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研究生:蔡政志
研究生(外文):Cheng-Chih Tsai
論文名稱:乳酸菌之分子鑑定及其特性與機能性評估
論文名稱(外文):Molecular Identification of Lactic Acid Bacteria and Characterization as well as Functional Evaluation
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:博士
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:207
中文關鍵詞:乳酸菌幽門桿菌沙門氏菌雙歧桿菌28天餵食毒性試驗聚合酶鏈鎖反應
外文關鍵詞:lactic acid bacteriaHelicobacter pyloriSalmonella TyphimuriumBifidobacterium spp.28-day feeding toxicity studypolymerase chain reaction
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乳酸菌廣泛用於食品和飼料,其分子檢測和機能性的研究廣受探討。本研究主要分為兩大部分,第一部分為雙歧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特性與機能性評估。在分子鑑定方面,針對16S rRNA和16S-23S ITS基因序列設計特異性引子組,可以分別檢測Bifidobacterium angulatum、B. animalis、B. breve、B. dentium、B. minimum、B. adolescentis和B. asteroides。使用國外文獻之引子組Lm3/P0,從336株分離自嬰兒、成人糞便和發酵產品之乳酸菌,檢測出44株Bifidobacterium菌株,並發現3株為B. adolescentis,5株為B. angulatum,1株B. animalis和2株B. minimum。
在抑制幽門桿菌之機能性評估方面,本研究為從嬰兒糞便篩選出抗幽門桿菌之乳酸菌Enterococcus faecium TM39。其具有吸附人類上皮細胞Int 407、結腸上皮細胞Caco-2、人類子宮頸癌上皮細胞HeLa和人類胃癌細胞TSGH 9201之能力,以及具耐酸耐膽鹽能力。TM39上清液在體外試驗可明顯抑制幽門桿菌及其尿素酶活性,其抑制物質除乳酸外,還包含一些目前還未知的物質。TM39乳酸菌菌體與上清液可降低幽門桿菌吸附TSGH 9201細胞。在TM39毒性試驗方面,體外試驗證明TM39菌株不具vancomycin抗性和對人類胃癌細胞TSGH 9201及人類腸道上皮細胞Int-407不具侵入性。分別連續28天餵食TM39不同濃度1 × 1012, 5 × 1011和2 × 1010 CFU/ kg,結果證實大鼠的行為、生長、飲食、飲水、血液分析、臨床血清生化分析、器官重量和組織病理觀察並無顯著影響。因此,TM39菌株可安全用於抗幽門桿菌之用途上。
在抑制沙門氏菌侵入之機能性方面,豬腸分離之L. acidophilus LA5和雞腸分離之L. fermentum LF33,對Int-407、Caco-2和BALB/ c小鼠腸道上皮細胞皆具吸附能力。植物來源乳酸菌皆不會吸附Int-407和Caco-2細胞,其中L. plantarum BCRC10069對BALB/ c小鼠之腸道上皮細胞具有吸附能力。所以,在抑制沙門氏菌侵入Int-407、小鼠肝和脾之感染能力時,乳酸菌LA5、LF33和LP69皆可抑制。在LA5毒性試驗方面,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。實驗結果證明Wistar大鼠攝取大量乳酸菌L. acidophilus LA5也不會有任何顯著的毒性產生。
Probiotic products from lactic acid bacteria (LAB) are widely used as human food and animal feed supplements. Molecular identification and potential evaluation are widely used methods. Two major topics were included in this study, one was the development of identification methods for Bifidobacterium spp., and the other was evaluation of the potential for lactic acid bacteria strains screened in this study. For molecular identification, we developed specific polymerase chain reaction (PCR) primers from 16S rRNA or 16S-23S ITS, and used of them for the identification of lactic acid bacteria species include Bifidobacterium angulatum, B. animalis, B. breve, B. dentium, B. minimum, B. adolescentis and B. asteroides. 44 Bifidobacterium strains including 3 B. adolescentis, 5 B. angulatum, 1 B. animalis and 2 B. minimum strains were identified from 336 LAB strains isolated from infant, adult stool and ferment product using Lm3/P0 and other specific primers.
On evaluation of the potential of antagonist effect against Helicobacter pylori, a strain of Enterococcus faecium TM39 from infant feces was screened for anti-H. pylori use. The screening process was based on the capability of LAB strains to adhere to the human intestinal epithelial cell (Int 407), colonial enterocyte-like Caco-2 cell, human cervical epithelioid carcinoma cell (HeLa), and human gastric carcinoma cell (TSGH 9201), and the capability to tolerate acid and bile salts. The spent culture supernatant (SCS) of a selected strain TM39, ie, TM39-SCS, was found significantly inhibitory to the viability of H. pylori in vitro. It also inhibited the urease activity of H. pylori in vitro. For these antagonistic effects, in addition to pH and lactic acid, some factors in TM39-SCS might play the major role. Treatment of H. pylori with the SCS or cells of strain TM39 significantly reduced its binding to TSGH 9201 cells. For the toxicity study, we demonstrated that strain TM39 is not vancomycin resistant and is not invasive to TSGH and Int-407 cells in vitro. Cells of strain TM39 were oral dietary administrated at concentrations of 1×1012, 5×1011 and 2×1010 CFU/ kg to the rat for 28 days. There were no adverse effects on the general conditions and behavior, growth, feed and water consumption, hematology, clinical chemistry values, organ weights and histopathologic analysis. Results of this study demonstrated that consumption of strain E. faecium TM39 was not associated with any obvious signs of toxicity in Wistar rats even following consumption of large quantities. Therefore, the newly isolated E. faecium strain TM39 is with potential for anti-H. pylori use.
Strain L. acidophilus LA5 from porcine intestine and strain L. fermentum LF33 from poultry caecum, were found adherent to the Int-407 and Caco-2 cells, the intestinal epithelium of their host as well as BALB/c mice. On the other hand, all the strains from vegetable origins were unable to adhere to the Int-407 and Caco-2 cells. One strain from vegetable origin, ie, strain L. plantarum LP69, however, was able to adhere to the mice intestinal epithelial cells. On the antagonistic activity against Salmonella invasion to Int-407 cells and the mice livers and spleens, LAB strains LA5, LF33 and LP69 were found to have inhibitory effect. For the toxicity of the strain L. acidophilus LA5 in Wistar rats assayed by oral 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 strain L. acidophilus LA5 was also not associated with any obvious signs of toxicity in Wistar rats even following consumption of large quantities.
中文摘要…………………………………………………………………I
英文摘要……………………………………………………………….II
第一章、文獻整理………………………………………………………1
I、乳酸菌之分子檢測…………………………..…………….1
一、乳酸菌:分類與生理學……………………………….1
二、乳酸菌之歷史與分類…………………………………………2
三、分類和鑑定的新工具…………………………………………4
四、雙歧桿菌的其他用途…………………………..…………13
II、乳酸菌之保健功能………………………………………….14
一、乳酸菌在消化道的分佈……………………………………14
二、乳酸菌在益生菌之應用……………………………….…….…15
三、乳酸菌促進人體健康之保健機能………………………..25
III、幽門桿菌及其致病機制……………………………………..30
一、幽門桿菌概論……………………………………………..30
二、幽門桿菌的診斷方法…………………………………………..31
三、幽門桿菌引起之消化道疾病的治療…..………………………32
IV、乳酸菌對沙門氏菌之抑制……….……….…………………..…33
一、沙門氏菌之簡介…………………………………………….33
二、沙門氏菌之致病……………………...…………………....34
三、沙門氏菌侵入性之相關研究…………………...………...35
四、競爭性驅除……………………………..………………..35
五、乳酸菌抑制沙門氏菌侵入或感染之相關研究……………….36
V、乳酸菌的安全性評估……………………………..…………….36
一、乳酸菌安全性簡介…………………………………………..36
二、安全性評估方法研究………………………….……………..37
三、細胞毒性試驗……………………….………………………..40
四、抗生素感受性試驗……………………………...…………...40
五、侵入性評估…………………………………………….....…41
總結…………………….………………….…………………...41
第二章、雙歧桿菌16S rRNA和16S-23S ITS之PCR檢測系統之發展
壹、摘要………………………………………………….…………43
貳、前言………………………………………………………..44
參、材料與方法……………………………………........…45
實驗材料……………………………………………………….....45
實驗方法…………………………………………………………..48
(一) PCR引子組設計……………………….…………………..48
(二) 聚合酶鏈鎖反應 (PCR)………………………………...…..48
1. DNA之製備………………………………………............48
2. 雙歧桿菌不同種之PCR特異性試驗………………...........49
3. 以PCR檢測雙歧桿菌B. adolescentis和 B. asteroides
之靈敏度試驗……………………………………………............50
4. 以P0/ Lm3引子組篩選分離自嬰兒糞便來源和乳酸菌
產品之菌株……………………………………………........……50
肆、結果與討論………………………………………….......……..50
(一) PCR引子組之設計…………………………………..……….........51
(二) 聚合酶鏈鎖反應 (PCR)…………………………………..............52
1. DNA之製備……………………………………………........……52
2. 利用16S rRNA之引子組Bang/ B16s-Re、Bani/ B16s-Re、
Bbre/ B16s-Re、Bden/ B16s-Re和Bmin/ B16s-Re之純菌檢
測系統..................................................…52
3. 利用16S-23S ITS rRNA引子組之純菌檢測系統…........……54
4. 利用P0/Lm3引子組篩選嬰兒、成人糞便和發酵產品
之雙岐桿菌………………...………………………………58
5. 利用可行性之引子組應用於檢測嬰兒、成人糞便和
發酵產品之雙岐桿菌不同species的菌株….......………………58
伍、結論 ……………………………………………………………59
表 ( 2-1~2-12 )…………………………………………………60
圖 ( 2-1~2-12 )…………………………………………………75
第三章、糞腸球菌 (Enterococcus faecium) TM39抑制幽門桿菌感染活性之體外試驗……..........................................…87
壹、 中文摘要………………………………………………....……87
貳、 前言.........................................88
參、材料與方法……………………………………………...……..89
實驗材料………………………………………………………......89
實驗方法…………………………………….......…………………90
1. 乳酸菌耐酸耐膽鹽...................................90
2. 組織培養之乳酸菌吸附試驗.......................90
3. 抗幽門桿菌試驗.....................................91
4. 尿素酶分析.........................................91
5. 乳酸菌抑制螢光標定幽門桿菌吸附人類細胞株...........92
6. TM39菌株之鑑定................................92
肆、結果與討論……………………………………………………92
1.吸附試驗、酸和膽鹽之耐性…...........…………93
2. TM39-SCS抗幽門桿菌之影響…...............………..93
3. TM39-SCS對幽門桿菌之尿素酶和吸附
TSGH 9201細胞之影響...................................94
4. TM39菌株鑑定.......................................94
伍、結論 …………………………………………………...………97
陸、英文摘要..........................................98
表 ( 3-1~3-3 )…………………………………….....…………......99
圖 ( 3-1~3-6 )……………………………………….....…………102
第四章、評估Enterococcus faecium TM39體外和體內毒性試驗
壹、中文摘要.........................................108
貳、前言……………………………………………………………109
參、 材料與方法………………...…………………..111
一、菌株和細胞培養生長環境…………………......……………111
二、TM39之抗生素敏感性試驗………………..……………111
三、TM39之侵入性試驗………………......………………...……113
四、28天餵食動物試驗………………………...............……113
肆、結果與討論…………………………………….....………….120
(一) 抗生素耐性........................................……120
(二) 侵入性試驗…...................................……120
(三) 外觀與活動力……..................................121
(四) 體重變化...........................……………………121
(五) 血液學檢驗…........................................…121
(六) 血清生化學檢驗...................................122
(七) 臟器重量.........................................122
(八) 組織病理檢驗.......................................122
伍、結論 …………………………………………………………125
陸、英文摘要..........................................126
表 ( 4-1~ 4-5 )…………………………………………………127
圖 ( 4-1~ 4-3 )…………………………………………………132
第五章、嗜酸乳桿菌Lactobacillus acidophilus LA5抑制沙門氏菌感染與侵入之體外和體內試驗
壹、中文摘要.........................................137
貳、前言……………………………………………………………138
參、材料與方法……………………...………………………..139
實驗材料....................................................139
實驗方法....................................................140
(一) 耐酸、耐膽鹽和腸道細胞株吸附試驗………......……………140
(二) 乳酸菌抑制S. typhimurium侵入腸道細胞株
Int 407之試驗........................................140
(三) 乳酸菌於BALB/c小鼠模式中抑制
S. typhimurium之感染試驗……..........................……141
(四) 乳酸菌在小鼠腸道上皮細胞之吸附……………………......143
(五) 以酵素連結免疫分析法 (ELISA)
測定TNF- 濃度...................................143
(六) 以酵素連結免疫分析法測定IL-6濃度.......................144
肆、結果與討論…………………………………….....………….145
一、耐酸性之評估......................................……145
二、耐膽鹽性之評估......................................……146
三、吸附腸上皮細胞株能力之評估……..........................146
四、乳酸菌抑制S. typhimurium侵入腸道細胞
株Int 407之試驗...................……………………147
五、乳酸菌於小鼠活體對Salmonella之抑制作用…................147
六、乳酸菌吸附於小鼠腸道上皮細胞之試驗......................148
七、乳酸菌抑制沙門氏菌侵入小鼠之TNF-α和IL-6測定............149
伍、結論 ……………………………………………………..……149
陸、英文摘要.........................................151
表 (5-1~ 5-4 )……………………………………………...……152
圖 (5-1~ 5-5 )……………………………………………...……156
第六章、利用28天餵食Wister大鼠試驗評估Lactobacillus acidophilus LA5之安全性
壹、中文摘要........................................162
貳、前言……………………………………………………..………163
參、材料與方法………..….......………....……..164
肆、結果與討論…………………………………….....……..…….164
(一) 外觀與活動力…….................................164
(二) 攝食量和飲水量...................……………………164
(三) 體重變化........................................165
(四) 血液學檢驗…......................................…165
(五) 血清生化學檢驗.........................................166
(六) 臟器重量.......................................168
(七) 組織病理檢驗....................................168
伍、結論 ………………………………………………….………170
陸、英文摘要.........................................171
表 (6-1~ 6-4 )…………………………………………….……172
圖 (6-1 )……………………………………..........…….…..…176
第七章、參考文獻……………………………………….…….………177
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