Biocontrol Strains Hd-087 High-yield Strain And Fermentation Conditions

Agricultural antibiotic is the principle part of biological pesticide, many countriesin the world attach great importance to agricultural antibiotics increasingly for itsvirtues, such as high erect low pollution, function selectively, decomposed easily, safetyfrom cancer, abnormality and mutation and no social effects of pollution.The Actinomycetes HD-087 was a wild type strain, which could inhibit the growthof Gibberella sanbinetti, and many other phytopathogenic fungi. In this work strainHD-087 was breeded and screened by mutagenesis breeding and genome shuffling toimprove the yield of antibiotic, furthermore, the fermentation conditions of the mutantand its fermentation liquid poperties were studied.First, six mutant strains with high yield, named UV-13,UV-20,MW-4,MW-25,N-3,N-16,were obtained by ultraviolet irradiation, microwave and NaNO₂ mutagenesis.The diameter of inhibition were increased 15.54%,14%,16.92%,23.53%,23.35%,20% respectively. They were used as a parent library for protoplast fusing in genomeshuffling.The conditions for protoplast preparation were optimized, that was: myceliumcultured 96h; Gly 0.6%; lysosome 4mg/mL; 32℃; lysis 1h. After ultraviolet irradiation150s and heat treatments 60min respectively, the protoplast of the parent library werefused, and follow this method, shuffled two rounds again, the high yield strainHD-087-18 was obtained by genome shuffling. Its antibiotic production increased 77%,and it showed genetic stable.The culture characteristic of strain HD-087-18 were compared with strain HD-087,the result showed that there were some differents between them, such as the shape of themycelium, the soluble pigment and colony form on solid medium. Extractionexperiment result showed that this antibiotic was water-solubility. The result of stabilityexperiment showed that the fermention liquid lost active when dealed with 100℃for10min, and it was stable at pH 3-9 and exposed in light. It had no inhibitory effect atdilution of 8 and kept stable after conserved under 54℃for 15d. The optimal medium combination based on the single factor experiment andorthogonal experiment was obtained, that is: starch 1%, soy powder 0.8%, yeast powder0.12%, CaCO₃0.4%.the fermention condition were composed of as follows: initial pH 6,seed age2d, inoculation2.5% with 40 mL medium in 250mL flask, 180r/min,28℃,cultured 5d.