Isolation and identification of an extracellular polysaccharide high-producing marine Bacillus and the study on its anti-tumor activity

【Objective】 The aim of the present study was to determine the biological activities, such as antitumor activity of the extracellular polysaccharide(EPS) from marine bacteria in Beibu Bay, Guangxi, and to provide reference basis for the research, development and application of the biological activity of the EPS secreted by bacteriafrom Beibu Bay.【Method】 The shallow soil samples of Maowei Sea mangrove in Qinzhou, Guangxi were collected. The marine bacteria with high production of EPS were screened by LB-aniline blue culture medium and phenol-sulfuric acid method, and the strains were identified by 16S rRNA sequence. The molecular weight and structure of the EPS were determined by high performance gel permeation chromatography and infrared spectroscopy. Finally, the cytotoxicity of the EPS to Vero cells and the inhibitory effect on the proliferation of HeLa cells were determined by thiazolyl blue(MTT) method.【Result】 The results showed that a strain named QLc-04, with EPS production of 0.2917 mg/mL was screened. The colony of QLc-04 formed on LB-aniline blue solid medium was white, opaque, slightly raised in the center and rough on the surface. QLc04 was identified to be Gram-positive bacilli. QLc-04 showed 99.45% to 99.82% 16S rRNA sequence similarity to Bacillus, with the highest similarity to B. thuringiensis BT62(CP044978) (99.82%), then 99.81% to B. cereus ATCC 14579^T (AE016877) and B. mycoides DSM 2048^T(ACMU01000002). In the phylogenetic tree based on 16S rRNA sequence similarity, QLc-04 and Bacillus were clustered into the same branch, so that QLc-04 was identified to be Bacillus. The molecular weight of QLc-04-EPS was about 179.9 kD and the EPS belonged to a polysaccharide with pyranoid structure. The QLc-04-EPS had no significant cytotoxicity to Vero cells. In the concentration between 10-300 μg/mL, the more QLc-04-EPS was used, the stronger proliferation ability of Vero cell was. However, when the concentration of QLc-04-EPS was more than 50 μg/mL, the inhibitory effect on HeLa cell proliferation was significantly enhanced(P<0.05).【Conclusion】 The production of EPS secreted by marine Bacillus QLc-04 is 0.2917 mg/mL after 36 h fermentation. QLc-04-EPSis a typical pyran polysaccharide with a molecular weight of 179.9 kD, and shows a significantly inhibitory effect on the proliferation of HeLa cells.