Screening And Identification Of Antibacterial Strains Isolated From Antarctic And Study On The Antibiotic Substances

Antarctica, due to its specific climate and geographical feature, is characterized by low temperature, acute seasonal changes in light and higher UV intensity. Antarctica is considered as one of the extreme environments on the earth for its extraordinary low temperature. Antarctica has many features and advantages which makes it a treasure house of resources for the discovering of new species and the precursor compounds for new drug.In this study, Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas solanacearum, Pseudomonas aeruginosa and Bacillus thuringiensis were used as indicator strains respectively, four antibacterial strains, named as 97, Z11, Z18 and Z19 were screened out from 580 bacteria strains isolated from Antarctic by method of agar diffusion. The growth curves, antibacterial curves and phylogenetic analysis of 4 antimicrobial strains were also studied. The results showed that all of the four strains reached the log phase when they were cultured 24 h, strain 97 was in the log phase when it was cultured after 48 h, and the remaining three strains Z11, Z18 and Z19, were in the stationary phase when they were cultured after 60 h. They had the highest antibacterial activity when it was cultured after 84 h, 96 h, 72 h and 72 h, respectively. Phylogenetic analysis indicated that these four strains belonged to genera of Rheinheimera, Psychrobacter, Pseudomonas and Psychrobacter, respectively.Single-factor experiment and orthogonal test were adopted to optimize the fermentation medium and conditions of Antarctic psychrotrophic bacterium Rheinheimera sp. 97, which exhibited stronger antibacterial activity. The results showed that the optimum fermentation medium contained tryptone 3.0 g/L, ammonium sulfate 1.0 g/L, starch 2.0 g/L, NaCl 15 g/L. The optimized fermentation conditions were as following: the initial pH of medium was 8.0, fermentation temperature was 10℃, liquid volume in erlenmeyer flask was 30% (V/V) and inoculation size was 1% (V/V). Under these optimal conditions, the antibacterial activity of Rheinheimera sp. 97 was increased by 22.2%.Studies on acid-base stability, thermal stability and polarity of bioactive substances produced by Rheinheimera sp. 97 were also conducted. The results showed that the antibiotic activity substances had a good stability when the pH at 5-10, when the fermentation culture was kept in 70℃for 30 min, the relative antibiotic activity was reduced to 50%. It showed the highest antibacterial activity when the concentrated solution was extracted by the ethyl acetate. Silica gel column chromatography and HPLC technique were used for the purification of the antibiotic activity substances, and 4 kinds of compounds with remarkable antibacterial activity were isolated. Identification of the 4 compounds by modern techniques such as mass spectrum (MS) nucleic magnetic resonance (NMR) indicated the 3 compounds were benzeneacetic acid,β-Sitosterol, 1-Formyl-2,5-dimethoxy-6,9,10-trimethylanthracene and, respectively. The structural elucidation of the fourth compound T-4-6-3 is being studied.