Screening Of Marine Symbiotic Microorganisms And Study Of Its Antimicrobial Active Products

Because of the unique environment of marine, the microorganisms and metabolites has become an important source for developing new antibacterial, antiviral, antitumor agents and preparing living bacteria agents. This project focus on isolation and identification of the marine symbiotic microorganisms with antibacterial activities from wild sea cucumber; Subsequently, the fermentation media and culture conditions of the marine strain HS-A38 were optimized, and the four active compounds are analysed from the crude extract of HS-A38. The main results were as follows:1. Using the agar block method and two sensitive bacteria as indicating strains,26 strains with antibacterial activities were isolated from 135 marine strains, accounting for 19.2% in total. After several rounds of screening,16 strains were identified with strong antibacterial activities. Among them,4.44% antagonistic Vibrio parahaemolyticus strains, 2.22% antagonistic Micrococcus lysodeikticus strains; 5.18% antagonistic the two of them, selected a plant named HS-A38 for further study.2. The physiological, biochemical and chemical properties of active crude ingredients produced by the strain HS-A38 were investigated in details. By using oxford cup approach to measure antibacterial activity, the results showed that the active substances has high temperature resistant, even at 121 ℃ they are still active. The active substances are also resistant to acid and alkali, active between the range of pH 2-12. The active substances could keep active in common organic solvents, especially in methanol.3. In the combination of thin layer chromatography and ninhydrin color printing with bioautography techniques, four substance were extracted from the precipitation of fermenting broth of the strains HS-A38. It is preliminarily deduced that these four compounds are all Straight-chain lipopeptide, and among them, compound 1 and 2 could inhibit Vibrio parahaemolyticus.4. By single factor experiment and response surface experiment design, The concentration of media ingredients for fermenting HS-A38 is as glucose 8.49 g/L, soybean powder 12.0 g/L, urea 0.625 g/L, yeast extract 4.0 g/L, K2HPO4 4.0 g/L, ZnSO4 0.053 g/L. The colony-forming units per milliliter (cfu/mL) were improved from 7.22 × 108 to 1.64 × 109 after medium optimization, and the yield of cfu/mL is increased by more than 2 times.