Studies On Excellular Polysaccharide Produced By Lactic Acid Bacterium

Seventy-two Gram-positive strains producing acid on BCP plate were obtained by repeatedly lining in order to identify and isolate lactic acid bacterium from intestine of cock, the surface of potato and many kinds of yogurt. To assay weather polysaccharides existed in their culture medium after dialysis and centrifugation by the method of Phenol-H₂SO₄ and ethanol precipitation, five strains, Z222, Z581, X1121, H₆,D₁21, producing exopolysaccharide were further screened. Based on the results of the cultural characterization, morphological, physical and biochemical test, such as lactic acid determination, hydrogen peroxidase, hydrolyzing starch test and movement detection, we concluded that strain Z₂22, X₁121 and D₁21 belong to Enterococcus, Z₅81 belongs to Pediococcus, and H₆ belongs to Leuconostoc according to Bergy's bacterial identification manual and classified identification of lactic acid bacterium. A 16Sr DNA sequence analysis of strain Z₂22 producing higher EPS production among those five strains was performed using Blast in GenBank. It has 99% homology with Enterococcus durans, thus strain Z222 was denominated as Enterococcus durans Z222 and became start strain for studying exopolysaccharide in this paper.In this paper, we studied effect of carbon source in medium and cultural condition on biomass of Enterococcus durans Z222 and EPS production in order to increase EPS production by optimizing conditions for fermentation. EPS production and biomass were different in synthetic medium GA and MRS. It was suggested that MRS is moresuitable for growth and EPS-producing of Enterococcus durans Z₂22 At the same time MRSC was obtained as improved medium by eliminating beef extract in MRS recipe for easily isolation and purification of EPS in the next step. In addition, that EPS was observed in GA without any macromolecular material including polysaccharide confirmed the EPS-producing ability of Enterococcus durans Z₂22For Enterococcus durans Z₂22, there were more biomass and EPS production in medium at 37°C than at 25°C. Different types of sugar and different original sugar concentration will influence biomass and EPS production based on related experiments. As a result, glucose is a more suitable carbon source than the others and the optimum concentration of glucose in original medium is 4%. And EPS production will alter with cultural time increasing, it reached maximum at 27 hours and stable value at 50 hours. So EPS should be recovered in 27 hours when fermentation.The growth of Enterococcus durans Z222 was carried out with a sub-cultured inoculum added in MRSC. The rough exopolysaccharide was obtained after the culture was centrifuged, condensed, removed protein, ethanol precipitation, dialyzed and lyophilized. Firstly, it was purified through CM-cellulose and two elutes EPS-CI and EPS-CII containing EPS were collected when determined with Phenol-H₂SO₄ method. Secondly, EPS-CI containing less protein was purified through DEAE-Sephadex A-25 ion-exchange chromatograph to give single EPS-DI peak containing EPS. Lastly Lyophilized EPS-DI was purified using Sephadex G-100 gel chromatograph. After all above purifying steps, EPS-I produced by Enterococcus durans Z222 was obtained.The EPS eluted as a single peak from a high performance liquid chromatography (HPLC), indicating the homogeneity of EPS-I and free from low-molecular-weightpolysaccharides. The result of element analysis showed that there are 41.08% carbon and 7.23% hydrogen in EPS-I without nitrogen, phosphorus and sulfur. It also suggested that EPS-I does not comprise protein, nucleotide, PC>42', SO42", acetyl-saccharide and amino-saccharide. Optical rotation of EPS-I, [oc]d, was +10° at 20°C. The molecular weight of the EPS-I was determined as 42 KDa by the light scattering method.The result of sugar composition analysis indicated that the polysaccharide is composed of glucose and mannose in mole ratio of 1:4. The results of the methylation analysis showed that EPS-I is composed of pentasaccharide repeating units, which possesses Glcp(l, 2)Manp(l, 4)Manp(l, 3)Manp(l" and 2,6)Manp(l residues in mole ratio of 1:1:1:1:1. The structure of EPS-I was extensively investigated by one and two-dimensional NMR in D2O, DQF-COSY and TOCSY spectra were used to analyze self-spin systems of sugar residues. HMQC spectrum was recorded to assign carbon resonances. NOESY experiment was performed to detect intra- and inter-residue NOE correlations. HMBC spectrum was used for revealing the long range connectivities (3Jc-h) between proton and carbon across the glycosidic linkages. The glyco-linkages and sequence of EPS-I was established on the basis of inter-residue NOEs among the residues. The further evidences for the glycosidic linkages were obtained from the inter-residue long-range C-H correlation (?Jc-h) between the anomeric proton and the glycosilated carbon in the HMBC spectrum. Based on the sugar composition analysis, methylation analysis, MS and ID and 2D NMR data of EPS-I, the primary structureof the excellular polysaccharide produced by Enterococcus durans Z222 was shown to Ii tbe built up by the following pentasaccharide repeating units: \-2 )a-D.14t1 oc-D-Man^1 a -D- GlcThe preliminary immunological efficacy of EPS-I against Siso planted in mice was carried out. The test results showed that EPS-I can significantly inhibit DTH (delayed-type hypersensitivity) induced by SRBC(sheep red blood cell), increase the weight of spleen and enhance the abilities of the antibody formation of spleen cell .Neutral red was given to peritoneal macrophages stimulated by different concentration EPS-I in order to detect the phagocytic activity. NK activity, ability of lymphocytes proliferation and IL-2 production by murine splenocyte were measured by colorimetric method which is based on the ability of viable cells to cleave MTT. EPS-I obviously promoted both lymphocytes proliferation of murine splenocyte in a dose-dependant manner and lymphocytes proliferation induced by Con A. EPS-I significantly enhanced phagocytic ability of mouse peritoneal macrophages when compared with control group. The ability of killing Yac-1 was increased when spleen cells were stimulated by EPS-I and the maximum NK% was up to 62.41%±2.54%. The ability of producing IL-2 of spleen cells stimulated by EPS-I was significantly higher than the control group. These results showed that EPS-I has immunological function in vitro.