【作者】 靳明亮；

【导师】 汪以真；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2011， 博士

【摘要】 本研究在Enterobacter cloacae Z0206多糖(EPS)发酵条件优化及发酵动力学研究的基础上,采用氯磺酸-吡啶法制备得到Z0206多糖的硫酸酯化衍生物,并对其结构进行了初步分析；在体外化学试验研究硫酸酯化多糖对超氧阴离子自由基、羟自由基以及1,1-二苯基苦基苯肼(1,1-Diphenyl-2-picrylhydrazyl, DPPH)自由基清除作用的基础上,通过建立H2O2诱导的RAW264.7小鼠巨噬细胞氧化损伤模型,研究了硫酸酯化多糖对细胞氧化损伤的保护作用,并采用蛋白质组学技术研究了硫酸酯化多糖发挥抗氧化功能的可能分子机制。主要研究结果如下：1.Z0206多糖发酵条件优化及发酵动力学研究采用单因素试验及正交试验,对E. cloacae Z0206发酵产糖的培养条件及培养基组成进行了筛选和优化。在此基础上,根据Box-Benhnken中心组合实验设计原理,以显著影响多糖产量的麦芽糖、蛋白胨和牛肉膏浓度为自变量、以多糖产量为响应值,采用三因素三水平的响应面分析方法,通过二次多项式回归方程求解以及响应面和等值线图分析,确定了E. cloacae Z0206发酵产糖的最佳培养基组成。E. cloacae Z0206产糖的最佳发酵条件为：初始pH：7.5；发酵温度：32℃；接种量：4%；K2HPO4·3H2O:0.3%; MgSO4·7H2O:0.15%；KNO3：0.05%；麦芽糖：3.72%；蛋白胨：0.51%；牛肉浸膏：0.56%。验证试验表明,在最佳发酵条件下,Z0206多糖的发酵产量达12.89 g/L。在E. cloacae Z0206发酵产糖的动力学特征研究方面,采用Logistic方程描述了Z0206多糖发酵过程中菌体生长的动力学过程；同时采用1-苯基-3-甲基-5-吡唑啉酮(PMP)柱前衍生化高效液相色谱法分析了不同发酵时间Z0206多糖的单糖组成变化,确定半乳糖和岩藻糖是发酵产生的特征性单糖。2.Z0206多糖硫酸酯化衍生物的制备及结构分析本研究在深层发酵、Sevag结合酶法脱蛋白、H2O2脱色、透析等处理纯化得到Z0206多糖的基础上,选用氯磺酸-吡啶法,采用L9(33)正交试验,研究了氯磺酸：吡啶、反应温度和反应时间对Z0206多糖硫酸酯化反应的影响,并制备得到不同取代度的硫酸酯化多糖。结果表明,影响Z0206多糖硫酸基团取代度的因素主次顺序为：氯磺酸：吡啶>反应时间>反应温度。随着氯磺酸比例的提高以及反应温度从50℃增加到70℃,硫酸酯化多糖的取代度也在提高。但当温度提高到90℃时,取代度反而下降。此外,当反应时间从1h延长到3h时,硫酸酯化多糖的取代度降低。同时,采用紫外光谱、傅里叶红外光谱、凝胶渗透色谱、核磁共振及粒度分布等对Z0206多糖硫酸酯化衍生物的结构进行了初步分析,结果表明：硫酸酯化多糖糖链连接上了硫酸基团,硫酸酯化取代部分发生在C-6位；酯化产物SEPS-2的重均分子量为1.954×104Da,平均粒径为62.69 nm；SEPS-4的重均分子量为18.221×104Da,平均粒径为94.47 nm。3.Z0206多糖硫酸酯化衍生物的抗氧化功能研究本试验首先采用体外化学试验研究了Z0206多糖EPS及其9种硫酸酯化多糖在体外对超氧阴离子自由基、羟自由基以及DPPH自由基的清除作用。研究结果表明,硫酸酯化修饰显著提高了多糖对体外超氧阴离子自由基及羟自由基的清除能力。同时,通过建立H2O2诱导的RAW264.7小鼠巨噬细胞氧化损伤模型,研究了硫酸酯化多糖SEPS-2和SEPS-4对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,0.658 mM的H2O2作用12h显著降低了细胞的相对存活率(p<0.05)；与H2O2单独作用组相比,0.5和2.0 gg/mL的EPS、SEPS-2、SEPS-4预处理,显著提高了氧化损伤细胞的相对存活率(p<0.05)；0.5μg/mL的SEPS-4预处理,显著提高了氧化损伤细胞中超氧化物歧化酶(SOD)及过氧化氢酶(CAT)的活性(p<0.05),并显著降低了氧化损伤细胞胞浆中Caspase-3的活性(p<0.05)；2.0μg/mL的SEPS-4预处理,使氧化损伤细胞的线粒体膜电位显著提高(p<0.05),细胞氧化损伤及DNA降解明显减轻。从细胞抗氧化的总体效果来看,取代度为0.17的硫酸酯化多糖SEPS-4的抗氧化活性优于多糖EPS及取代度为0.60的硫酸酯化多糖SEPS-2。结果提示,糖链上的硫酸基团可以促进多糖的抗氧化功能,适宜的取代度是硫酸酯化多糖发挥高抗氧化功能的前提。硫酸酯化多糖可以通过保护氧化损伤细胞的形态结构、提高抗氧化物酶的活性及线粒体膜电位、抑制Caspase-3的活化及DNA降解,对H2O2诱导的RAW264.7细胞氧化损伤实施保护作用,是一种有效的抗氧化剂。4.Z0206多糖硫酸酯化衍生物抗氧化差异蛋白质组学研究本研究通过双向电泳技术分析了H2O2诱导氧化损伤RAW264.7细胞及硫酸酯化多糖SEPS-4预处理实施氧化损伤保护的RAW264.7细胞中蛋白质组学之间的表达差异,并采用质谱分析对其中的差异蛋白点进行了鉴定。结果表明,两个试验组细胞蛋白点的等电点及分子量分布广泛,差异大于2倍的差异蛋白质点有132个。选择其中差异较大的30个蛋白进行MALDI-TOF/TOF质谱分析,将获得的肽质量指纹图谱(PMF)经Mascot数据库检索分析后,鉴定了其中26个差异蛋白点。结果表明,与H2O2单独处理相比,硫酸酯化多糖SEPS-4预处理使Peroxiredoxin-2、Eef1D、Eef1G、Phosphatidylethanolamine-binding protein等蛋白表达下调,使Heat-shock protein hsp84、Rho GDP-dissociation inhibitor 2、T complex protein 1、Alpha-tubulin isotype M-alpha-2、Enol等蛋白表达上调。这些蛋白涉及细胞的免疫和应激、骨架与结构、蛋白质代谢和修饰、能量代谢、信号传导等,可能在硫酸酯化多糖SEPS-4的保护机制中发挥重要作用。更多还原

【Abstract】 In this research, sulfated derivatives of exopolysaccharide (EPS) produced by Enterobacter cloacae Z0206 were prepared by chlorosulfonic acid-pyridine (CSA-Pyr) method and their chemical structures were analyzed based on fermentation optimization and kinetic modeling. The antioxidant activities, of EPS and its sulfated derivatives were evaluated in vitro, by scavenging abilities on superoxide radical, hydroxyl radical and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical. And the protective effects of sulfated derivatives against H2O2-induced RAW264.7 murine macrophages oxidative damage and the possible mechanism were studied using cell culture and proteomics technology. Results are as bellow:1. Technology optimization and kinetic characteristics of EPS production by fermentation of E. cloacae Z0206The medium composition and cultivation conditions of fermentation for EPS produced by E. cloacae Z0206 were optimized according to one-factor-at-a-time method and the orthogonal test. Then response surface methodology based on a three level Box-Behnken factorial design of experiments was employed to optimize the level of three factors significantly affecting the yield of EPS, i.e., concentration of maltose, tryptone and beef extract in the medium. The experimental data obtained were fitted to a second-order polynomial equation using multiple regression analysis and also analyzed by appropriate statistical methods. By solving the regression equation and analyzing the response surface contour plots, the optimal medium was determined. The optimal fermentation conditions were:initial pH 7.5, fermentation temperature 32℃, inoculation amount 4%, K2HPO4·3H2O 0.3%, MgSO4·7H2O 0.15%, KN030.05%, maltose 3.72%, tryptone 0.51% and beef extract 0.56%. Under the optimal conditions, EPS production was 12.89 g/L of fermentation liquor, which was also verified by validation experiments. Based on Logistic equation, the kinetic model describing cell growth was established. Meanwhile, the change of monosaccharide composition of EPS during the fermentation was analyzed by Precolumn Derivatization High Performance Liquid Chromatography. Galactose and fucose were determined as the characteristic monosaccharides during the fermentation.2. Sulfated modification and structural analysis of EPSEPS was prepared through fermentation, deproteinization, decolorization and dialysis. The nine sulfated derivatives of EPS, with various degrees of substitution (DS), were prepared by CSA-Pyr method which nine modification conditions were designed according to orthogonal test focusing on three affecting factors such as the ratio of CSAto Pyr, reaction temperature and reaction time. The results indicated that the extent of the impact of variables on DS followed the order:molar ratio of CSAto Pyr> reaction time> reaction temperature. With the increase of the molar ratio of CSAto Pyr and reaction temperature from 50℃to 70℃, DS of product increased very rapidly. However, the DS decreased when the temperature up to 90℃, The increased reaction time from 1 h to 3 h caused the decrease of DS. The chemical structure analysis of EPS and its sulfated derivatives SEPS indicated that sulfation of EPS were successful obtained, the SO3-group was partially sulfated at C-6 of the sugar unit. And the average molecular weight of EPS, SEPS-2 and SEPS-4 was 24.046×104 Da,1.954×104 Da and 18.221×104 Da. The average size of EPS, SEPS-2 and SEPS-4 was 163.50 nm,62.69 nm and 94.47 nm respectively.3. Study on the antioxidant activity of EPS and its sulfated derivativesThe antioxidant activities of EPS and its nine sulfated derivatives were evaluated in vitro, by scavenging abilities on superoxide radical, hydroxyl radical and DPPH radical. The results indicated that sulfated derivatives of EPS showed noticeable effects on scavenging superoxide radical and hydroxyl radical compared with native one. Then the protective effect of sulfated derivatives of EPS against H2O2-induced RAW264.7 murine macrophages oxidative damage and the possible mechanism were studied. The results showed that H2O2 treatment (0.658 mM) reduced the viability of cells by MTT analysis compared with control (p< 0.05). Addition of EPS, SEPS-2 and SEPS-4 (0.5 and 2.0μg/mL) increased the viability of oxidative damage cells significantly (p< 0.05). SEPS-4 (0.5μg/mL) significantly increased (p< 0.05) the activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), and significantly reduced (p< 0.05) the activation of Caspase-3 compared with H2O2-treated cells only. SEPS-4 (2.0μg/mL) treatment significantly increased (p< 0.05) the mitochondrial membrane potential, and reduced the DNA fragmentation and structure damage of cells induced by H2O2 obviously. Moreover, the antioxidant activity of sulfated derivative SEPS-4 (DS=0.17) was better than EPS and SEPS-2 (DS=0.60). The results indicated that sulfate modification could be considered as the effective approach to enhance the antioxidant activities of EPS, and a moderate DS of the sulfated derivatives was necessary for high biological activities. The sulfated polysaccharides could protect oxidative damage and apoptosis of RAW264.7 cells induced by H2O2 through protection of cell structure, improvements of antioxidant enzymes’activities and mitochondrial membrane potential, inhabitations of Caspase-3 activation and DNA fragmentation.4. Proteomic analysis of antioxidant effect of sulfated polysaccharide SEPS-4 on RAW264.7 cells induced by H2O2 A proteomic approach was used to indentify proteins involved in protective effect of sulfated polysaccharide SEPS-4 against H2O2-induced RAW264.7 murine macrophages oxidative damage. The cells were treated with 0.658 mM H2O2 to induce oxidative damage or pretreated with 2.0μg/mL SEPS-4 before H2O2 treatment. Two-dimensional gel electrophoresis (2-DGE) in combination of MALDI-TOF/TOF MAS was used to identify proteins whose expression level increased or decreased. The results showed that the isoelectric point and molecular weight of proteins distributed broadly, and 132 protein spots were differently expressed. Among the 132 differential protein spots,30 spots were analyzed by MALDI-TOF/TOF MS, and their peptide mass fingerprintings (PMF) were obtained. The PMF maps were searched in Mascot database, and 26 proteins were preliminarily identified. The results showed that SEPS-4 pretreatment downregulated the expression of Peroxiredoxin-2, Eef1D, Eef1G, Phosphatidylethanolamine-binding protein etc., and upregulated the expression of Heat-shock protein hsp84, Rho GDP-dissociation inhibitor 2, T complex protein 1, Alpha-tubulin isotype M-alpha-2, Enol etc.. Those proteins are involved in cell immune and stress, structure and skeleton, protein metabolism and modification, energy metabolism, signal transduction, and they may play important role in protective effect of sulfated polysaccharide SEPS-4 against H2O2-induced RAW264.7 oxidative damage.更多还原