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研究生:林佩佩
研究生(外文):Pai-Pai Lin
論文名稱:嬰兒糞便來源之乳酸菌抑制腸毒性及腸凝集性大腸桿菌生長能力之抑菌活性體外試驗
論文名稱(外文):Antagonistic activity against enterotoxigenic Escherichia coli and enteroaggregative Escherichia coli infection in vitro for lactic acid bacteria strains from healthy infant stool
指導教授:謝尤敏
指導教授(外文):You-Miin Hsieh
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:116
中文關鍵詞:腸毒性大腸桿菌腸凝集性大腸桿菌乳酸菌抑菌試驗
外文關鍵詞:Enterotoxigenic Escherichia coliantagonistic activitylactic acid bacteria
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腸毒性大腸桿菌 (Enterotoxigenic Escherichia coli, ETEC) 與腸凝集性大腸桿菌 (Enteroaggregative E. coli, EAggEC) 為主要造成人類下痢之病原菌之一,發生於開發中國家的幼童和從熱帶或亞熱帶地區的工業國家遊覽的旅客,也有造成學校和餐廳食物中毒之案件。益生菌可以抑制人體和動物腸道病原菌的生長,以及促進動物生長。可能的作用機制為益生菌產酸或代謝產物造成對病原菌的抑制。本研究從健康嬰兒糞便進行篩選具吸附能力的乳酸菌,再評估具吸附能力的乳酸菌抑制腸毒性大腸桿菌生長之抗菌活性。嬰兒來源之乳酸菌經益生菌所應具備特性進行篩選,包括耐酸和耐膽鹽試驗,並以人體腸道細胞株進行吸附性試驗,及利用 API 50CHL 菌種檢測系統做菌株的鑑定,結果發現 Lactobacillus acidophilus RY2、L. salivarius MM1 和 L. paracasei En4 具耐酸、耐膽鹽及抑制 ETEC 或 EAggEC 之潛力,其中,RY2 和 En4 對人類腸道細胞 C2BBel (Caco-2) 具良好吸附能力,再進一步評估乳酸菌抗菌活性經過加熱 (100℃, 15 分鐘) 和分解酵素後之抑制能力的影響,並進行乳酸菌上清液抑制 ETEC或 EAggEC 共培養之抑制能力分析。經觀察發現其乳酸菌上清液具耐熱性,且產酸能力影響抑菌能力。乳酸菌上清液抑制 ETEC 或 EAggEC 共培養之抑制能力分析結果,顯示其三株乳酸菌可於1小時內有效抑制大腸桿菌生長。最後進行發酵牛乳之製備與儲藏試驗,三株乳酸菌發酵牛乳於儲藏 4℃、14天後,L. acidophilus RY2、L. salivarius MM1 活菌數仍維持 107 CFU/mL,而 L. paracasei En4 活菌數降至 106 CFU/mL。L. acidophilus RY2、L. salivarius MM1 或 L. paracasei En4 可應用於發酵乳產品,以調控腸道中 ETEC 或 EAggEC 的菌量。
Enterotoxigenic Escherichia coli (ETEC) and Enteroaggregative E. coli (EAggEC) is a major cause of sporadic diarrhea disease in humans, affecting mainly children in developing countries and travelers from industrialized countries visiting tropical or subtropical areas. Probiotics have been shown to be inhibitory to the growth of a wide range of intestinal pathogens in human and animals. They are also able to promote the growth of animals. The possible mechanisms might include the production of acids and other by-products of the bacterial metabolism. In this study, we screen the adhesive strains of lactic acid bacteria (LAB) isolated from infant stool. Antagonistic activity was evaluated by inoculating wells among agar cultures of ETEC or EAggEC to assess zones of inhibition created by the lactobacilli cultures or spent culture supernant (SCS). Only a few isolates having high adhesive properties also possessed antagonistic activity. Identification of the isolates using API 50CHL strips showed that they belonged to different species of Lactobacillus. The objective of this study was to determine if adhesive LAB strains can exert inhibitory action on ETEC or EAggEC. Strains of Lactobacillus acdophilus RY2, Lactobacillus salivarius MM1 and Lactobacillus paracasei En4 were used in this study, based on their acid- and bile-tolerance. L. lactis RY2 and L. paracasei En4 could adhere to the cultured human intestinal C2BBel (Caco-2) cell line. After heating (100℃, 15 min), the SCS still had inhibitory effect on ETEC or EAggEC. The SCS were treated with lactate dehydrogenase, amylase, trypsin, chymotrypsin, pepsin, pronase, proteinase K or catalase, respectively. The treatment of lactate dehydrogenase reduce the inhibitory effect of SCS. The antagonistic activity reduced when the pH of SCS was adjusted to neutral (pH 7.2). Antimicrobial activity was performed by incubating the LAB-SCS with ETEC or EAggEC suspension. After 1 hour co-culture, the growth of ETEC and EAggEC was inhibited. The LAB was used as starter in skim milk fermentation. The fermented milk was stored at 4℃. There are 107 CFU/mL of LAB strains RY2 and MM1 after 14 days storage. The population of strain En4 reduced to 106 CFU/mL. This study suggests that L. acdophilus RY2, L. salivarius MM1 and L. paracasei En4 could be used as an effective control of ETEC or EAggEC on fermented milk processing.
中文摘要…………………………………………………………………………1
英文摘要…………………………………………………………………………2
第一章、前言…..…………..……………………………………………………3
圖 1 ………………………………………………………………………………4
第二章、文獻整理………………………………………………………………5
第一部份、保健性菌種乳酸菌之應用………………………………....………5
Ⅰ、乳酸菌分類及生理學……………………………………………………..5
一、乳酸菌的定義…………………………..………………………………5
二、乳酸菌的分類…………………………………………………………..5
II、乳酸菌之保健功能…………………………………………………….......7
一、乳酸菌在消化道的分佈…………………………………………...……7
二、乳酸菌作為益生菌之特性……………………………….…….……….8
三、乳酸菌促進人體健康之保健機能……………………………………..13
四、乳酸菌發酵食品之應用………………………………………………..16
第二部份、大腸桿菌的特性及對人類與動物之感染…………………………17
一、致病性大腸桿菌 (pathogenic E. coli) …………………………….......18
(一) 腸毒性大腸桿菌 (ETEC) …………………………………….……...19
(二) 腸出血性大腸桿菌 (EHEC)..……..…..………………………..........20
(三) 腸病原性大腸桿菌 (EPEC)…..……………………………………...20
(四) 腸侵入性大腸桿菌 (EIEC)…………………………………………..21
(五) 腸凝集性大腸桿菌 (EAggEC).…………………………………..........21
(六) 擴散附著性大腸桿菌 (DAEC)…...………………………………….21
(七) 尿道致病性大腸桿菌 (UPEC).…………………………………..........21
圖( 2-1~2-4 )……………………………………………………………………...23
表( 2-1~2-6 )………………………………………………………………………27
第三章、篩選具功能性之嬰兒來源乳酸菌…………………………………….33
壹、前言………………………………………………….…………………..33
貳、材料與方法………………………………………………………………34
一、實驗材料………………………………………………………............34
二、實驗方法……………………………………………………………….37
(一) 菌種、培養基和生長條件………………………………...………..37
(二) 孔洞擴散法之抑菌圈抑菌活性試驗……………………...………..38
(三) API 50CHL菌種鑑定………………………………..…....................38
(四) 腸道細胞株吸附性試驗..………………………………...................39
(五) 革蘭氏染色、鏡檢………………....….............................................40

(六) 耐酸性耐膽鹽試驗..………………………………...........................40
參、結果與討論………………………………………….......……………….41
(一) 抑制 ETEC 之孔洞擴散法試驗… …..………..………......................41
(二) 抑制 EAggEC 之孔洞擴散法試驗……………………...…….........41
(三) 腸道細胞株吸附性試驗..………………………………...................42
(四) 革蘭氏染色、鏡檢…………………………………………….…….43
(五) 耐酸性耐膽鹽試驗..…………………………………………………43
肆、結論 ……………………………………………………………………44
圖( 3-1~3-3 )………………………………………………………………….46
表( 3-1~3-9 )…………………………………………………………………49
第四章、乳酸菌抑制腸毒性大腸桿菌與腸凝集性大腸桿菌之體外試驗…...58
壹、前言........................................................................................................58
貳、材料與方法……………………………………………...……....………60
一、實驗材料………………………………………………………..........60
二、實驗方法……………………………………………………………….61
(一) 菌種、培養基和生長條件………………………………...……….61
(二) 乳酸菌上清液經加熱、酵素分解作用對抑菌能力之影響............61
(三) 定量乳酸菌產生之乳酸....................................................................61
(四) 乳酸菌上清液與 ETEC或 EAggEC 共培養之抑制能力分析…..62
(五) 腸凝集性大腸桿菌與抗微生物藥劑紙錠試驗................................63
(六) 發酵牛乳之儲藏試驗…………………………………...….............64
參、結果與討論…………………………………………………………………..64
(一) 乳酸菌上清液經加熱、酵素分解作用對抑菌能力之影響............64
(二) 定量乳酸菌產生之乳酸....................................................................65
(三) 乳酸菌上清液與 ETEC或 EAggEC 共培養之抑制能力分析….66
(四) 腸凝集性大腸桿菌與抗微生物藥劑紙錠試驗...............................66
(五) 發酵牛乳之儲藏試驗…………………………………...…............67
肆、結論 …………………………………………………...……………….67
圖( 4-1~4-9 )…………………………………….....…………..................... 69
表( 4-1~4-9 )……………………………………….....…………………. …78
參考文獻……………………………………….....………………………. ..87
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