Isolation,Purification And Identification Of Active Ingredient Produced By Marine Actinomycete P10-16

Unique growth environment of marine actinomycetes were recognized as an important source of natural products. Isolation, purification and structure identification provide important way to find new drugs. In our studies, an antifungal marine actinomycete P10-16 was isolated from marine microorganisms, which was collected by the First Institute of Oceanography, SOA. This paper not only dealt with the strain species characterization and optimization of fermentation, but also investigated on purification and structure identification of active secondary metabolites. The study found that metabolites were potential agricultural antibiotics. The results were as follow.1. P10-16 was isolated from marine microorganisms36 marine strains'fermentation liquor exhibited to antibacterial activties to Botrytis cinerea, Sphaerotheca fuligenea and Rhizoctonia solani, in 54 strains. Marine bacteria, marine fungi and marine actinomycete biological activity strain ratio respectively were 68.2%,62.5% and 70.8%. There were 4 strains more than 80% ofantibacterial activity. P10-16 is the only strain exhibited to three antibacterial activties.2. Identification of strain P10-16Determined by morphological observation, culture characteristics, physiological and biochemical characteristics and 16s rDNA sequence analysis, the P10-16 was classified with Streptomyces, and sequence homology of Streptomyces viridobrunneu was 99%.3. The initial optimization fermentation and active identification of P10-16Secondary metabolites of marine actinomycetes are closely connected with fermentation time and the salt concentration of medium. The polarity, thermal stability, acid stability, alkali stability and solubility in organic solvent of active ingredients are also the important parameters in the process of extraction. The initial optimization fermentation experiment, determined the fermentation time was 7 days, and the salt concentration in fermentation medium was 0.2%. The initialactive identification results show that the active ingredient in stable to heat and acid, best extraction effect in n-butanol, dissolve in all kinds of organic solvent. Theconditions provide important data support to following extraction work.4. Exploration of Secondary fermentation optimizationAfter 7 days fermentation (28 ?),we got two colors of fermented liquid. One was yellow acid fermented liquid (pH=6), another one was dark red with aromatic smell alkaline fermented liquid (pH=8). The former was quite effectivein vivo bioactivity, while the latter was not. Both fermented liquid have strong activity of rice blast fungus in vitro, but the latter activity was not stable. The latter accounted for over 90% of fermented liquid production, was not a good news for research. In order to got more effective metabolites, we pour yellow fermenting liquid into the fermentation medium. By observation and agricultural antibiotic activity test, determined the secondary fermentation time (3 days).5. Secondary metabolites of P10-16Rice blast bacteria as bioactivity-guided, as well as by centrifugal, solvent extraction, silica gel column chromatography, thin-layer chromatography, analysis HPLC and preparation HPLC, seven compounds are obtained in fermented supernatant (25L). By mass spectrometry and NMR methods analyzed structures. Five compounds are iturin A-2, iturin A-3, Iturin A-4, iturin A-6 and iturin A-7. One compound is DEHP, another compound is daidzein.6. Bacteriostatic activity of secondary metabolitesUnder the concentration of 500?g/ml, P10-16 fermented liquid crude extracting shown control effects (98%) on Botrytis cinereain vivo and shown no phytotoxicity. Under the concentration of 125?g/mL, extracting shown control effects (80%) on Botrytis cinereain vivo. Under the concentration of 500?g/mL, GJF-6-5-5 shown control effects (50%)on Botrytis cinereain vivo. Under the concentration of 1000?g/mL, GJF-6-2 shown control effects (70%) on Botrytis cinereain vivo. All natural products shown antimicrobial activity on rice blast and cucumber powdery mildewin vitro. GJF-6-5-5 (Po) MIC= 15.625 ?g/mL, GJF-6-2, GJF-6-3q, GJF-6-3h, GJF-6-4q and GJF-6-4h (Po) MIC= 62.5?g/mL, GJF-1-7 (Po) MIC=125?g/mL. All natural products shown antimicrobial activity on Botrytis cinereain vitro(MIC= 500?g/mL), shown no significant effects of other diseases (Hm, Gz and Cl).