Lactobacillus reuteri於酸性與膽鹽環境中之蛋白質體研究

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黃郁芳(Yu-Feng Huang) 查詢紙本館藏

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生命科學系

論文名稱

Lactobacillus reuteri於酸性與膽鹽環境中之蛋白質體研究
(The proteomic analsis of proteins induced by low pH and bile salts in Lactobacillus reuteri)

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摘要(中)

摘要
乳酸菌一般常用來作為益生菌與食品發酵微生物。在目前已知之腸內菌中，Lactobacillus reuteri為分佈較為廣泛之菌種。當乳酸菌藉由食物之攝取進入胃腸道，將會分別在胃部遭受酸之刺激與小腸中膽鹽之壓力。透過在不同程度的酸與不同膽鹽濃度之活性測試，發現pH 2.0與0.3%膽鹽濃度會對L. reuteri造成壓力。此菌屬耐酸性之乳酸菌，具相當高之產業價值，以pH 2.0處理3小時，存活率可達48%；而在含0.3%膽鹽環境則2小時後僅達3.0%。本研究擬以蛋白體學方法研究二種胃腸道壓力對此菌造成之蛋白質表現差異。以二維電泳分離蛋白質，經分析為被誘發者，續以介質輔助雷射揮離離子化飛行時間（matrix-assisted laser desorption ionization-time of flight, MALDI-TOF）質譜儀分析並透過在資料庫中比對?肽質量指紋以鑑定之。比較各實驗組中蛋白質之表現，分別在pH 2.0酸性環境、含0.3%膽鹽濃度與模擬腸道條件下，各有11、9與6個蛋白質點被誘發。在本研究中除了參與轉錄調節作用之?54與?S外，與蛋白質摺疊相關之GroEL以及參與脫氮作用之nitrous oxide reductase (NosL)亦在二種壓力環境中被誘發。酸休克蛋白、ornithine carbamoyltransferase、ATPase ?與?次單位只在酸性環境中被誘發。而保護蛋白GroES和ClpB、逆境蛋白AhpC、ATP-binding cassette (ABC) transporter則只有在處理膽鹽時增加其表現量。再者可以在膽鹽存在之環境中被誘發之D-alanine-D-alanine lygase是有關膽鹽可能造成細胞壁傷害之發現。

摘要(英)

Abstract
Lactic acid bacteria (LAB) are commonly used as probiotics or starter cultures in the fermented foods. Among them, Lactobacillus reuteri is the only enterolactobacillus known to be indigenous in a broad spectrum of hosts. When lactic acid bacteria were eaten with food, the microorganisms suffer the stresses of acid and bile salts in stomach and in duodenum, respectively. To investigate the proteome of stress response from lactic acid bacteria under these two types of stress, the viability of the bacterium under different pH and bile salts concentration was studied. Treated with Men-Rogosa-sharp (MRS) (pH 2.0) medium for 2 hours, 48% of Lactobacillus reuteri ATCC23272 survived. The survival rate was 3.0% under 0.3% bile salts in MRS (pH 5.7) medium for 2 hours. The proteomes of L. reuteri ATCC23272 treated with pH 2.0 or 0.3% bile salts were analyzed by two-dimensional polyacrylamide gel electrophoresis (2-DE) and quantified by Commassie blue staining. The peptide mass fingerprints of induced proteins were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry via searching the SWISS-PROT and TrEMBL databases. Comparing the protein patterns of cell extracts from MRS media, there are 11, 9, and 6 spots induced by pH 2.0, 0.3% bile salts and the combination of two conditions, respectively. In addition to ?54 and ?S involved in the regulation of transcription, protein involved in folding such as GroEL is induced by two types of stress. Nitrous oxide reductase (NosL) is also induced by two types of stress. The acid shock protein, ornithine carbamoyltransferase, ATPase ? and ? subunit are acid response proteins induced by acid only. And GroES, ClpB, alkyl hydroperoxide reductase (AhpC) and ATP-binding cassette (ABC) transporter are induced when L. reuteri is treated with bile salts. The induction of D-alanine-D-alanine lygase (Ddl) by bile salts indicates bile salts may damage bacterial cell wall.

關鍵字(中)

★ 蛋白質體學

關鍵字(英)

★ proteomics
★ Lactobacillus reuteri

論文目次

中文摘要………………...………..………………………………………..I
英文摘要….………………...…………………..………………………….II
目錄………..…………………………………………………………….…IV
圖目錄…………………………….…………………………………….…VI
表目錄…………………………….……………………………………....VII
縮寫與全名對照表………………………………………………………VIII
壹、緒論........................................................................................................ 1
一、蛋白質體學與微生物壓力反應........................................................ 1
二、乳酸菌與胃腸逆境............................................................................ 2
三、乳酸菌相關之逆境蛋白.................................................................... 6
四、研究大綱............................................................................................ 8
貳、實驗材料與方法................................................................................. 10
一、菌種與培養基.................................................................................. 10
二、二維電泳相關藥品配製.................................................................. 10
三、實驗方法.......................................................................................... 12
1. 生長曲線.......................................................................................... 12
2. 菌體存活率...................................................................................... 12
3. 二維電泳法...................................................................................... 13
4. 蛋白質偵測與分析.......................................................................... 16
5. 膠體內消化...................................................................................... 17
6. 質譜儀分析...................................................................................... 17
7. 資料庫之搜尋.................................................................................. 18
四、實驗儀器與藥品.............................................................................. 18
叁、結果...................................................................................................... 21
一、細菌生長.......................................................................................... 21
二、細菌形態.......................................................................................... 22
三、細胞粗萃液製備與二維電泳之改良.............................................. 22
四、二維電泳圖譜.................................................................................. 23
五、蛋白質鑑定...................................................................................... 25
肆、討論...................................................................................................... 27
一、轉譯調節.......................................................................................... 27
二、蛋白質摺疊...................................................................................... 27
三、酸反應蛋白...................................................................................... 28
四、其他功能蛋白.................................................................................. 29
伍、結論與建議......................................................................................... 32
陸、參考文獻.............................................................................................. 33
圖…………………………………………………………………………..39
表…………………………………………………………………………..55
附錄一、Peptide mass fingerprint 鑑定之結果…………………………..60
圖目錄
圖一、L. delbrueckii sp. bulgaricus 在酸刺激下之二維電泳圖譜….. 39
圖二、L. delbrueckii sp. Bulgaricus 經碳源轉換之二維電泳圖譜..…40
圖三、L. reuteri 於MRS（pH 5.7）培養液之生長曲線….…………41
圖四、L. reuteri 於不同pH 值之MRS 培養液之生長與存活率…....42
圖五、L. reuteri 於含不同濃度膽鹽之MRS 培養液之生長與存活……………………………………………………………….43
圖六、L. reuteri 培養於不同MRS 培養液之掃描式電子顯微鏡菌體
形態觀察………………………………….……………………44
圖七、未經核苷酸水解酶與去氧核酸水解酶處理之細胞粗萃液之二維電泳圖…………………………………………………….45
圖八、經核苷酸水解酶處理之細胞粗萃液之二維電泳圖………….46
圖九、L. reuteri 培養於MRS（pH 2.0）培養液之二維電泳圖………47
圖十、L. reuteri 培養於MRS（pH 5.7）培養液之二維電泳圖….….48
圖十一、L. reuteri 培養於含0.3%膽鹽MRS（pH 5.7）培養液之二維電泳圖…………………………………………………….49
圖十二、L. reuteri 經MRS（pH 2.0）與0.3%膽鹽MRS（pH 5.7）
培養液連續處理之二維電泳圖…………………………….50
圖十三、Spot 1 之MALDI-TOF 質譜儀分析圖譜與鑑定結果……..51
圖十四、L. reuteri 經不同處理所誘發之蛋白質點………………….52
圖十五、Argenine deiminase 系統…………………………………….53
圖十六、細菌細胞壁之peptidoglycan 合成途徑…………………….54
表目錄
表一、已發表之細菌基因體………………………………………… 55
表二、鑑定L. reuteri ATCC23272 由酸、膽鹽與模擬胃腸逆境連續
處理誘發表現之蛋白質………………………………………57
表三、L. reuteri ATCC23272 受酸及膽鹽誘發蛋白質之功能………59

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指導教授

黃雪莉(Shir-Li Huang)

審核日期

2002-7-18

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