臺灣博碩士論文加值系統

經由6G-果糖轉移酶催化蔗糖產生的新果寡糖是一種益生源。本研究主要是以法夫酵母（Xanthophyllomyces dendrorhous BCRC 22500）的液態醱酵生產胞內6G-果糖轉移酶。醱酵在五公升醱酵槽中進行，操作體積3公升，溫度20℃，轉速300 rpm，通氣速率2 vvm。另外改變其它醱酵條件，然後測定獲得的酵素活性和細胞重量。醱酵培養基的主要成分是酵母抽出物和蔗糖。醱酵過程因為產酸導致pH值下降，因此自動添加2 N 氫氧化鈉以控制酸鹼度。醱酵控制於不同pH時，發現pH 7是最適的酸鹼值。通常在低濃度時，酵素和細胞產量隨著酵母抽出物和蔗糖的濃度提高而增加，例如10 %蔗糖優於5 %蔗糖，然而3 %酵母抽出物卻優於4 %酵母抽出物。添加鋅離子是負面效果，而鎂離子具有正面效果。使用3 %酵母抽出物和10 %蔗糖為培養基，醱酵24小時得到每毫升17.6單位的酵素活性以及每公升58.9公克的濕細胞。為了降低成本，用酵母粉的鹼性水解物取代昂貴的酵母抽出物，使用3 %酵母粉的鹼性水解物、10 %蔗糖以及0.3 %硫酸鎂為培養基，可以得到每毫升15.9單位的酵素活性以及每公升53.3公克的濕細胞。

Neofructooligosaccharides (neoFOS), one of the prebiotics, can be produced by the catalytic reaction of 6G-fructosyltransferase on sucrose. In this work, production of intracellular 6G-fructosyltransferase by submerged culture of Xanthophyllomyces dendrorhous BCRC 22500 was investigated. The fermentation was performed in a 5-L fermenter with a working volume of 3 L at 20oC, 300 rpm and 2 vvm. Other fermentation conditions were varied and the biomass and enzyme activity were compared. The fermentation medium consisted mainly of yeast extract and sucrose. During the fermentation the pH of culture broth decreased due to the production of organic acid, therefore the pH was controlled by automatic addition of 2 N NaOH. When the fermentation was carried out at different pH, an optimal pH at 7.0 was obtained. Generally at low concentration, biomass and enzyme activity increased with the increase of yeast extract or sucrose. Initial concentration of sucrose at 10% was better than at 5%. However, yeast extract of 3% was better than of 4%. Zinc ion has negative effect whereas magnesium ion has positive effect. When the culture medium was composed of 3% yeast extract and 10% sucrose, after fermentation for 24 h 6G-fructosyltransferase activity at 17.6 U/mL (fresh cell 58.9 g/L) was achieved. Furthermore, in order to reduce the cost of culture medium, the expensive yeast extract was replaced by an alkaline hydrolysate of yeast powder. 6G-fructosyltransferase activity at 15.9 U/mL (fresh cell 53.3 g/L) was obtained when the fermentation medium was composed of 10% sucrose, 0.3% magnesium sulfate and an alkaline hydrolysate of 3% yeast powder.

誌謝i
中文摘要ii
英文摘要iii
目錄v
表索引ix
圖索引x
第一章 前言與研究目的1
1.1 前言1
1.2 研究目的3
第二章 文獻回顧4
2.1 果寡糖(Fructooligosaccharides, FOS) 簡介4
2.2 果寡糖 (FOS) 的來源6
2.3 果糖轉移酶 (Fructosyltransferase) 之簡介10
2.4 高效液相層析之簡介12
第三章 材料與方法14
3.1 實驗與層析儀器14
3.1.1 實驗儀器14
3.1.2 高效液相層析儀15
3.2 實驗材料16
3.2.1 實驗菌種來源16
3.2.2 實驗藥品16
3.3 實驗方法17
3.3.1 種菌活化17
3.3.2 種菌培養17
3.3.3 醱酵培養18
3.3.3.1 醱酵槽裝置18
3.3.3.2 接菌培養19
3.3.3.3 五公升醱酵槽操作條件20
3.3.4 醱酵槽培養方法20
3.4 樣品分析方法24
3.4.1 液態醱酵的酵素活性測定24
3.4.2 酵素活性單位 25
3.4.3 計算酵素活性 25
3.4.4 HPLC分析測定方法25
3.4.4.1 分析條件檢測器25
3.4.4.2製備標準品25
第四章 實驗結果與討論27
4.1 利用不同濃度蔗糖進行醱酵槽培養X. dendrorhous之生長曲線測定27
4.2鋅離子和鎂離子對醱酵生產6G-果糖轉移酶的影響29
4.3以10%(w/v)蔗糖進行醱酵生產6G-果糖轉移酶：不同pH值的影響31
4.4 利用不同濃度氮源進行醱酵槽培養X. dendrorhous之生長曲線測定36
4.4.1比較3%、4% 的Yeast extract兩個濃度及5% 蔗糖生產酵素36
4.4.2比較3%、4% 的Yeast extract兩個濃度及10%蔗糖生產酵素38
4.4.3比較 3%、4% Yeast extract和5%、10%蔗糖生產酵素40
4.5 利用不同濃度蔗糖進行醱酵槽培養X. dendrorhous之生長曲線測定42
第五章 結論45
第六章 參考文獻47

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