【作者】 闫景坤；

【导师】 李琳；

【作者基本信息】 华南理工大学 ， 制糖工程， 2010， 博士

【摘要】 冬虫夏草（简称虫草）[Cordyceps sinensis （Berk.）Sace.]是我国珍稀的药用真菌,具有广泛的生理活性和独特的药用价值,多糖为其中主要活性成分之一。本文旨在从人工培养的冬虫夏草菌（Cs-HK1）菌丝发酵液中分离虫草胞外多糖,确定胞外多糖提取和分离的最佳工艺条件,通过对粗多糖分级分离,获得各种胞外多糖级分,比较各级分的体外抗氧化活性和作用机制,确定抗氧化活性最佳的多糖级分,并进一步纯化,对抗氧化活性纯多糖的理化性质、化学结构、溶液性质和分子链构象进行深入分析,研究结果如下:采用乙醇沉淀法从人工培养冬虫夏草菌丝发酵液中提取胞外多糖,乙醇浓度95% （v/v）,乙醇添加倍数4.3倍,沉淀时间10.5 h和发酵液pH=6.8,胞外粗多糖的产率4.27 g/L,是目前所见报道的各类虫草菌胞外多糖提取的最高水平。化学组成为总糖50.1%、糖醛酸13.6%、蛋白28.4%。通过脱蛋白、脱色、透析预处理和离子交换柱层析分离分级,得到胞外多糖级分EPS-1和EPS-2。通过体外抗氧化筛选模型,发现胞外多糖EPS、EPS-1、EPS-2通过氢原子转移反应均能较好的清除O₂^·-、·OH和ABTS·+自由基,EPS-1的作用效果明显强于EPS及EPS-2,且对·OH自由基具有更好的清除作用,其IC₅₀值与Vc相当,是虫草菌胞外多糖的主要抗氧化活性组分。EPS-1经凝胶过滤得到纯胞外多糖EPS-1A,紫外光谱、比旋度和凝胶渗透色谱分析表明,胞外多糖EPS-1A为相对均一多糖组分,中性糖含量为99.0%,重均分子量（Mw）为4.0×10⁴ Da,比旋度为+6.5o （c 0.26, H₂O）,为非淀粉类,不含单糖、糖醛酸、蛋白质、核酸和多酚类物质的水溶中性多糖。通过酸水解、甲基化、高碘酸氧化、Smith降解、气相色谱（GC）、气质联用（GC-MS）色谱、红外光谱（IR）、1D和2D NMR等,确定EPS-1A的单糖组成为D-葡萄糖、D-甘露糖和D-半乳糖,分子摩尔比15.2:3.6:1.0,主链由（1→6）-α-D-葡萄糖和（1→6）-α-D-甘露糖组成,侧链出现在（1→6）-α-D-甘露糖残基的O-3位置,由（1→6）-α-D-葡萄糖、（1→6）-α-D-甘露糖和非还原性末端的β-D-半乳糖组成,为人工培养冬虫夏草菌（Cs-HK1）菌丝发酵液中发现的一新的多糖结构重复单元。刚果红反应揭示,EPS-1A在溶液中为无规线团链,通过不同溶液体系中特性粘度、比旋度和粒度分布的变化,推测该多糖在溶液中为柔顺无规线团链构象,在较大浓度易形成聚集。外界条件如温度、金属离子、变性剂和刚果红均不能改变多糖在溶液中的链构象,只是在不同程度上影响多糖分子的分子间或分子内氢键相互作用,使多糖变得松散、无序。粘度法和静、动态激光光散射分析EPS-1A在0.15 M NaCl水溶液中的构象参数特性粘度（[η]）、重均分子量（M_w）、均方根旋转半径（<S²>（1/2））、水动力学半径（Rh）和特征参数（ρ）分别为6.02 dL/g, 5.03×10⁴ g/mol, 133.80 nm, 145.35 nm和0.92,表明该多糖在水溶液呈柔顺无规线团“软球”状链构象,所形成“软球”的硬度要大于右旋糖酐的。原子力显微镜（AFM）显示EPS-1A为大小不等的球状结构,高度和直径分别为2-3 nm和10-50 nm,与右旋糖酐的相似。扫描电镜（SEM）显示胞外多糖EPS-1A的固态形貌为支化度较高的柔顺网状结构。更多还原

【Abstract】 Cordyceps sinensis （Berk.） Sace., generally called Cordyceps or Dongchongxiacao （winter worm-summer grass） in Chinese, is a rare medicinal fungus in China and has a wide range of physiological activities and unique medicinal values. Polysaccharides are regarded as the one of major active ingredients. The aim of this research project was to isolate exopolysaccharides from mycelial fermentation of a Cordyceps sinensis fungus Cs-HK1 and to determine the optimal condition for extraction and isolation of exopolysaccharides. Various exopolysacharide fractions which were isolated and fractionated from crude exopolysaccharides were subjected to compare the antioxidant activities in vitro and mechanism, and the best antioxidant activity of polysaccharide fractions were determined and further purified. Physicochemical properties, chemical structure, solution properties and molecular chain conformation in aqueous solution of purified exopolysaccharide owned better antioxidant activity were analyzed. The results are as follows:Ethanol precipitation was used to isolate the exopolysaccharides from mycelial fermentation of cultured Cordyceps sinensis （Cs-HK1）, and the extraction conditions were concentration of ethanol 95% （v/v）, multiples of ethanol 4.3, sedimentation time 10.5 h, and pH of fermentation broth 6.8, and the yield of crude exopolysaccharide was about 4.27 g/L. This is the highest level in extraction of exopolysaccharides from reported various Cordyceps. Chemical constituents were total sugar 50.1%, uronic acid 13.6%, and protein 28.4%.Removals of protein, decolorization, dialysis and ion-exchange column chromatography were used for isolation and fractionation of crude exopolysaccharides to yield two fractions, namely EPS-1 and EPS-2. In vitro trials of antioxidant activities were chosen to evaluate the free-radical scavenging activity. The results showed that exopolysaccharides EPS, EPS-1, and EPS-2 could moderately scavenge O₂^·-,·OH, and ABTS·+ radicals by the hydrogen atom transfer reaction. Amongst, EPS-1 had stronger effects on free radicals than EPS and EPS-2’s, and had significant free-radical scavenging activity on·OH. The IC₅₀ value of EPS-1 on·OH was closed to the vitamin C （Vc）’s, and was regarded as the major antioxidant component in exopolysaccharide of Cordyceps.EPS-1 was subjected to gel filter chromatography to yield a purified exopolysaccharide EPS-1A. Analytical results of UV spectrum, specific rotation and gel permeation chromatography （GPC） suggested that EPS-1A was a relatively homogeneous component, neutral sugar content of 99.0%, the weight-average molecular weight （Mw） of 4.0×10⁴ Da and the specific rotation of +6.5o （c 0.26, H₂O）, a non-starch water-soluble and neutral polysaccharide which did not contain monosaccharides, uronic acids, protein, nucleic acids and polyphenols.The structural characteristics of EPS-1A were elucidated with acid hydrolysis, methylation, periodate oxidation, Smith degradation, gas chromatography （GC）, gas chromatography-mass spectrograph （GC-MS）, infrared spectroscopy （IR）, 1D and 2D NMR, etc. EPS-1A was composed of D-glucose, D-mannose and D-galactose at 15.2:3.6:1.0, its backbone was composed of （1→6）-α-D-glucose and （1→6）-α-D-mannose, and branching occurred at O-3 position of （1→6）-α-D-mannose residue of the backbone with （1→6）-α-D-glucose, （1→6）-α-D-mannose and terminated withβ-D-galactose. This is a new structure of repeat units found in mycelial fermentation of Cordyceps sinensis （Cs-HK1）.Congo red reaction revealed that EPS-1A was a random coil chain in different aqueous solutions. Changes of intrinsic viscosity, specific rotation and particle size distribution in various aqueous solutions suggested that EPS-1A was a flexible random coil chain in aqueous solutions and formed aggregates at higher concentrations. Factors including temperature, metallic ion, denaturing agent and Congo red could not alter the chain conformation in aqueous solution, but influence the inter- or intra-molecular hydrogen bond interactions of polysaccharide molecules in some extent, so that polysaccharides became loose and disordered.Viscosity method and static and dynamic laser light scattering technology were used to determine intrinsic viscosity （[η]）, weight-average molecular weight （Mw）, radii of gyration (<S²>^1/2), hydrodynamic radium （Rh） and structural parameter （ρ） of the EPS-1A, and the obtained results were 6.02 dL/g, 5.03×10⁴g/mol, 133.80nm, 145.35nm, and 0.92 respectively, suggesting that EPS-1A exhibited a flexible random coil“soft sphere”chain conformation in aqueous solution, and the hardness of“soft sphere”was slightly greater than dextran’s. The chain conformation of EPS-1A was observed by atomic force microscopy （AFM） showing spherical structure of different sizes which was similar to dextran’s, and the height and diameter of the spheres ranged from 10 to 50 nm and 2 to 3 nm, respectively. The solid-state morphology of EPS-1A was also observed by scanning electron microscopy （SEM） showing a flexible network structure with a high degree of branching.更多还原