| Scientists are paying more and more attention to microbial polysaccharides since it is simply to separate and its origin is extensive.Marine environment can offer a tremendous diversity of microorganisms which can be a rich source of potentials polysaccharide. Those polysaccharide, which included Capsular polysaccharide(CPS) and Exopolysaccharides(EPS) exhibit potentials biological activities, such as immunological activity, antitumor activity, antibacterial activity, and antioxidant activity. In compared to CPS, the production of EPS is higher and easier to isolate. In order to provide a platform for further research on the EPS from marine bacteria, a reach on the screening and isolation of EPS marine bacteria was carried out in this study and the potential immunological activity of the EPS was also studied.Firstiy, then use the aniline blue LB solid culture medium on exopolysaccharide production bacteria at the first screening. Then using conventional fermentation medium Screening again, eventually won two strains producing extracellular polysaccharide production good strains what Respectively named QLc-04 and BHc-09.The sequences of 16 Sr DNA comparison shows that QLc-04 fungus belongs to the genus Bacillus(Bacillus sp.), while the BHc-09 bacterium belongs to the Vibrio genera(Vibrio sp.).Later, take Optimization toextracellular polysaccharide fermentation conditions of carbon source, nitrogen source and inorganicmetal ions on the two strains of bacteria. The result ishow that: the best fermentation condition of QLc-04 bacteria to 1L fermented liquid is adding 6g lactose, 4.5g yeast powder, 0.15 g Zn2SO4, BHc-09 bacteria optimum fermentation conditions of 1L fermented liquid is added to 6g starch, Yeast powder 4.5g, 0.05 g Mg2SO4. Under the optimized conditions, after 30 ℃, and 180 r/min after fermentation 36 h, QLc-04 bacterial extracellular polysaccharide(polysaccharide 04) yield can be up to 0.4759 mg/m L, and BHc-09 bacterial extracellular polysaccharide(polysaccharide 09) can be up to 0.4917 mg/m L.Secondly, 04 polysaccharide and 09 polysaccharide respectively carried on the preliminary analysis of physical and chemical properties.The results of infrared spectrum analysis show that two kinds of polysaccharide has a characteristic absorption peak, and extracellular polysaccharide of BHc- 09 bacteria have uronic acid absorption peak,which can determine 04 polysaccharide and 09 for different extracellular polysaccharide. Using high performance liquid chromatography(HPLC)to measure the molecular weight of 04 polysaccharide is 179.9KDa, and09 polysaccharide molecular about 184.5KDa. At last, by scanning electron microscopy(sem) as you can see, 04 polysaccharide is an irregular globular, and 09 polysaccharide present strip or flat..Thirdly, this study also investigated in vitro immunological activity of 04 polysaccharides and 09 polysaccharides by the mice T and B lymphocytes in vitro proliferation, and tested the Inhibitory effect about04 polysaccharide and 09 polysaccharides to Hela inhibition of tumour cells which in vitro cultured. As a result, 04 polysaccharide and 09 polysaccharide have promote T, B lymphocyte proliferation in mice and play the role of promoting the cell phagocytosis, 04 B Polysaccharide accelerate effect cells in vitro proliferation is better than its in vitro proliferation of T cells. The effect of proliferation on mice thymus T cell in polysaccharide concentration is 100μg/m L, reached maximum. And the effect of B cell proliferation was enhanced with the increase of polysaccharide concentrations; and the effect of 09 polysaccharide to T and B cells in vitro proliferation is enhanced with the increase of concentration. 04 polysaccharide and 09 polysaccharide in theconcentration of up to 50μg/m L, achieve the function of neutral red of phagocyte, after has a weakening trend, but is still higher than the control group. The experiments on Tumor cells in vitro inhibition have confirmed that 04 polysaccharide and 09 has anti-tumor cell proliferation activity,could significantly inhibit the proliferation of tumor cells in vitro, and enhancement was reduced with the increase of polysaccharide concentrations, the prior polysaccharide shows better results.Finally, the animal experiment to choose the commercial chicken of IBD poultry research techniques which were mature methods, and evaluate the effect about 04 polysaccharides and 09 polysaccharides and tapping the immune enhancing in animals. Results show that the experimental chicks what immune and injected the 04 polysaccharide and09 polysaccharide in the amount of antibodies to IBDV and IL-1 number significantly higher than that of immune no injection of polysaccharide of the experimental group, and the experimental group injected 04 polysaccharide and 09 only secretion of IL-1 level higher than the blank group. The result of protective immune experiment of IBDV, attacked by poison,the incidence of control group was 100%, mortality rate was12.5%, whereas the incidence was 30% in B87 immune group. With polysaccharide 04 and 09 group they were 20%, the result is significantly lower than B87 group.The immune organ index shows that bursa of fabricius and thymus in B87 immune group are shrink, but as usual in group B87 joint polysaccharide 04 and 09. It means polysaccharide04 and 09 can repair the damage of immune organs by immune caccine.It’s the same consequence with the key result of FCM and PR-PCR. So the final results showed that 04 polysaccharides and 09 polysaccharide can enhance the immune ability of chicks, and with the B87 vaccine strain combination can improve the chicks poison protection against infection vv IBDV.To sum up, in the optimization of culture conditions the institute screening QLc-04 bacteria(Bacillus sp.) and BHc-09 bacteria(Vibrio sp.)has the high capacity of extracellular polysaccharides. This two kinds of bacteria polysaccharides has good immunological activity. The results of this study was to further study the activity of extracellularpolysaccharide Marine bacteria and the bacteria diversity in the Beibu Gulf. Screening of the active polysaccharide immune related diseases also enhance theoretical and material basis of the related diseases of immune enhancement scheme in future. |
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