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研究生:劉振凰
研究生(外文):Tseng-Huang Liu
論文名稱:利用tufgene和PCR技術於乳酸桿菌之檢測與乳酸菌之安全性評估
論文名稱(外文):Use of tuf Gene and PCR Technique on the Detection of Lactobacillus Species and Safety Evaluation of Lactic Acid Bacteria
指導教授:曾浩洋曾浩洋引用關係
指導教授(外文):Hau-Yang Tsen
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品科學系
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:2004
畢業學年度:92
語文別:中文
論文頁數:89
中文關鍵詞:tuf 基因乳酸桿菌安全性評估聚合酶鏈鎖反應
外文關鍵詞:tuf geneLactobacillusSafety EvaluationPCR
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乳酸菌廣泛用於食品和動物飼料中,其分子檢測和機能性的研究廣為探討。本研究主要分兩個部分,第一部分為乳酸桿菌分子鑑定與分類;第二部分為研究室篩選之乳酸菌進行安全性評估。在分子鑑定方面,針對tuf基因序列設計特異性引子組,可以分別檢測Lactobacillus acidophilus、L. agilis、L. crispatus和L. ruminis。以上自行設計之4組引子組,從194株分離自嬰兒糞便之臨床菌株,分別檢測出2株L. acidophilus菌株、2株L. crispatus以及4株L. ruminis。另以自行設計的Lacituf-1/Lacituf-4引子組直接檢測市售標榜內含L. acidophilus之乳製產品以及飼料產品,檢測結果皆具有特異性。在安全性評估方面,從豬腸分離之益生菌Lactobacillus acidophilus LA5可以抑制Salmonella typhimurium侵入感染,Salmonella typhimurium為主要食品病原菌,會造成人類之沙門桿菌病症和動物下痢病例。本研究利用連續28天餵食Wistar大鼠三組不同濃度乳酸菌,分別為2.1×1011、1.05×1011和4.2×109 CFU/ kg大鼠體重。實驗結果發現大鼠在行為、生長、攝食、飲水、血液檢驗、臨床生化數值、主要器官重和組織切片觀察皆無嚴重有害的影響。證明Wistar大鼠攝取大量乳酸菌L. acidophilus LA5並不會有任何明顯的毒性產生。
Probiotic products from lactic acid bacteria (LAB) are widely used as human food and animal feed supplements. In addition to the evaluation of probiotic functions, molecular diagnostic methods are used for the identification of LAB cells. Two major subjects were included in this study. One was the development of DNA methods for identification of some Lactobacillus species. The other was evaluation of the safety for lactic acid bacteria strains screened with good probiotic properties. For molecular identification, we developed specific polymerase chain reaction (PCR) primers based on the DNA sequence of tuf gene, and used of these primers for the identification of lactic acid bacteria species including Lactobacillus acidophilus、L. agilis、L. crispatus and L. ruminis. 194 clinical strains isolated from infant stool samples were PCR assayed and 2 L. acidophilus、2 L. crispatus and 4 L. ruminis were identified. Furthermore, we screened lactic acid bacteria (LAB) strains from porcine intestine and evaluate their functional properties in previously study. We found a strain of Lactobacillus acidophilus LA5 as a probiotic to prevent the infection of Salmonella typhimurium is one of the major food pathogen which may cause human salmonellosis and animal diarrhea cases. The present study was conducted to evaluate the toxicity of the strain L. acidophilus LA5 in Wistar rats on dietary administration at concentrations of 2.1×1011, 1.05×1011 and 4.2×109 CFU/ kg for 28 days. There were no adverse effects on the general condition and behavior, growth, feed and water consumption, hematology, clinical chemistry values, organ weights and histopathologic analysis. Results of this study demonstrated that consumption of the strain L. acidophilus LA5 was not associated with any obvious signs of toxicity in Wistar rats even following consumption of large quantities.
目 次
頁次
中文摘要……………………………………………………………………….1
英文摘要……………………………………………………………………….2
第一章、文獻整理…………………………………………………………….3
Ⅰ、乳酸菌之分子檢測……………………………………………………….3
第一部分、乳酸菌………………………………………………………..….3
(一) 乳酸菌定義…………………………………………………………....3
(二) 乳酸菌的分類…………………………………………………………3
第二部分、乳酸菌之檢測鑑定……………………………………………..5
第三部分、應用分子生物學方法作為乳酸菌分類和鑑定的新工具……..6
(一) 限制片段長度多形性 (retriction fragment length polymorphism, RFLP)…………………………………………………………………...7
(二) 聚合酶鏈鎖反應 ( Polymerase chain reaction, PCR)……………….7
(三) 多套式聚合酶鏈鎖反應 (Multiplex-Polymerase chain reaction) 及其應用………………………………………………………………..9
(四) 隨機擴增多形性DNA(Randomly Amplified Polymorphic DNA, RAPD)………………………………………………………………9
(五) DNA探針分析法 (DNA probe method)…………………………...10
(六) 其他………………………………………………………………….11
第四部分、利用tuf 基因建立有效乳酸菌菌株之分子標幟………..……11
(一) 導論…………………………………………………………………..11
(二) tuf 基因應用於分子檢測相關之研究……………………………...13
II、乳酸菌的安全性評估…………………………………………………….16
第一部份、乳酸菌之保健功能…………………………………………….16
(一) 乳酸菌在消化道的分佈……………………………………………...16
(二) 乳酸菌在益生菌之應用……………………………………………...16
第二部分、乳酸菌安全性評估…………………………………………….18
(一) 乳酸菌安全性簡介…………………………………………………...18
(二) 安全性評估方法研究………………………………………………...19
第二章、乳酸桿菌tuf gene之PCR檢測系統之發展………………………23
壹、摘要…………………………………………………………………….23
貳、前言…………………………………………………………………….24
參、材料與方法……………………………………………………………25
實驗材料………………………………………………………………… 25
實驗方法………………………………………………………………….27
(一) PCR引子組設計……………………………………………….27
(二) 聚合酶鏈鎖反應 (PCR)……………………………………….28
1. DNA之製備………………………………………………………...28
2. 乳酸桿菌不同種之PCR特異性試驗………………………………29
3. 以PCR檢測乳酸桿菌L. acidophilus、L. agilis 、L. crispatus和 L. ruminis之靈敏度試驗………………………………………………29
4. 以LaITS-1/LaITS-2引子組篩選分離自嬰兒糞便來源之臨床菌株……………………………………………………………………..30
5. 利用可行性引子組應用於檢測嬰兒糞便來源之乳酸桿菌不同species的菌株………………………………………………………..30
6. 利用Lacituf-1/Lacituf-4引子組檢測市售乳酸菌產品和動物飼料..........................................................................................................30
肆、結果與討論…………………………………………………………….31
(一) PCR引子組之設計…………………………………………….31
(二) 聚合酶鏈鎖反應 (PCR)……………………………………….33
1. DNA之製備………………………………………………………...33
2. 利用tuf 基因之引子組Lacituf-1/Lacituf-4、Lagituf-1/Lagituf-2,Lcrituf-1/Lcrituf-2和Lrumtuf-1/Lrumtuf-2之純菌檢測系統……..33
3. Lacituf-1/Lacituf-4、Lagituf-1/Lagituf-2,Lcrituf-1/Lcrituf-2和Lrumtuf-1/Lrumtuf-2引子組之PCR靈敏度試驗………………….35
4. 利用LaITS-1/LaITS-2引子組篩選嬰兒糞便來源之乳酸桿菌…..36
5. 利用Lacituf-1/Lacituf-4、Lagituf-1/Lagituf-2,Lcrituf-1/Lcrituf-2和Lrumtuf-1/Lrumtuf-2引子組篩選嬰兒糞便之乳酸桿菌不同species之菌株……………………………………………………………….37
6. 利用Lacituf-1/Lacituf-4引子組檢測市售乳酸菌產品和動物飼料…………………………………………………………………….37
伍、結論……………………………………………………………………38
表 (2-1~2-13)……………………………………………………………...39
圖 (2-1~2-7)……………………………………………………………….53
第 三 章、利用28天餵食Wister大鼠試驗評估Enterococcus faecium TM39之安全性………………………………………………………….59
壹、中文摘要……………………………………………………………….59
貳、前言…………………………………………………………………….60
參、材料與方法…………………………………………………………61
一、28天餵食動物試驗……………………………………………..61
肆、結果與討論…………………………………………………………67
(一) 外觀與活動力…………………………………………………..67
(二) 攝食量與飲水量………………………………………………..67
(三) 體重變化………………………………………………………..68
(四) 血液學檢驗……………………………………………………..69
(五) 血清生化學檢驗………………………………………………..70
(六) 臟器重量………………………………………………………..71
(七) 組織病理檢驗…………………………………………………..72
(八) 討論……………………………………………………………..72
伍、結論…………………………………………………………………73
陸、英文摘要……………………………………………………………… 74
表(3-1~3-4)…………………………………………………………………..75
圖(3-1~3-2)…………………………………………………………………..79
第四章、參考文獻 .........................................................................................80
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