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研究生:于佳茜
研究生(外文):Chia-Chen Yu
論文名稱:利用雙叉桿菌半乳醣苷酶生產半乳寡醣之研究
論文名稱(外文):Study on production of galacto-oligosaccahrides byβ-galactosidase from bifidobacteria
指導教授:游若篍
指導教授(外文):Roch-Chui Yu
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:90
中文關鍵詞:雙叉桿菌半乳醣██半乳寡醣水解反應轉醣反應產氣莢膜桿菌超音波破碎益生菌
外文關鍵詞:bifidobacteriaβ-galactosidasegalacto-oligosaccharideshydrosylationtransgalactosylationclostridiumultrasonic treatmentprobiotics
相關次數:
  • 被引用被引用:5
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本研究利用萃取人體腸駐型益生菌(probiotics)-雙叉桿菌(bifidobacteria)所產生之半乳醣██,並以乳醣為原料進行半乳寡醣的生產研究。結果顯示利用超音波破碎含Triton X-100的懸浮菌液(50mg/ ml),可將所選用的五株腸駐型雙叉桿菌Bifidobacterium adolescentis CCRC 14606、B. bifidum CCRC 14615、B. infantis CCRC 15416、B. infantis CCRC 14633、B. longum CCRC 14634中之半乳醣██萃取出來。利用萃取出的粗酵素液進行轉醣反應活性的探討,結果以CCRC 14634在轉醣反應之表現最好,約可得到5%左右的半乳寡醣產量,主要產物皆為三醣。再以粗酵素液進行反應條件的探討,CCRC 14634之酵素在pH 4-6間活性都很穩定,最適轉醣反應pH值為6.0;而溫度在25-55℃間酵素亦很穩定,最適轉醣反應溫度為45℃;在最適反應時間上,當反應5小時後可得最大寡醣產量,時間再延長寡醣將被水解,分析達到寡醣最大產量時的乳醣反應率約70%。而在基質濃度試驗中可得轉醣反應之Km值為1.3 M。在反應液中添加二價金屬離子並不能促進轉醣反應之作用;添加EDTA及Cu2+、Zn2+等反而會使酵素失活。利用管柱層析方式可純化半乳醣██,利用SDS-PAGE及膠體管柱層析,可得知分子量為42kDa。實驗中以CCRC 14634之半乳醣██於pH 6.0的磷酸緩衝液中,添加1.0M乳糖溶液,於50℃下反應5小時,進行半乳寡醣之大量製備,並將製備出的半乳寡醣進行菌體利用性試驗,發現自製之半乳寡醣可促進雙叉桿菌(Bifidobacterium longum CCRC 14634)生長作用,且能使雙叉桿菌產酸,快速降低培養基中之pH值,最終可達pH 4.1左右;但其對產氣莢膜桿菌(Clostridium per-fringenes CCRC 10913)之生長,也同樣有促進作用。
In order to produce galacto-oligosaccharides which is useful for probiotics, this study was conducted to study the characters ofβ-galactosidase from bifidobacteria and use lactose substrate to produce galacto-oligosaccharides. Beta-galactosidase from Bifidobacterium ado-lescentis CCRC 14606、B. bifidum CCRC 14615、B. infantis CCRC 15416、B. infantis CCRC 14633、B. longum CCRC 14634 were extracted by ultrasonic treatment in the presence of Triton X-100. Crude enzyme of CCRC 14634 was found to be the most efficient producer of galacto-oligosaccharides. The yield was about 5% of total sugar and trioligo-saccharide was main product. Crude β-galactosidase was stable over pH 4-6, with maximal transgalactosylated activity at pH 6. The optimal temperature for transgalactosylation was 45℃. The maximum production was obtained after 5 hours of reaction as 70% of initial lactose was transformed by this enzyme. In the trans-galactosylated reaction, the Km value of crude enzyme was 1.3 M for lactose. EDTA, copper, cobalt, magnesium, stannum and zinc could not improve transgalactosylation. Moreover, EDTA, copper, and zinc inhibited the enzyme activity. Using chromatography could purifiedβ-galactosidase, the molecular weight was estimated to be 42 kDa by SDS-PAGE and gel filtration. Finally, crude β-galactosidase of CCRC 14634 and suitable reaction conditions was used to prepare galacto-oligosaccharides. The prepared galacto-oligosaccharides was found to be the best carbon source for Bifido-bacterium longum CCRC 14634 when compared to other commercial galacto-oligosaccharides, lactose, and glucose. However, the prepared galacto-oligosaccharides also stimulated the growth of Clostridium perfringenes CCRC 10913.
中文摘要
英文摘要
壹﹑前言
貳﹑文獻整理
(壹)雙叉桿菌
一﹑雙叉桿菌之分類、分佈及特性
二﹑雙叉桿菌之乳酸發酵與代謝路徑
三﹑雙叉桿菌對人體健康的影響
(1) 維持腸道內正常微生物族群
(2) 活化免疫系統
(3) 抗癌
(4) 促進蛋白質分解
(5) 促進乳酸之代謝
(6) 合成維生素B群
(7) 改善乳糖不耐症
(8) 降低血液中膽固醇的量
(9) 改善腹瀉與便秘
(10) 抗氧化
四﹑雙叉桿菌生長促進因子
(1) Bifidogenic factor I(雙叉桿菌生長促進因子I)
(2) Bifidogenic factor II(雙叉桿菌生長促進因子II)
(3) Oligosaccharides(寡醣)
五﹑雙叉桿菌之應用
(1) 選擇合適之雙叉桿菌
(2) 雙叉桿菌產品之菌數要求
(3) 對氧氣的忍受度
(貳)乳糖
(參)半乳寡醣
一﹑半乳寡醣之結構
二﹑半乳寡醣之性質
(1) 加工特性
(2) 生理機能性
三﹑半乳寡醣之生產
四﹑β-galactosidase之作用機制與反應條件
(1) 酵素來源
(2) 基質特性與濃度
(3) 酵素狀態
(4) 反應條件
(5) 乳糖轉換率
五﹑寡醣市場與開發
參﹑材料與方法
(壹)實驗材料
一﹑試驗菌株
二﹑培養基與藥品
(1) 培養基
(2) 藥品
三﹑主要儀器設備及器材
(貳)實驗方法
一﹑雙叉乳酸桿菌之保存與更新
二﹑雙叉乳酸桿菌接種原之製備
三﹑半乳醣██粗酵素液的萃取
四﹑蛋白質濃度分析
五﹑半乳醣██水解活性分析
六﹑半乳醣██轉醣活性分析
七﹑半乳醣██作用特性
(1) 最適反應時間
(2) 最適反應溫度
(3) 最適作用pH值
(4) 乳糖濃度
(5) 金屬離子與EDTA的影響
(6) 添加單糖(葡萄糖、半乳糖)的影響
八﹑半乳醣██的純化
九﹑蛋白質膠體電泳
十﹑雙叉桿菌與產氣莢膜桿菌對不同碳源之利用性檢測
十一﹑HPLC分析結果之計算方式
十二﹑統計分析方法
肆﹑結果與討論
一﹑雙叉桿菌不同菌株間在水解與轉糖反應活性的測定
(1) 反應條件之探討
(2) 半乳醣██基質活性的探討
二﹑半乳醣██最適反應條件的探討
(1) 最適反應時間
(2) 最適反應pH值
(3) 最適反應溫度
(4) 基質濃度變化
(5) 添加金屬離子
(6) 添加單糖的效應
三﹑半乳醣██的純化
四﹑半乳寡醣對於雙叉桿菌與產氣莢膜桿菌的影響
伍﹑結論
陸﹑參考文獻
圖次
圖一﹑雙叉桿菌與同型或異型發酵細菌對葡萄糖代謝途徑之差異
圖二﹑腸內菌群與人體健康之關係
圖三﹑商品化半乳寡醣之產品特性
圖四﹑半乳寡醣之合成機制
圖五﹑乳糖經雙叉桿菌半乳醣██粗酵素液作用後所產生之半乳寡醣、
葡萄糖、半乳醣及殘留乳糖之層析圖
圖六﹑雙叉桿菌半乳醣██以乳糖為基質,隨反應時間之變化三醣以上
寡醣生成情形
圖七﹑雙叉桿菌半乳醣██以乳糖為基質,隨反應時間探討基質與生成
物之變化情形
圖八﹑雙叉桿菌半乳醣██生成三醣以上寡醣與乳醣反應率之關係
圖九﹑不同反應pH值對雙叉桿菌半乳醣██酵素活性的影響
圖十﹑不同反應溫度對雙叉桿菌半乳醣██酵素活性的影響
圖十一﹑不同基質濃度對雙叉桿菌半乳醣██酵素活性的影響
圖十二﹑添加單糖對B. longum CCRC 14634半乳醣██酵素活性的影響
圖十三﹑利用SephacrylTM S-300膠體層析進行B. longum CCRC 14634
半乳醣██之純化
圖十四﹑利用Q sepharose fast flow進行B. longum CCRC 14634半乳
醣██的離子交換純化
圖十五﹑B. longum CCRC 14634之半乳醣██之native-PAGE
圖十六﹑B. longum CCRC 14634之半乳醣██之SDS-PAGE
圖十七﹑利用SDS-PAGE進行B. longum CCRC 14634之半乳醣██分子量
標定
圖十八﹑利用膠體層析法進行B. longum CCRC 14634之半乳醣██分子
量標定
圖十九﹑不同碳源的添加對雙叉桿菌與產氣莢膜桿菌生長的影響
圖二十﹑葡萄糖、乳糖、自製半乳寡醣與商品化半乳寡醣等不同碳源對
雙叉桿菌、產氣莢膜桿菌生長及產酸的影響
表次
表一﹑雙叉桿菌之酵素活性分析
表二﹑各種形式乳糖之物理性質特性
表三﹑半乳醣██利用乳糖生產半乳寡醣之結構
表四﹑不同酵素來源與反應條件下獲取最大寡醣產量
表五﹑雙叉桿菌之半乳醣██活性
表六﹑金屬離子對雙叉桿菌半乳醣██酵素活性的影響
表七﹑B. longum CCRC 14634半乳醣██的純化
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