Identification of a Antibiotics-producing Strain and Optimization of Its Fermentation Conditions
|Keywords||antifungal-antibiotics 16S rRNA single-factor methodology response surfacemethod (RSM) fermentation kinetietics|
In recent years, deep fungal infections increase gradually, which threaten sufferers’health and lives. The discovery and development of efficient, safe, low-toxic and broad-spectrum antifungal drugs become even more important. A strain with high inhibitory effect on Candida albicans ATCC64548was screened from East China Sea Soil. Research work on strain identification, fermentation process optimization, antifungal antibiotic structure characterization and antimicrobial efficacy was conducted in this thesis. The strain was identified as Bacillus subtilis based on the taxonomic studies including phenotype determination,16S rRNA gene sequencing and BLAST analysis in Genbank database. It was preserved in China General Microbiological Culture Collection Center, and the identification number was B. subtilis CGMCC4140.After testing the physiological and biochemical characteristics of the strain is found, actinomycetes, and then determined by16S rRNA sequence and Genbank database found with the Streptomyces sp.102P41-la homology is99%, according to the characteristics of the strain of Streptomyces named (Streptomyces) ZJU088. The strain is preserved in China General microbiological collection management center, No. CGMCC No.4889.Using single factor and response surface method to optimize the strain in shake flask fermentation process, and get the best fermentation condition:initial pH value7,8%inoculation quantity (v/v), speed of200r/min, liquid volume of40mL/250mL, temperature28℃, time7d of fermentation; fermentation conditions in shake flask fermentation of antibiotics. The single factor optimization, the optimal culture conditions for:age24h, speed of200r/min, temperature28℃, initial pH value7, medium volume in flask with40mL/250mL,8%inoculation quantity (v/v); fermentation base after optimization (g/L):soluble starch40,8.25soybean meal, yeast powder13.75, K2HPO42, NaCl2, MgSO41, CaCO30.5, CuSO40.02, FeSO40.02, MnSO40.02. Shake flask fermentation experiments were averaged three times in the experimental optimal fermentation conditions, the measured antibiotic titer was5926.43U/mL,20.57times higher than the initial value288.09U/mL.Through the Plackett-Burman experiment, the steepest optimization of culture medium was climbing experiments and Box-Behnken experiments after (g/L):2.54soybean meal, starch26.8, yeast extract3.96, K2HPO42, NaCl2, MgSO41, CaCO30.5, CuSO40.02, FeSO40.02, MnS040.02, initial pH value7. The fermentation liquid was15977.25U/mL, results are in good agreement with the model values, antibiotic titer of2.69times after single factor optimization.In5L fermentor batch fermentation experiments on strain. Using single factor method to optimize the fermentation conditions of fermentation tank to obtain better:stirring speed300r/min, ventilation volume1.5vvm, initial pH value7, fermentation temperature28℃,8%inoculation quantity (v/v), volume60%(v/v), the maximum5D titer was18542.04U/mL. The strains in5L fermentor’s batch fermentation kinetics process, and established the dynamic equation of cell growth, product production and substrate consumption. The material has a good inhibitory effect on human pathogenic fungus Candida albicans, broad antibacterial spectrum, has good prospects for development of new drugs.