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研究生:張雅琪
研究生(外文):Ya-Chi Chang
論文名稱:嗜鹽菌Haloferaxmediterranei生產類胡蘿蔔素之最適化探討
論文名稱(外文):Study on the optimization of carotenoids production by Haloferax mediterranei
指導教授:林銘澤
指導教授(外文):Ming-Tse Lin
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:136
中文關鍵詞:醱酵類胡蘿蔔素Haloferax mediterranei實驗設計最適化
外文關鍵詞:experiment designfermentationcarotenoidsHaloferax mediterraneioptimal
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根據饋料醱酵結果,培養基中導電度控制於1300 μs/cm (10% NaCl) 以下,或將培養後的Haloferax mediterranei菌體,加入含有5% NaCl的培養基中,可以誘導出較多的類胡蘿蔔素產量。
而後,為了瞭解無機鹽的種類及其濃度對菌體生長及類胡蘿蔔素生產之影響;因此運用了Plackett-Burman design (PBD)、陡升法 (Method of steepest ascent) 及中心混層設計等實驗設計,找出培養基中最適的無機鹽類及添加的濃度。從PBD結果得知,NaCl、MgCl2及MgSO4會明顯影響類胡蘿蔔素的生產,而後再由陡升法、以及中心混層設計找出類胡蘿蔔素最適生產的鹽類濃度分別為NaCl 79.95 、MgCl2 10.44 與MgSO4 19.26 g/L,其可獲得最佳之類胡蘿蔔素產量為 1208 �慊/L。於此之外,再由PBD實驗設計鹽類範圍NaCl (156和234 g/L)、MgCl2 (0和6.09 g/L)、MgSO4 (0和9.78 g/L)、CaCl2 (0和0.5068 g/L)、KCl (0和4 g/L)、NaHCO3 (0和0.2 g/L)、NaBr (0和0.5 g/L)、NH4Cl (0和2 g/L)、KH2PO4 (0和0.3 g/L)、FeCl3 (0和0.005 g/L)和Ttrisodium citrate (0和0.05 g/L) 結果得知MgCl2、MgSO4、NaHCO3會顯著促進菌體之生長,而NaCl則會抑制菌體之生長。最後再由陡升法設計得到鹽類添加量為NaCl 117、MgCl2 7.735、MgSO4 11.932、NaHCO3 0.222 g/L時,可達到較佳的細胞生長速率μm (1.04±0.04 OD520nmh-1)及細胞濃度 (9.29±0.05 OD520nm)。
由不同有機溶劑萃取研究發現,當菌體乾重對丙酮的重量比例低於0.5,且於室溫下萃取十分鐘即可萃取得菌塊內所含的類胡蘿蔔素。再將類胡蘿蔔素經矽膠色層分析分離純化後,經TLC、HPLC分離及光譜分析,推測具有三種主要類胡蘿蔔素。
According to the results of feed-batch fermentation, below 1300 μs/cm conductivity could obtain highest yield of cartotenoid. Besides, Haloferax mediterranei could be induced more pigment when transferred to another medium that contained 5% NaCl.
A statistical methodology, combining Plackett-Burman design (PBD) with method of steepest ascent and central composite design, was applied to optimize the inorganic salt components and their concentrations in liquid fermentative medium for cell growth and cartotenoids production of Haloferax mediterran. Among the eleven inorganic salts investigated with PBD, NaCl, MgCl2, and MgSO4 were selected because of their effects on the production of cartotenoids. By methods of steepest ascent analysis and central composite design, the concentrations of inorganic salts were NaCl 79.95 g/L, MgCl2 10.44 g/L, and MgSO4 19.26g/L for the optimal production of cartotenoids that was 1208 �慊/L. In addtion, NaCl, MgCl2, MgSO4, and NaHCO3 were also effective on cell growth by PBD method. From the steepest ascent analysis, the better combination of inorganic salts were NaCl 117, MgCl2 7.735, MgSO4 11.932, and NaHCO3 0.222 g/L for the cell growth rate (μm, 1.04±0.04 OD520nmh-1) and maximum cell concentration (9.29±0.05 OD520nm).
Using different solvents to extract cartotenoids, the highest yield was obtained by acetone extraction. Meanwhile, the time needed for the acetone-extraction of cartotenoid was less than 10 min. as the solid-solvent ratio ≦ 0.5. Thee major carotenoids were separated and identified by silica gel chomatography, TLC, HPLC, and molecular spectrum.
目錄
誌謝 i
摘要 ii
英文摘要 iv
目錄 vi
圖目錄 ix
表目錄 xi
壹、緒論 1
1.1前言 1
1.2 研究目的 2
貳、文獻回顧 4
2.1類胡蘿蔔素之特性與應用 4
2.1.1類胡蘿蔔素之特性 4
2.1.2 類胡蘿蔔之來源 6
2.1.3 類胡蘿蔔素之應用 11
2.2 古生菌 32
2.2.1古生菌的分類 32
2.2.2 嗜鹽菌之介紹 33
2.3實驗設計法之原理與應用 38
2.3.1 實驗設計法使用目的 38
2.3.2 反應曲面法 (Response surface methodology, RSM) 39
2.3.3 二水準因數設計 (Two-level factorial design) 42
2.3.4 陡升路徑法 (Method of path of steepest ascent, PSA) 43
2.3.5 中心混層設計 (central composite design, CCD) 44
2.3.6 利用實驗設計法增加類胡蘿蔔素產量之應用 47
參、材料與方法 49
3.1 實驗儀器 49
3.2 實驗材料 51
3.2.1 菌種來源 51
3.2.2 藥品 51
3.3 實驗架構 52
3.4 實驗方法 53
3.4.1 菌種保存與培養方法 53
3.4.2 醱酵培養 54
3.4.3 利用實驗設計法找出最適菌體色素生產條件 59
3.4.4 實驗樣本分析 60
3.4.5 H. mediterranei類胡蘿蔔素之分析 62
肆、結果與討論 66
4.1 Haloferax mediterranei之類胡蘿蔔素的最適化萃取條件探討 66
4.1.1 不同有機溶劑萃取之比較 66
4.1.2丙酮最適化萃取條件之探討 67
4.2 不同鹽濃度誘導Haloferax mediterranei色素之生產 71
4.2.1控制導電度之批次饋料醱酵 71
4.2.2 利用二階段搖瓶培養誘導Haloferax mediterranei色素表現 77
4.3 最適嗜鹽菌類胡蘿蔔素生產之培養基中鹽類濃度之探討 79
4.3.1 Plackett-Burman Design 79
4.3.2 利用陡升路徑法尋找極值 85
4.3.3 中心混成實驗設計 88
4.4 最適嗜鹽菌生長之培養基中鹽類濃度之探討 97
4.4.1 Plackett-Burman設計法 97
4.4.2 利用陡升路徑法尋找極值 99
4.4.3 尋找最適嗜鹽菌生長之培養基鹽類濃度範圍 102
4.5 嗜鹽菌類胡蘿蔔素之純化與分析 108
伍、結論 115
陸、參考文獻 117
柒、附錄 127
Appendix 1. Advantech Genie Software的醱酵控制圖型與程序設計 127
Appendix 2. 醱酵槽菌體濃度吸光值與細胞乾重之標準曲線圖 132
Appendix 3. 搖瓶菌體濃度吸光值與細胞乾重之標準曲線圖 133
方俊仁,2008,由Haloferax spp.生產C50類胡蘿蔔素之研究。碩士論文,台灣大學農業化學研究所。
朱方儀,2002,以Liposomes模式探討五種含氧類胡蘿蔔素之抗氧化功能。碩士論文,中山醫學大學營養科學研究所。
何宜倩,2008,三種天然物之保肝及抗肝癌細胞活性之研究。博士論文,大同大學生物工程研究所。
呂世欽,2004,白殭菌蛋白質分解酵素發酵製備之研究探討。碩士論文,朝陽科技大學應用化學系。
林惠敏,2005,嗜鹽菌Haloferax meditteranei PHA生合成之探討。碩士論文,大同大學生物工程研究所。
周建良,2005,醣脂類生物界面活性劑rhamnolipid醱酵基質最適化及生產策略之研究。碩士論文,成功大學化學工程系。
許淑惠,2004,利用擠壓米康澱粉於改變導電度下生產耐鹽菌之紅色色素的研究。碩士論文,大同大學生物工程研究所。
陳炳輝。2000。類胡蘿蔔素的特性與應用。科學發展月刊。28(8): 599-604。
陳嘉佩,2003,台灣芒果的類胡蘿蔔素分析和乾燥方法對其安定性的影響。碩士論文,輔仁大學食品營養學系。
黃韋成,2003,以反應曲面法探討鱈魚魚漿製品最適化研究。碩士論文,屏東科技大學食品科學系。
楊正峯,2007,以類胡蘿蔔素穩定之微脂粒包覆去氧核醣核酸水解酶之研究。碩士論文,大同大學生物工程研究所。
薛顯能,2006,嗜鹽菌Haloferax meditteranei紅色色素之抗氧化性及褪色性反應研究。碩士論文,大同大學生物工程研究所。
蕭辛癸,2000,利用氰細菌生產胡蘿蔔素與脂肪酸之探討。碩士論文,海洋大學食品科學所。
Asker, D., and Y. Ohta. 1999. Production of canthaxanthin by extremely Halophilic bacteria. J. Biosci. Bioeng. 88: 617-621.
Aementa-Lopez, R., I. Guerrero, and S. Huerta. 2002. Astaxanthin extraction from shrimp waste by lactic fermentation and enzymatic hydrolysis of the carotenoprotein complex. J. Food Sci. 67: 1002-1006.
Bauernfeind, J.C. 1972. Carotenoid vitamin A precursors and analogs in foods and Feeds. J. Agric. Food Chem. 20: 456-473.
Bhaskar, N., P. V. Suresh, P. Z. Sakhare, and N. M. Sacchindra. 2007. Shrimp biowaste fermentation with Pediococcus acidolactici CFR2182: Optimization of fermentation conditions by response surface methodology and effect of optimized conditions on deproteination/ demineralization and carotenoid recovery. Enzyme Microb. Technol. 40: 1427-1434.
Box, G. E., W. Hunter, J. S. Hunter. 1978. Statistics for experimenters. John Wiley and Sons, New York.
Britton, G., S. Liaaen-Jensen, and H. Phander. 1998. Carotenoids. Volume 3: Biosynthesis and metabolism. Birkhäuser Verlag. Switzerland. p. 94-140.
Britton, G., S. Liaaen-Jensen, and H. Pfander. 2004. Carotenoids Handbook. Birkhäuser Verlag. Switzerland. p.453-456.
Buzzini, P., A. Martini, M. Gaetani, B. Turchetti, U. M. Pagnoni, P. Davoli. 2005. Optimization of carotenoid production by Rhodotorula graminis DBVPG 7021 as a function of trace element concentration by means of response surface analysis. Enzyme Microb. Technol. 36: 687-692.
Chen, B.H. 1992. Studies on the stability of carotenoids in garland chrysanthemum as affected by microwave and conventional heating. J. Food Protec. 55: 296-300.
Chen, B. H., and Y. Y. Chen. 1993. Stability of chlorophylls and carotenoids in sweet potato leaves during microwave cooking. J. Agric. Food Chem. 41: 1315-1320.
Chen, D., Y. Han, and Z. Gu. 2006. Application of statistical methodology to the optimization of fermentative medium for carotenoids production by Rhodobacter sphaeroides. Process Biochem. 41: 1773-1778.
Chou, H. E., and W. M. Breene. 1972. Oxidative decoloration of β-carotene in low-moisture model systems. J. Food Sci. 37: 66-68.
Don, T. M., C. W. Chen, and T. H. Chan. 2006. Preparation and characterization of poly (hydroxyalkanoate) from the fermentation of Haloferax mediterranei. J. Biomater. Sci. Polymer Edn. 17: 1425-1438.
D'Souza, S. E., W. Altekar, and S. F. D'Souza. 1997. Adaptive response of Haloferax mediterranei to low concentrations of NaCl (< 20%) in the growth medium. Arch. Microbiol. 168: 68-71.
Edge, R., D. J. McGarvey, and T. G. Truscott. 1997. The carotenoids as anti-oxidants-a review. J. Photochem. Photobiol. B, Biol. 41: 189-200.
EI-Sayed, Wael S. M., S. Takaichi, H. Saida, M. Kamekura, M. Abu-Shady, H. Seki, and T. Kuwabara. 2002. 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 Physiol. 43: 379-383.
Fraser, N. J., H. Hashimoto, and R. J. Cogdell. 2001. Carotenoids and bacterial photosynthesis: The story so far…. Photosyn. Res. 70: 249-256.
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 genomic. BMC Genomics. 5: 3.
Goodwin T. W. 1992. Distribution of carotenoids. Methods Enzymol. 213: 167-175.
Gu, Z., D. Chen, Y. Han, Z. Chen, and F. Gu. 2007. Optimization of carotenoids extraction from Rhodobacter sphaeroides. Food Sci. Technol. 41: 1082-1088.
Holt, J. G., J. T. Staley, and M. P. Bryant. 2000. Archaeaobacteria. Bergery’s manual of systematic bacteriology, Vol 3. Lippincott Williams & Wilkins, New York. p. 2226-2228.
Lee, S. H., and D. B. Min. 1990. Effects, quenching mechanisms, and kinetics of carotenoids in chlorophyll-sensitized photoxidation of soybean oil. J. Agric. Food Chem. 38: 1630-1634.
Liebler, D. C., and T. D. McClure. 1996. Antioxidant reactions of beta-carotene: identification of carotenoid-radical adducts. Chem. Res. Toxicol. 9: 8-11.
Lillo, J. G. and F. Rodriguez-Valera. 1990. Effect of culture conditions on poly (β-hydroxybutyric acid) production by Haloferax mediterranei. Appl. Environ. Microbiol. 56: 2517-2521.
Johnson, E. A., and W. J. Lewis. 1979. Astaxanthin formation by the yeast Phaffia rhodozyma. J. Gen. Microbiol. 115: 173-183.
Kelly, M. and Liaaen-Jensen S. 1967. Bacterial carotenoids XXVI. C50-Carotenoids. II. Bacterioruberin. Acta Chem. Scand. 21: 2578-2580.
Kelly, M., Norgard S., and Liaaen-Jensen S. 1970. Bacterial carotenoids XXXI. C50-Carotenoids. V. Carotenoids of Halobacterium salinarium, especially bacterioruberin. Acta Chem. Scand. 24: 2169-2182.
Kushner, D. J. 1978. Life in high salt and solute concentrations: halophilic bacteria. In D. J. Kushner, (ed) Microbial life in extreme environments. Academic Press, London. p. 317-214.
Kushwaha, S. C., Kramer J. K. G. and Kates M. 1975. Isolation and characterization of C50-carotenoid pigments and other polar isoprenoids from Halobacterium cutirubrum. Biochim. Biophys. Acta. 398: 303-314.
Kushwaha, S. C. and M. Kates. 1976. Effect of nicotine on biosynthesis of C50 carotenoids in Halobacterium cutirubrum. Can. J. Biochem. 54: 824-829.
Miller, N. J., J. Sampson, L. P. Candeias, P. M. BramLey, C. A. Rice-Evans. 1996. Antioxidant activities of carotenes and xanthophylls. FEBS Lett. 384: 240-242.
Montgomery, D.C. 2005. Design and analysis of experiments. John Wiley and Sons, New York.
Obón, J. M., A. Manjón, and J. L. Iborra. 1996. Comparative thermostability of glucose dehydrogenase from Haloferax mediterranei. Effects of salts and polyols. Enzyme Microb. Technol. 19: 352-360.
Paolini, M., S. Z. Abdel-Rahman, A. Sapone, G. F. Pedulli, P. Perocco, G. Cantelli-Forti, and M. S. Legator. 2003. β-carotene: a cancer chemopreventive agent or a co-carcinogen?. Mutat. Res. 543: 195-200.
Pesek, C. A., and J. J. Warthesen. 1988. Characterization of the photodegration of β-carotene in aqueous model systems. J. Food Sci. 53: 1517-1520.
Philip, T., and T. S. Chen. 1988. Separation and quantitative analysis of some carotenoid fatty acid easters by liquid chromatography. J. Chromatogr. 435: 113-126.
Rao, A. V., and L. G. Rao. 2007. Carotenoids and human health. Pharmacol. Res. 55: 207-216.
Rønnekleiv, M., and S. Liaaen-Jensen. 1995. Bacterial carotenoids 53, C50- carotenoids 23 ; carotenoids of Haloferax volcanii versus other Halophilic bacteria. Biochem. Syst. Ecol. 23: 627-734.
Sachindra, N. M., N. Bhaskar, and N. S. Mahendrakar. 2005. Carotenoids in different body components of Indian shrimps. J. Sci. Food. Agric. 85: 167-172.
Sachindra, N. M., and N. S. Mahendrakar. 2005. Process optimization for extraction of carotenoids from shrimp waste with vegetable oils. Bioresour. Technol. 96: 1195-1200.
Sachindra, N. M., N. Bhaskar, and N. S. Mahendrakar. 2006. Recovery of carotenoids form shrimp waste in organic solvents. Waste Manag. 26: 1092-1098.
Sachindra, N. M., N. Bhaskar, G. S. Siddegowda, A. D. Sathisha, and P. V. Suresh, 2007. Recovery of carotenoids from ensilaged shrimp waste. Bioresour. Technol. 98: 1642-1646.
Sandmann, G. 2001. Carotenoid biosynthesis and biotechnological application. Arch. Biochem. Biophys. 385: 4-12.
Scheidegger, R., A. K. Pande, P. L. Bounds, and W. H. Koppenol. 1998. The reaction of peroxynitrite with zeaxanthin. Nitric. Oxide. 2:8-16
Sharma, S. K., and M. L. Maguer. 1996. Kinetics of lycopene degradation in tomato pulp solids under different processing and storage conditions. Food Res. Intern. 29: 309-315.
Shahmohammadi, H. R., E. Asgarani, H. Terato, T. Saito, Y. Ohyama, K, Gekko, O. Yamamoto, and H. Ide. 1998. Protective roles of bacterioruberin and intracellular KCl in the resistance of Halobacterium salinarium against DNA-damaging agents. J. Radiat. Res. 39: 251-262.
Sujak, A., W. Okulski, and W. Gruszedcki. 2000. Organisation of xanthophylls pigments lutein and zeaxanthin in lipid membranes formed with dipalmitoylphophatidylcholine. Biochem. Biophys. Acta. 1509: 255-263.
Sung, L.-S., and S.-Y. Huang. 2000. Medium optimization of transformed root cultures of stizolobium hassjoo producing L-DOPA with response surface methodology. Biotechnol. Prog. 16: 1135-1140.
Strand, A., S. Shivaji, and S. Liaaen-Jensen. 1997. Bacterial carotenoids 55. C50-carotenoids 25. Revised structures of carotenoids associated with membranes in psychrotrophic Micrococcus roseus. Biochem. Syst. Ecol. 25: 547-552.
Tee, E. S. 1992. Carotenoids and retinoids in human nutrition. Crit Rev Food Sci Nutr. 31: 103-163.
Tinoi, J., N. Rakariyatham, and R.L. Deming. 2005. Simplex optimization of carotenoid production by Rhodobacter glutinis using hydrolyzed mung bean waste flour as substrate. Process Biochem. 40: 2551-2557.
Williams, A. W., T. W. Boileau, J. R. Zhou, S. K. Clinton, J. W. Erdman. 2000. Beta-carotene modulates human prostate cancer cell growth and may undergo intracellular metabolism to retinol. J. Nutr. 130: 728-732.
Willis, M. S., F, H. Wians. 2003. The role of nutrition in preventing prostate cancer: a review of the proposed mechanism of action various dietary substances. Clin. Chim. Acta. 330: 57-83.
Young, A. J. 1993. In carotenoids in photosynthesis (ed. Young A. and Britton G.). Chapman and Hall. London. p. 16-72.
Zwietering, M. H., I. Jongenburger, F. M. Rombouts, and K. Van’t Riet. 1990. Modeling of the bacterial growth curve. Appl. Environ. Microbiol. 56: 1875-1881.
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