Screening Of Antimicrobial Strains Of Mangrove In Qinzhou Bay Of Guangxi And Isolation And Identification Of Active Compounds

In the "sea ——land “interface intertidal mangrove wetland ecosystem due to the natural environment characteristics of the salinization of soil, swamp, organic matter content and high strength of radiation, which has obvious superiority in the research and development of bio active substances. Mangrove ecosystem is not only the treasure trove of microorganisms found new species and genetic resources, but also to discover new drug lead compounds active substance such as treasure of resources.In this study, we used Staphylococcus aureu as indicator strain, 12 antibacterial strains, named 1-2-3, 1-3-1, 2-2, 2-3, 3-4-1, 4-4-1, 5-2, 5-8, 6-4-2, 6-9, 7-3-3 and 8-3-21 were screened out from 476 bacteria strains which were isolated from mangrove of Guang Xi by the method of agar diffusion. Among them, 5-8 the inhibitory intensity maximum. The Bacteriostatic spectrum, growth curves, antibacterial curves and phylogenetic analysis of strain 5-8 were determined.. The results showed that strain 5-8metabolites had strong inhibitory activity against Fusarium oxysporum, Phytophthora copsici, Verticillium dahlia Thanatephorus cucumeris and Escherichia coli.. Strain 5-8 in cultured 12 h entered the logarithmic phase, into the stable growth period in 60 H after culture; antimicrobial activity reaches a maximum value at 120 h. Development of molecular identification and system analysis shows that, the mangroves the strains 5-8isolated from the mangroves belonged to genus of Bacteria, Proteobacteria,Gammaproteobacteria and ErwiniaOn the basis of optimum fermentation condition from single-factor experiment, we used Plackett-Burrnan design to screen the significant factors influencing the antibacterial activity including fermentation temperature, salinity and peptone. Path of steepest ascent was undertaken to approach the optimal region of significant factors,Box-Behnken design and response surface analysis were adopted to further investigate mutual interaction between variables. Experimental results showed that the optimized fermentation conditions were as following, the fermentation temperature was 26.004 ℃,salinity was 0.9648%, Peptone concentration was 0.2696%, Glucose concentration was0.6%,the starting pH of medium was 8.5,liquid volume in erlenmeyer flask was30%(V/V)and inoculation amount was 1%(V/V).Under the optimized fermentation conditions, the antibacterial activity of Ewinia 5-8 was increased by 8.95%.The stability and polarity of strain Erwinia.sp.5-8 fermentation liquid active material suggested that, under the conditions of the storage time(0-8 weeks),temperature(30-60 ℃), pH(6.0-10.0), fermentation liquid activity was maintained at above 90%, the antimicrobial substances produced by ethyl acetate extract was the best.Using active tracking detection method, throng ethyl acetate extraction, silica gel column chromatography, thin layer chromatography, high performance liquid Chromatography to obtained two pure compounds named T2-8-4, T4-11-2 which has obvious antibacterial activity. Based on the spectral data(1H NMR,13 C NMR,COSY,HMBC,HSQC), the structure of T2-8-4 was dibutyl phthalate, the structure of T4-11-2 was flazine.