Study On Solid Fermentation Transformation Of Canrenone By Rhizopus Sp. UJS-0602 And The Comprehensive Utilization Of Fermentation Residue

Canrenone is a kind of steroid hormone drug,clinically useful for the treatment of cardiovascular diseases such as edema,heart failure,high blood pressure.The treatment effect is remarkable.However,if it is used to treat heart disease,there may be dangerous consequences because of overdose.The side effects of another kind of steroid hormone drug named eplerenone are significantly reduced when it is used to treat cardiovascular diseases,while the canrenone hydroxylation product 11α-hydroxy canrenone is the key pharma ceuticalinter mediate of synthesizing eplerenone.The conversion of canrenone to 11α-hydroxy canrenone by microbiological transformation was characterized by simple steps,few by-products and mild conditions.The preliminary study on transformation of canrenone by liquid fermentation has been completed in our research group.However,there are some limitations of liquid fermentation such as:canrenone is a kind of steroid compound,its water-insoluble properties will affect the transformation ratio to some extent.In the process of liquid fermentation,surfactants are often added to facilitate the dissolution of the substrate,which can complicate the subsequent extraction and purification of the products,so steps to remove surfactants need to be added.At the same time,the extraction process requires the use of water-immiscible organic solvent like ethyl acetate as an extracting agent.Ethyl acetate is toxic,harmful and easy to have adverse effects on human body and environment.Using solid fermentation can simplify the process of substrate transformation and product extraction,in addition,solid fermentation is eco-friendly.Therefore,canrenone transformed into its hydroxylation product 11α-hydroxy canrenone by Rhizopus sp.UJS-0602 with the method of solid fermentation has been studied in the research.Optimization of the medium component in solid fermentation has been carried out.Fermentation kinetic equations have been established by measuring the variation of biomass,the content of the product and substrate during the fermentation process.The extraction conditions of the product have been selected and optimized.The structure of final product has been identified by comparing the spectrum of the substrate and the product in order to further determine the product was 11α-hydroxy canrenone.Furthermore,residue after the fermentation has been used by means of comprehensive utilization.Main conclusions are as follows:（1）Canrenone was transformed by Rhizopus sp.UJS-0602 with the method of solid fermentation.Taking transformation ratio as an indicator,single factor tests were used to study the effect of medium components on transformation ratio.Medium components included the additive levels of carbon source,nitrogen source,inorganic salt,nutrient factor,water and substrate.Results showed that glucose,（NH₄）₂SO₄ and glycine were the optimal carbon source,nitrogen source and nutrient factor respectively.The addition of inorganic salts could inhibit the transformation process.Optimum additions of glucose,（NH₄）₂SO₄ and glycine were 1.0%,0.8%and 0.8%respectively,the best ratio of material to water was 1:1,the optimal amount of substrate was 10%（m/m）.Based on single factor tests,orthogonal experiments were designed to optimize the composition of solid fermentation transformation medium with addition of carbon source,nitrogen source,nutrient factor,material-to-water ratio and substrate content.The optimum conditions were as follows:1%of glucose,0.6%of ammonium sulfate,0.8%of glycine,material to water ratio was 1:1,substrate addition was 12%.Under the best condition,the transformation ratio could reach a level of 94.21%.（2）Through the analysis of solid fermentation and metabolism process of Rhizopus sp.UJS-0602,it was confirmed that the fermentation type of 11α-hydroxy canrenone belongs to part growth associated type.Therefore,by using the Logistic equation,Luedeking-Piret equation and material balance equation,cell growth kinetics model,product synthesis kinetics model and substrate consumption kinetics model were fitted and established.Results showed that the kinetics model of solid fermentation by Rhizopus sp.UJS-0602 fitted well.（3）The optimal conditions for product extraction were determined by orthogonalexperiments:the finial product was extracted by means of condensation reflux and the optimal extraction conditions were as follows:the concentration of ethanol was 80%（V/V）and its volume was 100 m L,pH=2,extraction temperature was 90℃,extraction hours were 5 h,extraction times were 4.Under this condition,the extraction rate can reach a level of 87.33%.The crude product was purified by silica gel column chromatography and recrystallized.The structure of product was identifiedbyhighperformanceliquidchromatography-massspectrometry（HPLC-MS）,Fourier transform infrared spectroscopy（FT-IR）and nuclear magnetic resonance（NMR）.Results showed that the product was 11α-hydroxy canrenone.After fermentation,the comprehensive utilization of matrix residue was preliminarily explored.The optimum conditions for fermenting rice wine by substrate residue were determined through orthogonal experiment:the fermentation temperature was 28℃,the fermentation hours was 60,the amount of distiller’s yeast was 2%.Under this condition,the sensory score of rice wine was 84 and the alcoholicity was 3.5%vol.