天然冬虫夏草和发酵虫草菌粉的化学组成及其多糖精细结构表征

发布时间：2017-12-28 22:25

本文关键词：天然冬虫夏草和发酵虫草菌粉的化学组成及其多糖精细结构表征　出处：《南昌大学》2017年博士论文　论文类型：学位论文

【摘要】：冬虫夏草(Cordyceps sinensis(Berk.)Sace.)是我国一种珍贵的药食两用真菌,具有广泛的生理活性和独特的药用价值,其含有多种活性成分,如多糖、氨基酸、蛋白质、核苷类、虫草酸等。由于天然冬虫夏草的生长环境特殊,每年产量有限,无法满足日益增长的市场需求,因此,国内外众多研究机构从野生冬虫夏草中分离出菌株,然后结合固液态发酵技术从而获得相应的发酵菌丝体和发酵产物。但是这些发酵菌丝体是否与天然冬虫夏草具有相似的化学组成,是否保持相似的生物活性不得而知。因此,本文综合采用多种化学和现代仪器分析方法对天然冬虫夏草和发酵虫草菌粉的化学组成和活性成分含量(包括水分、灰分、脂肪、多糖、虫草酸和核苷类物质含量,以及氨基酸组成、脂肪酸组成和矿物元素组成)进行了系统且全面的分析和比较。其次,分别提取和纯化出天然冬虫夏草和发酵虫草菌粉的多糖成分,通过对其理化性质和化学结构进行深入分析,为冬虫夏草多糖构效关系的研究奠定理论基础。现将主要研究结果归纳如下:1.天然冬虫夏草和发酵虫草菌粉中最主要的氨基酸为谷氨酸,并且发酵虫草菌粉中的必需氨基酸比例较高。天然冬虫夏草主要脂肪酸是油酸(50%),而发酵虫草菌粉的则为亚油酸(42.67%)。除了Cr、Mo、Sn、V和Cd,其他无机元素在天然冬虫夏草和发酵虫草菌粉中的含量具有显著性差异。发酵虫草菌粉多糖含量最高(10.43%),虫草酸含量最低(4.75%)。此外,发酵虫草菌粉中腺嘌呤和腺苷分别为0.5和2.61 mg/g,均显著高于天然冬虫夏草,两者鸟苷含量无显著性差异,且均未检测到虫草素。发酵虫草菌粉的水提物具有较好的DPPH、羟自由基清除能力和还原能力。2.干燥处理和贮存过程对天然冬虫夏草氨基酸组成和脂肪酸组成无明显影响。长时间贮存会导致海藻糖和虫草酸含量均显著增加,但是对多糖含量无显著影响,而干燥处理导致海藻糖含量显著增加,多糖含量显著降低,但是对虫草酸含量无显著影响。新鲜样品中鸟苷、尿苷、次黄嘌呤和腺嘌呤含量均最高,但是干燥处理和贮存过程会导致腺苷含量显著增加。3.不同产地天然冬虫夏草水提多糖化学结构类似,但是与发酵虫草菌粉多糖相比差异显著。主要包括:a)天然冬虫夏草水提多糖以分子量为960 kDa左右的高分子量组分为主(60%),而发酵虫草菌粉多糖则主要由分子量为28 kDa左右相对低的组分组成;b)天然冬虫夏草多糖主要由葡萄糖组成,含有少量的半乳糖和甘露糖,而发酵虫草菌粉主要由葡萄糖、半乳糖和甘露糖组成,还有少量的阿拉伯糖和半乳糖醛酸;c)天然冬虫夏草多糖的主链主要由1,4-linked Glcp组成,C-6为主要分支点,而发酵虫草菌粉多糖的糖苷键组成复杂,主要的糖残基为1,4-linked Galp和1,4-linked Glcp。天然冬虫夏草和发酵虫草菌粉多糖均具有清除DPPH和羟自由基的能力,以及铁离子还原能力,这可能是由于酚类物质、氨基酸等多个因素共同作用的结果。4.采用单糖组成、甲基化及气质联用仪(GC-MS)分析、特异性糖苷酶水解结合基质辅助激光解吸飞行时间质谱仪(MALDI-TOF)分析和一维和二维核磁共振波谱(1D/2D NMR)技术对天然冬虫夏草水提多糖NCSP-50的化学结构进行表征。NCSP-50为α-(1→4)-葡聚糖,分支在C-6位,分子量为9.76×105 Da,推断其重复结构单元如下:此外,通过体外RAW 264.7细胞免疫实验证明了NCSP-50是一种与淀粉类多糖不同的,具有显著免疫调节作用的活性多糖。5.采用部分酸水解、甲基化及GC-MS分析和1D/2D NMR对天然冬虫夏草碱提低分子量多糖组分ANCSP-S50(7207 Da)的化学结构进行表征。该多糖组分为一种新颖的α-(1→6)-半乳甘露聚糖,主要由甘露聚糖主链和半乳呋喃糖短链两部分组成,其中半乳呋喃糖链由β-(1→5)-Galf和β-(1→6)-Galf交替连接或β-(1→6)-Galf残基连接至甘露糖残基的O-2和O-4,端基为β-T-Galf。甘露聚糖部分是以α-(1→6)-甘露糖残基线性连接,在O-2和O-4位有分支,其中O-2与α-(1→2)-甘露糖残基或半乳呋喃糖链相连,O-4与半乳呋喃糖链相连。由此推断出这种半乳甘露聚糖的可能结构如下:6.通过甲基化结合GC-MS分析和1D/2D NMR对天然冬虫夏草碱提高分子量多糖高分子量组分ANCSP-50的化学结构进行分析。结果表明:ANCSP-50是一种β-(1→3)-葡聚糖,在葡萄糖的C-6位置分支,分子量为4.77×105 Da,特性粘度[η]为2.80 dL/g,推断可能的结构如下:采用动态光散射(DLS)和静态光散射(SLS),结合高分子稀溶液理论对该多糖在不同溶液体系下(0.5 M NaOH和DMSO溶液)的构象特征进行探讨,结果显示ANCSP-50在0.5 M Na OH溶液中的Mw、Rg和A2分别为424 kDa、17.3 nm、5.0×10-3 cm3 mol/g2,而在DMSO中分别为2200 kDa、56.7 nm、1.79×10-5 cm3 mol/g2。ANCSP-50在0.5 M NaOH溶液中的Rh为27.9 nm,而在DMSO中形成大的聚集体,表明多糖在0.5 M NaOH溶液可以消除聚集体的形成,得到单分子分布溶液且呈现超支化链构象。7.单糖组成、甲基化结合GC-MS分析和1D/2D NMR分析表明发酵虫草菌粉多糖是一种主要由葡萄糖、半乳糖和甘露糖组成结构复杂的高分子。因此,在凝胶柱分离的基础上,继续用离子交换柱层析,通过不同浓度Na Cl洗脱,得到的主要组分CSP2-F0.05,继续对其进行深入分析,从而获得几个可能的连接片段,结果如下,综上,本文较为系统的比较了天然冬虫夏草和发酵虫草菌粉两种不同来源冬虫夏草的化学组成和活性成分含量及其水提物的体外抗氧化活性,同时对其不同多糖组分的化学结构进行解析。结果显示不同来源冬虫夏草的化学成分含量差异明显,尤其是多糖、虫草酸、腺苷、腺嘌呤等有效成分含量,天然冬虫夏草多糖主要包括α-(1→4)-葡聚糖(NCSP-50),β-(1→3)-葡聚糖(ANCSP-S50)和α-(1→6)-半乳甘露聚糖(ANCSP-50),而发酵虫草菌粉多糖组成复杂,是一种含有半乳糖、葡萄糖和甘露糖的杂多糖。
[Abstract]:Cordyceps sinensis (Cordyceps sinensis (Berk.) Sace.) is a valuable medicinal and edible fungus in China. It has a wide range of physiological activities and unique medicinal value. It contains a variety of active ingredients, such as polysaccharides, amino acids, proteins, nucleosides, cordycepic acid and so on. Because of the special natural environment for the growth of Cordyceps sinensis, the annual output is limited, unable to meet the increasing market demand, therefore, many research institutions at home and abroad strains isolated from wild Cordyceps sinensis, then combined with solid and liquid fermentation technology and fermentation mycelia and corresponding fermentation products. However, it is not known whether these fermented mycelium have similar chemical composition with the natural Cordyceps, and it is not possible to maintain similar biological activity. Therefore, this paper uses a variety of chemical and modern instrument analysis method of natural Cordyceps sinensis and Fermented Cordyceps powder, chemical composition and content of active ingredients (including moisture, ash, fat, polysaccharides, cordycepic acid and nucleosides content and amino acid composition, fatty acid composition and mineral elements) were systematically and comprehensively and the analysis. Secondly, the polysaccharides from natural Cordyceps sinensis and Cordyceps militaris powder were extracted and purified respectively, and their physicochemical properties and chemical structure were analyzed in depth, so as to lay a theoretical foundation for the study of the structure activity relationship of Cordyceps sinensis polysaccharides. The main results are summarized as follows: 1.. The most important amino acids in natural Cordyceps sinensis and Cordyceps militaris powder are glutamic acid, and the proportion of essential amino acids in Cordyceps sinensis powder is high. The main fatty acids of the natural Cordyceps are oleic acid (50%), while the Fermented Cordyceps are linoleic acid (42.67%). In addition to Cr, Mo, Sn, V and Cd, the contents of other inorganic elements in the natural Cordyceps and Fermented Cordyceps were significantly different. The content of polysaccharide in Fermented Cordyceps was the highest (10.43%), and the content of oxalic acid was the lowest (4.75%). In addition, adenosine and adenosine in Fermented Cordyceps sinensis powder were 0.5 and 2.61 mg/g, respectively, which were significantly higher than those of natural Cordyceps sinensis, but there was no significant difference in guanosine content between them, and no cordycepin was detected. The water extracts of the Fermented Cordyceps powder have good DPPH, hydroxyl radical scavenging ability and reduction ability. 2. the drying process and storage process had no obvious effect on the composition of amino acid and fatty acid composition of the natural Cordyceps sinensis. Long term storage resulted in significant increase in trehalose and oxalic acid content, but no significant effect on polysaccharide content. Dry treatment resulted in a significant increase in trehalose content and a significant decrease in polysaccharide content, but it had no significant effect on the content of cordycepic acid. Fresh samples of guanosine, uridine, hypoxanthine and adenine content were the highest, but the drying and storage process will lead to significantly increase the content of adenosine. 3. different areas of natural Cordyceps sinensis polysaccharide chemical structure is similar, but with the fermentation Cordyceps polysaccharide significantly. Mainly include: a) with high molecular weight natural Cordyceps polysaccharide group with molecular weight of about 960 kDa. (60%), and Fermented Cordyceps sinensis polysaccharide mainly by the molecular weight is about 28 kDa lower component; b) natural Cordyceps polysaccharide was composed of glucose group, containing a small amount of galactose and mannose, and fermented Cordyceps powder mainly composed of glucose, galactose and mannose, and a small amount of Arabia sugar and galacturonic acid; c) natural Cordyceps polysaccharide backbone is mainly composed of 1,4-linked Glcp, C-6 as the main branch point, and the fermentation of Cordyceps polysaccharide glycoside composition complex. The principal sugar residue is 1,4-linked Galp and 1,4-linked Glcp. Both natural Cordyceps sinensis and Fermented Cordyceps sinensis polysaccharide have the ability to scavenge DPPH and hydroxyl radical and iron reduction ability, which may be due to many factors such as phenols and amino acids. 4. the monosaccharide composition, methylation and gas chromatography-mass spectrometry (GC-MS) analysis, specific glycosidase hydrolysis combined with matrix assisted laser desorption time-of-flight mass spectrometer (MALDI-TOF) analysis and 2D nuclear magnetic resonance spectroscopy (1D/2D NMR) the chemical structure of natural Cordyceps sinensis polysaccharide NCSP-50 were characterized. NCSP-50 alpha (1 - 4) - glucan, branch in C-6, the molecular weight of 9.76 * 105 Da, deduce the repeating structural unit are as follows: in addition, the in vitro RAW 264.7 cell immunity experiment proved that NCSP-50 is a kind of starch and different polysaccharides, has significant immunomodulatory effects of active polysaccharides. 5., we used partial acid hydrolysis, methylation and GC-MS analysis and 1D/2D NMR to characterize the chemical structure of the low molecular weight polysaccharides ANCSP-S50 (7207 Da) extracted from natural Cordyceps sinensis. The polysaccharide component is a novel alpha - (1 - 6) mannogalactan, mainly composed of mannose and galactose backbone furanoses short chain is composed of two parts, which induced by beta chain furanoses (1 - 5) -Galf and beta (1 - 6) -Galf alternately connected or beta - (1 - 6) -Galf residues linked to mannose residues O-2 and O-4, group -T-Galf beta. Mannan is partly connected by alpha - (1 - 6) mannose residues, and has branches in O-2 and O-4 sites, where O-2 is connected to alpha - (1 - 2) mannose residues or galactomannan chains, O-4 is linked to galactomannose chains. It is concluded that the structure of galactomannan is as follows: 6., through methylation and GC-MS analysis and 1D/2D NMR, we can analyze the chemical structure of high molecular weight polysaccharide ANCSP-50 from natural Cordyceps sinensis. The results show that ANCSP-50 is a beta (1 - 3) - glucan, branched glucose in the C-6 position, the molecular weight of 4.77 * 105 Da, intrinsic viscosity is 2.80 dL/g, the following may infer the structure by dynamic light scattering (DLS) and static light scattering (SLS), combined with polymer dilute solution theory of the polysaccharides in different solution system (0.5 M NaOH and DMSO solution) discussed the conformational characteristics, the results showed that ANCSP-50 in 0
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS218;TQ920.1

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