| Androstenedione(referred to as AD),is an endogenous steroid hormone,which can be secreted by the human gonads and adrenal glands,and is an androgen synthesis in the body.The direct precursor of testosterone in the pathway is also the precursor of estrogen in the estrogen synthesis pathway.It plays a vital role in rationally controlling the balance of androgens and estrogens.In addition,AD is also an important intermediate for the synthesis of steroid drugs,and almost all steroid drugs can be synthesized through AD.In this paper,the seed medium formulation was optimized firstly.Through single factor experiment and orthogonal experiment,the optimum seed medium combination and cultivation time were obtained.Then,in view of the hydrophobicity of phytosterols,the effects of organic solvents,deep eutectic solvents,surfactants,cyclodextrins and other co-solvents and two-phase systems on the AD produced by fermentation were determined.Orthogonal experiment was selected to optimize the fermentation of the two-phase system.When the concentration of phytosterol was 10 g/L,the organic phase was soybean oil,the proportion was 16%,the highest AD yield reached 42.15%,and the AD concentration was 2.83 g/L.The effects of fermentation conditions such as the temperature of the shake flask,the speed of rotation,the amount of liquid filled,the amount of inoculation,the initial p H value,and the cultivation time on AD production were studied.The optimized fermentation conditions are as follows: temperature 30?,initial p H 8.0,speed 200 rpm,inoculation volume 20%,liquid volume 150 m L.After 6 days of fermentation,AD yield reached 50.37%,AD concentration was 3.39 g/L,the substrate conversion rate was 76.42%.After optimization,the yield of AD increased 19.50% in compared to the former(42.15%).The thesis systematically studied the process conditions of AD production by resting cell method.The substrate is difficult to dissolve in water,so different types of co-solvents such as organic solvents,deep eutectic solvents,surfactants,cyclodextrins were selected to study the effect on the conversion results.The hydroxypropyl-?-cyclodextrin as a co-solvent,when its molar ratio to sterol is 1:1,AD yield was 48.51%,AD concentration was 3.24 g/L.The response surface method was used to optimize the AD production process by the resting cell method.Firstly,the Plackett-Burman experiment design was used to screen out the three main factors affecting the yield of AD: phytosterol concentration,hydroxypropyl-?-cyclodextrin concentration and cell concentration.On this basis,the steepest climbing method was used to approximate the range of the maximum response value,then the response surface analysis method was used to determine the interaction and optimal conditions between these major factors.The results showed that when the phytosterol concentration was 19.41 g/L,the hydroxypropyl-?-cyclodextrin concentration was 80.14 g/L,and the cell concentration was 110.55 g/L,the yield of AD reached 67.25%.The actual average yield was closed to the predicted yield.After optimization,the yield of AD increased 38.66% in compared to the former,the AD content reached 8.68 g/L,and the production intensity was 2.17 g/(L d).Finally,the AD extraction process was also studied in the article.Through a series of optimization of extraction,concentration,and crystallization,AD products were detected by GC,IR and NMR.The purity of the product can reach 98.31%.The product meets the quality standards of AD products. |
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