosgenin were studied by single factor test and orthogonal test,and the addition amount of three enzymes were optimized.The optimal addition amount of three enzymes were as follows: cellulase 50 U/g,xylanase 100 U/g,pectinase 120 U/g.At the basis of the optimal combination of temperature and time and the optimal addition of three enzymes,the yield of diosgenin was 0.845%,enhancing 69% than the yield of diosgenin of prefermentation without any enzyme at constant temperature of 58?.(2)The amlyme content was determined in the Dioscorea zingiberensis C.H.Wright.Its content was 31.11%.According to the comparison test of ?-amylase and pullulanase addition sequence,it was confirmed that the enzyme hydrolysis was better when ?-amylase was added first and then pullulanase was added during liquefaction.The single factor test and orthogonal test were used to optimize the enzymolysis process of starch,and the final optimal conditions were as follows: ?-amylase 80 U/g raw material,?-amylase enzymolysis time 2.0h,pullulanase enzymolysis time 2.0h,pullulanase 50U/g raw material,glucoamylase enzyme amount 190U/g raw material,glucoamylase enzymolysis time 2.0h.Under this optimal condition,the hydrolysis rate of the starch was 91.87%.(3)A strain producing enzyme which can convert dioscin into diosgenin was screened form soil by the plate separation method.The enzyme activities were measured after the strains were inoculated in the shake flask.Its initial enzyme activity was 8.414U/mL.Its growth curve was measured,whose logarithmic growth phase was 4-19 h.(4)The fermentation conditions of the strain producing enzyme were optimized by the single factor experiment.The final optimal conditions were as follows: inoculation amount 5%(v/v),liquid volume 50mL/250 mL,initial medium pH 6.5,fermentation temperature 31?,shaker Speed 180r/min,fermentation period 48 h.Then the single factor tests,Plackett-Burman design test,steepest climbing test and central composite design test were used to optimize the fermentation medium.The optimal fermentation medium components were as follows: glucose 17.8 g/L,peptone 16 g/L,sodium dihydrogen phosphate.6.4 g/L,manganese sulfate 6.2 g/L,potassium dihydrogen phosphate 3.0 g/L,and magnesium sulfate 1.0 g/L.Under this condition,the repeatability test was performed and the average enzyme activity obtained was 79.985 U/mL,which was basically consistent with the predicted value.