臺灣博碩士論文加值系統

β-(1,3)/(1,6)-葡聚醣是以 β-(1,3)醣苷為主鏈、β-(1,6)醣苷為分枝的多醣類，β-(1,3)/(1,6)-葡聚醣經人體吸收具有提高免疫系統之能力，固醇類是扮演著維持細胞膜完整性及細胞膜上酶類活性的重要角色，此兩種化合物是醫藥工業上很重要之原料。本研究敘述麵包酵母菌中 β-(1,3)/(1,6)-葡聚醣及固醇類的分離及純化方法及其特性研究，其分離步驟：(1)氫氧化鈉水溶液中加熱，(2)醋酸水溶液中加熱，(3)酸性甲醇溶液萃取，(4)鹼性甲醇溶液萃取。每步驟所分離純化後之產品均經 FTIR分析，發現氫氧化鈉水溶液中加熱可去除核酸、甘露聚糖及蛋白質等物質，進一步分離純化可得單一成分蛋白質物質，在醋酸水溶液中加熱步驟可分離出幾丁質，在酸性甲醇溶液中萃取步驟可去除脂溶性蛋白質及醣苷物質，鹼性甲醇溶液萃取步驟可分離出脂質及固醇類，進一步分離出固醇類及 β-(1,3)/(1,6)-葡聚醣。固醇類在FTIR分析有麥角固醇的1458及1377 cm-1等特定吸收峰，從 HPLC發現此固醇類為三種以上物質混和物，在 1H NMR中也發現有麥角固醇位移峰。β-(1,3)/(1,6)-葡聚醣在 FTIR分析有891 cm-1的特定吸收峰，從 FTIR與 1H NMR分析中發現，β-(1,3)/(1,6)-葡聚醣純化後其純度很高。本研究設計之分離純化步驟，是以最簡單製程來生產出純度最高的產品為目的，其中 β-1,3/1,6-葡聚醣及固醇類產率分別為8.2％及0.68％。

-(1,3)/(1,6)-glucan is a polysaccharide and a main chain of (1-3)-linked β-D-glucopyranosyl units with a single β-D-glucopyranosyl unit linked (1-6). β-(1,3)/(1,6)-glucan has been shown to have immunopharmcological activity in human beings. Sterols are essential for structural features such as integrality of cell membrane and activity of membrane-bond enzymes. These two kinds of chemical compounds are very important raw materials on medical industry. This study presents a process for isolation and characterization of β-(1,3)/(1,6)-glucan and sterols from baker’s yeast (Saccharomyces cerevisiae). The steps of the isolation of baker’s yeast are (A) in hot alkali; (B) in hot acetic acid; (C) extracting with acidic methanol-solution and (D) extracting with alkaline methanol-solution. The products of each step are analyzed by the FTIR. We found that the proteins, nucleic acid and mannan can be removed from baker’s yeast in hot alkali. The pure protein can be separated from the alkaline solution. The chitin in the insoluble solid can dissolve in hot acetic acid and fat-soluble proteins and glycosides can also dissolve in acidic methanol-solution. Sterols and lipids are extracted from alkaline methanol-solution. Finally the β-(1,3)/(1,6)-glucan is obtained from the insoluble solid. The significant spectral 1458 and 1377cm-1 is recognized as the ergosterol in the FTIR analysis of sterols. From the HPLC analysis, the sterols compose of more than three kinds of mixtures. The significant spectral 891 cm-1 is attributed to a β-(1,3)/(1,6)-glucan by FTIR analysis. From the FTIR and NMR analytical data, the purified β-(1,3)/(1,6)-glucan is found to have exclusively high purity. This research designs the simplest process of separation and the purification, the purpose is to produce the high purity products of β-(1,3)/(1,6)-glucan and sterols. β-(1,3)/(1,6)-glucan and sterols are obtained in a high purity and a yield of 8.2 % and 0.68% respectively.

中文摘要 Ⅰ
英文摘要 Ⅱ
誌謝 Ⅲ
目錄 Ⅳ
表目錄 Ⅵ
圖目錄 Ⅶ
第一章 文獻整理 1
1.1 酵母菌 1
1.1.1 酵母菌細胞壁分析 2
1.1.2 酵母菌細胞膜分析 3
1.1.3 酵母菌細胞質成分 4
1.2 麥角固醇化學結構 4
1.3 麥角固醇功能及應用 5
1.3.1 以麥角固醇為原料之藥物 6
1.3.2 麥角固醇化妝品方面的應用 7
1.4 β-1,3/1,6-葡聚醣化學結構 7
1.5 β-1,3/1,6-葡聚醣功能及應用 9
1.5.1 β-1,3/1,6-葡聚醣在醫藥方面應用 9
1.5.2 β-1,3/1,6-葡聚醣在化妝品方面的應用 10
1.6 麥角固醇與 alkali-insoluble葡聚醣製法文獻回顧 10
1.6.1 麥角固醇之製法 10
1.6.2 Alkali-insoluble葡聚醣之製法 15
1.7 研究動機及目的 19
第二章 實驗方法 20
2.1 酵母菌來源 20
2.2 實驗藥品 20
2.3 實驗產物分佈概述 21
2.4 Alkali-soluble物質之分析 23
2.5 Acid-methanol soluble物質之分析 25
2.6 Alkali-methanol soluble物質之固醇類分離純化 27
2.7 β-1,3/1,6-葡聚醣分離純化過程 29
2.8 分離純化之研究所需儀器 32
第三章 結果與討論 34
3.1 Alkali-soluble物質之分析 35
3.1.1 Alkali-soluble物質之 FTIR分析 35
3.1.2 Alkali-soluble proteins之 HPLC分析 42
3.1.3 Alkali-soluble proteins之 1H NMR分析 43
3.2 Acid-methanol soluble物質之 FTIR分析 50
3.2.1 Ether-soluble物質之 FTIR分析 51
3.2.2 Ether-insoluble物質 52
3.3 Alkali-methanol soluble物質分離出固醇類之分析 53
3.3.1 固醇類之 FTIR光譜分析 54
3.3.2 分離純化固醇類與麥角固醇和維他命 D2數據比較 58
3.3.3 固醇類之 HPLC分析 60
3.3.4 分離純化固醇類之 1H NMR分析 61
3.4 β-1,3/1,6-葡聚醣之分析 64
3.4.1 β-1,3/1,6-葡聚醣之 FTIR分析 64
3.4.1.1 β-1,3/1,6-葡聚醣之 FTIR分析 65
3.4.1.2 純化後 β-1,3/1,6-葡聚糖與 Mohaček-Grošev之β-1,3/1,6-葡聚醣和 Lanigan產品之 β-1,3/1,6-葡
聚醣的 FTIR波數比較 69
3.4.2 β-1,3/1,6-葡聚醣之 1H NMR檢測 73
3.5 分離純化過程中各成份產率分布 74
3.5.1 酵母菌以氫氧化鈉水溶液分離純化之產率分布 75
3.5.2 Alkali-insoluble物質以醋酸水溶液分離純化之產率
分布 75
3.5.3 Acid-insoluble物質在酸性甲醇溶液分離純化之產率
分布 75
3.5.4 Acid-methanol insoluble物質在鹼性甲醇溶液分離純
化之產率分布 76
第四章 結論 78
參考文獻 79

[1]Freimund, S., Sauter, M., Kappeli, O., Dutler, H. A new non-degrading isolation process for 1,3-β-D-glucan of high purity from baker’s yeast Saccharomyces cerevisiae. Carbohydr. Polym. 2003, 54, 159-171.
[2]香港教育學院 http://www.ied.edu.hk/has/bio/dlo/fungi/fun_char.htm.
[3]Galichet, A., Sockaligum, G. D., Belarbi, A., Manfait, M. FTIR spectroscopic analysis of Saccharomyces cerevisiae cell walls: study of an anomalous strain exhibiting a pink-colored cell phenotype. FEMS Microbiol Lett. 2001, 197, 179-186.
[4]Freimund, S., Sauter, M. Isolation of glucan particles and uses thereof. U.S. Patent Application. 2006, 0134759.
[5]John, V. H., Kerry, M. P., Brij, V. Mass spectrometric determination of ergosterol in a prairie natural wetland. J. Chromatogr. 2002, 958, 149-156.
[6]宋文霞, 于雪雲, 關鳳梅. 酵母中麥角固醇含量測定的研究. 山東商業職業技術學院學報 2005, 5(4), 86-88.
[7]路玲玲, 檀建新, 馬雯等. 高溫和高酒精濃度下的酵母菌特性. 中國釀造. 2004, 9, 5-7.
[8]Rober, K. M., Daryl, K. G., Peter, A. M. Rodwell.Harper’s Biochemistry. Norwalk, Conn. 1996, 15-32, 34-35.
[9]Shang, F., Wen, S., Wang, X. Effect of nitrogen limitation on the ergosterol production by fed-batch culture of Saccharomyces cerevisiae. J. biotechnol. 2006, 122, 285-292.
[10]王亞平, 任 超. 紫外線抗佝僂病的機制及紫外線療法. 錦州醫學院學報 1997, 18, 45-46.
[11]張步暖. 酵母在醫藥工程中的應用. 山東醫藥工程 1997, 16(2), 41-42.
[12]馮祥華. New Perspectives of Vitamin D Analogues in Clinical Therapy.內科學誌 1992, 13, 167-174.
[13]潘一紅. 全球化粧保養品產業及技術發展趨勢. 化工資訊與商情 2005, 25.
[14]倪捷兒. 植物甾醇的氣相色譜分析. 浙江科技學院學報2002, 14(1), 14-16.
[15]芝蘭健康網http://www.cheeryland.com/m_element/element_detail.asp?ckid=9&cid=363.
[16]水野 卓. 巴西磨菇的抗癌奇蹟. 世茂出版社 1999, 初版, 36-56.
[17]胡曉忠, 明 杰, 甄寶貴. 用酸鹼法崇麵包酵母中提取-(1-3)-D-葡聚糖. 工業微生物 2000, 30(1), 28-31.
[18]謝曉環. 由麵包酵母菌(Saccharomyces cerevisiae)純化-(1,3)/(1,6)-glucan之研究. 高雄應用科技大學化學工程系 2004, 碩士論文.
[19]Tarasova. Method for preparing ergosterol and ubiquinone-9 in a single process. U. S. Patent. 1976, 3965130.
[20]Tan, T., Zhang, M., Gao, H. Ergosterol production by fed-bacth fermentation of saccharomyces cerevisiae. Enzyme and Microbial Techonology. 2003, 33, 366-370.
[21]孫百申, 周立平, 陳旭峰. HPLC法測定紅曲霉發酵樣品中麥角固醇的含量. 中國食品添加劑 2004, 2, 89-92.
[22]張惠波, 施立欽, 朱建華. 麥角固醇提取及分離純化研究. 中國科技信息 2005, 12, 138.
[23]李惠珍, 許旭萍, 謝華玲. 正紅菇的麥角固醇及多醣提取法的研究.中國食用菌 1998, 17, 37-39.
[24]Kelly, G. E. Process for glucan preparation and therapeutic uses of glucan. U. S. Patent. 2001, 6242594.
[25]Davidson, D. Glucan preparation. U. S. Patent. 1997, 5622939.
[26]Jordan, Frank, M.,Gault, Ruth, A., Hunter, Kenneth, W. Method for preparing small particle size glucan in a dry material. U. S. Patent. 2002, 6476003.
[27]Guoyou. Li, Bogang. Li, Guangye. Liu. Sterols from Aspergillus Ochraceus 43. Chin. J. Appl. Eenviron. 2005, 11, 67-70.
[28]李潔莉, 陸 玲, 倉一華. 猴頭菇及提取物有關腦醇類化合物初探.中國生化藥物雜誌 2001, 22, 124-126.
[29]Ping Gong, Xin Guan, Ernst Witter. A rapid method to extract ergosterol from soil by physical disruption. Applied soil Ecology. 2001, 17, 285-289.
[30]吳 克, 揚本宏, 劉 斌等. Trichoderma viride 菌生物量測定及其纖維素脢合成特徵. 食品與發酵工程 2002, 28, 9-12.
[31]Abramson, D., Smith, D. M. Determination of ergosterol in canola (Brassia napus L.) by liquid chromatography. J. of Stored Product Research. 2003, 39, 185-191.
[32]張毅偉. 靈芝中具β-(1→6)分支之(1→3)-β-D-聚葡萄糖的性質檢測.國立台灣大學食品科技研究所 2003, 博士論文.
[33]劉雅峰, 潘 勤. 真菌竹黃中的過氧化麥角腦醇的分離. 天津中醫學院學報 2004, 23(1), 15-16.
[34]成大化工系 http://www.che.ncku.edu.tw/stude_4.php
[35]Mohaček-Grošev, V., Božac, R., Puppels, G. J. Vibrational spectroscopic characterization of wild growing mushrooms and toadstools. Spectrochimica Acta Part A 2001, 57, 2815-2829.
[36]Nguyen, T, H., Fleet, G, H., Rogers, P, L. Composition of the cell walls of several yeast species. Appl. Microbiol. Biotechnol. 1998, 50, 206-212.
[37]Šandula, J., Kogan, G., Kačuráková, M., Machová, Eva. Microbial (1→3)-β-D-glucans,their reparation, physico-chemical characterization and immunomodu-latory activity. Carbohydr. Polym. 1999, 38, 247-253.
[38]堀口博. 赤外吸光圖說總覽. 三共出版株會社 1973, 265-270.
[39]Lin S. Y., Li M. J., Liang R. C. Non-destructive analysis of the conformational changes in human lens lipid and protein structures of the immature cataracts associated with glaucoma. Spectrohimica Acta Part A 1998, 54, 1509-1517.
[40]Wüthrich, Kurt. NMR of Proteins and Nucleic Acids. Johns wiley andsons. 1986.
[41]亞利桑那州立大學http://asnmr4.la.asu.edunmrpdfgramlab_2004_week1.pdf
[42]Feuillat, M. Yeast Macromolecules: Origin, Composition, and Enological Interest. Am J. Enol. Vitic. 2003, 54, 211-213.
[43]Anthonle, K. Emulsifier from yeast. European Patent Application 1997, 0790316.
[44]Wang, Y. J., Yao, S. J., Guan, Y. X. A novel process for preparation of (1→3)--D-glucan sulphate by a heterogeneous reaction and its structural elucidation. Carbohydr. Polym. 2005, 59, 93-99.
[45]程文明, 楊柏珍, 李春如. 文冠果果殼中兩種甾醇成分的結構研究. Chinese Traditional and Herbal Drug 2001, 32(3), 119~201.
[46]傅建龍. 一種新的海洋甾醇的分離和結構鑑定. 華南理工大學學報1994, 22(3), 120-122.
[47]Chemexper網站FTIR資料庫. http://chemeth.chemexper.com
[48]Gottlieb, H. E., Kotlyar, V., Nudelman, A. NMR chemical shifts of common laboratory solvents as trace impurities. J. Org. Chem. 1997, 62, 7512-7515.
[49]藍文健, 林翠梧, 蘇鏡娛. 圓裂短足軟珊瑚Cladiella krempfi中的甾醇. 高等學校化學學報 2003, 24, 2019-2021.　
[50]江勇, 劉蕾. 遠志的化學成分研究Ⅲ. 中國天然藥物 2003, 1, 142-145.
[51]Barrow1, K. D., Netting, A. G., Chilcott, T. C. Effects of singlet oxygen on membrane sterols in the yeast Saccharomyces cerevisiae. Eur. J. Biochem. 2000, 267, 1607-1618.
[52]徐石海, 賀煥華, 曾隴梅. 海綿 Spongia obigue中兩種新的多羥基甾醇的分離與結構鑑定. Chinese Traditional and Herbal Drug. 2001, 24, 172-174.
[53]SIGMA網站FTIR資料庫. http://www.sigmaaldrich.com
[54]Kollar, R., Reinhold, B. B., Petrakova, E. Architectureof the yeast cell wall. L(1→6)-glucan interconnects mannoprotein,L(1→3)-glucan, and chitin. J. Biol. Chem. 1997, 272, 17762-17775.
[55]Lanigan產品 http://www.aboutbetaglucan.com/best-beta-glucan.asp
[56]Lanigan產品http://www.aboutbetaglucan.com/analysis.asp
[57]Ohno, N., Miura, T., Miura, N. N. Structure and biological activities of hypochlorite oxidized zymosan. Carbohydr. Polym. 2001, 44, 339 -349.
[58]Kim, Y. T., Kim. E. H., Cheong, C. Structural characterization of -D-(1,3)/(1,6)-linked glucans using NMR spectroscopy. Carbohydr. Res. 2000, 328, 331-341.
[59]Carbonero, E. R., Sassaki, G. L. Comparative studies of the polysaccharides isolated from lichenized fungi of the genus Cladonia: signi¢cance as chemotypes. FEMS Microbiol. Lett. 2001, 194, 65-69.