Studies On Screening And Active Components Of Mutant Strains Of Streptomyces Roseoflavus Strain Men-myco-93-63

Streptomyces roseoflavus strain Men-myco-93-63 is a biological agent isolated frompotato scab declined soil, which showed strong inhibition on many plant pathogens andstrong antagonistic effect. The objective of the research was to screen producing strainfrom 734 mutant strains, analyze its active components from solid medium by HPLC andTLC.At first, using Verticillium dahliae as a indicator, there are 10 producing mutant strainsof 176 which were stored in -20℃20% glycerol and 6 producing mutant strains from 558which were stored in slant medium. Mutant strain U90" was found as a high producingmutant strain by artifical passage for 5 and HPLC detecting. The optimal solvent wasscreened during ten impregnants, the crude extraction of distilled water was no-inhibition,the crude extraction of ethyl acetate and acetone has comparative activity, the inhibition ofthe crude extraction of ethyl acetate was optimal, so the active components can bedissolved in ester relative to water, other impregnants has slightly inhibition. Simultaneity,the crude extractions was abstersion by distilled water, then it was discovered that thewater part was no-inhibition to Verticillium dahliae. For the further research, it was foundthat the active components consisted in both of mycelium and the culture medium of themutant strain U90". Though the test of mix impregnants, the optimal impregnant was theformulation of ethyl acetate and acetone with a proportion of 3:1 (v/v).The test of the paper chromatography method to the mutant strain U90" showed thatthe inhibitory activity belonged to the six part antibiotic according to the paperchromatography method class curve. The active components of the mutant strain U90"were detected by HPLC, three kinds of activity components against to Verticillium dahliaewere found. The UV spectrum of these component showed that the maximum absorbwavelength (λmax) was 275.7nm, 360nm and 198.1nm respectively. The activity of275.7nm absorb wavelength was analyzed with HPLC, and three kinds of conditions werefound: (1) the mobile phase, 50%-80% (acetonitrile: water), 1%/min, sample 5μL, pH6. (2) the mobile phase, 50％-60％(acetonitrile: water), the flow rate, 0.25％/min,sample 5μL, pH6. (3) the mobile phase, 20％-50％-60％(acetonitrile: water), the flowrate, 2％/min, sample 3βL, pH 7. When the prepared samples of 275.7nm and 360nmabsorb wavelength were detected, the result showed that the activity of 275.7nm and360nm were deduced tautomers. The crude antibiotic was analyzed with TLC and theresults showed that it was the suitable developing solvent while the ratio of methyl alcoholand chloroform was 1:6 and 9 components could be separated on silica gel plate. The fivecomponent had the inhibition to Verticillium dahliae and the componentⅢwas the mostactive agent. The UV spectrum of componentⅢshowed that the maximum absorbwavelength (λmax)was 194.6 nm and 258.0nm.