臺灣博碩士論文加值系統

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。

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.

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

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