Shandong Chain Fungal Mutation Breeding And Optimization Of Fermentation Conditions

Streptomyces strain 24# was isolated from our lab to produce secondary metabolites which could strongly antibiotic to 30 plant pathogenic fungi, such as Helminthosporium sorokinum, fusarium graminearum Schw., Valsa mali, etc. The secondary metabolites produced by the strain had malformation effect on these plant pathogenic fungi. The 16S rDNA sequence analysis and the traditional identification methods was used to identify the strain. Based on test results, this strain was identified as Streptomyces thermocarbonxydus var shandaensis.To culture antibiotic overproducing strains, the study include using different physical and chemical factors to induce Streptomyces thermocarbonxydus var shandaensis to mutation. After original strain were mutagensised by UV combined LiCl,⁶⁰Co, DES, 9 high-yield strains were obtained. Using Oxford Plate Assay method, the antibiotic effect measured by the diameter of antibiotic circle of mutational strains includingUV—19, Co—38, DES—21 were respectively 1.875, 1.68, 1.52 times than the original strain. By analyzing and comparing imitative effects of different imitative factors,it was concluded that the imitative effects of UV combined LiCl were better than other factors which the study used.The mutagenic experiments defined four mutants as parent strains,then by the means of protoplast fusion technique, the fusion experiments obtained high-yield strains: P4-2, P4-3, P4-4, P4-5, P4-40. By Minimum Inhibitory Concentration experiment, it found that the antibiotic production of these high-yield strains were about 8 times than the original strain.After excerpted the optima formula of fermentation medium, the optimal medium and cultural conditions were ascertained.The optimal medium was composed of maize powder10%, wheat bran broth 8%% NaCl 0.05%, K₂HPO₄ 0.1%, MgSO₄0.15% or 0.2%. And the optimal cultural conditions involved:the optimum period was 7d, the optimum temperature was 30℃, the initial pH of the medium was 6.5—8.0. And the shaking-flask experiments were done in 500ml,flasks containing 120ml medium with 10 % inoculation quantity orbital shaking at 150r/min.