簡易檢索 / 詳目顯示

回結果列表
研究生： 沈建瑋
Shen, Chien-Wei
論文名稱： 以批次培養探討Mucor rouxii 生產γ-次亞麻油酸之研究
Studies on the γ-Linolenic Acid Production from Mucor rouxii in Batch Mode
指導教授： 吳文騰
Wu, Wen-Teng
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年： 2007
畢業學年度： 95
語文別： 中文
論文頁數： 79
中文關鍵詞： 油質性真菌 、γ-次亞麻油酸 、多元不飽和脂肪酸
外文關鍵詞： polyunsaturated fatty acids, γ-Linolenic acid, oleaginous microorganism, Mucor rouxii
相關次數： 點閱：173  下載：1 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 摘要

    Mucor rouxii BCRC 30546是一可生產大量多元不飽和脂肪酸（Polyunsaturated Fatty Acids, PUFAs）之油質性真菌。這些多元不飽和脂肪酸油脂裡，富含了人體不可或缺的γ-次亞麻油酸 （γ-Linolenic Acid,GLA），目前以微生物生產GLA油脂之商業化產品並不多見，故以微生物生產GLA有其發展潛力。
    大部分的油脂性微生物在累積油脂時，菌體的生長與油脂累積間的關係並非正相關，本研究利用批式培養的模式定性觀察到M. rouxii在累積油脂時的代謝型態不同於其他油脂性真菌，因而進一步採取在生化程序中常用的非線性Monod模型：dL/dt=α dX/dt+βX，來描述菌體生長與油脂累積間的關係並定量出菌體生長與總油脂產量間正相關的程度，經由實驗數據與生長動態模型間的擬合，分別得到α與β的值分別為0.2633以及0.0020，由於α大於β，証明了此一菌體在累積油脂時確為接近生長正相關的模式（growth associated），而非一般常見的非生長相關。
    藉著此一發現，可以推測初始培養基的組成將同時影響到後續菌體的生長與油脂累積的情況，因而利用曲面擬合的方式來決定最適化初始培養營養源的濃度。比較菌體乾重、細胞內油脂含量與總GLA產量的實驗值與預測值，可以發現其相對誤差分別為0.452%、2.287%與5.233%左右，實驗結果顯示出此模型可以有效的計算出最適培養基的濃度、菌體量與GLA的量。

    Abstract

    Mucor rouxii BCRC 30546 is an oleaginous fungus producing considerable quantities of polyunsaturated fatty acids (PUFAs). γ-Linolenic acid (GLA) is one of essential fatty acids in the fungus. It has potential for using oleaginous microorganism to produce GLA due to there are few single cell oils rich in GLA commercialized presently.
    In order to achieve lipid accumulation, the fermentation strategies of most oleaginous microorganisms are cultivated in the non-growth associated mode. But the strain Mucor rouxii cultivated in this study expresses the production lipid with the growth associated mode. Since this situation is rare, it is necessary to quantity the degree of growth association. In biochemical process, Monod model is used to describe the microbial growth kinetics, and the product model can be express as: dL/dt=α dX/dt+βX. If α is larger than β, this biosystem is determined as growth associated mode. Since the actual value of α and β in M. rouxii fermentation are estimated as 0.2633 and 0.0020 respectively, it implies that this system should be defined as growth associated mode.
    Based on this result, it indicates that the total lipid content is associated with the biomass growth, which is strongly influenced by the initial culture conditions. For getting the optimal initial culture conditions for biomass growing and lipid production, the curve fitting method is employed to optimize the initial concentrations of carbon and nitrogen sources. After the optimal initial conditions are calculated and conducted in the experimental verification, the relative error between prediction and experimental results are 0.452%、2.287% and 5.233% for biomass yield、cellular lipid content and total GLA content.

    目錄 目錄.....................................................Ⅰ 表目錄...................................................Ⅴ 圖目錄...................................................Ⅵ 第一章 緒論..............................................1 1.1 前言................................................1 1.2 研究目的............................................4 1.3 研究動機............................................5 1.4 多元不飽和脂肪酸與γ-次亞麻油酸......................7 1.4.1 多元不飽和脂肪酸之簡介...........................7 1.4.2 不飽和脂肪酸的應用...............................9 1.4.3 γ-次亞麻油酸（γ-Linolenic acid，GLA）...........10 1.5 利用微生物生產多元不飽和脂肪酸的優勢...............13 1.5.1 多元不飽和脂肪酸之微生物條件....................14 1.5.2 油質性真菌 Mucor rouxii.........................16 1.5.3 微生物產油機制..................................17 1.6 油質性真菌產油量之培養.............................20 1.7 論文之章節.........................................23 第二章 研究材料與方法...................................24 2.1 研究材料...........................................24 2.1.1 菌株來源........................................24 2.1.2 培養基..........................................24 2.1.3 儀器設備........................................27 2.2 研究方法...........................................28 2.2.1 菌種保存........................................28 2.2.2 前培養條件......................................29 2.2.3 微生物生長狀況影響的探討........................29 2.2.4 兩階段溫度實驗..................................30 2.2.5 不同起始碳氮源濃度實驗..........................31 2.3 分析方法...........................................32 2.3.1 細胞乾重分析方法................................32 2.3.2 葡萄糖濃度分析方法..............................32 2.3.3 脂質萃取與定義方法..............................34 2.3.4 脂肪酸分析條件..................................37 第三章 實驗結果與討論...................................38 3.1 Mucor rouxii之脂肪酸分析結果.......................39 3.2 批次實驗...........................................40 3.2.1 碳源限制下對微生物生長狀況影響的探討............40 3.2.2 氮源限制下對微生物生長狀況影響的探討............44 3.2.3 探討溫度變化對微生物生長的影響..................48 第四章 微生物生長模型...................................55 4.1 微生物生長與代謝物模型.............................55 4.1.1 模型的定義......................................55 4.1.2 模型的建立與參數估計............................57 4.1.3 模型的驗證......................................59 4.2 最適化條件的探討...................................65 4.2.1 迴歸模型參數之估計..............................65 4.2.2 二階曲面擬合之極值分析..........................68 4.2.3 菌體乾重的二階曲面函數和極值分析................69 4.2.4 細胞內油脂含量的二階曲面函數和極值分析..........70 4.2.5 γ-次亞麻油酸總產量的二階曲面函數和極值分析......71 4.2.6 實驗的驗證......................................72 第五章 結論與未來展望...................................74 5.1 結論...............................................74 5.2 未來展望...........................................75 參考文獻.................................................76 表目錄 表 1.1 商業化之多元不飽和脂肪酸產品及公司.................3 表 1.2 含PUFA 之脂質的商品在生物醫學及藥物食品上的應用....9 表 1.3 GLA在臨床醫療上之應用............................12 表 1.4 良好工業化生產PUFA之微生物條件....................15 表 2.1 液態培養碳氮源組成................................25 表 2.2 液態培養基鹽類組成................................26 表 2.3 實驗儀器及設備....................................27 表 3.1 不同初始碳氮源濃對於微生物生長的影響(培養48小時)..52 表 3.2 不同初始碳氮源濃對於微生物生長的影響(培養72小時)..53 表 4.1 模型中之參數......................................56 表 4.2 參數估計值........................................58 表 4.3 本系統實際模擬出的判定係數(R2)....................64 表 4.4 假設本系統為完全高度相關模擬出的判定係數(R2)......64 表 4.5 實驗值與預測值的相對誤差..........................72 表 4.6 利用γ-次亞麻油酸總產量交叉驗證細胞乾重與細胞內油脂 含量..............................................73 圖目錄 圖 1.1 油脂微生物累積油脂過程.............................6 圖 1.2 多元不飽和脂肪酸的生物合成途徑.....................8 圖 1.3 γ-次亞麻油酸結構..................................10 圖 1.4 citrate/malate cycle..............................18 圖 1.5 油質（脂肪酸）的累積機制..........................19 圖 2.1 葡萄糖檢量線......................................33 圖 2.2 脂肪酸分析之樣品前處理流程圖......................36 圖 3.1 脂肪酸GC分析圖：(a) Mucor rouxii油脂之脂肪酸成分； (b)定量用C15:0標準品；(c)定性用脂肪酸標準品.......39 圖 3.2 碳源限制的培養情況下，葡萄糖濃度消耗與細胞乾重隨 時間變化的過程....................................42 圖 3.3 碳源限制的培養情況下，細胞乾重與細胞體內油脂含量 隨時間變化的過程..................................43 圖 3.4 氮源限制的培養情況下，葡萄糖濃度消耗與細胞乾重隨 時間變化的過程....................................46 圖 3.5 氮源限制的培養情況下，細胞乾重與細胞體內油脂含量 隨時間變化的過程..................................47 圖 3.6 不同溫度變化下細胞乾重隨時間變化的過程............49 圖 3.7 不同溫度變化下細胞體內油脂含量隨時間變化的過程 ...50 圖 3.8 細胞體內油脂含量隨初始基質碳氮比不同的變化........54 圖 4.1 細胞乾重隨時間變化的過程..........................60 圖 4.2 細胞內油脂含量隨時間變化的過程....................61 圖 4.3 葡萄糖濃度隨時間變化的過程........................62 圖 4.4 細胞內油脂含量隨時間變化的過程(β=0)...............63

    參考文獻

    Alonso D. L. and Maroto F. G. "Plants as Chemical Factories for the Production of Polyunsaturated Fatty Acids." Biotechnology Advances 18: 481-497(2000).

    Bajpai P., Bajpai P. K., Ward O. P. "Optimization of Culture Condition for Production of Eicosapentaenoic Acid by Mortierella Elongata NRRL 5513." Journal of Industrial Microbiology 9: 1-18(1992).

    Carvalho Patrícia de Oliveira, Oliveira Joaquim Gilberto de "Enhancement of Gamma-Linolenic Acid Production by the Fungus Mucor sp LB-54 by Growth Temperature." Revista de Microbiologia 30: 170-175(1999).

    Certik M. and Shimizu S. "Biosynthesis and Regulation of Microbial Polyunsaturated Fatty Acid Production." Journal of Bioscience and Bioengineering 87(1): 1-14(1999).

    Eroshin V. K., Satroutdinov A. D., Dedyukhina E. G., Chistyakova T. I. "Arachidonic Acid Production by Mortierella Alpina with Growth-Coupled Lipid Synthesis." Process Biochemistry 35: 1171-1175(2000).

    Galbraith J. C. and Smith J. E. "Filamentous Growth of Aspergillus Niger in Submerged Shake Culture." Trans. Br. Mycol. Soc. 52: 237–246(1969).

    Gellerman J. L. and Schlenk K. "Methyl Directed Desaturation of Arachidonic Acid to Eicosapentaenoic Acid in the Fungus Saprolegnia Parasitica." Biochimica et Biophysica Acta 573: 23-30(1979).

    Gill L. and Valivety R. "Polyunsaturated Fatty Acids,Part1:Occurrence,Biological Activities and Applications " TIBTECH 15: 401-409(1997).

    Hansson L. and Dostàlek M. "Effect of Culture Conditions on Mycelial Growth and Production of γ-linolenic Acid by the Fungus Mortierella Ramanniana." Applied Microbiology and Biotechnology 28: 240-246(1988).

    Horrobin D. F. and Huang Y. S. "The Role of Linolenic Acid and Its Metabolites in the Lowering of Plasma Cholesterol and the Prevention of Cardiovascular Disease." International Journal of Cardiology 17: 241-255 (1987).

    Jantti J., Seppala E., Vapaatalo H., Isomaki H. "Evening Primrose Oil and Olive Oil in Treatment of Rheumatoid Arthritis." Clinical rheumatology 8: 238-244(1989).

    Jiang W. G., Hisocx S., Hallett M. B., Scott C., Horrobin D. F., Puntis M .C. A. "Inhibition of Hepatocyte Growth Factor-Induced Motility & In-Vitro Invation of Human Colon-Cancer Cells by Gamma-Linolenic Acid." British Journal of Cancer: BJC 71(4): 744-752(1995).

    Kavadia A., Komaitis M., Chevalot I., Blanchard F., Mark I., Aggelis G. "Lipid and γ-Linolenic Acid Accumulation in Strains of Zygomycetes Growing on Glucose." Journal of the American Oil Chemists' Society 78: 341-346(2001).

    Lindberg A. M. and Hansson L. "Production of γ-Linolenic Acid by the Fungus Mucor Rouxii on Cheap Nitrogen and Carbon Sources." Applied Microbiology and Bioteclmology 36: 26-28(1991).

    McGregor L., Smith A. D., Sidey M., Belin J., Zilkha K. J. "Effects of Dietary Linoleic Acid and Gamma Linolenic Acid on Platelets of Patients with Multiple Sclerosis." Acta Neurologica Scandinavica 80(1): 23-27(1989).

    McMurrough I., Flores-Carreon A., Bartnicki-Garcia S. "Pathway of Chitin Synthesis and Cellular Localization of Chitin Synthetase in Mucor rouxii." The Journal of Biological Chemistry 246: 3999-4007(1971).

    Owen P. and Singh A. "Omega-3/6 Fatty Acids: Alternative Sources of Production." Process Biochemistry 40: 3627–3652(2005).

    Papanikolaou S., Sarantou S., Komaitis M., Aggelis G. Repression of Reserve Lipid Turnover in Cunninghamella Echinulata and Mortierella Isabellina Cultivated in Multiple-Limited Media." Journal of Applied Microbiology 97: 867–875(2004).

    Papanikolaou S., Komaitis M., Aggelis G. "Single Cell Oil (SCO) Production by Mortierella Isabellina Grown on High-Sugar Content Media." Bioresource Technology 95: 287–291(2004).

    Puolakka J., Mäkäräinen L., Viinikka L., Ylikorkala O. Biochemical and clinical effects of treating the premenstrual syndrome with prostaglandin synthesis precursors." Journal of Reproductive Medicine 30: 149-153(1985).

    Ratledge C. "Yeasts,moluds,algae and bacteria as sources of lipids. " Technological advances in Improved and alternative sources of Lipids. Kamel B. S. and Kakuda Y. (1994).

    Ratledge C. "Fatty Acid Biosynthesis in Microorganisms being Used for Single Cell Oil Production." Biochimie 86: 807–815(2004).

    Romero J. A. and Ruiz-Herrera J. "Purification of Fatty Acid Synthetase from the Yeast Phase of Mucor Rouxii." FEMS Microbiology Letters 44: 243-248(1987).

    Sajbidor J., Certik M., Dobronova S. "Influence of Different Carbon Sources on Growth, Lipid Content and Fatty Acid Composition in Four Strains Belonging to Mucorales." Biotechno!ogy Letters 10: 347-350(1988).

    Schalin-Karrila M., Mattila L., JANSEN C. T., Uotila P. "Evening Primrose Oil in the Treatment of Atopic Eczema: Effect on Clinical Status, Plasma Phospholipid Fatty Acids and Circulating Blood Prostaglandins." British Journal of Dermatolog 117: 11-19(1987).

    Shimizu S., Kawashima H., Akimoto K., Shinmen Y., Yamada H. "Production of Eicosapentaenoic Acid by Mortierella Fungi." Journal of the American Oil Chemists' Society 65: 1445-1459(1988).

    Teresa Lopes da Silva, Pinheiro H. M., José Carlos Roseiro "Stress-Induced Morphological and Physiological Changes in γ-Linolenic Acid Production by Mucor Fragilis in Batch and Continuous Cultures." Enzyme and Microbial Technology 32: 880–888(2003).

    Somashekar D., Venkateshwaran G., Sambaiah K., Lokesh B. R. "Effect of Culture Conditions on Lipid and Gamma-Linolenic Acid Production by Mucoraceous Fungi." Process Biochemistry 38: 1719-1724(2002).

    White S. A., Farina P. R., Fultons I. "Production and Isolation of Chitosan from Mucor Rouxii." Applied and Environmental Microbiology 38: 323-328(1979).

    Yan G. and Viraraghavan T. "Heavy-Metal Removal from Aqueous Solution by Fungus Mucor Rouxii." Water Research 37: 4486–4496(2003).

    Yongmanitchai W. and Ward O. P. "Growth and Omega-3 Fatty Acid Production by Phaeodactylum Tricornnyum under Different Culture Conditions." Applied and Environmental Microbiology 57: 419-426(1991).

    柯銀府 蘇力菌醱酵系統之動態分析, 清華大學化學工程研究所碩士論文(2000).

    宋逸詩 以影像分析技術探討Mucor rouxii 生產γ-次亞麻油酸之研究, 清華大學化學工程研究所碩士論文(2005).

    下載圖示
    2008-08-02公開
    QR CODE