嗜鹽菌Haloferax mediterranei生產類胡蘿蔔素之最適化探討

Study on the optimization of carotenoids production by Haloferax mediterranei

張雅琪(Ya-Chi Chang)

指導教授 ： 林銘澤

大同大學/工程學院/生物工程學系所/碩士(2008年)

摘要

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 g/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。

關鍵字

醱酵 ； 類胡蘿蔔素 ； Haloferax mediterranei ； 實驗設計 ； 最適化

並列摘要

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 g/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.

並列關鍵字

experiment design ； fermentation ； carotenoids ； Haloferax mediterranei ； optimal

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