【作者】 徐春兰；

【导师】 汪以真；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2008， 博士

【摘要】 本研究通过对从肉灵芝上分离、纯化的产多糖菌种进行了耐硒驯化,得到了产多糖耐硒菌种Z0206,并对其进行了鉴定；通过深层富硒发酵制备了富硒蛋白多糖；进一步利用现代分离纯化技术,得到富硒多糖及其主要组分,并对其结构进行了分析；通过建立环磷酰胺所致的免疫抑制小鼠模型和H202诱导的RAW264.7细胞氧化损伤模型,研究了富硒多糖的免疫调节功能和抗氧化活性及其作用机制。其主要研究结果如下：1.产多糖耐硒菌种的驯化和鉴定通过平板划线法和浓度梯度法对产多糖菌种进行了耐硒驯化,得到产多糖耐硒菌种Z0206,并运用形态学、生理生化特征和16S rDNA基因序列分析相结合的方法对其进行了鉴定。鉴定结果表明,产多糖耐硒菌种Z0206属于E.cloacae。将测序获得的16S rDNA基因序列登录GenBank,获得登录号为EU647232,并与其相似性较高的序列构建了系统发育树。2. E.cloacaeZ0206富硒多糖的制备通过发酵培养基和发酵条件的优化得到E.cloacae Z0206富硒蛋白多糖的发酵条件：采用PDA加富培养基,接种量2%,培养温度30℃,pH值7.0,转速200 rpm,培养第6h加硒,加硒浓度20μg/mL,发酵时间48h,起始葡萄糖浓度2%,第24h流加2%葡萄糖；利用优化的发酵条件进行深层富硒发酵,发酵液通过蒸发浓缩、离心、乙醇沉淀等制备得到了富硒蛋白多糖(SPPS):产量为26.7g/L,含硒量为65.7mg/kg；进一步经Sevag结合酶法脱蛋白、H2O2脱色、透析等处理纯化得到富硒多糖(SPS)。3. E.cloacae Z0206富硒多糖的结构分析将纯化得到的富硒多糖SPS经离子交换柱层析(DEAE-52)和分离型凝胶柱层析(Sephadex G-100)分离纯化得到两个组分,其中组分1(SPS-1)占76%,采用液相色谱、红外光谱、核磁共振等对SPS-1的结构进行初步分析。结果表明,SPS-1是由葡萄糖、半乳糖和甘露糖组成的均一性杂多糖,摩尔比为8.530：0.061：0.706,分子量为23.9KD,分散系数为1.3；存在吡喃糖环构型和α、β糖甙键构型,并具有三股螺旋结构,具有较长的侧链和较多的分枝,SPS-1平均粒径为47.49nm,表面电荷为11.9mV,脱水温度85.98℃,气化温度为256.57℃。4.E.cloacae Z0206富硒多糖的免疫调节功能研究本试验通过建立坏磷酰胺所致的ICR小鼠免疫低下模型,研究了细菌E.cloacae Z0206富硒多糖对免疫低下小鼠的免疫调节作用及其作用机制。结果表明,与空白对照组相比,环磷酰胺(50mg/kg.B.W)对免疫器官重、部分细胞和体液免疫指标具有显著的抑制作用(p<0.05),即建模成功：与环磷酰胺作用组相比,200 mg/kg和400 mg/kg的富硒蛋白多糖(SPPS),400 mg/kg的富硒多糖(SPS)和多糖(PS)均显著提高了脾脏和胸腺指数(p<0.05),缓解环磷酰胺引起的脾脏和胸腺萎缩,显著提高了ConA和LPS诱导的脾脏T、B淋巴细胞增殖(p<0.05)和细胞因子(IL-2、TNF-α)的mRNA水平(p<0.05)；与环磷酰胺作用组相比,400 mg/kg的SPPS和SPS预防性灌胃后,血清和细胞培养液中IL-2和TNF-α水平显著升高(p<0.05),400 mg/kg的PS和200 mg/kg的SPPS提高了血清中TNF-α水平、培养液中IL-2和TNF-α的水平(p<0.05)；400mg/kg的SPPS、SPS和PS预防组的CD4+、CD4+/CD8+和NK细胞活性均显著高于环磷酰胺组(p<0.05),CD8+细胞活性显著低于环磷酰胺组(p<0.05),其中400mg/kg的SPPS组使其恢复到正常水平；200 mg/kg的SPPS,400 mg/kg的SPPS、SPS和PS显著提高了免疫低下小鼠血清溶血素水平(p<0.05)和抗体形成细胞数(p<0.05),其中经400mg/kg的SPPS灌胃预防均可使血清溶血素半数溶血值和抗体形成细胞数目恢复到正常水平。从其作用效果来看：富硒蛋白多糖>富硒多糖>多糖。提示：富硒多糖可通过调节细胞免疫和体液免疫,而发挥的免疫调节作用；硒、蛋白和多糖三组分在发挥免疫调节作用方面可能具有较强的协同作用。5. E.cloacae Z0206富硒多糖的抗氧化活性研究本试验通过建立H2O2诱导小鼠巨噬细胞系RAW264.7细胞氧化损伤模型,研究了细菌E.cloacae Z0206富硒多糖对RAW264.7细胞氧化损伤的保护作用及其作用机制。结果表明,与对照组相比,H2O2显著降低RAW264.7细胞的存活率(p<0.05)：与H2O2单独作用组相比,200μg/ml和400μg/ml的富硒蛋白多糖(SPPS),400μg/ml的富硒多糖(SPS)和多糖(PS)显著提高了SOD和GSH-Px活性,抑制ROS、MDA的产生和LDH的释放,维持细胞线粒体膜的完整性,抑制跨膜电位的耗散、Caspase-3的活化和DNA的降解(p<0.05)：与H2O2单独处理组相比,不同浓度的(200μg/ml和400μg/ml)的SPPS和SPS,400μg/ml的PS显著提高了Bcl-2基因的mRNA水平(p<0.05),降低了Bax基因的mRNA表达水平(p<0.05)；与H2O2单独处理组相比,SPPS (200μg/ml)和SPS(400μg/ml)显著增加了Bcl-2基因的蛋白表达水平和Bcl-2/Bax(p<0.05),降低了Bax基因的蛋白表达水平,PS (400μg/ml)显著提高了Bcl-2基因的蛋白表达水平(p<0.05)；与H2O2作用组相比,200μg/ml和400μg/ml的SPPS、SPS和PS均显著提高了血红素氧化酶(HO-1)的mRNA和蛋白表达水平。从抗氧化效果来看：富硒蛋白多糖>富硒多糖>多糖；对于SPPS,浓度过高,其抗氧化活性显著下降,而SPS和PS则表现出明显的量效关系。提示：富硒蛋白多糖和富硒多糖可能通过清除活性氧,提高抗氧化酶活性、诱导HO-1的表达以及抑制凋亡信号通路而对H2O2引起的RAW264.7细胞的氧化损伤实施保护作用,是一种有效的抗氧化剂；硒、蛋白和多糖三组分可能在发挥抗氧化功能方面具有较强的协同作用。更多还原

【Abstract】 The Selenium-tolerant strain Z0206 Rosenth producing a large quantity of viscous exopolysaccharide isolated from Daphniphyllum oldhami (Hemsl.) belong to E.cloacae by phylogenetic analysis. We prepared crude Se-enriched exopolysaccharide through fermentation with E.cloacae Z0206 in enriched-selenium fermentation culture. Se-enriched exopolysaccharide was isolated and purified from crude Se-enriched exopolysaccharide; we further analyzed the structure of Se-enriched exopolysaccharide; On base of those, we investigated its immunomodulatory function on immunosuppressed mice and the protective effect against H2O2-induced Raw 264.7 murine macrophages oxidative damage and the possible mechanism of action. Results are as follows:1. Screening and Classification for Se-tolerant Strain Producing ExopolysaccharidesThe strain Z0206 producing exopolysaccharides isolated from Daphniphyllum oldhami (Hemsl.) Rosenth was identified as Selenium-tolerant strain through screening and domesticated by increasing the Se-concentration. It belongs to E.cloacae by identification with traditional physiological and biochemical methods, together with the 16S rDNA sequence homology and phylogenetic analysis. We obtained the accession number (EU647232) of E.cloacae Z0206 by submitting its 16S rDNA sequence to GenBank.2. Preparation of Enriched-Selenium ExpolysaccharidesThe experiment was aimed to prepared enriched-selenium exopolysaccharides. We first optimized culture medium and culture conditions for selenium expolysaccharide production. And the optimal culture medium was obtained, which contains tomato extract 200mL/L, glucose 20g/L, yeast extract 2g/L. Meanwhile the optimized culture conditions are:culture volume 100ml/500mL flash, inoculation amount 2%, initial pH 7.0, shake culture 48h,200rpm,30℃. Se-added time 6th h, Se-concentration 20μg/ml. The crude enriched-Selenium exopolysaccharides was prepared through fermentation with E.cloacae Z0206 in enriched-selenium fermentation culture medium. Yield of crude exopolysaccharide was 26.7 g/L, Se-enriched amount was 65.7 mg/kg. The enriched-selenium exopolysaccharide was isolated and purified by Sevag and enzymes method, frozen thaw method and with activated carbon, yield of purified exopolysaccharide was 17.5 g/L, Se-enriched amount was 35.7 mg/kg.3. Structural Analysis of Enriched-Selenium ExpolysaccharideThe obtained purified exopolysaccharide was loaded on a column of anion-exchange (DEAE-52) and gel-permeation chromatography (Sephadex G-100), concentrated, dialyzed and lyophilized. Finally obtained compound was named SPS-1. The structural characteristics was analysed by HMR, FTIR and HPLC methods. The results showed that SPS-1 was homogeneity and made up of Glucose, galactose and mannose with the molarity rate of 8.530:0.061:0.706. Its molecular weight is about 2.39kD. SPS-1 is a neutral heteropolysaccharide, and SPS-1 containedα,β-type glycosidic linkages, and may contain a-D glucopyranose, mammopyranoside. The molecule of SPS-1 had long dypass chain and more branch chains. The results of X-ray and DSC indicated that the state structure of the exopolysaccharides was noncrystal Elements. The particle size and zeta potential were 47.49nm and 11.9mV respectively. The dehydration temperature and gasification temperature were 85.98℃and 256.57℃respectively.4. Study on the Immune Function of Enriched-Selenium ExpolysaccharidesThe experiment was to investigate the immunomodulatory activity of selenium exopolysaccharide produced by E.cloacae Z0206 in enriched-selenium fermentation culture by establish of immunosuppressed mice model. Results indicated that CP showed suppressive effects on immune organs weight and cellularity and other parameters of humoral immunity (p<0.05) compared to control. SPPS, PPS and SPS treatment (400 mg/kg B.W.) significantly (p<0.05) can relieve immunodepression and significantly (p<0.05) increase the spleen and thymus index, decrease CD8+ cells and stimulate the proliferation of T and B lymphocytes compared to CP-treated animals alone. SPPS and PPS treatment (400 mg/kg B.W.) significantly increased (p<0.05) IL-2, TNF-αlevels in serum and spleencyte culture. In quantitative hemolysis of sheep red blood cell (SRBC) (QHS) assay and PFC response in CP-treated animals, SPPS, PPS and SPS treatment (400 mg/kg B.W.) showed protection in CP-treated animals. Additionally, treatment with SPPS (400 mg/kg B.W.) was more effective than other treatment groups. SPPS treatment itself produced no toxicity. The administration of SPPS to CP-exposed animals resulted in improved humoral and cellular responses. SPPS may be developed into a new kind of immunomudulation agent.5. Study on the Antioxidative Activity of Enriched-Selenium ExpolysaccharidesThe experiment was conducted to investigate the protective effect of selenium exopolysaccharide produced by E. cloacae Z0206 against H2O2-induced Raw 264.7 murine macrophages oxidative damage and the possible mechanism of action. Results showed that H2O2 addition reduced the viability of cells by MTT analysis compared to control (p<0.05). Addition of SPPS (200μg/ml and 400μg/ml), and SPS (400μg/ml), PS (400μg/ml) increased (p<0.05) superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, reduced (p<0.05) the production of intracellular peroxide (MDA), DNA ladders, reactive oxygen species(ROS) and the release percentage of Lactate Dehydrogenase (LDH), and protected cells from H2O2-induced a decrease in the mitochondrial membrane potential, activation of Caspase-3 (p<0.05) compared to H2O2-treated cells only. SPPS, SPS and PS (200μg/ml and 400μg/ml) upregulated Bax, downregulated Bcl-2 mRNA and proteins levels compared to H2O2-treated cells only. Additionally, treatment with SPPS, SPS, and PS (200μg/ml and 400μg/ml) significantly increased HO-1 mRNA and protein levels compared to H2O2-treated cells only (p<0.05). Moreover, treatment with SPPS (200μg/ml) was more effective than other treatment groups. These data suggested that induction of HO-1 protein, increase of antioxidant enzyme activities, reduction of intracellular ROS production and mitochondria dysfunction with blocking apoptotic events may participate in the protective mechanism of enriched-selenium polysaccharide on H2O2-induced apoptosis.更多还原