【作者】 刘俊；

【导师】 曾晓雄；

【作者基本信息】 南京农业大学 ， 生物工程， 2010， 博士

【摘要】 作为一种特殊的生物资源,植物内生菌存活于健康植物组织内部,而不使宿主植物表现出明显感染症状。近年来的研究表明,植物内生菌可产生具有抗肿瘤、抗菌、抗炎症、抗病毒、免疫调节、降血糖和抗氧化等多种生物活性的次生代谢产物。由植物内生菌产生的胞外多糖在内生菌与植物的相互作用方面起着重要的作用：胞外多糖可以保护内生菌免受环境脱水,并在内生菌入侵植物时起着信号分子的功能。尽管目前对胞外多糖在内生菌与植物相互作用方面的认识在不断深入,但关于植物内生菌胞外多糖发酵条件、结构和生物活性的报道还很少。多粘类芽孢杆菌(Paenibacillus polymyxa EJS-3)是从药用植物蔓生百部的根部组织中分离得到的植物内生细菌,本文系统研究了P.polymyxa EJS-3胞外多糖的发酵条件、结构特征、化学修饰及其抗氧化活性,主要研究结果如下：1.首先,利用单因素方法研究了影响P.polymyxa EJS-3胞外多糖的产量的诸多因素(如初始pH值、温度、碳源、氮源、无机盐等),结果表明,胞外多糖合成的最佳初始pH值和温度分别为8.0和24℃；产胞外多糖的最适碳源、氮源和无机盐分别是蔗糖、酵母膏、KH2PO4和CaCl2；值得注意的是,蔗糖浓度是影响胞外多糖产量的最重要因素,当蔗糖浓度为160 g/L时,胞外多糖产量最高(22.82 g/L)。随后,利用响应面法对培养基组成进行优化：部分因子设计的实验结果表明,蔗糖、酵母膏和CaCl2是影响胞外多糖产量的显著因子；其中,蔗糖和酵母膏对胞外多糖产量具有正效应,而CaCl2对胞外多糖产量具有负效应；基于部分因子设计和最陡爬坡实验的结果,进行了中心组合设计以得到最优的培养基组成,结果表明,最佳培养基组合为蔗糖188.2g/L、酵母膏25.8 g/L、K2HPO45 g/L和CaCl2 0.34 g/L,相应的最高胞外多糖产量为35.26g/L,是利用单因素优化结果的1..55倍。2.通过发酵液稀释、离心、乙醇沉淀和透析等获得胞外多糖的粗提物,然后利用大孔树脂通过静态和动态吸附实验对P.polymyxa EJS-3胞外多糖乙醇初提物进行脱色脱蛋白研究。静态吸附实验的结果表明,在六种大孔树脂中,S-8树脂的脱色率和蛋白去除率最高；在最佳的静态吸附条件下(pH值6.0、温度35℃、吸附时间70 min),S-8树脂对粗多糖溶液的脱色率、蛋白去除率和多糖保留率分别为76.8%、78.9%和69.8%。而动态吸附实验的结果表明,在最佳的动态吸附实验条件下(流速2 BV/h、160ml的2.5 mg/ml的粗多糖溶液),S-8树脂对粗多糖溶液的脱色率、蛋白去除率和多糖保留率分别为84.6%、91.7%和81.3%。经S-8树脂脱色脱蛋白后的粗多糖样品依次使用DEAE-52和Sephadex G-100进行分级纯化后主要得到两个组分(EPS-1和EPS-2),根据Sephadex G-100纯化后的多糖干重和DEAE-Cellulose 52柱层析前的粗多糖干重的比例可知,EPS-1和EPS-2的得率分别为53.6%和4.8%。在高效液相色谱上,EPS-1和EPS-2均显示单一和对称的色谱峰,分子量分别为1.22×106和8.69×105Da。此外,12反应、CTAB反应、α-萘酚反应和蒽酮反应均证明样品中多糖的存在。3.通过多种方法对EPS-1和EPS-2的结构特征进行了测定。对多糖的紫外扫描结果表明EPS-1和EPS-2在200-400 nm的波长范围内无最大吸收。单糖组成分析的结果表明EPS-1和EPS-2均是由甘露糖、果糖和葡萄糖组成的,其单糖组成(甘露糖：果糖：葡萄糖)的摩尔比分别为2.59：29.83：1和4.23：36.59：1。红外光谱分析EPS-1和EPS-2的红外光谱图基本相似：3422 cm-1处的峰是多糖O-H键的伸缩振动峰；2936 cm-1处的峰是多糖C-H键的伸缩振动峰,同时,可以看到在2936 cm-1处有两个吸收峰,进一步说明了果糖基的存在；在1500和1200 cm-1之间存在的峰对应于C-H键的变角振动峰；而1128和1014 cm-1之间存在的峰对应多糖骨架C-O-C和C-O-H的伸缩振动峰,同时,在1100和1010 cm-1之间仅有两个吸收峰,进一步证明了EPS-1和EPS-2主要是由呋喃糖组成的；在926 cm-1处的吸收峰是由果糖基的吡喃环的伸缩振动引起的；811 cm-1处的峰是甘露糖残基的特征吸收峰。高碘酸氧化-Smith降解的结果说明EPS-1和EPS-2中的葡萄糖和甘露糖基不含有1→3和1→4连接的糖苷键。甲基化反应和气质联用分析的结果说明,EPS-1和EPS-2均是由(2→6)-连接的β-D-果糖基构成的主链,以及位于支链末端的果糖基和甘露糖基,而少量的葡萄糖基则一般位于主链的末端。而13C核磁共振的结果表明EPS-1和EPS-2均是由(2→6)-连接的β-D-果糖基的主链,和位于果糖基1位的甘露糖和葡萄糖基的支链构成的典型的果聚糖结构,且EPS-1和EPS-2的分支度分别为10%和13%。而刚果红反应的结果表明,两种多糖都不含有三螺旋结构。4.外抗氧化实验说明,胞外多糖具有较弱的还原能力和铁离子还原力；中等的H202清除能力、DPPH自由基清除能力和脂质体过氧化抑制效果；较强的超氧自由基清除能力、羟基自由基清除能力和金属离子的螯合能力。多糖样品的体外抗氧化活性的顺序为粗多糖>EPS-2>EPS-1.体内抗氧化实验说明,给由D-半乳糖诱导的衰老小鼠灌胃胞外多糖可以有效地提高小鼠的脾指数和肝脏指数,从而增强小鼠的免疫功能。此外,给衰老小鼠灌胃胞外多糖还能有效地提高体内的抗氧化酶和TAOC的活性,而降低体内的脂质体过氧化的水平。因而,由P. polymyxa EJS-3产生的胞外多糖具有较强的体外和体内抗氧化活性,可以作为天然的抗氧化剂用于食品和医药工业。5.通过乙酰化、磷酸化和苯甲酰化制备了不同的胞外多糖的衍生物。与未衍生化的胞外多糖相比,衍生物的总糖含量均降低；此外,衍生物的分子量也均降低。红外光谱和核磁共振谱均证明了胞外多糖的衍生物已被成功制备；且胞外多糖的乙酰化和磷酸化取代度分别为0.53和0.048。体外实验表明,通过化学修饰可显著提高胞外多糖的抗氧化和抗肿瘤活性,且修饰物的体外活性呈现浓度正相关。不同取代物的活性也不相同,其中超氧自由基清除活性的顺序为B-L>Ac-L>P-L>胞外多糖,羟基自由的清除活性顺序为P-L>Ac-L>B-L≈胞外多糖,还原力大小顺序为Ac-L>P-L> B-L>胞外多糖。此外,胞外多糖及其衍生物对肿瘤细胞BGC-823的抑制率顺序为P-L>B-L>Ac-L>胞外多糖。更多还原

【Abstract】 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 1→3 or 1→4 linked glycosidic bond. Methylation and GC-MS analysis showed that EPS-1 and EPS-2 were both composed ofβ-(2→>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 (2→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.更多还原