Fermentation Conditions, Structures, Chemical Modification And Antioxidant Activity Of Exopolysaccharides From Paenibacillus Polymyxa

As a slightly opened reservoir of special bio-resources, endophytes (microorganisms that reside in the internal tissues of living plants without causing any overt negative effects) have been demonstrated to be excellent producers of bioactive and structurally novel metabolites. A lot of novel bioactive products such as antibiotics, antiviral, anticancer, antidiabetic as well as antioxidant agents have been isolated from endophytes. Exopolysaccharides (EPS) produced by endophytes are also important metabolites, which play key roles in plant-endophyte interactions. They provide the endophytes additional protection against desiccation and serve as molecular signals during plant invasion. Despite the growing understanding on the role of EPS in the establishment of interactions, only few reports on the culture conditions, structural characterizations and biological activities of EPS from endophytes are available to date. Paenibacillus polymyxa (previously Bacillus polymyxa) EJS-3 was an endophytic bacterium strain isolated from the root tissue of Stemona japonica (Blume) Miquel, a traditional Chinese medicine. In this paper, the culture conditions, structural characterizations, chemical modification and antioxidant activities of EPS from P. polymyxa EJS-3 were investigated systematically. The main results are as follows:1. Effects of various culture conditions (initial pH, temperature, carbon, nitrogen and mineral sources) on EPS production were investigated firstly by single factor method. For EPS production, the preferable culture conditions were 24℃and pH 8 for 60 h with sucrose, yeast extract, KH2PO4 and CaCl2 as the carbon, nitrogen and mineral sources, respectively. Notably, sucrose concentration was the prominent factor, and the maximum yield of EPS (22.82 g/L) was obtained at a sucrose concentration of 160 g/L. Then, response surface methodology (RSM) was applied for the medium optimization of EPS production from P. polymyxa EJS-3. Experimental data of fractional factorial design (FFD) showed that the significant variables affecting EPS production were the levels of sucrose, yeast extract and CaCl2.From the regression analysis of the variables, sucrose and yeast extract were found to display positive effects on EPS production, whereas CaCl2 display a negative effect on EPS production. Based on FFD and steepest ascent experiments, three variables including sucrose, yeast extract and CaCl2 were further investigated for their optimum concentrations by central composite design (CCD). As results, the optimal medium compositions were determined as following (g/L):sucrose 188.2, yeast extract 25.8, K2HPO4 5 and CaCl20.34, with a corresponding yield of 35.26 g/L. The yield of EPS was about 1.55-fold compared with that (22.82 g/L) using the medium optimized by single factor method.2. The crude EPS extract was firstly obtained from the fermentation broth of P. polymyxa EJS-3 by properly dilution, centrifugation, ethanol precipitation and dialyzation. Then, a simple method for simultaneous decoloration and deproteinization of crude EPS extract using macroporous resin was developed through statistic and dynamic adsorption tests. As results, S-8 resin was found to have the highest decoloration and deproteinization ratios among various resins tested. Under the optimized statistic adsorption conditions (pH 6.0, 35℃and adsorption time of 70 min), the decoloration, deproteinization and polysaccharide recovery ratios of S-8 resin were 76.8%,78.9% and 69.0%, respectively. Further investigation into the dynamic adsorption experiments showed that under the optimized dynamic adsorption conditions (flow rate of 2 BV/h,160 ml of 2.5 mg/ml crude levan solution), the decoloration, deproteinization and polysaccharide recovery ratios of S-8 resin were 84.6%,91.7% and 81.3%, respectively. The decolorized and deproteinized EPS obtained from S-8 resin was further purified by chromatography of DEAE-52 and Sephadex G-100 to afford two polysaccharide fractions (EPS-1 and EPS-2). The recovery rates of EPS-1 and EPS-2 based on the amount of crude EPS were 53.6% and 4.8%, respectively. EPS-1 and EPS-2 both showed only one symmetrical peak on HPLC with the molecular weights estimated to be 1.22×106 and 8.69×105 Da, respectively. In addition, the results of indine, Fehling’s, CTAB,α-Naphthol and Anthrone reactions all indicated the existence of polysaccharides in the samples.3. The structural characterizations of EPS-1 and EPS-2 were investigated by various methods. UV-vis spectra showed no absorbance in the range of 200～400 nm. Monosaccharide compositional analysis showed that both EPS-1 and EPS-2 were composed of mannose, fructose and glucose in a molar ratio of 2.59:29.83:1 and 4.23:36.59:1, respectively. The FT-IR spectra of EPS-1 and EPS-2 were similar: the strong band at 3422 cm-1 was assigned to the hydroxyl stretching vibration of the polysaccharide. The band at 2936 cm-1 was due to C-H stretching vibration and the band at 1645 cm-1 was due to the bound water. The bands in the region of 1500 and 1200 cm-1 were assigned to C-H deformation vibration. The bands between 1128 and 1014 cm-1 corresponded to C-O-C and C-O-H stretching vibration. A characteristic absorption at 926 cm-1 was resulted from the stretching vibration of pyran ring. The obvious absorption at 811 cm-1 revealed the existence of mannose residue. And two peaks in the region of 3000 and 2800 cm-1 were observed, indicating the existence of fructose residue. The results of periodte oxidation-Smith degradation indicated that both EPS-1 and EPS-2 did not contain 1â†'3 or 1â†'4 linked glycosidic bond. Methylation and GC-MS analysis showed that EPS-1 and EPS-2 were both composed ofβ-(2â†'>6)-linked backbone of fructose residues, which substituted at C-1 by fructose and mannose residues. In addition,13C NMR spectroscopy confirmed that EPS-1 and EPS-2 were both levan type polysaccharides, which were consisted of (2->6)-linkedβ-D-Fruf residues with (2â†'1)-linkedβ-D-Fruf andβ-D-Manp branches. The branching degrees of EPS-1 and EPS-2 were 10% and 13%, respectively. However, helix-coil transition assay EPS-1 and EPS-2 did not exist helix conformation in their structures.4. The antioxidant activities of EPS from Paenibacillus polymyxa EJS-3 were evaluated by various methods in vitro and in vivo. In antioxidant assays in vitro, both crude EPS and its purified fractions (EPS-1 and EPS-2) were found to have moderate reducing power, DPPH radical scavenging activity, hydrogen peroxide scavenging activity, lipid peroxidation inhibition effect, and strong ferrous ion chelating activity, superoxide radical scavenging activity as well as hydroxyl radical scavenging activity. And the antioxidant activities in vitro of EPS decreased in the order of crude EPS> EPS-2> EPS-1. In antioxidant assays in vivo, mice were subcutaneously injected with D-galactose (D-Gal) for 6 weeks and administered EPS-1 via gavage simultaneously. As results, administration of EPS-1 significantly increased the thymus and spleen indices of D-Gal induced aging mice. Moreover, EPS-1 administration significantly enhanced the activities of antioxidant enzymes (SOD, GSH-Px and CAT) and total antioxidant capacity, whereas decreased the levels of malondialdehyde in both serums and livers of aging mice. These results suggested that EPS had potent antioxidant activities and could be explored as novel natural antioxidant.5. The different derivatives of EPS were prepared by means of acetylation, phosphorylation and benzoylation. As compared to native levan, the total sugar contents of all derivatives decreased after modification. In addition, the average molecular weights of all derivatives also decreased compared to native levan. The FT-IR and NMR spectra showed that all the derivatives were successfully prepared. The degree of acetylation and phosphorylation were determined to be 0.53 and 0.048, respectively. Antioxidant and antitumor activity assay in vitro showed that certain derivatives exhibited stronger activities than native EPS. In antioxidant activity in vitro assay, different derivaitves showed different activities. For superoxide radical, the scavenging activity decreased in the order of B-L> Ac-L> P-L> EPS, for hydroxyl radical, the scavenging activity decreased in the order of P-L> Ac-L> B-L≈EPS, and the reducing power decreased in the order of Ac-L> P-L> B-L> EPS. In addition, antitumor activity in vitro on BGC-823 decreased in the order of P-L> B-L> Ac-L> EPS.