Screening And Identification Of Marine Actinomycetes Producing Antibacterial Subatances And Researches On The Active Substances

Due to the unique environmental qualities of the ocean,the marine microorganisms have special physiological mechanisms,genotypes,and great potential producing new bioactive substances.Marine actinomycetes were isolated from the Bohai intertidal zone sea mud sediments samples in Qinhuangdao Shandong Fort bridge for further research.The main research contents were as follows:248 strains marine actinomycetes were isolated from sea mud sediments samples.The active strains were screened by tube plate method.34 strains of marine actinomycetes generated antimicrobial activity substances against 3 tested bacteria,accounting for 13.7% of the total.According to E.coli,Candida albicans and Methicillin-resistant staphylococcus aureus,the strains producing active substances were 21,7 and 13,respectively and the rate were 8.5%,2.8% and 5%.The strain MA-21 was chosen for subsequent research.The strain MA-21 was classified by morphological characteristics,culture characteristics,physiological and biochemical experiments and 16 S r DNA sequence analysis.Phylogenetic tree was constructed by the Mega 5.0 software with the 16 S rDNA sequence of 1436 bp.The strain MA-21 may be identified as Streptomyces griseorubens with morphology,cultivation,physiological and biochemical experiments and 16 S rDNA sequence analysis.The strain MA-21 had salt tolerance to some extent.The beef extract peptone mediumwas selected as the fermentation medium.The optimized fermentation conditions of strains were 3d,initial pH 7.0,28?,fluid volume 28%,and inoculum size 9%.Antimicrobial activity of the substances produced by the strain MA-21 was not affected much after the strain was incubated 10 times once every month.These results showed that the strain MA-21 had good hereditary stability.The antibacterial substance was stable under neutral conditions and could not tolerate heat,which could be dissolved in distilled water,methanol,ethanol and ethyl acetate.The minimum inhibitory concentration of crude extract with antibacterial substances was 3.3?g/mL.1.567 g crude extract was obtained from 21 L fermentation broth of strain MA-21 through extraction.Abide by the antibacterial activity,8 antibacterial components were obtained by means of silica gel column chromatography,Sephadex LH-20 column chromatography and preparative thin-layer chromatography,which was named as MA-21-D1,MA-21-D3,MA-21-G1 A,MA-21-G1 B,MA-21-G2,MA-21-H2,MA-21-I1,MA-21-I2,respectively.