臺灣博碩士論文加值系統

玉米黑穗菌是一種寄生在玉米上的寄生菌，在玉米上生成膨大的腫瘤組織，在墨西哥當地被視為一種傳統美食。研究指出，此菌會代謝色胺酸，生成如indole-3-acetic acid(IAA)以及pityriarubin類化合物，其分別在動物實驗和細胞實驗上證實具有抗發炎效果。因此，本研究即期望利用此菌，以IAA為指標，使用攪拌式發酵槽生產具有抗發炎活性的玉米黑穗菌發酵液。經過一系列的最適化研究，結果發現在培養溫度24 ℃、起始pH 4、培養基組成為含有1 g/L色胺酸的PDB培養基額外添加20 g/L葡萄糖、扇葉攪拌速率900 rpm、通氣量0.5 vvm的條件下，經過32小時的發酵時間，可生產出含有30.8±4.5 μg/ml IAA (HPLC定量)的玉米黑穗菌發酵液，並以RAW264.7細胞模式證明此發酵液在10%的添加量下，其抑制發炎因子NO的比例達81.0±0.3%。

Ustilago maydis is a pathogen infecting on corn and induces tumor tissue formation. Althoug it is a pathogen, it is seen as a traditional cusine in Mexico. It can metabolize trytophan into indole-3-acetic acid (IAA) and pityriarubin with anti-inflammation bioactivity. Therefore, the goal of this research was to produce fermentation broth of Ustilago maydis possessing anti-inflammation function using indole-3-acetic acid as marker in a 5 L stirred tank fermentor. After optimization process, it was found that the fermentation process would produce 30.8±4.5 μg/ml IAA in the broth using PDB medium supplemented with 1g/L tryptophan and 20 g/L glucose with initial pH at 4. The optimal fermentation conditions in the stirred tank bioreactor are: agitation rate at 900 rpm; aeration rate at 0.5 vvm, and 24℃ for 32 h. Moreover, the broth (10%) can inhibit nitrite production up to 81.0±0.3% in RAW264.7 inflammation cell model.

第一章、文獻整理 1
第一節、玉米黑穗菌介紹 1
(一)玉米黑穗菌之簡介 1
(二)玉米黑穗菌之生長史 1
(三)玉米黑穗菌之一般成份 4
(四)玉米黑穗菌之活性物質 6
(a)多醣體 6
(b)糖脂質 6
(c)螯鐵蛋白(Siderophore) 6
(d) Pityriarubin 6
(e)Indole-3-acetic acid (IAA) 7
(五)玉米黑穗菌之液態培養製程 10
(a)碳源 10
(b)氮源 10
(c)溫度 10
(d)pH值 10
第二節、Indole-3-acetic acid (IAA)介紹 13
(一)IAA的生合成 13
(二)玉米黑穗菌代謝tryptophan生成IAA 14
第三節、生化反應器介紹 17
(一)生化反應器簡介 17
(二)以攪拌式發酵槽生產玉米黑穗菌代謝產物實例 18
第四節、發炎反應 19
(一)發炎反應簡介 19
(a)發炎反應 19
(b)巨噬細胞的活化 19
(二)發炎因子介紹 22
(a)促發炎細胞激素( pro-inflammatory cytokines) 22
(b)高反應性含氧分子( reactive oxygen species, ROS ) 22
第二章、研究目的 24
第三章、實驗架構 25
第一節、以搖瓶培養方式探討不同溫度和起始pH對IAA產量的影響 25
第二節、以發酵槽探討生產Indole-3-acetic acid的條件 26
第三節、RAW264.7細胞模式探討發酵液的抗發炎活性與HPLC活性成份分析 27
第四章、材料與方法 28
第一節、實驗材料 28
(一)實驗菌株 28
(二)實驗細胞株 28
(三)藥品 28
(四)器材與儀器 28
第二節、實驗方法 29
(一)菌種的保存與活化 29
(a)微生物培養基配製 29
(b)菌種活化 29
(c)菌種保存 29
(二)利用搖瓶與發酵槽培養玉米黑穗菌 29
(a)樣品培養基配製 29
(b)搖瓶培養 30
(c)發酵槽培養 30
(三)分析方法 30
(a)菌體生長情形測定 30
(b)菌體乾重測定 30
(c)pH測定 30
(d)還原糖測定 30
(e)IAA測定 31
(f)High-performance liquid chromatograph分析 32
(四)RAW264.7細胞培養與保存 33
(a)細胞培養基配製 33
(b)細胞之活化 33
(c)細胞之繼代 33
(d)細胞之冷凍保存 33
(五)RAW264.7細胞實驗 34
(a)細胞實驗樣品製備 34
(b)細胞計數 34
(c)NO抑制率測定 34
(d)細胞存活率---MTT assay 35
第五章、結果與討論 36
第一節、以搖瓶培養探討不同溫度和起始pH對IAA產量及其NO抑制率的影響 36
(一)以搖瓶培養方式探討不同溫度和起始pH對IAA產量的影響 36
(a)菌體生長情形變化(OD600) 36
(b)pH變化情形 37
(c)還原糖消耗變化 37
(d)IAA產量變化 38
(二)以搖瓶方式生產出之發酵液對於RAW264.7的NO抑制率影響 49
(a)24 ℃搖瓶生產出之發酵液對NO抑制率與細胞存活率之影響 49
(b) 30 ℃搖瓶生產出之發酵液對NO抑制率與細胞存活率之影響 52
(三) 小結 55
第二節、以發酵槽探討生產Indole-3-acetic acid的條件 56
(一)探討額外添加萄葡糖對於IAA產量的影響 56
(a)額外添加葡萄糖對菌體生長影響(以OD600和菌體乾重為指標) 56
(b)pH變化情形 56
(c)溶氧變化情形 57
(d)還原糖消耗情形 57
(e)IAA生產情形 57
(二)探討不同通氣量對於IAA產量的影響 65
(a)菌體生長情形變化(OD600和菌體乾重) 65
(b)pH變化情形 65
(c)溶氧變化情形 66
(d)還原糖消耗情形 66
(e)IAA變化情形 66
(三)探討不同扇葉轉速對於IAA產量的影響 74
(a) 菌體生長情形變化(OD600和菌體乾重) 74
(b)pH變化情形 74
(c)溶氧變化情形 75
(d)還原糖消耗情形 75
(e)IAA變化情形 76
(四)小結 83
第三節、RAW264.7細胞模式探討發酵液的抗發炎活性與HPLC活性成份分析 84
(一)HPLC活性成份分析 84
(a)Tryptophan與IAA之HPLC定量 84
(b)HPLC圖譜分析和全波長分析 85
(二)以RAW264.7細胞模式探討發酵液的抗發炎活性 94
(a)發酵樣品之NO抑制率 94
(b)發酵樣品對RAW264.7細胞存活率影響 94
(c)G20-0.5-900產率最佳樣品與NO抑制率之劑量關係 94
(三)小結 98
第六章、結論 99
第七章、參考文獻 100

1.白日霞; 薛業, 一種水溶黑粉菌多糖的結構和抗腫瘤活性研究(英文). 中國藥物化學雜志 1999, 20-23.
2.張寶艷; 高增貴; 莊敬華; 張小飛; 趙輝, 玉米絲黑穗病菌菌絲在寄主體內的生長過程. 種子 2008, 8-9.
3.連耘愷. 黑龍菇菌種發酵液生物活性探討. 南台科技大學, 台南縣, 2005.
4.陳智偉, 在多重網板氣舉式反應器中以饋料批次培養生產幾丁聚醣. 國立清華大學, 新竹市, 2000.
5.董純婷, 網狀內管氣舉式反應器混合效能之探討. 國立清華大學, 新竹市, 2001.
6.Banuett, F., Pathogenic development in Ustilago maydis: a progression of morphological transitions that results in tumor formation and teliospore production. Mycol. Ser. 2002, 15, 349-398.
7.Bartnicki-Garcia, S., III. Mold-yeast dimorphism of mucor. Microbiol. Mol. Biol. Rev. 1963, 27, 293.
8.Brandl, M.; Lindow, S., Cloning and characterization of a locus encoding an indolepyruvate decarboxylase involved in indole-3-acetic acid synthesis in Erwinia herbicola. Appl. Environ. Microbiol. 1996, 62, 4121.
9.Brandl, M. T.; Lindow, S. E., Environmental signals modulate the expression of an indole-3-acetic acid biosynthetic gene in Erwinia herbicola. Mol. Plant. Microbe Interact. 1997, 10, 499-505.
10.Broek, A. V.; Gysegom, P.; Ona, O.; Hendrickx, N.; Prinsen, E.; Van Impe, J.; Vanderleyden, J., Transcriptional analysis of the Azospirillum brasilense indole-3-pyruvate decarboxylase gene and identification of a cis-acting sequence involved in auxin responsive expression. Mol. Plant. Microbe Interact. 2005, 18, 311-323.
11.Budde, A. D.; Leong, S. A., Characterization of siderophores from Ustilago maydis. Mycopathologia 1989, 108, 125-133.
12.Chung, K. R.; Tzeng, D. D., Biosynthesis of indole-3-acetic acid by the gall-inducing fungus Ustilago esculenta. J. Biol. Sci. 2004, 4, 744-750.
13.Cohen, J., The immunopathogenesis of sepsis. Nature 2002, 420, 885-891.
14.Cornejo-Mazon, M.; Jaramillo-Flores, M. E.; Villa-Tanaca, L.; Hernandez-Sanchez, H., Optimization of Biomass Production by Ustilago maydis in Submerged Culture using Taguchi Experimental Design. Biotechnology 2008, 7, 818-821.
15.Costacurta, A.; Keijers, V.; Vanderleyden, J., Molecular cloning and sequence analysis of an Azospirilium brasilense indole-3-pyruvate decarboxylase gene. Mol. Genet. Genomics 1994, 243, 463-472.
16.Fluharty, A. L.; O''Brien, J. S., A mannose-and erythritol-containing glycolipid from Ustilago maydis. Biochemistry (Mosc). 1969, 8, 2627-2632.
17.Frautz, B.; Lang, S.; Wagner, F., Formation of cellobiose lipids by growing and resting cells of Ustilago maydis. Biotechnol. Lett 1986, 8, 757-762.
18.Gallay, P.; Heumann, D.; Le Roy, D.; Barras, C.; Glauser, M., Mode of action of anti-lipopolysaccharide-binding protein antibodies for prevention of endotoxemic shock in mice. Proc. Natl. Acad. Sci. 1994, 91, 7922.
19.Glickmann, E.; Dessaux, Y., A critical examination of the specificity of the Salkowski reagent for indolic compounds produced by phytopathogenic bacteria. Appl. Environ. Microbiol. 1995, 61, 793.
20.Goldsby, R.; Kindt, T.; Osborne, B.; Kuby, J., Leukocyte migration and inflammation. Immunology, 5th ed. WH Freeman & Co., New York, NY 2003, 338-360.
21.Graziewicz, M.; Wink, D. A.; Laval, F., Nitric oxide inhibits DNA ligase activity: potential mechanisms for NO-mediated DNA damage. Carcinogenesis 1996, 17, 2501.
22.Haskins, R., Biochemistry of the Ustilaginales: I. Preliminary cultural studies of Ustilago zeae. Can. J. Res. 1950, 28, 213-223.
23.Isoda, H.; Kitamoto, D.; Shinmoto, H.; Matsumura, M.; Nakahara, T., Microbial extracellular glycolipid induction of differentiation and inhibition of the protein kinase C activity of human promyelocytic leukemia cell line HL60. Biosci. Biotechnol. Biochem. 1997, 61, 609.
24.Isoda, H.; Shinmoto, H.; Kitamoto, D.; Matsumura, M.; Nakahara, T., Differentiation of human promyelocytic leukemia cell line HL60 by microbial extracellular glycolipids. Lipids 1997, 32, 263-271.
25.Isoda, H.; Shinmoto, H.; Matsumura, M.; Nakahara, T., Succinoyl trehalose lipid induced differentiation of human monocytoid leukemic cell line U937 into monocyte-macrophages. Cytotechnology 1995, 19, 79-88.
26.Jaiswal, M.; LaRusso, N. F.; Burgart, L. J.; Gores, G. J., Inflammatory cytokines induce DNA damage and inhibit DNA repair in cholangiocarcinoma cells by a nitric oxide-dependent mechanism. Cancer Res. 2000, 60, 184.
27.Jensen, J. B.; Egsgaard, H.; Van Onckelen, H.; Jochimsen, B. U., Catabolism of indole-3-acetic acid and 4-and 5-chloroindole-3-acetic acid in Bradyrhizobium japonicum. J. Bacteriol. 1995, 177, 5762-5766.
28.Jones, L.; Abdalla, D.; Freitas, J., Effects of indole-3-acetic acid on croton oil-and arachidonic acid-induced mouse ear edema. Inflamm. Res. 1995, 44, 372-375.
29.Karpuzoglu, E.; Ahmed, S. A., Estrogen regulation of nitric oxide and inducible nitric oxide synthase (iNOS) in immune cells: implications for immunity, autoimmune diseases, and apoptosis. Nitric Oxide 2006, 15, 177-186.
30. Kobayashi, G. S., Medoff, G., Maresca, B., Sacco, M. & Kumar, B. V. (1985). Studies on phase transition in the dimorphic pathogen Histoplasma capsdatum. In Fungal Dimorphism, with Emphasis on Fungi Pathogenic for Humans, pp. 69-91. Edited by P. J. Szaniszlo. New York: Plenum Press.
31.Koga, J.; Adachi, T.; Hidaka, H., Molecular cloning of the gene for indolepyruvate decarboxylase from Enterobacter cloacae. Mol. Gen. Genet. 1991, 226, 10-16.
32.Kramer, H. J.; Kessler, D.; Hipler, U. C.; Irlinger, B.; Hort, W.; Bodeker, R. H.; Steglich, W.; Mayser, P., Pityriarubins, novel highly selective inhibitors of respiratory burst from cultures of the yeast Malassezia furfur: comparison with the bisindolylmaleimide arcyriarubin A. Chembiochem 2005, 6, 2290-2297.
33.Kurz, M. et al.,. Ustilipids, acylated beta-D-mannopyranosyl D-erythritols from Ustilago maydis and Geotrichum candidum. J. Antibiot. (Tokyo) 2003, 56, 91–101.
34.Lemieux, R., The biochemistry of the Ustilaginales: III. The degradation products and proof of the chemical heterogeneity of ustilagic acid. Can. J. Chem. 1951, 29, 415-425.
35.Lizarraga-Guerra, R.; Lopez, M. G., Monosaccharide and alditol contents of huitlacoche (Ustilago maydis). J. Food Compost. Anal. 1998, 11, 333-339.
36.Luderitz, O.; Freudenberg, M. A.; Galanos, C.; Lehmann, V.; Rietschel, E. T.; Shaw, D. H., Lipopolysaccharides of gram-negative bacteria. Curr. Top. Membr. 1982, 17, 79-151.
37.Marti’ nez, V.; Osuna, J.; Paredes-Lo’ pez, O.; Guevara, F., Production of indole-3-acetic acid by several wild-type strains of Ustilago maydis. World J. Microbiol. Biotechnol. 1997, 13, 295-298.
38.Miller, S. I.; Ernst, R. K.; Bader, M. W., LPS, TLR4 and infectious disease diversity. Nat. Rev. Microbiol. 2005, 3, 36-46.
39.Minghetti, L., Cyclooxygenase-2 (COX-2) in inflammatory and degenerative brain diseases. J. Neuropathol. Exp. Neurol. 2004, 63, 901.
40.Morrissette, N.; Gold, E.; Aderem, A., The macrophage--a cell for all seasons. Trends Cell Biol. 1999, 9, 199.
41.Mourao, L. R. M. B.; Santana, R. S. S.; Paulo, L. M.; Pugine, S. M. P.; Chaible, L. M.; Fukumasu, H.; Dagli, M. L. Z.; de Melo, M. P., Protective action of indole‐3‐acetic acid on induced hepatocarcinoma in mice. Cell Biochem. Funct. 2009, 27, 16-22.
42.Navarre, D.; Damann, K., Synthesis of indole-3-acetic acid by Ustilago maydis, abstr. A784. Phytopathology 1990, 80, 1055.
43.Ona, O.; Impe, J.; Prinsen, E.; Vanderleyden, J., Growth and indole‐3‐acetic acid biosynthesis of Azospirillum brasilense Sp245 is environmentally controlled. FEMS Microbiol. Lett. 2005, 246, 125-132.
44.Ona, O.; Smets, I.; Gysegom, P.; Bernaerts, K.; Van Impe, J.; Prinsen, E.; VANDERLEYDEN, J., The effect of pH on indole-3-acetic acid (IAA) biosynthesis of Azospirillum brasilense Sp7. Symbiosis 2003, 35, 199-208.
45.Patten, C. L.; Glick, B. R., Bacterial biosynthesis of indole-3-acetic acid. Can. J. Microbiol. 1996, 42, 207-220.
46.Patten, C. L.; Glick, B. R., Role of Pseudomonas putida indoleacetic acid in development of the host plant root system. Appl. Environ. Microbiol. 2002, 68, 3795.
47.Prinsen, E.; Costacurta, A.; Michiels, K.; Vanderleyden, J.; Van Onckelen, H., Azospirillum brasilense indole-3-acetic acid biosynthesis: evidence for a non-tryptophan dependent pathway. Mol. Plant Microbe Interact. 1993, 6, 609-609.
48.Raetz, C. R. H., Biochemistry of endotoxins. Annu. Rev. Biochem. 1990, 59, 129-170.
49.Reed, R.; Holder, M., The antibacterial spectrum of ustilagic acid. Can. J. Med. Sci. 1953, 31, 505.
50.Rodriguez, C.; Dominguez, A., The growth characteristics of Saccharomycopsis lipolytica: morphology and induction of mycelium formation. Can. J. Microbiol. 1984, 30, 605-612.
51.Roxburgh, J.; Spencer, J.; Sallans, H., Submerged Culture Fermentation, Factors Affecting the Production of Ustilagic Acid by Ustilago Zeae. J. Agric. Food. Chem. 1954, 2, 1121-1124.
52.Ruiz-Herrera, J.; Leon, C. G.; Guevara-Olvera, L.; Carabez-Trejo, A., Yeast-mycelial dimorphism of haploid and diploid strains of Ustilago maydis. Microbiology 1995, 141, 695.
53.Ruiz-Herrera, J.; Martinez-Espinoza, A. D., The fungus Ustilago maydis, from the aztec cuisine to the research laboratory. Int. Microbiol. 1998, 1, 149-158.
54. San-Blas, F. & San-Blas, G. (1985). Paracoccidioides brasifiensis. In Fungal Dimorphism with Emphasis on Fungi Pathogenic for Humans, p p. 93-120. Edited by P. J. Szaniszlo. New York: Plenum Press.
55.Sanchez‐Marroquin, A.; Ledezma, M.; Barreiro, J., Oxygen transfer and scale‐up in lysine production by Ustilago maydis mutant. Biotechnol. Bioeng. 1971, 13, 419-429.
56.Sanchez-Marroquin, A.; Ledezma, M.; Carreno, R., Sugar substrates for L-lysine fermentation by Ustilago maydis. Appl. Environ. Microbiol. 1970, 20, 687.
57. Soll, D. R. (1985). Candida afbicans. In Fungal Dimorphism with Emphasis on Fungi Pathogenic for Humans, pp. 167-195. Edited by P. J. Szaniszlo. New York: Plenum Press.
58.Sosa-Morales, M.; Guevara-Lara, F.; Martinez-Juarez, V.; Paredes-Lopez, O., Production of indole-3-acetic acid by mutant strains of Ustilago maydis (maize smut/huitlacoche). Appl. Microbiol. Biotechnol. 1997, 48, 726-729.
59.Spaepen, S.; Vanderleyden, J.; Remans, R., Indole‐3‐acetic acid in microbial and microorganism‐plant signaling. FEMS Microbiol. Rev. 2007, 31, 425-448.
60.Sporn, M.; Roberts, A. B., Peptide growth factors and inflammation, tissue repair, and cancer. J. Clin. Invest. 1986, 78, 329.
61.Teng, S. F.; Sproule, K.; Husain, A.; Lowe, C. R., Affinity chromatography on immobilized "biomimetic" ligands. Synthesis, immobilization and chromatographic assessment of an immunoglobulin G-binding ligand. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2000, 740, 1-15.
62.Theunis, M.; Kobayashi, H.; Broughton, W. J.; Prinsen, E., Flavonoids, NodD1, NodD2, and nod-box NB15 modulate expression of the y4wEFG locus that is required for indole-3-acetic acid synthesis in Rhizobium sp. strain NGR234. Mol. Plant. Microbe Interact. 2004, 17, 1153-1161.
63.Thippeswamy, T.; McKay, J.; Quinn, J.; Morris, R., Nitric oxide, a biological double-faced janus-Is this good or bad? Histol. Histopathol. 2006, 21, 445-58.
64.Trikha, M.; Corringham, R.; Klein, B.; Rossi, J. F., Targeted anti-interleukin-6 monoclonal antibody therapy for cancer. Clin. Cancer Res. 2003, 9, 4653-4665.
65.Tsurumi, S.; Wada, S., Identification of 3-hydroxy-2-indolone-3-acetylaspartic acid as a new indole-3-acetic acid metabolite in Vicia roots. Plant Cell Physiol. 1986, 27, 559.
66.Valverde-Gonzalez, M. E., Estudios Sobre la Infeccion de Ustilago maydis (Huitlacoche) ysus Caracteristicas Alimentarias, M.Sc. Thesis,CINVESTAV-IPN, Irapuato, Mexico, 1992.
67.van Horssen, R.; ten Hagen, T. L. M.; Eggermont, A. M. M., TNF-α in cancer treatment: molecular insights, antitumor effects, and clinical utility. Oncologist 2006, 11, 397-408.
68.Wolf, F. T., The production of indole acetic acid by Ustilago zeae, and its possible significance in tumor formation. Proc. Natl. Acad. Sci. U. S. A. 1952, 38, 106-111.
69.Wolf, F. T., The utilization of carbon and nitrogen compounds by Ustilago zeae. Mycologia 1953, 45, 516-522.
70.Yen, G. C.; Lai, H. H., Inhibition of reactive nitrogen species effects in vitro and in vivo by isoflavones and soy-based food extracts. J. Agric. Food. Chem. 2003, 51, 7892-7900.
71.Zimmer, W.; Wesche, M.; Timmermans, L., Identification and isolation of the indole-3-pyruvate decarboxylase gene from Azospirillum brasilense Sp7: sequencing and functional analysis of the gene locus. Curr. Microbiol. 1998, 36, 327-331.
72.Zuther, K.; Mayser, P.; Hettwer, U.; Wu, W.; Spiteller, P.; Kindler, B. L. J.; Karlovsky, P.; Basse, C. W.; Schirawski, J., The tryptophan aminotransferase Tam1 catalyses the single biosynthetic step for tryptophan‐dependent pigment synthesis in Ustilago maydis. Mol. Microbiol. 2008, 68, 152-172.