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研究生： 陳怡倩
Yi-Qing Chen
論文名稱： 利用批式液態培養來探討檸檬酸對裂褶菌生長及其多醣體生成影響之研究
指導教授： 徐敬衡
Chin-Hung Shu
口試委員:
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程與材料工程學系
Department of Chemical & Materials Engineering
畢業學年度： 89
語文別： 中文
論文頁數： 98
中文關鍵詞： 檸檬酸 、裂褶菌多醣 、裂褶菌
外文關鍵詞： schizophyllum commune, schizophyllan
相關次數： 點閱：13  下載：0 
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    • 裂褶菌多醣能有效提高生物體的免疫機能，活化老鼠體內巨噬細胞的吞噬能力，以及提升對小白鼠Sarcoma 180的腫瘤抑制率，可與西藥搭配治療用於幫助增強癌症病人的免疫系統以及抵抗腫瘤的能力，因此具有相當潛力被開發為未來醫療用品或作為近年來國內頗熱門的保健食品，視為未來的明星產品。
    為了增加裂褶菌多醣的產量，本研究以液態培養發酵的方式以期增加多醣產量，然而影響發酵的環境因子很多，其中本實驗選擇以檸檬酸為探討變因，其原因來自曾有文獻指出(Jana et al., 1995 )添加檸檬酸於微生物的發酵環境能提高菌體耗氧速率以促進菌體產生大量ATP以供產物多醣生成所需之能量來源，且有關檸檬酸對真菌發酵的探討研究不多，所以本研究將以檸檬酸探討其對裂褶菌菌絲生長與多醣生成所造成的影響， 同時並引用數學模式來計算不同系統所對應的動力學參數值，試著從參數裡推測檸檬酸與裂褶菌代謝機制的關係。
    本研究分別以搖瓶振盪及小型批式發酵槽的培養方式，採用之菌株為裂褶菌Schizophyllum commune ATCC 38548進行培養，以討論檸檬酸對菌絲生長及其多醣體生成的影響。
    實驗結果分為兩大部分，第一部份為搖瓶振盪的結果，首先為篩選碳源部份，本實驗以單位菌絲對多醣之轉化率(Yp/x)的值作比較，結果以添加檸檬酸的轉化率最高，其值為0.26，因此以檸檬酸探討其濃度梯度對裂褶菌菌絲產量以及多醣轉化率的關係，結果添加檸檬酸的實驗組其多醣轉化率(Yp/x)約為控制組的2.2倍；而以多醣總產量而言，以添加檸檬酸比例為0.1∼0.3％的產量最高 ，但是若添加檸檬酸濃度大於0.5%則會因為過分抑制菌絲生長而導致多醣產量減少，因此在搖瓶實驗裡添加適量的檸檬酸有促進多醣產量生成的結果。第二部份為發酵槽的實驗結果，目的探討檸檬酸對裂褶菌發酵系統的動力學參數值的關係，其結果發現隨著檸檬酸濃度升高，則動力學參數m值呈現上升的趨勢，因此從實驗的結果可以推論得到檸檬酸在裂褶菌發酵系統中是扮演促進多醣的生成愈趨近為ㄧ次代謝產物的角色。

    目錄 目錄Ⅰ 圖索引Ⅵ 表索引Ⅸ 第一章 緒論1 1.1研究背景1 1.2研究目的3 第二章 文獻回顧4 2.1 真菌多醣簡介4 2.1.1 真菌多醣的合成4 2.1.2 β-D-葡聚糖的免疫功能5 2.1.3真菌多醣之商業應用價值12 2.2裂褶菌14 2.3裂褶菌多醣15 2.3.1 簡介15 2.3.2裂褶菌水溶性多醣的應用17 2.4深層發酵培養20 2.5影響發酵的物理化學因子21 2.5.1培養基21 2.5.1.1碳源21 2.5.1.2氮源28 2.5.1.3碳氮比28 2.5.1.4無機鹽類29 2.5.1.5其他添加物30 2.5.2攪拌速率31 2.5.3溫度31 2.5.4 pH效應32 2.5.5黏度(viscosity)33 2.5.6溶氧值 (DO值)34 2.6 發酵動態行為35 2.6.1描述動態行為之數學模式36 2.6.1.1 菌絲生長37 2.6.1.2 產物生成38 2.6.1.3 基質消耗速率39 第三章 材料與方法40 3.1 實驗架構40 3.2實驗材料41 3.2.1 實驗試藥41 3.2.2 實驗裝置43 3.2.2.1發酵槽外部裝置圖43 3.2.2.2 發酵槽內部裝置圖44 3.2.2.3 實驗其他操作設備與規格45 3.3實驗方法47 3.3.1菌株(strain)47 3.3.2培養基組成47 3.3.3操作條件49 3.3.3.1 前培養49 3.3.3.2 主培養50 3.4 分析方法51 3.4.1發酵液處理流程51 3.4.2 菌體乾重處理51 3.4.3 應用高效率液相色層層析儀分析發酵液的成份52 3.4.4 裂褶菌多醣濃度分析54 第四章 結果與討論57 4.1 裂褶菌菌絲形態57 4.2 搖瓶實驗58 4.2.1 篩選適當碳源58 4.2.2 檸檬酸濃度之影響62 4.3 發酵槽培養試驗66 4.3.1 環境因子66 4.3.1.1控制發酵環境的pH值與否對產物的影響66 4.3.1.2 攪拌速率的影響69 4.3.2 檸檬酸效應70 4.3.2.1菌絲顆粒大小的比較70 4.3.2.2 菌絲生長73 4.3.3 檸檬酸濃度對多醣產量的影響77 4.3.3.1 多醣轉化率77 4.3.3.2多醣分子量分布79 4.3.3.3 多醣中性單糖成份測定82 4.3.3.4 發酵液代謝產物之分析83 4.4 動力學參數84 4.4.1 以Logistic Equation Model 模擬菌絲生長曲線84 4.4.2 產物動力學參數 m、n85 4.4.3 基質消耗動力學參數α、β之影響87 第五章 結論與建議89 5.1 結論89 5.2建議90 參考文獻91

    1.李明彥, “松杉靈芝浸漬發酵的培養條件對產物的影響“,國立臺灣大學農業化學研究所,碩士,1989。
    2.梁志欽, “松杉靈芝浸漬培養產生1,3-β-glucanase對菌體外醣的影響“, 國立臺灣大學農業化學研究所,碩士,1991。
    3.馬榮村,“ 10-氧-反-烯酸(ODA)對食藥用菇類菌絲生長之影響“,國立中興大學食品科學系,碩士,1999。
    4.柯銀府,“蘇力菌醱酵系統之動態分析 ” ,國立清華大學化學工程研究所,碩士,2000。
    5.張正芳,蘇茂森,陳宏遠,廖一久:“由Schizophyllum commune萃取之Beta-1,3-glucan應用於強化草蝦苗活力之研究 ”。水產研究。1993,3(2) , 125-132。
    6.游英欽, “以搖瓶振盪及小型發酵槽培養,探討培養基組成及物理化學因子對於靈芝多醣生成及生長形態變化之影響“,國立交通大學生物科技研究所,碩士,1995。
    7.黃賜源, “靈芝液態培養及氣舉式生化反應器應用之研究”, 私立東海大學化學工程研究所,碩士,1996。
    8.楊鴻銘,“ 製程研究與放大 ”, 化工, 44(1), 16-25, 1997。
    9.賴慶亮，“菇的化學、生化學 ”。國立編譯館。13-374，1997。
    10.網站”培基免疫革新網”，千禧龍國際生物科技股份有限公司網頁。
    11.Adachi, Y. ; Ohno, N. ; Ohsawa, M. ; Oikawa, S. ; Yadomae, T. : Macrophage activation in vitro by chemically cross-linked (1-3)-β-D-glucans. Chemical & Pharmaceutical Bulletin.1990,38, 988-922.
    12.Aouadi, S. ; Heyraud, A. ; Seigle-Murandi, F. ; Steiman, R. ; Kraus, J. ; Franz, G. : Structure and properties of an extracellular polysaccharides from Laetisaria arvalis evaluation of its antitumor avtivity. Carbohydrate Polymers. 1991, 16,155-165.
    13.Boa, J. M. ; LeDuy, A. : Pullulan from Peat Hydrolyzate Fermentation Kinetics. Biotechnology and Bioengineering. 1987, XXX, 463-470.
    14.Carlos Roseiro, J. ; Girio, F. M. ; Kara, A. ; Amaral Collaco, M. T. : Kinetic and metabolic effects of nitrogen, magnesium and sulphur restriction in Xanthomonas campestris batch cultures. Journal of Applied Bacteriology.1993, 75, 381-386.
    15.Carolan, G. ; Catley, B. J. ; Mcdougal, F. J. : The Location of Tetrasaccharide Units in Pullulan. Carbohydrate Research.1983, 114( 2), 237-243.
    16.Chain, E. B. ; Gualandi, G. ; Morisi, G. : Aeration studies. IV. Aeration condition in 3000-liter submerged fermentation with various microorganisms. Biotechnology and Bioengineering. 1966, 8, 595-619.
    17.Cogan, T. M. : Co-metabolism of citrate and glucose by Leuconostoc spp.: Effects on growth, substrates and products. Journal of Applied Bacteriology. 1987, 63, 551-558.
    18.Ellison, J. The Brewer.601-606,1973.
    19.Fevre, M. ; Rougier, M. : Beta-1-3-Glucan and Beta-1-4-Glucan Synthesis by Membrane-Fractions from the Fungus Saprolegnia. PLANTA.1981, 151( 3), 232-241.
    20.Forage, R. G. ; Harrison, D. E. F. ; Pitt, D. E. : Effect of environment on microbial activity. Comprehensive Biotechnology. 1985, 1, 253-279.
    21.Fujimoto, S. ; Orita, K.; Kimura, T.; Kondo, T.; Taguchi, T.; Yoshida, K.; Ogawa, N.; Furue, H. : Clinical evaluation of SPG (schizophyllan) as a therapeutic adjuvant after surgery of gastric cancer-controlled study by an envelope method. Gan To Kagaku Ryoho. 1983, 10, 1135-1145.
    22.Fujimoto, S. ; Furue, H. ; Kimura, T. ; Kondo,T.; Orita, K. ; Taguchi, T. ; Yoshida, K. ; Ogawa, N. : Clinical evaluation of schizophyllan adjuvant immunochemotherapy for patients with resectable gastric cancer-a randomized controlled trial. Jsn. J. Surg. 1984, 14, 286-292.
    23.Goel, A. ; Lee, J.; Domach, M. M. ; Ataai, M. M. : Suppressed acid formation by cofeeding of glucose and citrate in Bacillus cultures : Emergence of pyruvate kinase as a potential metabolic engineering site. Biotechnology Progress. 1995, 11, 380-385.
    24.Hamazaki, Y. ; Kuramoto, M. ; Okamura, K. ; Yajima, A. ; Higashi-iwai, H. ; Suzuki, M. : Studies on immunotherapy of uterine cervical cancer by administration of schizophyllan (SPG) . Nippon Sanka Fujinka Gakkai Zasshi. 1980, 32, 929-935.
    25.Ikekawa, T.; Uehara, N.; Maeda, Y.; Nakanishi, M.; Fukuoka, F. : Antitumor activity of aqueous extracts of edible mushroom. Cancer Research. 1969, 29, 734-735.
    26.Jana, A. K. ; Ghosh, P. : Xanthan biosynthesis in continuous culture : citric acid as an energy source. Journal of Fermentation and Bioengineering. 1995, 80, 485-491.
    27.Jana, A. K.; Ghosh, P. : Effect of citric acid on the biosynthesis and composition of xanthan. The Journal of General and Applied Microbiology.. 1999, 45, 115-120.
    28.Kang, A.; Wang, Y.; Harvey, L. M; McNeil B.: Effect of air flow rate on scleroglucan synthesis by Sclerotium glucanicum in an airlift bioreactor with an internal loop. Bioprocess Engineering. 2000, 23, 69-74.
    29.Kitamura, S.; Hori, T.; Kurita, K.; Takeo, K.; Hara, C.; Itoh, W.; Tabata, K.; Elgsaeter, A.; Stokke, B. T. : an Antitumor Branched β-(1à3)-D-glucan from a water extract of fruiting bodies of Cryptoporus Volvatus. Carbohydrate Research. 1994, 263, 111-121.
    30.Komatsu, N. ; Nagumo, N. ; Okubo, S. ; Koike, K. : Protective effect of schizophyllan on bacterial infections of mouse. Jan. J. Abtibiot. 1973, 26, 277-283.
    31.Lacroix, C. ; LeDuy, A. ; Noel, G. ; Choplin, L. : Effect of pH on the batch fermentation of pullulan from sucrose medium. Biotechnology and Bioengineering. 1985, XXVII, 202-207.
    32.Lazic, M. L. ; Veljkovic, V. B. ; Vucetic, J. I. ; Vrvic, M. M. : Effect of pH and aeration on dextran production by Leuconostoc mesenteroides. Enzyme and Microbial Technology. 1993, 15, 334-338.
    33.Matsuoka, H. ; Seo, Y. ; Wakasugi, H. ; Saito, T. ; Tomoda, H. : Lentinan potentiates immunity and prolongs the survival time of some patients. Anticancer Research.1997, 17, 2751-2755.
    34.Misaki, A. ; Kawaguchi, K. ; Miyaji, H. ; Magae, H. ; Hokkoku, S. ; Kakuta, M. ; Sasaki, T. : Structure of pestalotan, a highly branched (1-3)-β-D-glucan elaborated by Pestalotia sp. 815, and the enhancement of its antitumor activity by polyol modification of the side chains. Carbohydrate Research. 1984, 129, 209-227.
    35.Miyazaki, T. ; Yadomae, T. ; Yamada, H. ; Oikawa, N. : An endo-(1-3)- β-D-glucanase from Mucorhiemalis. Carbohydrate Research. 1977, 55, 65-72.
    36.Mohammad, F.H.A.; Badr-Eldin, S.M.; El-Tayeb, O.M.; Abdel-Rahman, O.A. Polysaccharides production by Aureobasidium Pullulans III. The influence of initial sucrose concentration on batch kinetics. Biomass and Bioenergy. 1995, 8, 121-129.
    37.Moraine, R. A. ; Rogovin, P. : Kinetics of the xanthan fermentation . Biotechnology and Bioengineering. 1973, 15, 225-237.
    38.Mulchandani, A. ; Luong, J. H. T. : Batch Kinetics of Microbial Polysaccharide Biosynthesis. Biotechnology and Bioengineering. 1988, 32, 639-646.
    39.Munzberg, J. ; Rau, U.; Wagner, F. : Investigations on the regioselective hydrolysis of a branched β-1,3-glucan. Carbohydrate Polymers. 1995, 27, 271-276.
    40.Nakano, T. ; Oka, K. ; Hanba, K. ; Morita, S. : Intratumoral administration of sizofiran activates Langerhans cell and T-cell infiltration in cervical cancer. Clinical of Immunology and Immunopathology. 1996, 79, 79-86.
    41.Okamura, K. ; Suzuki, M. ; Chihara, T.; Fujiwara, A. ; Fukuda, T. ; Goto, S. ; Ichinohe, K. ; Jimi, S. ; Kasamatsu, T. ; Kawai, N. : Clinical evaluation of schizophyllan combined with irradiation in patients with cervical cancer-a randomized controlled study. Cancer. 1986, 58, 865-872.
    42.Peters, H. U. ; Herbst, H. ; Hesselink, P. G. M. ; Lunsdorf, H. : The influence of agitation rate on xanthan production by Xanthomonas Campestris. Biotechnology and Bioengineering. 1989, 34, 1393-1397.
    43.Prokop, A. ; Rapp, P. ; Wagner, F. : Production of extracellular β-1,3-/β-1,6-Glucan by Mono- and Dikaryons of Schizophyllum commune. Experimental mycology. 1992, 16, 197-206.
    44.Quigley, D.R. ; Selitrennikoff, C.P. : Beta-(1-)3) Glucan Synthase Activity of Neurospora-Crassa - Stabilization and Partial Characterization.Experimental mycology.1984, 8(3), 202-214.
    45.Rau, U. ; Gura, E. ; Olszewski, E. ; Wagner, F. : Enhanced glucan formation of filamentous fungi by effective mixing, oxygen limitation and fed-batch processing. Journal of Industrial Microbiology. 1992, 9, 19-26.
    46.Schilling, B. M. ; Rau, U. ; Fankhauser, P. Modeling and scale-up of the unsterile scleroglucan production process with Sclerotium rolfsii ATCC 15205. Bioprocess Engineering. 1999, 20, 195-201.
    47.Sone, Y. ; Okuda, R. ; Wada, N. ; Kishida, E. ; Misaki, A. : Structure and antitumor activities of polysaccharides isolated from fruiting body and growing culture of mycelium of Ganoderm alucidum. Agricultural and Biological Chemistry. 1985,49, 2641-2653.
    48.Souw, P. ; Demain A. L. : Role of citrate in Xanthan production by Xanthomonas campestris. Journal of Fermentation Technology.1980, 58, 411-416.
    49.Sonnleitner, B. : Dynamic adaptation of microbes. Journal of Biotechnology. 1998,65,47-60.
    50.Stasinopoulos, S. J. ; Seviour, R. J. : Exopolysaccharide production by Acremonium persicinum in stirred-tank and air-lift fermentors. Applied Microbiology Biotechnology. 1992, 36, 465-468.
    51.Tako, M. : Molecular Origin for the Thermal Stability of Schizophyllan. Polymer Gels and Networks. 1996, 4, 303-313.
    52.Taurhesia, S. ; McNeil, B. : Pysicochemical factors affecting the formation of the biological response modifier Scleroglucan. Journal of Chemical Technology and Biotechnology. 1994, 59, 157-163.
    53.Tseng, T.C. ; Shiao, M.S., ; Shieh,Y.S. ; Hao, Y.Y. : Study on Ganoderma lucidum 1. Liquid Culture and Chemical Composition of Mycelium. Botanical Bulletin of Academia Sinica. 1984, 25,149-157.
    54.Wang, Y. ; McNeil, B. : pH effects on exopolysaccharide and oxlic acid production in cultures of Sclerotium glucanium. Enzyme and Microbial Technology. 1995, 17, 124-130.
    55.Wang, Y.; McNeil, B. : Scleroglucan. Critical Reviews in Biotechnology. 1996, 16(3), 185-215.
    56.Wessels, J. G. H. ; Sietsma, J. H. : Wall structure and growth in Schizophyllum commune. Chemical structure of the hyphal wall.
    57.Yan, J. ; Vaclav, V. ; Xia, Y. ; Coxon, A. ; Carroll, M. C. ; Mayadas, T. N. ; Ross, G.D. : The Journal of Immunology.1999, 163, 3045-3052.
    58.Yang, F. C. ; Liau, C. B. : The influence of environmental conditions on polysaccharide formation by Ganoderma lucidum in submerged cultures. Process Biochemistry. 1998, 33, 547-553.
    59.Yoshioka, Y. ; Uehara, N. ; Saito, H. : Conformation-Dependent Change in Antitumor-Activity of Linear and Branched (1-)3)-Beta-D-Glucans on the Basis of Conformational Elucidation by C-13 Nuclear-Magnetic-Resonance Spectroscopy. Chemical & Pharmaceutical Bulletin. 1992, 40, 1221-1226.
    60.Young, S. H. ; Jacobs, R. R. : Sodium hydroxide-induced conformational change in schizophyllan detected by the fluorescence dye, aniline blue. Carbohydrate Research. 1998, 310, 91-99.

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