Isolation, Identification And Biological Properties Of The Marine Actinomycetes Strain DL-4 Against Diverse Aquatic Pathogens

This thesis was aimed to isolate and purify actinomycetes from the sea mud samples, which came from Dalian, Shandong, Hainan and Shanxi province. Ninety strains marine-derived actinomycetes were isolated from the above samples. To screen strains producing antibacterial product, we use agar diffusion method and flat hole-trap method against Eschenchia coli, Staphlococcus aureus, Aeromonas punctata f.instestinalis, Vibrio anguillarum, Aeromonas hydrophila at primary screening phase, and use excavating-aperture bioassay test at examination phase. One high antibacterial strain DL-4 was choosen.16S rDNA gene sequence of the strain DL-4 was determined. The sequence was compared with 16SrDNA gene sequences in data of GenBank, which was similar with Streptomyces fradiae. The result indicated that the strain FA-F-5 had a near system development with Streptomyces rubrolavendulae by constructing a system evolution tree of 16S rDNA. Binding morphological characteristics, cultural charachteristics and physic-biochemical charachteristics, the strain DL-4 was belonged to Streptomyces fradiae.In order to increase yield of antibacterial substances, the compositions of medium and fermentation conditions were optimized. It showed the optimum medium composition: starch 10g/L, peptone5g/L. Optimization of fermentation showed that initial pH was 8.0, inoculation quantity was 5%, the optimum temperature was 37℃and fermentation time was 96h.The experiment showed that the strongest potency of fermentation liquid was 75.469ug/ml equivalenting to Gentamycin and The MIC and MBC of fermentation liquid was 9.43μg/mL and 18.87μg/mL equivalenting to E coli respectively.This study has conduct an investigation in the stability of secondary metabolites, it showed that the the strain DL-4 has a strong hereditary stability. The fermentive liquor was dealed with different temperature, pH, light and storage time. The results of the stability test indicated that active substance of fermentation liquid of the strain DL-4 was high stable for hyperthermy, pH, light and storage. The results of paper chromatography demonstrate the bioactive substance may be a large polar water-soluble substancesThe purification procedure of the fatsoluble antibiotic DL-4 was elucidated by research on fermentation liquid pretreatment, rotary evaporator concentrating, organic solvent extracting, D201 absorbent resin, polyamide column chromatography, sephadex LH-20 and reversed phase column isolating. the crude crystal of antibiotic from fermentation liquid of the strain DL-4 was obtained.