Identification And Fermentation Of A Bacillus Subtilis (Strain321122) From The East China Sea And Characterization Of Its Secondary Metabolites

The dissertation describes the isolation of a marine Bacillus subtilis (strain 321122) from the East China Sea, as well as the genomics identification of the strain based on its 16S rDNA, the optimization of fermentation conditions, the separation and purification of its metabolites, and the screening for its biological activities.The 28 sediment and 11 seawater samples were collected on the differrent spots more than 30 nautical miles away from the seacoast of the East China Sea, from which 4 sediment and 4 seawater samples were randomly selected. Using different media, 1041 colonies were obtained by the infinite dilution on plate followed by 219 colonies with antimitotic activity screened out using the Pyricularia oryzae inhibition model. The proportion of bioactive colonies was above 20%, suggesting that the East China Sea shared with the rich resources of medicinal microorganisms.The strain 321122 isolated from the middle-level seawater in the East China Sea, can inhibit the growth of Pyriularia oryzae, as well as induce strong morphological abnormalities of its hypha. The strain exhibited better stability of heritance and growth. The bioactive secondary metabolites in its fermentation broth were stable under the heating and alkaline conditions. By the analysis of the 16S rDNA sequence of the strain 321122, it had been primarily identified to be a Bacillus subtilis. The study of morphology, physiological and biochemical characteristics showed it was a slight halophilic strain, and can produce amylase, fluidify glutin. and showed the positive reaction in the litmus milk test, suggesting some of its metabolic pathway of the strain 321122 had probably changed under the sea environment.Fermentation conditions were reasonably optimized in the research, including fermentation temperature, fermentation time, inoculation dose, the makeup and pH of culture medium and so on. The bioactive secondary metabolites in the fermentation broth were isolated by several chromatographic techniques, such as TLC, Sephadex LH-20, and HPLC. More than 20 compounds were obtained. Three of them showed antagonistic effects on the plant pathogenic fungi. The chemical structures of compounds had been elucidated by the physicochemical properties, MS, ¹H-NMR, DEPT ¹³C-NMR. The 11 compounds were indentified to be: cyclo (L-Ala-L-Leu),cyclo (L-Ala-L-Ile), cyclo (L-Pro-D-Leu), cyclo (Pro-He), cyclo (L-Val-L-Leu), cyclo (Tyr-Leu), L-Trp, cyclo (Val-Val), uridine, cyclo (Trp-Ala), and cyclo (Phe-Gly). The elucidation of compounds had been going on. The data of NMR, MS suggested the another three bioactive compounds probably were cyclopeptides.The detailed structures of 11 compounds were described as following:uridinecyclo (Trp-Ala)cyclo (Phe-Gly)...