臺灣博碩士論文加值系統

Ectoine為許多嗜鹽菌或耐鹽菌在高鹽環境下，為維持及平衡細胞內外滲透壓所合成的一種相容性溶質(compatible solute)。研究顯示ectoine為兼具抗紫外線輻射、抗皮膚老化、保濕、治療阿茲海默症(Alzheimer’s disease)等多功能之天然細胞保護劑，屬於多功能之全方位生技化妝品。基於ectoine生產菌株主要為嗜鹽菌或耐鹽菌，必須在高鹽 (高滲透壓)之培養基進行培養以生產ectoine。因此，預期調控培養基質之滲透壓，將有效影響微生物代謝合成ectoine之程度與機制。因此本研究擬自高鹽環境所獲得之ectoine優質化生產菌Marinococcus sp.進行最適化培養，並觀察及評估不同培養基所產生之滲透壓及營養度對Marinococcus sp.合成ectoine能力之影響。首先藉由調整培養基之鹽份(salinity)濃度來調控其滲透壓，以觀察其對ectoine產能之影響。研究證實當YAMS(Yeast extract-Ammonium acetate Mineral Salts)及YTMS(Yeast extract-Tryptone Mineral Salts)培養基中，鹽份(sodium chloride，NaCl)濃度分別為3M及2.5M時，ectoine合成達最高產量分別為23及27 mg/L，此時相對應之滲透壓分別為7025及6010 mmol/kg，菌體生長分別為0.74及1.24 g/L，另LBMS(Luria-Bertani Mineral Salts)培養基中，NaCl濃度為2M時，ectoine產量高達574 mg/L，相對應之滲透壓為5240 mmol/kg，菌體生長為9.34 g/L，由上述結論顯示：低滲透壓，不利於菌體合成ectoine；反之，當高滲透壓之環境，卻有助於產物ectoine之生合成。
本研究同時測定不同培養基之間的滲透壓下降速率，實驗證實在滲透壓、ectoine之生合成與營養度三者之間，唯一具高度相關性之參數為滲透壓下降速率與ectoine之生合成，而不同培養基具不同之滲透壓下降速率，但具有共同之趨勢，當滲透壓下降速率高達一定值，ectoine產率便開始趨於平穩下降，此時便出現對應於ectoine產量下之最佳滲透壓下降速率數值，當YAMS、LBMS及YTMS培養基中，ectoine產量之最大值，分別為23、27及574 mg/L，此時對應滲透壓下降速率之最佳值分別為234、200及174 mmol/kg/min，最終將選擇ectoine產量具最大值之LBMS培養基進行探討。接著以LBMS培養基，培養完之菌體放置不同種類及濃度之滲透壓誘導劑－蔗糖(sucrose)、葡萄糖(glucose)、甘露醣醇(mannitol)、氯化鈉(NaCl)、谷氨酸(glutamate)中，探討ectoine之釋放量，發現添加sucrose、glucose、mannitol之釋放溶液其滲透壓較低，與培養基中所產生之滲透壓差大，所以ectoine的釋放量明顯較NaCl及glutamate高。另外，分別添加不同種類之滲透保護劑，其添加ectoine保護劑於培養基中，可促進菌體生長及ectoine產量，分別高達8.26 g/L及1706 mg/L，且明顯高於控制組。最後，本研究以5L醱酵槽進行製程放大，同時添加滲透保護劑ectoine，並結合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44小時後更高達2528 mg/L，ectoine產率則有1379 mg/L/Day之多。

To master the osmotic stress of saline environments, halophilic bacterium accumulate highly water-soluble organic osmolytes, so-called compatible solutes. Ectoine, the compatible solute that was first discovered in Ectothiorhodospira halochloris, is one of the most commonly found osmolytes in nature. Ectoine is a natural cell protective agent that it can against UV radiation, protect skin from wrinkle, moisture and treatment of Alzheimer's disease. Therefore ectoine is widely used in cosmetic biotechnology. A large amount of salts (High osmotic pressure) must be added to the medium for the production of ectoine in the current process. Therefore, the expected regulation of culture substrates osmotic pressure will affect the extent and mechanism of the microbial metabolic synthesis of ectoine. This study was obtained from high salt environment of ectoine production of quality strains Marinococcus sp. for optimal culture, and evaluation of different medium by osmolarity and nutrition synthesis of ectoine abilities in Marinococcus sp. First by the adjustment of medium concentration of salt (salinity) to regulate their osmotic pressure, to observe the effects of its ectoine production. In YAMS (Yeast the extract-Ammonium acetate, Mineral Salts) and YTMS (Yeast the extract-tryptone Mineral Salts) medium, the concentration of salt (sodium chloride, NaCl) 3M and 2.5M, respectively, ectoine synthesis highest yields were 23 and 27 ppm, that corresponds to the osmotic pressure of 7025 and 6010 mmol/kg, cell growth were 0.74 and 1.24 g/L, another LBMS (Luria,-Bertani Mineral Salts) medium, NaCl concentration of 2M, ectoine yield reach to 574 ppm, corresponding to the osmotic pressure of 5240 mmol/kg, cell dry weight can reach to 9.34 g / L, Shown by the above conclusions: the low osmotic pressure, contribute to cell growth, but is not conducive to bacterial synthesis of ectoine. In contrast, high osmotic pressure of the environment, detrimental to cell growth, but it helps the product of ectoine biosynthesis.
In this study, determination of osmolality decreasing rate between the different medium, the experiments confirmed that in the osmolality, ectoine the biosynthesis and nutrition among, only with a high degree of correlation parameters for the osmolality decreasing rate and ectoine biosynthesis different medium with different osmolality decreasing rate, but have a common trend when the osmolality decreasing rate reach a critical value, ectoine production began to tend to a steady decrease at this time, the osmolality decreasing rate of the optimal value corresponds to ectoine production,when YAMS、LBMS and YTMS medium, ectoine yield the maximum value, respectively, 23,27 and 574 ppm, the corresponding osmolality decreasing rate of the optimal value were 234,200 and 174 mmol / kg / min, will eventually choose the ectoine yield a maximum LBMS medium. Then, the LBMS medium, cultured bacteria into the osmolality of different types and concentrations of inducer (sucrose, glucose, mannitol, NaCl, glutamate) in the explore the release of ectoine, found that adding the release of sucrose, glucose, mannitol solution, its osmolality low and medium osmolality difference between the ectoine the release was significantly higher than NaCl and glutamate. In addition,different types of medium were added osmoprotectant ectoine add protective agent in the medium to promote cell growth and ectoine production, reach to 8.26 g/L and 1706 ppm respectively, and significantly higher than the control group.
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 and productivity even reach to 2528 mg/L and 1379 mg/L/Day via the fed-batch fermentation strategy, which the feed timing of yeast extract was depended on pH and DO factors.

中文摘要 iv
英文摘要 vii
誌謝 ix
表目錄 xiv
圖目錄 xv
附錄 xvii
第一章　前言 1
1.1 微生物調控滲透壓機制 1
1.2 Marinococcus sp.菌種介紹 3
1.3 Ectoine簡介 4
1.3.1 生合成途徑 4
1.4 Ectoines的生產 7
1.5 Ectoine的應用 8
1.5.1 保溼 8
1.5.2 抗UV照射並防止肌膚老化 9
1.6 鹽度對電導度及滲透壓之影響 10
1.7 研究目的與策略 10
第二章 材料與方法 12
2.1 實驗材料 12
2.1.1 實驗菌株 12
2.1.2 實驗藥品 12
2.1.3 實驗儀器 13
2.1.4 培養基 14
2.2 實驗方法 15
2.2.1 批次醱酵實驗 15
2.2.1.1 菌株之保存 15
2.2.1.2 菌株之培養 15
2.2.2 水之萃取對於ectoine之釋放實驗 [ bacteria milking (Sauer and Galinski, 1998) ] 16
2.2.3 饋料批次醱酵策略－結合pH和DO之特性 (Wei, Yuan et al., 2011) 16
2.3 分析方法 17
2.3.1 測定培養基之電導度與滲透壓 17
2.3.2 菌體乾重之測定 17
2.3.3 ectoine之濃度分析 18
第三章 結果與討論 21
3.1 論文研究架構 21
3.2 培養基中電導度對 Marinococcus sp.代謝ectoine之影響 22
3.2.1 培養基中添加不同濃度之 NaCl 22
3.2.2 培養基中個別添加主培養基成份中之離子 23
3.3 不同培養基中滲透壓對 Marinococcus sp.代謝ectoine之影響 25
3.3.1 培養基中個別添加主培養基成份中之離子 25
3.3.2 YAMS培養基中添加不同濃度之 NaCl 27
3.3.3 YTMS培養基中添加不同濃度之 NaCl 27
3.3.4 LBMS培養基中添加不同濃度之 NaCl 28
3.3.5 菌體經osmotic downshock過程後，胞內所含之殘留量 29
3.4 不同培養基中Ectoine釋放過程滲透壓之變化 34
3.5 不同培養基之滲透壓、Ectoine生合成及營養度之相互關係 35
3.5.1 YAMS培養基 35
3.5.2 YTMS培養基 36
3.5.3 LBMS培養基 36
3.5.4 三種不同培養基間之相關性 37
3.6 Ectoine釋放過程中，添加不同滲透誘導劑之影響性 41
3.7 培養基中添加不同滲透誘導劑，對胞內Ectoine累積之影響 45
3.8培養基中，添加不同種類之滲透保護劑 48
3.9 醱酵槽之應用 49
3.9.1 批次醱酵 (Batch) 49
3.9.2 饋料批次醱酵－結合pH與DO之特性 (Fed-batch) 50
第四章 結論與未來展望 53
4.1 培養基中電導度之探討 53
4.2 培養基中滲透壓之研究 54
4.3 滲透壓之調控策略 54
4.4 醱酵槽之應用 56
4.5 未來展望 56
第五章 參考文獻 58

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