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系統識別號 U0026-2407201418374300
論文名稱(中文) Halobacterium salinarum 饋批次發酵進料策略與生產菌色素對清除自由基之研究
論文名稱(英文) Investigation on the feeding strategy for the fed-batch fermentation of Halobacterium salinarum and the bacterial pigment production for the scavenging of free radicals
校院名稱 成功大學
系所名稱(中) 化學工程學系
系所名稱(英) Department of Chemical Engineering
學年度 102
學期 2
出版年 103
研究生(中文) 許哲耕
研究生(英文) Jhe-Geng Hsu
學號 N36011146
學位類別 碩士
語文別 中文
論文頁數 63頁
口試委員 指導教授-許梅娟
口試委員-吳文騰
口試委員-鄧熙聖
口試委員-周明顯
中文關鍵字 Halobacterium salinarum  饋料批次發酵  自由基移除  抗氧化 
英文關鍵字 Halobacterium salinarum  fed-batch fermentation  free radical scavenging  antioxidant 
學科別分類
中文摘要 Halobacterium salinarum 是一種能在高鹽環境下生長的古嗜鹽菌 (Halobacteriaceae),其分泌的菌色素具有抗氧化的功能。由於生物老化、食物腐敗等多項日常生活中的反應,都和氧化作用脫離不了關係,因此,藉由培養 H. salinarum 以萃取具有抗氧化力的菌色素,是相當有發展潛力的。
本研究探討有效培養 H. salinarum 的方法,先在批次條件下,針對培養溫度及培養基各種成份進行探討,試圖歸納出一個比較良好的批次發酵環境,之後再依此條件進行饋批次發酵,以尋求 H. salinarum 更佳的飽和濃度。之後萃取其分泌的菌色素,以 2, 2’-二苯基-1-苦味胼基 (2,2’-diphenyl-1-picrylhydrazyl radical, DPPH•) 做為自由基提供來源,以進行移除自由基 (free radical scavenging) 的測試。
溫度 37 oC、液態培養基添加 0.1 g/L 葡萄糖、0.5 g/L 谷胺酸鈉、3 g/L 檸檬酸鈉、7.5 g/L 酪蛋白胺基酸、3 g/L 酵母萃取物和 200 g/L 氯化鈉將有助於提升菌體培養的飽和濃度。以此進行 1 L 起始體積培養,並且每 12 小時間隔 24 小時進料含 200 g/L 氯化鈉的培養液,同時使菌體的收穫和其所對應的菌色素移除自由基測試有好的表現。
英文摘要 Investigation on the feeding strategy for the fed-batch fermentation of Halobacterium salinarum and the bacterial pigment production for the scavenging of free radicals
Author: Jhe- Geng Hsu
Advisor: Mei- Jywan Syu
Department of Chemistry engineering, National Cheng Kung University
SUMMARY
Halobacterium salinarum is an ancient Halobacteriaceae which is able to grow at a high salt environment. It can secrete bacterial pigments which is quite potential with antioxidant application. The purpose of this study is to investigate a efficient way to culture H. salinarum. First, I culture H. salinarum in batch system to make sure which temperature and medium composition can let H. salinarum grows best. Then, I culture H. salinarum in fed-batch system with condition which makes H. salinarum grows best and investigate the best feeding way. In order to have a better H. salinarum saturated cell density, the culturing was set at 37 oC with 0.1 g/L glucose, 0.5 g/L Na-glutamate, 3 g/L Na3-citrite, 7.5 g/L casamino acid, 3 g/L yeast extract, and 200 g/L sodium chloride in medium. H. salinarum grows well during the initial volume test of 1 L with the above conditions by feeding medium containing 0.1 g/L glucose, 0.5 g/L Na-glutamate, and 200 g/L sodium chloride. And so does the free radical scavenging test of the pigment extracted from this kind of culture.
Keywords: Halobacterium salinarum, fed-batch fermentation, free radical scavenging, antioxidant
INTRODUCTION
Halobacterium salinarum is an ancient Halobacteriaceae which is able to grow at a high salt environment. H. salinarum can secrete bacterial pigments with antioxidant function. Many reactions in daily life, like biological aging and food spoilage, are inseparable from oxidation. Therefore, it is quite potential to extract the secreted orange pigment as antioxidants from H. salinarum fermentation.
H. salinarum is a rod shape halophilic archaea. It needs at least 1.5 M sodium chloride for culturing H. salinarum and the optimal sodium chloride concentration is 2.0 M ~5.2 M.
The result of H. salinarum culturing and the quality of pigments H. salinarum secreted may be influence by many conditions such as temperature, light, salt concentration, and organic nutrient concentration. This study cultures H. salinarum and changes culturing temperature, salt concentration, and organic nutrient concentration to make sure which condition can let H. salinarum grows best and secretes pigment which has highest free radical scavenging effect.
MATERIALS AND METHODS
The strains of the extremophile microorganisms H. salinarum was bought from bioresource collection and research center (BCRC), Hsinchu, Taiwan. The culture medium contained sodium chloride, magnesium sulfate, potassium chloride, sodium citrate, yeast extract, casamino acid, Iron(II) chloride, Manganese chloride and Na-glutamate.
The pigments were extracted from H. salinarum by methol: acetone (2:7) and used in free radical scavenging activity test. The pigment was measured by 1, 1-diphenyl-2-picryl-hydrazil (DPPH), solution of DPPH (0.3 mM) in methanol was prepared. 0.75 mL of pigment solution was added to 0.3 mL DPPH solution. he mixture was shaken vigorously and stand at room temperature for 30 min. Then the absorbance was measured at 517 nm by using UV-Visible spectrophotometer. The DPPH scavenging effect was calculated by equation:
DPPH scavenging effect (%) = ((A_0-A))/A_0 ×100%
RESULTS AND DISCUSSION
The effective methods of cultivating H. salinarum were investigated in this study. The first step is to explore the effect of a variety of medium ingredients and the incubation temperature on its batch culture, trying to sum up a relatively good fermentation environment for the further fed-batch experiments. They are to seek a better H. salinarum saturation concentration for the free radical scavenging test, which 2,2'-diphenyl-1- picrylhydrazyl radical (DPPH •) provides a source of free radicals, by its secreted pigment. The result of H. salinarum culture and affect by all conditions are listed in table 1.
Table 1. Results of culturing H. salinarum with different condition
  high concentration medium concentration low concentration
yeast extract 5 g/L grows better
(3 g/L)
casamino acid grows better
(7.5 g/L) 5 g/L no obvious change
(2 g/L)
sodium citrate no obvious change
(5 g/L) 3 g/L no obvious change
(1 g/L)
Na-glutamate grows worse
(3 g/L) 1 g/L grows better
(0.5 g/L)
glucose pigment changed
(1.0 g/L) pigment changed
(0.5 g/L) initial grow rate increase
(0.1 g/L )
sodium chloride 250 g/L grows better
(200 g/L) grows worse
(100g/L )

In order to have a better H. salinarum saturated cell density, the culturing was set at 37 oC with 0.1 g/L glucose, 0.5 g/L Na-glutamate, 3 g/L Na3-citrite, 7.5 g/L casamino acid, 3 g/L yeast extract, and 200 g/L sodium chloride in medium. H. salinarum grows well during the initial volume test of 1 L with the above conditions by feeding medium containing 0.1 g/L glucose, 0.5 g/L Na-glutamate, and 200 g/L sodium chloride. And so does the free radical scavenging test of the pigment extracted from this kind of culture.
After set best batch culture condition, this study use this condition to culture H. salinarum by fed-batch culture and try different feeding way to expect the increase of H. salinarum broth saturation concentration and free radical scavenging effect of the pigment which it secrets.
The result of fed-batch culture shows that H. salinarum can grows better when the culture is starting from initial volume 1 L and stop feeding 24 hour for each feeding 12 hour.
CONCLUSION
H. salinarum grows best in batch culture when the medium composed with 0.1 g/L glucose, 0.5 g/L Na-glutamate, 3 g/L Na3-citrite, 7.5 g/L casamino acid, 3 g/L yeast extract, and 200 g/L sodium chloride.
When culture H. salinarum in fed-batch system with above condition, the better way to let H. salinarum grows better is starting from initial volume 1 L and stop feeding 24 hour for each feeding 12 hour.
論文目次 中文摘要 I
Abstract………………………………………………………………………………………………………………………...…II
致謝…………………………………………………………………………………………………………………….. V
目錄…………………………………………………………………………………………………………………………..…..Ⅵ
表目錄……………………………………………………………………………………………………………………………Ⅷ
圖目錄 ..……………………………………………………………………………………………………………………....IX
第一章 緒論 1
1-1 前言 1
1-2 實驗動機和目的 2
第二章 文獻回顧 3
2-1 古嗜鹽菌簡介 3
2-2 Halobacterium salinarum 簡介 3
2-3 影響 Halobacterium salinarum 生長之因素 5
2-3-1 溫度 5
2-3-2 光 5
2-3-3 鹽分濃度 8
2-3-4 其他無機鹽類濃度 8
2-3-5有機養分濃度 9
2-4 嗜鹽菌之培養策略 10
2-4-1 批次培養 10
2-4-2 饋料批次培養 10
2-4-3 連續式培養 11
2-5 抗氧化實驗原理簡介 11
2-5-1 氧化反應的原理簡介 11
2-5-2 抗氧化劑簡介 12
2-5-3 抗氧化測定方式簡介 14
第三章 實驗器材與方法 22
3-1 實驗材料 22
3-1-1 使用菌株 22
3-1-2 培養基組成 22
3-1-3 微量金屬 22
3-2 實驗方法 23
3-2-1 前培養 23
3-2-2 正式培養 23
3-2-3 色素萃取 23
3-3 實驗分析方法 23
3-3-1 嗜鹽菌濃度測定 23
3-3-2 測定自由基移除能力 24
3-4 實驗藥品 26
3-5 實驗儀器 26
第四章 結果與討論 27
4-1 不同批次培養因子對 H. salinarum 生長之探討 27
4-1-1不同培養體積 27
4-1-2 不同培養溫度 28
4-1-3 不同碳、氮源 29
4-1-4 不同無機成份 33
4-2 饋料批次培養不同因子對 H. salinarum 生長之探討 35
4-2-1 起始培養體積 35
4-2-2 進料策略 36
4-2-3進料成份有無氯化鈉 42
4-3 H. salinarum 萃取菌色素之 DPPH 自由基清除試驗 45
4-3-1 不同生長因子影響 45
4-3-2 不同進料策略影響 46
第五章 結論 59
參考文獻 60
參考文獻 1.Elif Oztetik, Ayse Cakir, New food for an old mouth: New enzyme for an ancient archaea, Enzyme and Microbial Technology, 2014, 55, 58-64.
2.Scott B. Bintrim, Timothy J. Donohue, Jo Handelsman, Gary P. Roberts, Robert M. Goodman, Molecular phylogeny of Archaea from soil, Microbiology, 1997, 94, 277–282.
3.Ken F. Jarrell, Sonja-Verena Albers, The archaellum: an old motility structure with a new name, Trends in Microbiology, 20, No. 2012, 7, 307-312.
4.Aharon Oren, The order halobacteriales, Prokaryote, 2006, 3,113-164.
5.Jessie L. Robinson, Brandy Pyzyna, Rachelle G. Atrasz, Christine A. Henderson, Kira L. Morrill, Anna Mae Burd, Erik DeSoucy, Rex E. Fogleman III, John B. Naylor, Sarah M. Steele, Dawn R. Elliott, Kathryn J. Leyva, and Richard F. Shand, Growth kinetics of extremely halophilic Archaea (family: Halobacteriaceae) as revealed by arrhenius plots, J. Bacteriol, 2005, 187(3), 923-929.
6.Stefan Leuko, Mark J. Raftery, Brendan P. Burns, Malcolm R. Walter, Brett A. Neilan, Global protein-level responses of Halobacterium salinarum NRC-1 to prolonged changes in external sodium chloride concentrations, Journal of Proteome Research, 2009, 8, 2218-2225.
7.Sean F. Gilmore, Andrew I. Yao, Zipora Tietel, Tobias Kind, Marc T. Facciotti, Atul N. Parikh, Role of squalene in the organization of monolayers derived from lipid extracts of Halobacterium salinarum, Langmuir, 2013, 29, 7922-7930.
8.Hans-Jűrgen Busse, Helga Stan-Lotter, Halobacterium noricense sp. nov., an archaeal isolate from a bore core of an alpine Permian salt deposit, classification of Halobacterium sp. NRC-1 as a strain of H. salinarum and emended description of H. salinarum, Extremophiles, 2004, 8, 431-439
9.Wailap Victor Ng, Sean P. Kennedy, Gregory G. Mahairas, Brian Berquist, Min Pan, Hem Dutt Shukla, Stephen R. Lasky, Nitin S. Baliga, Vesteinn Thorsson, Jennifer Sbrogna, Steven Swartzell, Douglas Weir, John Hall, Timothy A. Dahl, Russell Welti, Young Ah Goo, Brent Leithauser, Kim Keller, Randy Cruz, Michael J. Danson, David W. Hough, Deborah G. Maddocks, Peter E. Jablonski, Mark P. Krebs, Christine M. Angevine, Heather Dale, Thomas A. Isenbarger, Ronald F. Peck, Mechthild Pohlschroder, John L. Spudich, Kwang-Hwan Jung, Maqsudul Alam, Tracey Freitas, Shaobin Hou, Charles J. Daniels, Patrick P. Dennis, Arina D. Omer, Holger Ebhardt, Todd M. Lowe, Ping Liang, Monica Riley, Leroy Hood, Shiladitya DasSarma, Genome sequence of Halobacterium species NRC-1, Proc Natl Acad Sci USA, 2000, 97(22), 12176-12181.
10.Wael S. M. El-Sayed, Shinichi Takaichi, Haruo Saida, Masahiro Kamekura, Mohamed Abu-Shady Humitake Seki, Tomohiko Kuwabara, Effects of light and low oxygen tension on pigment biosynthesis in Halobacterium salinarum, revealed by a novel method to quantify both retinal and carotenoids, Plant Cell Physoil, 2002, 43(4), 379-383.
11.Muthu Manikandan, Lejla Pašic, Vijayaraghavan Kannan, Optimization of growth media for obtaining high-cell density cultures of halophilic Archaea (family Halobacteriaceae) by response surface methodology, Bioresource Technology, 2009, 100, 3107-3112.
12.D. Oesterhelt, G. Krippahl, Phototrophic growth of halobacteria and its use for isolation of photosynthetically-deficient mutants. Ann. Microbiol. (Inst. Pasteur), 1983, 134, 137-150.
13.Antoinette M. Dummer, Jessica C. Bonsall, Jacob B. Cihla, Stephanie M. Lawry, Gabriela C. Johnson, Ronald F. Peck, Bacterioopsin-mediated regulation of bacterioruberin biosynthesis in Halobacterium salinarum, Journal of Bacteriology, 2011, 193 (20), 5658-5667.
14.In M. Dworkin, S. Falkow, E. Rosenberg, K.-H. Schleifer, and E. Stackebrandt (ed.), The prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, identification, applications, 3rd ed. Springer-Verlag, New York, N.Y. Oren. A. 2001. The order Halobacteriales, p. 2, 6–19.
15.Ratnakar Deole, Jean Challacombe, Douglas W. Raiford, Wouter D. Hoff, An extremely halophilic proteobacterium combines a highly acidic proteome with a low cytoplasmic potassium content, The Journal of Biological Chemistry, 2013, 288, 581-588.
16.Chun-Jen Fang, Kuo-Lung Ku, Min-Hsiung Lee, Nan-Wei Su, Influence of nutritive factors on C50 carotenoids production by Haloferax mediterranei ATCC 33500 with two-stage cultivation, Bioresource Technology, 2010, 101, 6487-6493
17.蘇逸祥,培養綠球藻 Chlorella vulgaris 之進料策略研究以及纖維素分解酵素切解藻壁生成還原單醣之探討,國立成功大學,碩士論文,2012。
18.李孟哲,以澱粉為碳源探討 α-澱粉水解酵素之發酵環境,國立成功大學,碩士論文,2000。
19.K.P. Ho, C.Y. Tam, B. Zhou, Growth and carotenoid production of Phaffia Rhodozyma in fed-batch cultures with different feeding methods, Biotechnology Letters, 1999, 21: 175–178.
20.Chiara Schiraldi, Mariateresa Giuliano, and Mario De Rosa, Perspectives on biotechnological applications of archaea, Archaea, 2002, 1, 75–86
21.P. Calo, T. de Mlguel, C. Sielro, J.B. Velazquez' , T.G. Villa, Ketocarotenoids in halobacteria:3-hydroxy-echinenone and trans-astaxanthin, Journal of Applied Bacteriology, 1995, 79, 282-285.
22.Tuanjai Noipa, Supalax Srijaranai, Thawatchai Tuntulani, Wittaya Ngeontae, New approach for evaluation of the antioxidant capacity based on scavenging DPPH free radical in micelle systems, Food Research International, 2011, 44, 798-806.
23.J. M. C. Gutteridge, B. Halliwell, Free radicals and antioxidants in the year 2000: A historical look to the future, Annals of the New York Academy of Sciences, 2000, 899, 136-147.
24.Hülya Bayır, Reactive oxygen species, Crit. Care Med., 2005, 33 (12), 498-501.
25.Sarvajeet Singh Gill, Narendra Tuteja, Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants, Plant Physiology and Biochemistry, 2010, 48, 909-930.
26.C. W. Mulholland, J. J. Strain, Serum total free radical trapping ability in acute myocardial infarction, Clinical Biochemistry, 1991, 24, 437-441.
27.J. M. Olmedo, J. A. Yiannias, E. B. Windgassen, M. K. Gornet. Scurvy: a disease almost forgotten. Int. J. Dermatol, 2006, 45 (8), 909-913.
28.Jara Pérez-Jime’nez, Sara Arranz, Maria Tabernero, M. Elena Díaz- Rubio, José Serrano, Isabel Goñi, Fulgencio Saura-Calixto, Updated methodology to determine antioxidant capacity in plant foods, oils and beverages: extraction, measurement and expression of results, Food Research International, 2008, 41, 274-285.
29.Sasidharan P, Raja R, Karthik C, Ranandkumar Sharma, Indra Arulselvi P, Isolation and characterization of yellow pigment producing Exiguobacterium sps, J Biochem Tech, 2013, 4 (4), 632-635
30.Fernanda Mandelli, Viviane S. Miranda, Eliseu Rodrigues, Adriana Z. Mercadante, Identification of carotenoids with high antioxidant capacity produced by extremophile microorganisms, World J Microbiol Biotechnol, 2012, 28, 1781–1790
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