跳到主要內容

臺灣博碩士論文加值系統

(18.97.9.172) 您好!臺灣時間:2025/02/14 03:30
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:袁芳偉
研究生(外文):Fang-Wei Yuan
論文名稱:利用本土嗜鹽海洋球菌Marinococcussp.醱酵生產肌膚抗皺成分Ectoine之研究
論文名稱(外文):Process development of a skin-care component – Ectoine using Marinococcus sp.
指導教授:魏毓宏
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技暨生物資訊研究所
學門:生命科學學門
學類:生物科技學類
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:86
中文關鍵詞:EctoineMarinococcus sp.培養基最適化回應曲面實驗設計法兩階段醱酵策略
外文關鍵詞:EctoineMarinococcus sp.RSMtwo stage fermentation strategy
相關次數:
  • 被引用被引用:4
  • 點閱點閱:624
  • 評分評分:
  • 下載下載:17
  • 收藏至我的研究室書目清單書目收藏:1
本研究所使用之菌株Marinococcus sp.係由台灣南部高鹽環境下篩選而得,且經菌種分析後確定其為一新穎之菌株。而Marinococcus sp.之產物透過13C-NMR、1H-NMR與FAB-MS之分析確定其為ectoine。當Marinococcus sp.培養於初始之複合培養基 (YCMS) 時,其ectoine產量僅有0.4 g/L,但經由培養條件最適化並利用回應曲面法 (Response surface methodology,RSM) 進行培養基最適化探討後,除ectoine產量可高達3.5 g/L之外,產率更達2.3 g/L/Day之多。
之後,透過不同醱酵策略之結合對於ectoine之生產更有進一步之突破。首先,針對Marinococcus sp.在環境滲透壓急遽下降時進行探討,結果顯示,除細胞不會因滲透壓之急遽改變而破裂或萎縮外,同時,細胞內所累積之ectoine更能釋放至環境中。因此,利用特性所進行之連續萃取醱酵策略,已將ectoine產率提升至超過3.0 g/L/Day;另外,於重複批次策略中,Marinococcus sp.培養至93小時更有最高產量達7.0 g/L。而將上述兩策略進行結合之兩階段醱酵生產策略,ectoine最高產量仍然可高達6.0 g/L,但在產率方面已比單階段之策略更大幅提升至10.0 g/L/Day之多,且Marinococcus sp.在經過多次萃取與長時間培養後,依然可維持菌體量與生產ectoine之活性,此對於未來若要大量且連續生產ectoine將會是一大優勢。
最後,本研究以5 L醱酵槽進行製程放大,而批次醱酵之結果顯示,不論是利用轉速或純氧控制溶氧 (dissolved oxygen,DO),當Marinococcus sp.培養至30小時左右ectoine產量皆可達3.3 g/L之多,至於ectoine產率與content則大約為2.6 g/L/Day與11 %,這些結果皆與搖瓶實驗具有一致性。此外,結合pH與DO作為yeast extract饋料時機之饋料批次醱酵策略,ectoine產量於Marinococcus sp.培養34小時後更高達5.6 g/L,ectoine產率則有3.9 g/L/Day之多。
綜合上述之結果,本研究所進行一系列Marinococcus sp.醱酵生產ectoine策略之探討,已成功提升ectoine之產量與產率。於搖瓶策略中,ectoine最高產量為7.0 g/L,而最佳產率更超過10.0 g/L/Day;而於醱酵槽策略中,ectoine最高產量與產率則為5.6 g/L與3.9 g/L/Day。特別於ectoine產率部份,更為目前文獻最佳値2.0 g/L/Day之5倍。
A halophilic bacterium isolated from a salt environment located in southern Taiwan was identified as a Marinococcus sp. The bacterium could synthesize and accumulate intracellular ectoine as a compatible solute to resist osmotic stress in a hyper-osmotic environment. The chemical structure and molecular mass of the purified product was confirmed by 13C-NMR, 1H-NMR, and FAB-MS. The fermentation medium composition was optimized via response surface methodology (RSM) to improve ectoine production from Marinococcus sp. With the optimization strategy, the ectoine production was enhanced from 0.6 g/L to 3.5g/L.
Subsequently, in order to break through the ectoine production, so use the different fermentation strategies. First, ectoine accumulated by Marinococcus sp. to cope with hyperosmotic pressure environment, interested, the ectoine are released from the microorganisms when they are exposed to osmotic downshock. Via this characteristic, in this study developed a continue extracted fermentation strategy and the ectoine productivity was advance to 3.0 g/L/Day; in addition, the ectoine concentration even reach to 7.0 g/L when Marinococcus sp. incubated for 93 hours through the repeat-batch fermentation strategy. So combine the continue extracted fermentation strategy and repeat-batch fermentation strategy, the result was amazingly. In the two stage fermentation strategy, the maximum ectoine concentration was not only reached to 6.0 g/L but also the ectoine productivity was more than 10.0 g/L/Day. By the way, although the Marinococcus sp. incubated for a long time and extracted repeatedly but the microorganisms still maintain the ability of ectoine produce, and it is an advantage for the ectoine production.
Finally, to achieve a large scale of supply for ectoine in compliance with the demands, the 5 L fermentor has been used. The results show that ectoine concentration, content and productivity can reach to 3.3 g/L, 11% and 2.6 g/L/Day when the Marinococcus sp. incubated for 30 hours, respectively. Moreover, the ectoine concentration and productivity even reach to 5.6 g/L and 3.9 g/L/Day via the fed-batch fermentation strategy, which the feed timing of yeast extract was depended on pH and DO factors.
The ectoine production by Marinococcus sp. has successes development in this study. Where the maximum ectoine concentration was reach to 7.0 g/L as well as the ectoine productivity even more than 10.0 g/L/Day through the different fermentation strategies in flask cultured. In addition, the ectoine concentration and productivity were also reaching to 5.6 g/L and 3.9 g/L/Day. Especially, the maximum ectoine productivity in this study was the highest in the ectoine production field.
第一章 前言………………………………………………… 1
1.1 Marinococcus sp.……………………………….……… 1
1.2 Ectoine之簡介………………………………….……….. 1
1.3 回應曲面法 (Response surface methodology,RSM)… 4
1.3.1 二水準因子設計………………………….……… 5
1.3.2 陡升路徑法……………………………….……… 5
1.3.3 中心混成設計……………………………………… 6
1.4 研究動機與目的………………………………….…… 6
第二章 材料與方法………………………………………… 10
2.1 實驗材料………………………………………… 10
2.1.1 實驗菌株………………………………….……… 10
2.1.2 培養基……………………………………………… 10
2.2 實驗方法………………………………………… 10
2.2.1 批次醱酵實驗……………………………………… 10
2.2.1.1 菌株之保存…………………………………… 10
2.2.1.2 菌株之培養……………...……………………… 11
2.2.2 回應曲面法(Response surface methodology,RSM) 11
2.2.2.1 實驗設計………………...……………………… 11
2.2.3 連續萃取醱酵實驗………………………………… 12
2.2.3.1 重複批次與菌液濃縮之實驗…………… 12
2.2.3.2 水之萃取體積對於ectoine釋放量之影響…… 13
2.2.3.3 連續醱酵策略………………………………… 13
2.2.3.4 掃描式電子顯微鏡(Scanning electron microscope, SEM) ……………………………… 14
2.3 分析方法………………………………………… 14
2.3.1 菌體濃度…………………………………………… 14
2.3.2 ectoine之濃度分析………………………………… 15
2.3.3 ectoine之粗萃取…………………………………… 15
2.3.4 Ectoine之定性分析……………………………… 16
第三章 結果與討論………………………………………… 19
3.1 論文研究架構……………………………………… 19
3.2 Ectoine醱酵生產菌株之篩選……………………… 20
3.3 Ectoine之定性與定量分析………………………… 21
3.3.1 定性分析…………………………………………… 21
3.3.2 定量分析…………………………………………… 22
3.4 溫度、pH、轉速與NaCl濃度對於Marinococcus sp.代謝ectoine之影響………………………………… 23
3.4.1 溫度、pH、轉速……………………………………… 23
3.4.2 NaCl濃度對於Marinococcus sp.代謝ectoine之影響…………………………………………………… 25
3.5 不同碳、氮源對於Marinococcus sp.代謝ectoine之影響………………………………………………… 26
3.6 利用回應曲面法(Response surface methodology,RSM)進行最適化培養基之開發…………………… 27
3.6.1 二水準因子設計 (Two-level factorial design)…… 28
3.6.2 陡升路徑法 (Method of path of steepest) ………… 29
3.6.3 回應曲面法 (Response surface methodology) … 29
3.6.4 最適化培養基條件之確認………………………… 31
3.7 Ectoine連續釋放醱酵策略 (bacteria milking)……… 32
3.7.1 Ectoine萃取溶劑之篩選………………………… 32
3.7.2 掃描式電子顯微鏡 (Scanning electron microscope, SEM) 與水萃取體積對於Ectoine釋放率之影響………………………………………… 33
3.7.3 水萃取體積之篩選………………………………… 35
3.7.4 重複批次醱酵策略 (repeat-batch stratery)……… 36
3.7.5 連續釋放醱酵策略與重複批次醱酵策略之結合… 37
3.7.6 連續釋放醱酵策略與菌液濃縮策略之結合… 39
3.7.7 不同醱酵策略對於Marinococcus sp.生產ectoine能力之比較………………………………………… 41
3.8 醱酵槽之應用…………………………………………… 41
3.8.1 批次醱酵 (Batch)………………………………… 41
3.8.2 饋料批次醱酵-利用pH之特性 (Fed-batch) …… 44
3.8.3 饋料批次醱酵-結合pH與DO之特性 (Fed-batch) 45
3.8.4 批次與饋料批次策略對Marinococcus sp.生產ectoine能力之比較………………………………… 46
第四章 結論與未來展望…………………………………… 75
4.1 結論……………………………………………… 75
4.1.1 一新穎之ectoine醱酵生產菌株:Marinococcus sp.………………………………………………… 75
4.1.2 最適化培養基之開發……………………………… 75
4.1.3 連續萃取醱酵策略之探討………………………… 76
4.1.4 醱酵槽之應用……………………………………… 80
4.2 未來展望………………………………………… 81
參考文獻………………………..……………………………… 82
[1] Hao MV, Kocur M, Komagata K. Marinococcus gen.nov.,a new genus for motile cocci with meso-diaminopimclic acid in the cell wall;and Marinococcus albus sp.nov.,and Marinococcus halophilus (Novitsky and Kushner)comb.nov. J Gen Appl Microbiol 1984; 30: 449-459.
[2] Marquez MC, Ventosa A, Ruiz-Berraquero F. Marinococcus hispanicus, a new species of moderately halophilic gram-positive cocci. International Journal of Systematic Bacteriology 1990; 40 (2): 165-169.
[3] Novitsky TJ, Kushner DJ. Planococcus halophilus sp. nov., a facultatively halophilic coccus. Int J Syst Bacteriol 1976; 26: 53-57.
[4] Li WJ, Schumann P, Zhang YQ, Chen GZ, Tian XP, Xu LH, Stackebrandt E, Jiang CL. Marinococcus halotolerans sp. nov., isolated from Qinghai, north-west China. Int J Syst Evol Microbiol 2005; 55: 1801-1804.
[5] Kraegeloh A, Kunte HJ. Novel insights into the role of potassium for osmoregulation in Halomonas elongata. Extremophiles 2002; 6: 453-462.
[6] Larsen H. The halobacteria''s confusion to biology. Antonie van Leeuwenhoek 1973; 33: 383-396.
[7] Eisenberg H, Wachtel EJ. Structural studies of halophilic proteins, ribosomes, and organelles of bacteria adapted to extreme salt concentrations. Annu Rev Biophys Biophys Chem 1987; 16: 69-92.
[8] Lai MC, Sowers KR, Robertson DE, Roberts MF, Gunsalus RP. Distribution of compatible solutes in the halophilic methanogenic archaebacteria. J Bacteriol 1991; 173: 5352-5358.
[9] Csonka LN. Physiological and genetic responses of bacteria to osmotic stress. Microbiol Rev 1989; 53: 121-147.
[10] Severin J, Wohlfarth A, Galinski EA. The predominant role of recently discovered tetrahydropyrimidines for the osmoadaptation of halophilic bacteria. J Gen Microbiol 1992; 138: 1629-1638.
[11] Galinski EA, Truper HG. Microbial behaviour in salt-stressed ecosystems. FEMS Microbiol Rev 1994; 15: 95-108.
[12] Roberts MF. Organic compatible solutes of halotolerant and halophilic microorganisms. Saline Systems 2005; 1: 5.
[13] Perlman DF, Goldstein L. Organic osmolyte channels in cell volume regulation in vertebrates. J Exp Zool 1999; 283: 725-733.
[14] Edwards MD, Booth IR, Miller S. Gating the bacterial mechanosensitive channels: MscS a new paradigm? Curr Opin Microbiol 2004; 7: 163-167.
[15] Sauer T, Galinski EA. Bacterial milking: A novel bioprocess for production of compatible solutes. Biotechnol Bioeng 1998; 57: 306-313.
[16] Blount P, Moe PC. Bacterial mechanosensitive channels: integrating physiology, structure and function. Trends Microbiol 1999; 7: 420-424.
[17] Brown AD, Simpson JR. Water relations of sugar-tolerant yeasts: the role of intracellular polyols. J Gen Microbiol 1972; 72: 589-591.
[18] Lippert K, Galinski EA. Enzyme stabilization be ectoine-type compatible solutes: protection against heating, freezing and drying. Applied Microbiology and Biotechnology 1992; 37: 61-65.
[19] Louis P, Truper HG, Galinski EA. Survival of Escherichia coil during drying and storage in the presence of compatible solutes. Appl Microbiol Biotechnol 1994; 41: 684-688.
[20] Nau-Wagner G, Boch J, Le Good JA, Bremer E. High-affinity transport of choline-O-sulfate and its use as a compatible solute in Bacillus subtilis. Appl Environ Microbiol 1999; 65: 560-568.
[21] Kappes R, Bremer E. Response of Bacillus subtilis to high osmolarity : uptack of carnitine, crotonobetaine and ?butyrobetaine via the ABC transport system OpuC. Microbiology 1998; 144: 83-90.
[22] Pflughoeft KJ, Kierek K, Watnick PI. Role of ectoine in Vibrio cholerae osmoadaptation. Appl Environ Microbiol 2003; 69: 5919-5927.
[23] Galinski EA, Pfeiffer HP, Truper HG. 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid. A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira. Eur J Biochem 1985; 149: 135-139.
[24] Garcia-Estepa R, Canovas D, Iglesias-Guerra F, Ventosa A, Csonka LN, Nieto JJ, Vargas C. Osmoprotection of Salmonella enterica serovar Typhimurium by Ngamma-acetyldiaminobutyrate, the precursor of the compatible solute ectoine. Syst Appl Microbiol 2006; 29: 626-633.
[25] Nagata S, Wang YB. Accumulation of ectoine in the halotolerant Brevibacterium sp. JCM 6894. J Biosci Bioeng 2001; 91: 288-293.
[26] Ono H, Okuda M, Tongpim S, Imai KO, Shinmyo A, Sakuda S, Kaneko Y, Murooka Y, Takano M. Accumulation of compatible solutes, ectoine and hydroxyectoine, in a moderate halophile, Halomonas elongata KS3 isolated from dry salty land in Thailand. Journal of Fermentation and Bioengineering 1998; 85 (4): 362-368.
[27] Nagata S, Wang C. Effect of duration of osmotic downshock and coexisting glutamate on survival and uptake of ectoine in halotolerant Brevibacterium sp. JCM 6894. J Biosci Bioeng 2006; 101: 57-62.
[28] Nagata S, Wang C. Efficient utilization of ectoine by halophilic Brevibacterium species and Escherichia coli subjected to osmotic downshock. J Biosci Bioeng 2005; 99: 61-67.
[29] Nagata S, Wang Y, Oshima A, Zhang L, Miyake H, Sasaki H, Ishida A. Efficient cyclic system to yield ectoine using Brevibacterium sp. JCM 6894 subjected to osmotic downshock. Biotechnol Bioeng 2008; 99: 941-948.
[30] Onraedt A, De MC, Walcarius B, Soetaert W, Vandamme E. Ectoine accumulation in Brevibacterium epidermis. Biotechnol Lett 2004; 26: 1481-1485.
[31] Nakayama H, Yoshida K, Ono H, Murooka Y, Shinmyo A. Ectoine, the compatible solute of Halomonas elongata, confers hyperosmotic tolerance in cultured tobacco cells. Plant Physiol 2000; 122: 1239-1247.
[32] Nagata S, Wang YB. Interrelation between synthesis and uptake of ectoine for the growth of the halotolerant Brevibacterium species JCM 6894 at high osmolarity. Microbios 2001; 104: 7-15.
[33] Wang YB, Ikeuchi T, Jin Y, Nagata S. Uptake and utilization of ectoine by halotolerant Brevibacterium sp. JCM 6894 subjected to osmotic downshock. J Biosci Bioeng 2002; 94: 440-446.
[34] Onraedt AE, Walcarius BA, Soetaert WK, Vandamme EJ. Optimization of ectoine synthesis through fed-batch fermentation of Brevibacterium epidermis. Biotechnol Prog 2005; 21: 1206-1212.
[35] Ono H, Sawada K, Khunajakr N, Tao T, Yamamoto M, Hiramoto M, Shinmyo A, Takano M, Murooka Y. Characterization of biosynthetic enzymes for ectoine as a compatible solute in a moderately halophilic eubacterium, Halomonas elongata. J Bacteriol 1999; 181: 91-99.
[36] Schubert T, Maskow T, Benndorf D, Harms H, Breuer U. Continuous synthesis and excretion of the compatible solute ectoine by a transgenic, nonhalophilic bacterium. Appl Environ Microbiol 2007; 73: 3343-3347.
[37] Bursy J, Pierik AJ, Pica N, Bremer E. Osmotically induced synthesis of the compatible solute hydroxyectoine is mediated by an evolutionarily conserved ectoine hydroxylase. J Biol Chem 2007; 282: 31147-31155.
[38] Malin G, Lapidot A. Induction of synthesis of tetrahydropyrimidine derivatives in Streptomyces strains and their effect on Escherichia coli in response to osmotic and heat stress. J Bacteriol 1996; 178: 385-395.
[39] Farwick M, Siewe RM, Kramer R. Glycine betaine uptake after hyperosmotic shift in Corynebacterium glutamicum. J Bacteriol 1995; 177: 4690-4695.
[40] Sauer T, Schwarz T, Galinski EA. Ectoine-biotechnische produktion und mogliche anwendungsbereiche. GIT Labor-Fachzeitschrift 1995; 10: 892-896.
[41] 蒙哥馬利 (Montgomery, Douglas C.):實驗設計與分析 (2003)
[42] 韓竹婷:重金屬汙染環境中抗高濃度類金屬碲微生物之分離與探討,私立元智大學生物科技暨生物資訊研究所,碩士論文,(2007)。
[43] 林冠穎:海洋分離之Oceanicola marinus新種與ectoine生產之Marinococcus菌株特性研究,國立高雄海洋科技大學水產食品科學所,碩士論文,(2007)。
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊