Screening And Identification Of Marine Microorganisms For Producing Natural Bioactive Substances

The marine microorganisms have become an important source for developing new bioactive substances because of their unique environment and diversity of species. This project was done to study on screening and identifying of marine strains producing bioactive substances, purification of bioactive substances and analysis of their physicochemical properties. The main results were as follows:1.200marine bacteria strains were isolated from sea cucumber intestine and were screened for antimicrobial activity strains with the methods of ager block screening and filter disc. The results showed that13of the screened strains possessed antimicrobial activity, accounting for6.5%of total isolated strains. These13marine bacteria strains could inhibit Gram positive and negative tested strains at the same time.2. The three bacteria strains, HS-A38, A8-5and A8-20, were selected for species identification. Based on morphological and16S rDNA sequence analysis, the strains of HS-A38, A8-5, and A8-20were identified as Bacillus subtilis, B. vallismortis, and B. amyloliquefaciens, respectively. Eventually, the strain HS-A38with a higher antimicrobial activity was selected for further study.3. Two bioactive substances (1and2) were purified from B. subtilis HS-A38with the methods of fractional sedimentation with ammonium sulfate, CM-52ion-exchange chromatography and Sephadex G-75column. The SDS-PAGE analysis indicated that the relative molecular weight of these two bioactive substances were41kDa and28kDa, respectively. Substance1could only inhibit Gram positive strains, whereas substance2could inhibit both the Gram positive and negative bacteria.4. To gain physical and chemical properties of bioactive substance2, the effects of different temperatures, pH and organic solvents on inhibition activity were studied using the purified substance2. The results showed that it was thermal stable, remaining about80%of initial activity after heating at121℃for30min. It was also stable to acid and alkali, while the inhibition activity could still be kept at the range of pH2-12. The substance2was tolerant to common organic solvents, especially in methanol its inhibition activity was not affected. The minimal inhibitory concentration (MIC) of substance2was detected and the result showed that its MIC was64μg/ml.