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研究生:葉哲宏
研究生(外文):CHE-HUNG YEH
論文名稱:開發快速抓取啤酒中乳酸菌之免疫磁珠分離法
論文名稱(外文):Development of Immunoseparation Method for The Rapid Capture of LAB in Beers
指導教授:李世傑李世傑引用關係李泰林李泰林引用關係曾浩洋曾浩洋引用關係
指導教授(外文):SHIH-CHIEH SLEETAI-LIN LIHAO-YANG TSENG
學位類別:碩士
校院名稱:大葉大學
系所名稱:分子生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:92
中文關鍵詞:免疫磁珠乳酸菌生產線微生物食品啤酒晶片
外文關鍵詞:immunomagnetic separation(IMS)Lactobacillus sppBeer chip
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中文摘要

近年來已陸續發展出許多種鑑定乳酸菌的方法,如利用生化、培養形態等方法,然而傳
統生化學原理所發展出來之乳酸菌檢驗方法。以往在食品及生物產業用來檢測其中所含
微生物的方法,大都以傳統方法鑑定,但此種方法在檢測時必需先花費相當時間,以
LAB 言,可能需要一星期去培養所要檢測的微生物。其中光花在培養微生物的時間上,
就已經佔去了大半的實驗時間。
在已知之分子檢測法中,對於大量菌株具有同時檢測之能力者,則以生物晶片為首選。本碩士論文的工作,即利用晶宇生技公司的生物晶片,去檢測啤酒中的造成品質敗
壞的乳酸菌,包括Lactobacillus中的brevis、case、plantarum以及Pediococcus spp.etc.
且為建立線上監控系統 ,我們採用免疫磁珠方法吸附濃縮這些菌株,進而以生物
晶片檢測之。我們先以兔子免疫,生產上述乳酸菌的抗體。這些抗體純化後,再與磁珠(magnetic beads) 結合,形成免疫磁珠(Immunomagnetic bead);我們再使用這
些免疫磁珠,運用於這些啤酒中特定LAB的吸附、濃縮。進而將吸附的 LAB 以生物晶
片檢測,如此,完成啤酒晶片生產線上監督之監督系統。
ABSTRACT

In recent years, developement of the molecular diagnostic methods for bacteria identification has been noteworthy. Conventional methods for detection of LAB are laborious and time consuming and may require 1 week . Of the molecular dctection methods, when many LAB species are subjected to identification, the biochip method could be used. To establish the online monitoring system for the detection of the LAB cells in beers during brewing, in my thesis work, the immunomagentic beads method specific for LAB species in beers will be developed for the collection and concentration of lactic acid bacteria cells including Lactobacillus brevis, L. casei, L. plantarum and Pediococcus damnosus. These four lactic acid bacteria may spoil the beer and should be detected online during the brewing processes. The antibody to these LAB species was obtained from immunized rabbit and then purified. The purified antibody was linked to magneticbead. After obtaining the immunomagnetic bead, the LAB cells in beer were captured and detected using the biochip from Dr. Chip Company. By this way, the online monitoring system for detection of LAB during the beer brewing process could be establish.
目 錄

封面內頁
簽名頁
授權書.............................................................iii
中文摘要............................................................iv
英文摘要.............................................................v
誌謝................................................................vi
目錄...............................................................vii
圖目錄..............................................................xi
表目錄.............................................................xiv

1. 前言.............................................................1
2. 文獻整理.........................................................2
2.1 啤酒釀造過程乳酸菌的影響..........................................2
2.2 乳酸菌菌種簡介...................................................4
2.2.1 乳酸菌的定義...................................................4
2.2.2 乳酸菌的分類...................................................5
2.3 鑑定乳酸菌的方法.................................................6
2.3.1 傳統生理生化鑑定...............................................7
2.3.1.1 革蘭氏染色法.................................................7
2.3.1.2 Catalase試驗................................................7
2.3.1.3 API 50CHL菌種鑑定...........................................7
2.3.2 聚合酶連鎖反應(polymerase chain reaction;PCR) ................8
2.3.3 生物晶片法(Biochip method) ....................................9
2.3.4 免疫磁珠分離法(immunomagnetic separation;IMS) ...............10
2.4 抗體免疫原理....................................................11
2.4.1 何謂免疫系統..................................................11
2.4.1.1 基本特性...................................................11
2.4.1.2 抗體抗原的結構..............................................12
2.4.1.3 抗體的運作方式..............................................12
2.4.2 抗體的種類....................................................13
3. 材料與方法.......................................................15
3.1 材料...........................................................15
3.1.1 菌種.........................................................15
3.1.2 動物.........................................................15
3.1.3 藥品.........................................................15
3.1.4 試驗材料.....................................................17
3.1.5 儀器.........................................................17
3.2 方法...........................................................18
3.2.1 菌株收集培養..................................................19
3.2.2 抗原乳劑處理..................................................19
3.2.2.1 抗原處理...................................................19
3.2.2.2 免疫.......................................................20
3.2.3 抗血清製備....................................................20
3.2.3.1 採血.......................................................20
3.2.3.2 血清製備...................................................20
3.2.4 抗血清力價測試................................................21
3.2.5 免疫球蛋白純化................................................22
3.2.6 IgG抗體蛋白片段大小鑑定........................................22
3.2.6.1 SDS-PAGE電泳膠片配製........................................22
3.2.6.2 電泳步驟...................................................23
3.2.6.3 膠體染色...................................................24
3.2.7 利用FPLC(Fast Protein Liquid Chromatography)製備抗體蛋白......25
3.2.7.1 蛋白精製純化................................................25
3.2.7.2 AKTA purifier儀器清洗、保養.................................25
3.2.8 免疫磁珠表面抗體接合反應.......................................26
3.2.8.1 國產免疫磁珠(Tanbead U-118、U-128) .........................26
3.2.8.2 進口免疫磁珠(Dynabead M-280) ...............................26
3.2.9 免疫磁珠表面菌體分離...........................................27
3.2.9.1 標準菌液磁珠吸附............................................27
3.2.9.2 模擬工廠末端製備完成啤酒液微量菌磁珠吸附.......................27
3.2.10 免疫磁珠表面菌體分離液研究分析.................................28
3.2.10.1 菌量測定..................................................28
3.2.10.2 菌種測定..................................................28
3.2.11 免疫磁珠清洗保存.............................................28
4. 結果............................................................30
4.1 乳酸菌的培養條件................................................30
4.2 利用紐西蘭長耳兔所製備抗體力價測試結果.............................30
4.3 利用AKTA purifier純化抗體蛋白質IgG結果...........................31
4.4 個別設計四種乳酸菌的特異性引子,並利用標準乳酸 菌株,以測試引子的靈敏
度與專一性.....................................................32
4.5 免疫磁珠與標準菌液(108 cfu/ml)作用後,利用PCR 引子擴增法測試結果....32
4.6 免疫磁珠與標準菌液(108 cfu/ml)作用後,利用生化 塗菌方法分析結果......33
4.7 利用市售生啤酒,模擬工廠端之乳酸菌微量測試結果......................33
4.8 直接利用兔子的抗血清與免疫磁珠結合與測試結果........................34
4.9 測試免疫磁珠可否重複使用,並使用於檢測同種乳酸菌株...................34
5. 討論............................................................35
6. 結論...........................................................37
參考文獻............................................................66
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