The Breeding Of Endophtic Bacillus Licheniformis SYt1 Producing Diosgenin And Its Optimization Of Fermentation Conditions

Diosgenin is an important basic raw material for the synthesis of steroidal drugs.It has extremely high medicinal value and economic benefits,and has great potential for development and broad application prospects.So,it has become a hot research topic all over the world.In this laboratory,endophtic Bacillus licheniformis SYt1 producing diosgenin was isolated from Dioscorea zingiberensis C.H.Wright.Its fermentation process and the extraction and separation process of diosgenin were explored.Moreover,adding the synergistic factors and metal ions that affect the fermentation process of SYt1 to the medium were studied.However,in the process of long-term cultivation,the strain degradation is very serious,and the yield of diosgenin is gradually reduced.Therefore,it is necessary to select strains and optimize the selected mutant strains.In this paper,the fermentation process of endophtic Bacillus licheniformis SYt1 producing diosgenin is studied,and diosgenin-producing endophyte SYt1 is cultivated many times by strain domestication technology.The diosgenin-producing endophyte SYt1 is mutagenized by ultraviolet and lithium chloride,and the selected mutants are optimized for fermentation conditions.Optimize the optimal culture conditions of the mutant strain,improve the production capacity and fermentation level of the strain,and finally increase the yield of diosgenin.Moreover,it provides evidence and relevant research bases for small-scale microbial fermentation to produce diosgenin.The main research contents and results are as follows:(1)Strain domesticationThe endophtic Bacillus licheniformis SYt1 producing diosgenin is continuously domesticated and cultured by adding powdered extract of different concentrations from rhizome of Dioscorea zingiberensis C.H.Wright to the culture medium.The results show that the yield of diosgenin of SYt1 which are continuously domesticated to the ninth generation reaches 79.8 mg/L,which is about one time higher than that of the unaltered strain.When the strain is domesticated each time in subsequent culture,the addition of extract added to the gradient is 0.03 g/L.(2)Mutation breedingUsing the ultraviolet mutagenesis treatment method,the starting strain SYt1 was directly mutagenized by bacterial suspension and single colony,and four mutant strains with high yield of diosgenin were screened as UV-25,UV-38,U3 and U4.The SYt1 mutant strain(U4)selected by ultraviolet mutagenesis was used as the starting strain,and it was subjected mutagenesis to lithium chloride-containing plate and bacterial suspension respectively,and combined with TLC,finally three high-yield mutant strains numbered L-12,UL-14 and UL-28 are screened out.The yield of diosgenin of three mutants reach135mg/L,148.3mg/L and 138.4mg/L,respectively,which is 120.2%,141.9% and 125.8%higher than that of the control group.It lays the foundation for the optimization of further fermentation conditions.(3)Optimization of fermentation conditionsThe single-factor experiment,orthogonal experimental design and quadratic general rotation center combination experimental design are used to optimize the fermentation conditions of the selected UL-14 mutant strain.The optimal result is: the optimum fermentation conditions for the UL-14 mutant strain are fermentation cycle 148 h,fermentation temperature 27 ℃,and shaking speed 205 rpm.Under these conditions,the yield of diosgenin predicted by the model is 191.9 mg/L.The yield of diosgenin verified by fermentation test is 189.7 mg/L,which is basically consistent with the yield of diosgenin predicted by the model,and is 27.8% higher than that before optimization.