Study On Identification And Secondary Metabolites Of Anti-microbial Activities Actinomycete JMC06001

The extraction of pharmaceuticals and their precursors form marine microorganisms is presently one of the foreland fields of the research and development of new medicines.The marine bioactive actinomycete strain JMC06001was isolated from a saline mud sample collected from Naozhou Island in the South China Sea. In this study, the taxonomic identification of the strain JMC06001was investigated, and the antimicrobial spectrum and secondary metabolites of the fermentation broth as well as the mycelium were observed, and then the fermentation medium was optimized by using the response surface methodology.The results showed that strain JMC06001grown well on most media tested, with white aeriel hyphae and pale yellow to pale brown substance hyphae. Yellow diffusible pigments were produced on oatmeal agar (ISP3), potato extract agar and glucose/asparagines agar, and pale brown to deep brown diffusible pigments were produced on yeast extract/malt extract agar (ISP2), glycerol/asparagine agar (ISP5), peptone-yeast ext-Fe agar(ISP6)and nutrient agar. Growth occurred at4℃～40℃and pH6.0～9.0, with optimum growth at28℃and pH7.0. The tolerant range of NaCl was0～1.5mol/L, with best growth occurring in media containing0.2～0.5mol/L NaCl. The combination of morphology, physiological and biochemical characteristics, chemotaxonomic data and16S rRNA gene sequence analysis supported the view that strain JMC06001belong to the known species of the genus Steptomyces, Steptomyces peucetius.The antimicrobial spectrum of the fermentation broth as well as the mycelium of strain JMC06001were investigated with cylinder plate method using nine indicators (Sarcina lutea, Bacillus subtilis, Escherichia coli, Asperillus niger, Enterobacter aerogenes, Proteus vulgaris, Candida albicans, Penicillium sp., P. aeruginosa), and the stability of the bioactive materials was observed under several conditions (various temperatures, pH values, ultraviolet light). The antimicrobial substances were isolated by silica gel column and thin layer chromatography (TLC). The results suggested that strian JMC06001exhibited obvious broad antimicrobial spectrum, including Gram-negative and Gram-positive bacteria and fungi. The results of the stability tests showed that the most suitable effective temperature of antibacterial substances was40℃, and the most suitable pH value was about7.0, and that the antibacterial substances had fairly broad pH effective range, fairly good thermostability and relatively strong anti-ultraviolet ability. According to the silica gel column chromatography separation and TLC detection, the antimicrobial substances showed multitude components.The fermentation medium was optimized by using the response surface methodology, and the results showed that the optimum composition of the fermentation medium was as follows:glucose12g, maltose7g, peptone9g, soybean meal14g, NaCl37g, CaCO31g, broth A of compound salts20mL, broth B of compound salts1mL, and water1000mL, and the optimum original pH value of the fermentation medium was7.0. The diameter of inhibition zone against staphylococcus aureus produced by S. peucetius JMC06001strain in the optimal medium reach up to31.50mm, which was18.8%higher than that cultured with the original medium optimized by orthogonal experimental design.