Screening And Breeding Of Streptomyces YM And Optimizing Of Fermentation Conditions

In this thesis, the breeding and optimization of fermentation conditions of Streptomyces YM were studied to improve the yield of antibiotics, and provides conditions for furtner study and industrial production. First of all, Select breeding of high-yield antibiotics strain Streptomyces YM-3. In this paper, used sensitive Bacillus subtilis as idicator bacteria and oxford-cup method was used to detect antibiotic titer. Purified Streptomyces YM are numbered as YM-1. Phsical mutagenesis with UV, primary screening of agar block method and rescreening of flask fermentation were used to develop the original Streptomyces YM and high-yield antibacterial strain YM-2 was obtained. Then, by nitrous acid mutagenesis, primary screening and secondary screening, obtained YM-3 from YM-2. The inhibition zone of fermentation supernatant for YM-3 was 23.6mm. Compared with YM-1, YM-3 has a higher ability of producing antibiotics which is raised by 60.5%. It was viewed that the mutant strain YM-3 has good genetic stability after several passages.Based on mutant strain YM-3, media composition and fermentation conditions were optimizated to enhance the strain YM-3 production of antibacterial substances. Single factor experiments of carbon and nitrogen sources, inorganic salts etc were operated to analysis the effect on the YM-3 production of antibiotics, the results as follows: the composition as glucose 4.0%, peptone 1.5%, K₂HPO₄ 25.0×10-3%, NaCl 0.15% was conducive to the production of antibiotics. Three main factors, glucose, peptone and NaCl were determined by Plackett-Burman experiment. Then center combination experiment was operated and the optimal composition of three components was determined: glucose 4.68%, peptone 1.25%, NaCl 0.12%. Through the analysis of the single factor experiments and orthogonal experiments, the best fermentation conditions for YM-3 were determined as follows: the best age of inoculation 90h, inoculum size 12mL, the medium volume for flask 60mL/250mL, shaker speed 160r/min, shaking culture temperature 30℃, the initial medium pH 7.2. The diameter of bacteriostatic circle to 37.6mm.At the end of the thesis, the physicochemical properties of the antibacterial activity substances in fermentation liquid were preliminarily studied. It was indicated that antibiotics were stable under between pH8.0-12.0, while pH <5.0, the stability was low; in 90℃water bath for 30min, the activity decreased by10.1%, 60min decreased 32.9%, and under 100℃water bath for only 15min completely inactivated; antibacterial substances in the UV were stabile. The active substance was consist with two components by pH paper chromatography and Doskochilova system integration chromatography: one component may belong to polar, non-alkaline water-soluble, gold antimycin antibiotics, and the other may be acidic, strongly polar antibiotics.