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研究生:陳依瑩
研究生(外文):Yi-Ying Chen
論文名稱:嗜鹽菌硫化多醣之大量生產及其於抗氧化與醫學功能之探討
論文名稱(外文):The mass production of a halophilic bacteria’s sulfated polysaccharide and it’s antioxidant function with biomedical application
指導教授:陳志成陳志成引用關係
指導教授(外文):C. Will Chen
學位類別:碩士
校院名稱:大同大學
系所名稱:生物工程學系(所)
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:129
中文關鍵詞:多醣嗜鹽菌
外文關鍵詞:polysaccharidehalophilic bacteria
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多醣已廣泛的應用於食品、製藥與化妝品及其他工業上。多醣大部分的來源是從藻類或植物萃取所得,缺點在於萃取分離的成本過高。而利用微生物來生產胞外多醣則具有可大量生產與易分離的優點。已知極端嗜鹽菌Haloferax mediterranei所生產的胞外多醣,是一種含有硫酸根與甘露糖的多醣聚合物。因此本研究乃尋找醱酵培養H. mediterranei,生產胞外硫化多醣(exopolysaccharide,EPS)之最適培養基配方與其應用。實驗發現利用飼料級玉米粉為碳源,以溫度42℃,轉速180 rpm和碳源濃度15.5 g/L的條件下可得最佳的菌體量以及EPS產量,分別為1.84×106 cells/ml及5.9 g/L的多醣。同時發現EPS的分子量約為3.1×104 Da且含硫量約為7.5 %。而經由細胞培養試驗得知,此EPS以內差法求得對人類肺癌細胞(A549)和人類橫紋肌瘤細胞(RD)細胞達到50%毒性抑制的濃度(CC50)分別1.94及1.49 mg/mL;在抗氧化試驗可得知,EPS於43.7 mg/mL濃度下對清除DPPH自由基可達50 %的清除能力(IC50);於91.2 mg/mL濃度下對螯合亞鐵離子可達50 %的螯合能力;在抗病毒試驗得知,EPS在0.63 mg/mL濃度之下對腺病毒41型(AD41)有16.35%的些微抗病毒效果,當EPS濃度於1.25 mg/mL時對,腸病毒71型(EV71)有較明顯的抑制效果,為23.62 %;在抗菌試驗可得知,EPS於50 mg/mL濃度下對野生型大腸桿菌可達中度的抑菌活性(抑制圈13.17±0.3 mm),於1 mg/mL濃度下對金黃色葡萄球菌可達中度抑菌活性(抑制圈15.33±0.3 mm)。上述數據顯示,嗜鹽菌EPS具有可大量生產與醫學上的應用價值。
Polysaccharide recently has been used in some industries like food, pharmacy and cosmetics. Most polysaccharides can be extracted from algae or plants, however, the cost of extracting process is expensive. On the contrary, there is a benefit to produce and purify the mass quantity of exopolysaccharide(EPS) from microorganism. Haloferax mediterranei can produce EPS with functional qroup of sulfate and mannose units. In this stuy, we are seeking for the suitable medium and culture method to produce EPS by H. mediterranei, and also looking for the application of EPS. By the shaking-flask experiment, it is found that using 15.5 g/L field corn as the carbon source, and culture condition at 42℃ and rotational speed of 180 rpm, we could obtain the best quantity of cell concentration of 1.84×106 cells/ml and 5.9 g/L EPS. The molecular weight of EPS was measured as 3.1×104 Da, and sulfur content in the EPS was measured as 7.5%. By the cell culture test, the 50% toxicity(CC50) of EPS to the human lung A549 cell and Rhadomyosarcoma cell line were 1.94 and 1.49 mg/mL respectively by linear interpolation. In the anti-oxidation experiments, it is found that the 50% radical scavenging activity (IC50) to DPPH is 43.7 mg/mL, and the IC50 of Fe2+ chelating ability was 91.2 mg/mL. Besides, in the of anti-virus experiments, the concentrations of 0.63 mg/mL EPS shown a slight anti-virus activity (16.4%) to Adeno Virus (AD41). The anti-virus effect to Enterovirus EV71) is 23.62% when the concentrations of EPS is 1.250 mg/mL. By the antibacterial test, the concentration of EPS of 50 mg/ml shows a mild effect of inhibition (with inhibition zone of dia, 13.17±0.3 mm to E.coli). The concentration of 1 mg/ml EPS shows a mild inhibition to the growth of Staphylococcus aureus with inhibition zone of 15.33±0.3 mm). We conclude that the EPS from H. mediterranei could be mass produced and also shows the potential medical applications.
中文摘要 I
英文摘要 III
目錄 V
圖目錄 IX
表目錄 XI
第一章 緒論 1
1.1 前言 1
1.2 研究動機 2
第二章 文獻回顧 3
2.1 胞外多醣(exopolysaccharide, EPS) 3
2.2 古生菌 5
2.2.1 古生菌的分類 5
2.3嗜鹽菌的介紹 7
2.3.1 生理特性 7
2.3.2 能量來源 9
2.4 嗜鹽菌Haloferax mediterranei 的介紹 10
2.5 Haloferax mediterranei生產之硫化多醣 12
2.6 硫化多醣於醫藥領域上的應用 15
2.6.1 肝素(Heparin)的介紹 15
2.6.2 硫化多醣的抗凝血功能與結構 17
2.6.3 具潛力的抗病毒藥物-硫化多醣 21
2.7 抗氧化活性之測定原理 22
第三章 材料與方法 25
3.1 實驗儀器 25
3.2 實驗材料 26
3.2.1菌種來源 26
3.2.2 培養基成分 27
3.2.3 藥品 27
3.3 實驗架構 30
3.4 實驗方法 31
3.4.1 探討不同配方、濃度之搖瓶培養 31
3.4.2探討不同溫度之搖瓶試驗 32
3.4.3 醱酵槽培養 32
3.4.3.1醱酵槽配置 32
3.4.3.2 電極校正 33
3.4.3.3接菌 35
3.4.3.4 電腦監控系統 35
3.4.3.5 醱酵控制條件 35
3.5 分析方法 37
3.5.1菌體數之測定法 37
3.5.2 菌體乾重之測定法 39
3.5.3 總醣測定 40
3.5.4 胞外多醣的萃取與純化 41
3.5.5 分子量測定-凝膠滲透層析儀GPC (Gel Permeation Chromatography) 42
3.5.6 官能基測定(傅立葉轉換紅外線光譜分析-FTIR) 42
3.5.7 細胞抑制測試 44
3.5.8 抗病毒測試 45
3.5.9 抗菌測試 46
3.5.10 清除DPPH自由基能力測試 47
3.5.11 螯合亞鐵離子能力的測定 48
第四章 結果與討論 50
4.1 搖瓶實驗 50
4.1.1探討不同碳源濃度對菌體生長與多醣產量之影響 51
4.1.2 探討不同溫度對菌體生長與多醣產量之影響 65
4.3 批次醱酵 69
4.4 總醣測定法與實際EPS純化產量之關係 71
4.5 官能基分析 73
4.6胞外多醣含硫量之探討 78
4.7 硫化多醣對抗氧化試驗 82
4.7.1清除DPPH自由基能力測試 82
4.7.2 螯合亞鐵離子能力的測定 84
4.8 硫化多醣對細胞抑制試驗 86
4.9 硫化多醣對抗病毒試驗 91
4.10 硫化多醣之抗菌測試 94
第五章 結論 99
第六章 參考文獻 101
附錄 106

圖目錄
圖2.5.1 利用H. mediterraneie生產胞外多醣之FT-IR圖譜 13
圖2.5.2 H. mediterranei 胞外多醣的構造式 14
圖3.4.1 Thoma氏血球計數板構造及計數室 36
圖4.1.1 不同濃度葡萄糖對菌體生長之影響 52
圖4.1.2不同濃度的黃豆粉對菌體生長情形 53
圖4.1.3不同濃度的飼料級玉米粉對菌體生長之影響 54
圖4.1.4不同濃度葡萄糖對多醣產量之影響 55
圖4.1.5不同濃度黃豆粉對多醣產量之影響 56
圖4.1.6不同濃度飼料級玉米粉對多醣產量之影響 57
圖4.1.7 利用實驗設計軟體對不同濃度飼料級玉米粉之多醣產量的評估 59
圖4.1.8 利用飼料級玉米粉為碳源,隨機選取飼料級玉米粉濃度7.5 g/L與理論上可生產最大胞外多醣(EPS)之飼料級玉米粉(Field corn ) 15.5 g/L之菌體生長情形 60
圖4.1.9 利用飼料級玉米粉為碳源,隨機選取飼料級玉米粉濃度7.5 g/L與理論上可生產最大胞外多醣(EPS)之飼料級玉米粉(Field corn ) 15.5 g/L之多醣產量 61
圖4.1.10以15.5 g/L飼料級玉米粉為碳源改變不同溫度之菌體生長情形 64
圖4.1.11以15.5 g/L玉米粉為碳源改變不同溫度之多醣產量 65
圖4.3.1以15.5 g/L的飼料級玉米粉、溫度42℃進行批次醱酵之菌體濃度與多醣產量 68
圖4.5.1 利用葡萄糖為碳源生產胞外多醣之FT-IR圖譜 72
圖4.5.2 利用生黃豆粉為碳源生產胞外多醣之FT-IR圖譜 73
圖4.5.3 利用飼料級玉米粉為碳源生產胞外多醣之FT-IR圖譜 74
圖4.7.1硫化多醣清除DPPH自由基之情形 81
圖4.7.2 硫化多醣對螯合亞鐵離子之情形 83
圖4.8.1a硫化多醣對人類肺癌細胞(A549細胞株)抑制情形 85
圖4.8.1b硫化多醣對人類肺癌細胞(A549細胞株)抑制之線性關係 86
圖4.8.2硫化多醣對人類橫紋瘤細胞(RD細胞)抑制情形 87
圖4.8.2b硫化多醣對人類肺癌細胞(RD細胞)抑制之線性關係 88
圖4.9.1硫化多醣對腺病毒41型(AD41)的抗病毒能力 90
圖4.9.2硫化多醣對腸病毒71型(EV71)的抗病毒能力 91
圖4.10.1 不同濃度之EPS對E.coli的抑菌情形 93
圖4.10.1不同濃度之EPS對Staphylococcus aureus subsp.aureus的抑菌情形 94
表目錄
表2.1 市面上常見的硫化多醣 18
表4.1.1不同碳源及濃度之最大菌體量、生長速率與遲滯時間的比較 58
表4.1.2 利用ANOVA分析之EPS理論值與實際搖瓶醱酵所得EPS產量 62
表4.1.3不同溫度之最大菌體量、反曲點生長速率及遲滯時間的比較 66
表4.4.1 酚硫酸法之預估與實際產量的關係 70
表4.5.1 利用FTIR分析不同碳源所生產多醣之官能基鑑定 75
表4.6.1 培養基質與其胞外多醣主要的元素分析 78
表4.6.2 三種嗜鹽菌所生產硫化多醣之分子量、產量及硫含量 79
表4.9.1硫化多醣對大腸桿菌與金黃色葡萄球菌之抑菌活性 91
表4.9.2硫化多醣對大腸桿菌與金黃色葡萄球菌之最小抑菌濃度 92
財團法人中央畜產會(http://www.naif.org.tw/home.htm)
朱朝麟。2003。利用酵母菌pichia holstii醱酵生產胞外甘露多糖。大同大學生物工程研究所碩士論文。
沈萍。2003。微生物學。台北:五南圖書出版股份有限公司。
林明君。2002。新鮮及乾燥番茄甲醇萃取液之抗氧化性比較。大葉大學食品工程學研究所碩士論文。
張安強,張勁松,潘迎捷。2005。食藥用菌多醣的提取、分離純化與結構分析。實用菌學報。12:62-68.
張希文。2004。探討嗜鹽菌於不同導電度和以大豆水解液作為氮源醱酵生產Polyhydroxyalkanoate之研究。大同大學生物工程研究所碩士論文。
黃丁晏。2003。以擠壓農業原料醱酵生產polyhydroxyalkanoates 之研究。大同大學生物工程研究所碩士論文。
楊永吉。2000。反應性擠壓處理澱粉最為醱酵碳源於PH-stat 饋料控制程序中生產微生物高分子之研究。碩士論文。大同大學生物工程研究所。
謝秋蕙。2005。嗜鹽菌於批次饋料醱酵下之胞內與胞外多醣之研究。大同大學生物工程學研究所碩士論文。
薛顯能。2006。嗜鹽菌Haloferax mediterranei紅色色素之抗氧化及退色性反應研究。大同大學生物工程研究所碩士論文。

Anton J., I. Meseguer, and F. Rodrfguez-Valera.1988. Production of an Extracellular Polysaccharide by Haloferax mediterranei. Appl. Environ. Microbiol. 54:2381-2386.

Bauer A.W., Kirby, W.M. Sherris, J.C. and Turck M. 1996. Antibiotic
susceptibility testing by a stardized single disk method. The Americal
Journal of Clinical Pathology. 46:493-496.

Beijering, R.J.R., H. and Cate, J.W. 1996. Cate. Clinical applications of new antithrombotic agents. Ann. Hematoal. 72:177-183.

Blosi, M. S. 1958. Antioxidant determination by the use of a stable
free radical. Nature. 26: 1199-1200.

Boan I.F., J.C. Garcia-Quesada, J. Anton, F. Rodriguez-Valera, and A. Marcilla. 1998. Flow properties of haloarchaeal polysaccharides in aqueous solutions. Polymer. 39:6945-6950.

Boyer R.F., C.J. McCleary, 1987. Superoxide ion as a primary reductant in ascorbate-mediated ferritin iron release. Free Radic. Biol. Med. 3:
389-395.

Cerning, J. 1995. Production of exopolysaccharides by lactic acid bacteria and dairy propionibacteria. Le Lait. 75: 463-472.

Dubois, M., K. A. Gilles, J.K. Hamilton, P.A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350-356.

Fernandez-Castillo, R. F., F. Rodriguez-Valera, J. Gonzalez-Ramos, and F. Ruiz-Berraquero. 1986. Accumulation of poly(β-hydroxybutyrate)
by halobacteria. Appl. Environ. Microbiol. 51: 214-16.

Folkman, J. 1991. “Tumour angiogensis.” In “Cancer Medicine”. Ed Holland, J. F., Lea and Febiger, Philadelphia.

Folkman, J. and Brem, H., 1992. “Angiogensis and inflammation” In:“ Inflammation. Basic priciples and clinical correlates”. Goldstein, I. M. and Snyderman, R. S.,Raven Press, New York.

Goo, Y. A., J. Roach, G. Glusman, N. S. Baliga, K. Deutsch, M. Pan, S. Kennedy, S. DasSarma, W. V. Ng, and L. Hood. 2004. Low-pass
sequencing for microbial comparative genomics. BMC Genomics.
5:3.

Halliwell, B. and J. M. C.Gutteridge. 1989. Free radicals, ageing and disease. In: Free Radicals in Biology and Medicine, ed. by B. Halliwell, and J. C. Gutteridge.

Hayashi, T. K., M. Hayashi, and I. Kojima. 1996. Calcium spirulan, an inhibitor of enveloped virus replication, from a blue-green alga Spirulina platensis. J. Nat. Prod. 39:83-87.

Holt, J. G., J. T. Staley, and M. P. Bryant. 2000. Archaeabacteria. Bergery’s manual of Systematic Bacteriology, Vol 3. New York: Lippincott Williams & Wilkins. 2226-2228.

Jeske W and J. Fareed. 1999. In vitro studies on the biochemistry and pharmacology of low molecular weight heparins. Semin Thromb Hemost 25:27-33.

Kushner, D. J. 1978. Life in hight salt and solute concentrations: halophilic bacteria. In D. J. Kushner, (ed) Microbial Life in Extreme Environments. Academic Press. London. 214-317.

Lee, J. B., K. Hayashi, M. Hirata, E. Kuroda, E. Suzuki, Y. Kubo, and T. Hayashi. 2006. Antiviral sulfated polysaccharide from Navicula directa, a diatom collected from Deep-Sea water in Toyama Bay. Biol. Pharm. Bull. 29:2135-2139.

Lee, J.H. and I.Y. Lee. 2001. Optimization of uracil for curdlan (β-1→3-glucan) production by Agrobacterium sp. Biotechnol. Letter. 23:1131-1134.

Mata, J. A., V. Béjar, I. Llamas , S. Arias, P. Bressollier, R. Tallon, M. C. Urdaci, and E. Quesada. 2006. Exopolysaccharides produced by the recently described halophilic bacteria Halomonas ventosae and Halomonas anticariensis. Res. Microbiol. 157:827-835.

Martinez-Canovas, M.J., E. Quesada, I, Llamas and V. Bejar. 2004a. Halomonas ventosae sp. nov., moderately halophilic, denitrifying, exopolysaccharide-producing bacterium. Int. J. Syst. Evol. Microbiol. 54:733-737.

Martinez-Canovas, M.J., V. Bejar, F. Martinez-Checa, and E. Quesada. 2004b. Halomonas anticariensis sp. nov., from Fuente de Piedra, a saline-wetland wildfowl reserve in Malaga, southern Spain. Int. J. Syst. Evol. Microbiol. 54:1329-1332.

Mitsuhashi, S. and N. Murata. 1991. Inhibitory activity of Bifidobacteria. J. Jpn. Soc. Nutri. Food. Sci. 44:365-372

Monsan, P., S. Bozonnet, C. Albenne, G. Joucla, R.-M.Willemot, and M. Remaud-Simeon. 2001. Homopolysaccharides from lactic acid bacteria. Int. Dairy J. 11: 673-683.

Nole, G., A. Johnson and A. Znaiden. 2000. Antibacterial lotion testing:a practical approach to demonstrate the antibacterial efficacy of a
triclosancontaining leave-on moisturizer.,International Journal of
Cosmetic Science. 22:341-347.

Riccio, R., R. B. Kinnel, G. Bifulco, and P. J. Scheuer. 1996. Kakelokelose, a sulfated mannose polysaccharide with anti-HIV activity from the Pacific Tunicate Didemnum molle. Tetrahedron Lett. 37:1979-1982.

Rodriguez-Valera, F.,Juez, G. and kushner, D.J. 1983. Halobacterium mediterranei spec.nov.,anewcarbohydrate-utilizing extreme halophile. System. Appl. Microbiol. 4:369-381.

Rodriguez-Valera, F., F. Ruiz-Berraquero, and A. Romos-Cormenzana. 1980. Isolaction of extremely halophilic bacteria able to grow in defined inorganic media with single carbon sources. J. Gen.
Microbiol. 119: 535-538.

Ruas-Madiedo, P., Tuinier, R., Kanning, M. and Zoon, P. 2002. Role of exopolysaccharides produced by Lactococcus lactis subsp. cremoris on the viscosity of fermented milks. Int. Dairy J. 12: 689-695.

Sutherland, I.W. 1998. Novel and established application of microbial polysaccharides. Tibtech. January. 16: 41-46.

Thamas, J. 1995. The role of free radicals and antioxidants : How do we know that are working. Crit. Rev. Food Sci. Nutr. 35:21-39.
Torreblanca, M., F. Rodríguez Valera, G. Juez, A. Ventosa, M. Kamekura, and M. Kates. 1986. Classification of non-alkaliphilic halobacteria based on numerical taxonomy and polar lipid composition, and description of Haloarcula gen. nov. and Haloferax gen. nov. Syst. Appl. Microbiol. 8: 89-99.

Williams, W. B., M. E. Cuvelier, and C. Berset. 1995. Use of a free radical method to evaluate antioxidant activity.Lebensm-Wiss. Technol. 28: 25-30.

Yamaguchi T., H. Takamura, T. Matoba, and J. Terao, 1998. HPLC method for evaluation of the free radical-scavenging activity of foods by using 1,1-diphenyl-2-picrylhydrazyl. Biosci. Biotechnol. Biochem. 62: 1201-1204.

Yim, J. H., S. J. Kim, S. H. Ahn, and H. K. Lee. 2003. Optimal conditions for the production of sulfated polysaccharide by marine microalga Gyrodinium impudicum strain KG03. Biomolecular Engineering. 20:273-280.

Yoshida, T., Y. Yasuda, T. Mimura, Y. Kaneko, H. Nakashima, and N. Yamamoto, N. 1995. Synthesis of curdlan sulfates having inhibitory beffects in vitro against AIDS viruses HIV-1 and HIV-2. Carbohydr. Res. 276:425-436.
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