| Diosgenin,whose chemical name is 5 different spiral sterene-3-beta-alcohol,belongs to different spiral sterene alkyl derivatives.Diosgenin has the function of cleaning lung-heat,stopping cough,damp elimination and smoothing showering,activating meridians to stop pain,resolving toxin and dispersing swelling,also it can inhibit platelet aggregation,promote bile secretion and strengthen the bones at the same time.Diosgenin,which can synthesize thousands of kinds of steroid hormone drugs,such as anabolic hormones,corticosteroids,sex hormones,called "medicinal gold",has become a research hotspot in the world as its significantly high medicinal value and economic benefits.While the action of extracting diosgenin from Dioscorea genus destroys the plant resources and the environment,also restricts the development of relevant industries.In recent years,several purified endophytic strains which can produce diosgenin has been separated and purified from Dioscorea zingiberensis C.H.Wrigh,of which SYt1 has an optimal production of diosgenin and a diosgenin extraction method from the fermentation broth was established.In this study,we mainly focus on the optimal diosgenin producing endogenous bacteria-SYt1.That the synchronous optimization of fermentation culture medium compositions and culture conditions,the combining use of the single factor and multiple factor to achieve optimal fermentation condition of SYt1 bacteria high density cultivation by comparison and analysis and the increase of diosgenin production provide experimental basis and reasonable advice for the industrialization of microbial fermentation to produce diosgenin.(1)Experiment of single factorAfter the experiment of single factor for the carbon source,the nitrogen source,the inorganic salt,the inoculum amount,the fermentation temperature,the shaking speed and the fermentation time,the eight factors(sucrose,soybean meal,sodium chloride,sodium hydrogen phosphate two,magnesium sulfate,calcium carbonate,inoculation and fermentation time)were finally selected for further optimized P-B design.(2)P-B(Plackett-Burman)experimentAfter P-B experiment,the soybean meal(B),Na2HPO4(E)and fermentation time(L)were selected as three important factors influencing saponin yield.The main factors encoded multivariate linear regression equation:Y=64.72+20.28*B-5.76*E+5.34*L,where y is the response value of saponin yield while the coefficient of determination R2 equation was 0.7395.As there were 1.01%distortions in model results,a good correlation was shown.Then other five factors of fermentation as sucrose 30g/L,NaCl 5g/L,MgSO4 0.5g/L,GaCO340g/L and inoculation amount 12%,were determined according to the positive and negative effects,thus providing a basis for the subsequent optimization.(3)The steepest ascent experiment(Steepest Ascent)Through the analysis of the steepest ascent(Steepest Ascent)experimental results,saponin yield increased with the increase of the number of stages and peaked at 162.682mg/L at the last stage of experiment,improving 307.8 per cent than that of pre-optimization as 39.89mg/L.The best condition of the adjacent area of main influencing factors were found,at that time,the fermentation conditions were as follows,sucrose 3 0g/L,sodium chloride 22g/L,NaCl 5g/L,Na2HPO4 0.6g/L,MgSO4 0.5g/L,GaCO3 40g/L,inoculum amount 12%,fermentation temperature 28℃,shaking speed 150rpm,fermentation time 7d. |
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