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研究生:林嘉鎮
研究生(外文):Chia-Chan Lin
論文名稱:不同乳酸菌之特性探討、分子檢測鑑定及應用
論文名稱(外文):Characterization, molecular identification and application of lactic acid bacteria from various origins.
指導教授:曾浩洋曾浩洋引用關係
指導教授(外文):Hau-Yang Tsen, Ph. D.
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:2001
畢業學年度:89
語文別:中文
論文頁數:111
中文關鍵詞:乳酸菌幽門桿菌人體來源發酵
外文關鍵詞:Lactic acid bacteriaHelicobacter pylorihuman originfermentation
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乳酸菌為人體腸道之益生菌,作為益生菌之基本條件,包括具有耐酸性、耐膽鹽性、抑制病原菌的生長,且能附著於宿主的腸道上皮細胞。除了上述所具有之基本條件外,益生菌所被期待的特性,尚包括有為人體來源、對治癌及致病菌具拮抗作用、用於食品及臨床用途皆具安全性等功能。在本研究中的一方面,所使用的乳酸菌,為使用自嬰兒糞便分離出之乳酸菌,以進行益生菌之評估。實驗結果發現,對於人體細胞株Int 407及Caco-2吸附性方面,僅篩得四株乳酸菌 (實驗室編號為MRS12、MRS39、RS15及RS17) 對於Int 407及Caco-2具有明顯的附著性。其中三株菌株 (MRS39、RS15及RS17) 對於pH 2.0磷酸鹽緩衝溶液及對0.3 %的膽鹽具有耐受性;在抑菌病原菌分析方面,此三株菌株 (MRS39、RS15及RS17) 對於G(+)及G(-)病原菌具有高度的抑制能力。另外,更一進步探討以乳酸菌之上清液進行對Helicobacter pylori抑制能力之分析,結果顯示此三株菌株之上清液可明顯地抑制H. pylori的生長。
在另一方面,在以植物來源之應用在蔬菜牛奶汁的發酵方面,所使用三株菌株為Pediococcus pentosaceus MP 012、Lactobacillus plantarum CCRC 10069及L. fermentum CCRC 12190,都呈現快速生長的情形;而其pH值及產酸能力俱佳。在儲藏試驗方面,三株菌株之發酵乳在4℃冷藏14天後,仍保有近於107 CFU/ml以上的活菌數。
在小鼠腸道上皮細胞吸附試驗方面,結果發現,由動物性篩選出來之乳酸菌比植物性篩選出來之乳酸菌較具有吸附性的效果。
Lactic acid bacteria (LAB) strains may be used as probiotics in human intestines. The basic requirement for LAB as probiotics are the capability of tolerance to gastric juice and bile salts, the ability to inhibit pathogenic bacteria and the adherence to human intestinal epithelium cells. Beside, the other requirement for LAB as probiotics is that they should be from human origin. Also, their inhibitory effect on cancer and their safety for food and clinical use are also important. On one aspect, this project aims at the evaluation of the probiotic properties of LAB strains, isolated from infant stool samples. It was found that four LAB strains (MRS12、MRS39、RS15 and RS17) are able to adhere to the Int 407 and Caco-2 cell lines. Three of these four LAB strains (MRS39、RS15 and RS17) were tolerant to pH 2.0 and resistant to 0.3% bile salts. These three LAB strains also showed the inhibitory ability on G(+) or G(-) pathogenic bacteria. Furthermore, the inhibitory effect of LAB on Helicobacter pylori was also studied. It was found that the culture supernatant of these three LAB strains are obviously inhibitory to the growth of H. pylori.
On the other aspect of this project, we have used LAB strains isolated from plant origins for the preparation of fermented milk. It was found that three LAB strains from plant sources, i.e., Pediococcus pentosaceus MP 012, Lactobacillus plantarum CCRC 10069 and L. fermentum CCRC 12190, not only grow well and fast, but also showed the higher ability for acid production. As for the storage study, it was found that fermented milks prepared from these LAB strains contain about 107 CFU/ml of LAB cells after a 14 days’ storage at 4℃.
Finally, regarding the adhesion ability for LAB to the mice intestinal cells, it was found that LAB strains isolated from animal were higher adhesive than those from plant origins.
中文摘要…………………………………………………………………..………..1
英文摘要………………………………………………………………….….……..2
第一章、文獻整理…………………………………………………….….….…..3
一、乳酸菌………………………………………………………….…….……3
(一) 乳酸菌定義……………………………………………….……….….3
(二) 乳酸菌的分類…………………………………………….…………..3
(三) 乳酸菌和其他菌群在消化道的分佈………………………….……..4
二、乳酸菌在益生菌之應用…………………………………….…….………4
(一) 益生菌之定義…………………………………………….…………..4
(二) 益生菌應具備之特性……………………………………..………….5
(三) 乳酸菌作為益生菌之特性……………………………….….…….…5
三、乳酸菌促進人體健康之機能……………………………………….…….8
(一) 抑制致病菌……………………………………………………..….…8
(二) 緩和乳糖不耐症…………………………………………...…………8
(三) 改善營養價值及食物之消化性…………………...…………………9
(四) 降低膽固醇含量……………………………………...………………9
(五) 抗癌性………………………………………………………………...9
(六) 強化免疫系統………...……………………………………………...10
四、 乳酸菌對腸胃道耐受性之相關研究…………………………..……….10
(一) 乳酸菌之耐酸性研究……………………………………..………....10
(二) 乳酸菌之膽鹽耐性研究…………………………………..………....11
(三) 乳酸菌對腸道細胞附著性試驗……………...…………….………..11
五、乳酸菌與免疫系統………………………….…………………………..12
(一) 腸道免疫系統……………………………………………….………12
(二) 乳酸菌與腸道免疫之相關性…………………………….…….……13
(三) 巨噬細胞經激活後其形態功能的改變………………………....…..13
(四) 乳酸菌與免疫相關性之研究報告………………………………….14
六、乳酸菌對沙門氏菌之抑制………………………………………..…….16
(一) 沙門氏菌之簡介…………………………………………………….16
(二) 沙門氏菌之致病………………………………………….………….17
(三) 沙門氏菌侵入性之相關研究……………………………….……….18
(四) 競爭性驅除 (Competitive exclusion)…………………….…………18
(五) 乳酸菌抑制沙門氏菌侵入或感染之相關研究…………….……….19
七、乳酸菌對幽門桿菌之抑制……………………………….……………..19
(一)幽門桿菌 (Helicobacter pylori) 之簡介…………....………….…..19
(二)H. pylori造成的病理變化、致病機轉及毒力因子….……….……20
(三)H. pylori之診斷方法………………………………….……….……22
(四)乳酸菌抑制幽門桿菌感染之相關研究……………………….…....24
八、脈衝電場膠體電泳 (pulsed field gel electrophoresis, PFGE)…………..24
(一)完整DNA取之不易………………………………………………...25
(二)限制酶之採用………………………………………………………..25
(三)未知分子量的的決定………………………………………………..26
九、表一………………………………………………………………….…….27
表二………………………………………………………………….…….28
表三………………………………………………………………….…….28
第二章、人體來源乳酸菌作為益生菌之特性評估...……………….…...29
壹、前言…………………………………………………………….…………….29
貳、材料與方法………………………………………………….……………….29
一、實驗材料……………………………………………….………………….29
二、實驗方法……………………………………………………………….….32
(一)乳酸菌株之篩選…..…………………………………………………32
(二)腸道細胞株附著性 (adhesion) 試驗…………………….…………33
(三)菌株初步確認………………………………………………………..34
(四)耐酸性試驗…………………………………………………………..35
(五)模擬消化程序之處理………..………………………………………35
(六)抑菌性之評估………………………..………………………………36
(七)抑制幽門桿菌試驗…………….…………………………….………37
(八)API菌種鑑定……...…………………………………………………38
參、結果與討論…………………………………………………………………..38
(一)篩選出具吸附性之乳酸菌……………………..……………………38
(二)菌株之初步確認…………………………….……………………….39
(三)耐酸性之評估…………………...…………………………………...40
(四)模擬消化程序之處理………………………………………………..41
(五)抑菌性之評估………………………………………………………..42
(六)乳酸菌對幽門桿菌之抑制…………………………………………..43
(七)菌種鑑定………………………………………………………….….44
肆、結論….……………………………………………………………………….44
伍、表(2-1~2-7)…….…………………………………………………………….46
陸、圖(2-1~2-2)…..………………………………………………………………53
第三章、植物性來源乳酸菌應用於發酵乳品之評估………….……….55
壹、前言…………………………………………..…………………………….55
貳、材料與方法…………………………………….…………………………..55
一、實驗材料…………………………………….…………………………….55
二、實驗方法…………………………………….…………………………….56
(一) 乳酸菌之發酵試驗……………………………………….……...…..56
(二) 儲藏試驗……………………………………………………………..57
(三) 乳酸菌在小鼠腸道上皮細胞附著性之評估………...……………...57
參、結果與討論…………………………………………………………………58
(一)乳酸菌在蔬菜牛奶汁中之生長情形………………..………………58
(二)儲藏試驗………………………………………………….………….59
(三)乳酸菌吸附於上皮細胞之試驗………………………………….….59
肆、結論…………………………………………………………………………60
伍、圖(3-1 ~ 3-11)………………..……………..………………………………62
第四章 乳酸菌之脈衝電場膠體電泳 (PFGE) 圖譜…….……………....70
壹、前言…………………………………………………………………………70
貳、材料與方法…………………………………………………………………70
一、實驗材料…………………………………….…………………………….70
二、實驗方法…………………………………….…………………………….72
(一)於瓊脂洋菜膠塊中製備genomic DNA………………….………….72
(二)限制酶之剪切………………………………………………………..72
(三)PFGE之電泳條件………………………………..…...……………...72
(四)PFGE所使用之Marker………………………………………………73
(五)電腦分析……………………………………………………………..73
參、結果與討論…………………………………………………………………73
(一)L. acidophilus之NotI限制酶圖譜…………….………………..……73
(二)L. fermentum之NotI限制酶圖譜……….…………………………...73
(三)具有益生菌特性之乳酸菌之NotI限制酶圖譜……………….….….74
肆、表(4-1 ~ 4-3)………………..………………………………….……………75
伍、圖(4-1 ~ 4-7)…………………………………………………….…………..78
參考文獻…………………………………………………………………
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