本文關鍵詞：天然冬蟲夏草和發(fā)酵蟲草菌粉的化學組成及其多糖精細結構表征　出處：《南昌大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 天然冬蟲夏草 發(fā)酵蟲草菌粉 化學組成 活性成分 抗氧化活性 多糖 分離 純化 結構特征

【摘要】：冬蟲夏草(Cordyceps sinensis(Berk.)Sace.)是我國一種珍貴的藥食兩用真菌,具有廣泛的生理活性和獨特的藥用價值,其含有多種活性成分,如多糖、氨基酸、蛋白質、核苷類、蟲草酸等。由于天然冬蟲夏草的生長環(huán)境特殊,每年產量有限,無法滿足日益增長的市場需求,因此,國內外眾多研究機構從野生冬蟲夏草中分離出菌株,然后結合固液態(tài)發(fā)酵技術從而獲得相應的發(fā)酵菌絲體和發(fā)酵產物。但是這些發(fā)酵菌絲體是否與天然冬蟲夏草具有相似的化學組成,是否保持相似的生物活性不得而知。因此,本文綜合采用多種化學和現代儀器分析方法對天然冬蟲夏草和發(fā)酵蟲草菌粉的化學組成和活性成分含量(包括水分、灰分、脂肪、多糖、蟲草酸和核苷類物質含量,以及氨基酸組成、脂肪酸組成和礦物元素組成)進行了系統(tǒng)且全面的分析和比較。其次,分別提取和純化出天然冬蟲夏草和發(fā)酵蟲草菌粉的多糖成分,通過對其理化性質和化學結構進行深入分析,為冬蟲夏草多糖構效關系的研究奠定理論基礎�，F將主要研究結果歸納如下:1.天然冬蟲夏草和發(fā)酵蟲草菌粉中最主要的氨基酸為谷氨酸,并且發(fā)酵蟲草菌粉中的必需氨基酸比例較高。天然冬蟲夏草主要脂肪酸是油酸(50%),而發(fā)酵蟲草菌粉的則為亞油酸(42.67%)。除了Cr、Mo、Sn、V和Cd,其他無機元素在天然冬蟲夏草和發(fā)酵蟲草菌粉中的含量具有顯著性差異。發(fā)酵蟲草菌粉多糖含量最高(10.43%),蟲草酸含量最低(4.75%)。此外,發(fā)酵蟲草菌粉中腺嘌呤和腺苷分別為0.5和2.61 mg/g,均顯著高于天然冬蟲夏草,兩者鳥苷含量無顯著性差異,且均未檢測到蟲草素。發(fā)酵蟲草菌粉的水提物具有較好的DPPH、羥自由基清除能力和還原能力。2.干燥處理和貯存過程對天然冬蟲夏草氨基酸組成和脂肪酸組成無明顯影響。長時間貯存會導致海藻糖和蟲草酸含量均顯著增加,但是對多糖含量無顯著影響,而干燥處理導致海藻糖含量顯著增加,多糖含量顯著降低,但是對蟲草酸含量無顯著影響。新鮮樣品中鳥苷、尿苷、次黃嘌呤和腺嘌呤含量均最高,但是干燥處理和貯存過程會導致腺苷含量顯著增加。3.不同產地天然冬蟲夏草水提多糖化學結構類似,但是與發(fā)酵蟲草菌粉多糖相比差異顯著。主要包括:a)天然冬蟲夏草水提多糖以分子量為960 kDa左右的高分子量組分為主(60%),而發(fā)酵蟲草菌粉多糖則主要由分子量為28 kDa左右相對低的組分組成;b)天然冬蟲夏草多糖主要由葡萄糖組成,含有少量的半乳糖和甘露糖,而發(fā)酵蟲草菌粉主要由葡萄糖、半乳糖和甘露糖組成,還有少量的阿拉伯糖和半乳糖醛酸;c)天然冬蟲夏草多糖的主鏈主要由1,4-linked Glcp組成,C-6為主要分支點,而發(fā)酵蟲草菌粉多糖的糖苷鍵組成復雜,主要的糖殘基為1,4-linked Galp和1,4-linked Glcp。天然冬蟲夏草和發(fā)酵蟲草菌粉多糖均具有清除DPPH和羥自由基的能力,以及鐵離子還原能力,這可能是由于酚類物質、氨基酸等多個因素共同作用的結果。4.采用單糖組成、甲基化及氣質聯用儀(GC-MS)分析、特異性糖苷酶水解結合基質輔助激光解吸飛行時間質譜儀(MALDI-TOF)分析和一維和二維核磁共振波譜(1D/2D NMR)技術對天然冬蟲夏草水提多糖NCSP-50的化學結構進行表征。NCSP-50為α-(1→4)-葡聚糖,分支在C-6位,分子量為9.76×105 Da,推斷其重復結構單元如下:此外,通過體外RAW 264.7細胞免疫實驗證明了NCSP-50是一種與淀粉類多糖不同的,具有顯著免疫調節(jié)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對天然冬蟲夏草堿提低分子量多糖組分ANCSP-S50(7207 Da)的化學結構進行表征。該多糖組分為一種新穎的α-(1→6)-半乳甘露聚糖,主要由甘露聚糖主鏈和半乳呋喃糖短鏈兩部分組成,其中半乳呋喃糖鏈由β-(1→5)-Galf和β-(1→6)-Galf交替連接或β-(1→6)-Galf殘基連接至甘露糖殘基的O-2和O-4,端基為β-T-Galf。甘露聚糖部分是以α-(1→6)-甘露糖殘基線性連接,在O-2和O-4位有分支,其中O-2與α-(1→2)-甘露糖殘基或半乳呋喃糖鏈相連,O-4與半乳呋喃糖鏈相連。由此推斷出這種半乳甘露聚糖的可能結構如下:6.通過甲基化結合GC-MS分析和1D/2D NMR對天然冬蟲夏草堿提高分子量多糖高分子量組分ANCSP-50的化學結構進行分析。結果表明:ANCSP-50是一種β-(1→3)-葡聚糖,在葡萄糖的C-6位置分支,分子量為4.77×105 Da,特性粘度[η]為2.80 dL/g,推斷可能的結構如下:采用動態(tài)光散射(DLS)和靜態(tài)光散射(SLS),結合高分子稀溶液理論對該多糖在不同溶液體系下(0.5 M NaOH和DMSO溶液)的構象特征進行探討,結果顯示ANCSP-50在0.5 M Na OH溶液中的Mw、Rg和A2分別為424 kDa、17.3 nm、5.0×10-3 cm3 mol/g2,而在DMSO中分別為2200 kDa、56.7 nm、1.79×10-5 cm3 mol/g2。ANCSP-50在0.5 M NaOH溶液中的Rh為27.9 nm,而在DMSO中形成大的聚集體,表明多糖在0.5 M NaOH溶液可以消除聚集體的形成,得到單分子分布溶液且呈現超支化鏈構象。7.單糖組成、甲基化結合GC-MS分析和1D/2D NMR分析表明發(fā)酵蟲草菌粉多糖是一種主要由葡萄糖、半乳糖和甘露糖組成結構復雜的高分子。因此,在凝膠柱分離的基礎上,繼續(xù)用離子交換柱層析,通過不同濃度Na Cl洗脫,得到的主要組分CSP2-F0.05,繼續(xù)對其進行深入分析,從而獲得幾個可能的連接片段,結果如下,綜上,本文較為系統(tǒng)的比較了天然冬蟲夏草和發(fā)酵蟲草菌粉兩種不同來源冬蟲夏草的化學組成和活性成分含量及其水提物的體外抗氧化活性,同時對其不同多糖組分的化學結構進行解析。結果顯示不同來源冬蟲夏草的化學成分含量差異明顯,尤其是多糖、蟲草酸、腺苷、腺嘌呤等有效成分含量,天然冬蟲夏草多糖主要包括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),而發(fā)酵蟲草菌粉多糖組成復雜,是一種含有半乳糖、葡萄糖和甘露糖的雜多糖。
[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
【學位授予單位】：南昌大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：TS218;TQ920.1

【相似文獻】

相關期刊論文 前7條

1 張博;楊黎彬;李金娟;;高效液相色譜法測定蟲草菌粉中腺苷的含量[J];光譜實驗室;2011年02期

2 童應凱,鄺濤,吳雅欣,張慶勇,任靜;蟲草菌絲體與天然冬蟲夏草成分的比較[J];食品研究與開發(fā);1997年04期

3 彭科懷,趙年華;發(fā)酵蟲草菌粉制品中甘露醇的測定[J];職業(yè)衛(wèi)生與病傷;2005年03期

4 吳紅艷,陳飛,李莉,鄧淑芳,路廣玉;金龍消栓合劑中蟲草菌粉的薄層色譜鑒別研究[J];微生物學雜志;2002年06期

5 彭日煌,謝明勇,王遠興,章志明,陳軍輝;RP-HPLC測定蟲草菌粉中核苷類成分[J];食品與機械;2005年01期

6 沈曉云,李兆蘭,田軍;冬蟲夏草與蟲草菌絲有效成分分析比較[J];山西大學學報(自然科學版);1998年01期

7 帥再金;蟲草菌粉干燥采用旋轉閃蒸干燥機的探索[J];江西能源;2000年02期

相關會議論文 前5條

1 張Z，

本文編號：1347691