| Marine microorganisms have a wide range of antimicrobial strains, which are the important potential sources of antimicrobial substances. As a new field of the research of marine microbial resources and because of the uniqueness and extremeness of the marine environment, many biologically active substances the marine microorganisms excreted are relatively newly to us. Therefore, it is a huge potential to screen novel, highly active and low poisonous antimicrobial substances. In order to find an highly active and safe antimicrobial substance using as food preservtives and feed antibiotics in the future, these works were done:(1) to isolate, screen and identify marine microorganisms with novel and highly active antimicrobial substnaces from the marine microbial resources of Lianyungang;(2) to study antimicrobial activity, physics and chemistry nature, composition and the antibacterial mechanism of antimicrobial substances from strain GB-2. The results were described as follows:The 667 marine microorganisms were isolated from the marine samples collected from the sea area of Lianyungang. 246 strains of them(about 36.88% of the total isolates)had antimicrobial activity, consisting of 74 actinomycetes, 156 bacteria and 16 fungi and the isolation rate of antimicrobial active strains from actinomycetes was highest. After duplicate sieves, 11 strains were observed inhibiting Penicillium expansion and Saccharomyces cerevisia obviously, majority of these 11 strains exhibited different extend antagonism against Gram-positive bacteria and Gram-negative bacteria. The antimicrobial activity of these 11 stains demonstrated the potential application in future. Finally, the strain GB-2 of actinomycete from marine sediment collected in the inteltidal zone was determined for the next step of experimental strain because of its strongest and broad-spectrum antibacterial and antifungal activities.Then, the strain GB-2 was identified with classical and molecular methods. According to the morphological and cultural characteristics, the physiological and biochemical properties and chemical components of cell wall, it was indicated that strain GB-2 was belong to Streptomyces. In the 16SrRNA sepuence analysis, the sequence fragment of 1395bp 16S ribosomal RNA was amplified from total DNA of strain GB-2 by PCR, then the sequence analysis was down by the application of BLAST on the websites of NCBI and the phylogenetic tree was drawn by Bioedit 7.0~T and Treedrawing~T. The results showed that GB-2 shared 98% 16SrRNA homology with Streptomyces fradiae(AB184776). After compared with the characteristics of Streptomyces fradiae reported, strain GB-2 was identified as a varation species of Streptomyces fradiae.Influence of different water of medium on the production of antimicrobial substances was examined. And the results showed the high production of antimicrobial substances was significantly associated with aritificial seawater and the color of the fermentation broth was closely related to the salt concentration of the medium. Strain GB-2 displayed significant inhibitory effects not only on Gram-positive and Gram-negative food-borne pathogens and spoilage bacteria but also on the drug-resistant bacteria and the spoilage fungi in diets and plant pathogenic fungi. The results of stability studies of the fermentation broth of Streptomyces GB-2 showed that it was very stable at pH1 and pH12 under 121℃and had little change after ultraviolet treatment. The potential use of substance produced by strain GB-2 in bio-control, food and medical application was predicted.The purification of the antimicrobial substances produced strain GB-2 was studied. The antimicrobial substances were diffluent in the water, dissoluble in the methanol and alcohol, and insoluble in other organic solvents. The results of ultrafiltration of fermented liquid showed that, there were two components of antibacterial activity and antifugal activity respectively, and the molecular weight of antibacterial substance was≤1KDa. The antibacterial substance was proved to be a neutral and water-soluble according to paper chromatogram analysis. After purification on the Sephadex LH-20 and Sephadex G-15, the ultrafiltration fraction with molecular weight≤1KDa exhibited three series of antibacterial activity, among which peak a displayed the strongest activity to inhibit Bacillus cereus and E.coli. The HPLC analysis of peak a showed two series of peaks and the second peak was the one who had the antibacterial activity and might be an aminoglycoside antibiotic according to its property.Sensibility and inhibition effect of antibacterial substance produced by strain GB-2 to Bacillus cereus and E.coli was studied. The results showed that Bacillus cereus and E.coli were very sensitive to antibacterial substance and their MIC were 0.663μg/mL and 0.904μg/ml respectively. Antibacterial substance could strongly inhibit their growth-breeding. Studies of growth curve of thalline, membrane permeability showed that antibacterial substance added into the broth could result in the decrease of bacterial concentration and the increase of ultraviolet absorption, which indicated antibacterial substance could kill or lysis Bacillus cereus and E.coli. SEM and TEM results demonstrated that antibacterial substance could destroy integrity of cell membrance, which led to increasing of membrane permeability and leaking out of macromolecule in the cell, and then thallus cells break. This phenomenon only could be seen in thallus cells which were in the stage of growing. And there was little change of the resting cells and spores after antibacterial substance added.
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