臺灣博碩士論文加值系統

益生菌（probiotics）普遍存在人體及動物的腸道及生殖泌尿道，能產 生多種酸性代謝產物如乳酸（lactic acid）、醋酸（acetic acid）等物質使其 生存環境不利於其他微生物生長，並可產生數種抑菌物質例如細菌素 （bacteriocin）、過氧化氫（hydrogen peroxide，H2O2）及洛德因 （reuterin）等，具有維持宿主健康的功效，但卻對宿主不產生傷害，故美 國食品和藥物管理局（US Food and Drug Administration，FDA）將乳酸菌 （lactic acid bacteria，LAB）定義為一般公認是安全的（Generally Recognized As Safe，GRAS）微生物，其中 Lactobacillus spp.為最主要的菌 屬。乳酸菌也是目前人類最常用於保健的健康食品之一。然而好的腸道乳 酸菌除了須具備抑菌能力外，必須能通過胃腸道的嚴苛環境方能在腸道內 進行作用，故本研究主要目的在利用試管外實驗（in vitro）模擬乳酸菌經 過胃腸道所必須面臨的環境考驗，並初步測試對革蘭氏陽性菌及陰性菌的 抑制能力，藉以找尋未來可以用於人體的有功效之益生菌。 本實驗以 3.5 mg/ml 胃蛋白酶（pepsin）在 pH 2.5 的環境中，測試 8 種乳 酸桿菌屬共 57 株菌的抗性，接著測試菌株是否能耐受胃的酸性（pH 3.0），再以膽鹽 0.3% Oxgall 來模擬腸道中的膽汁環境，測試菌株食入人體 後能否存活，最後以瓊脂挖洞擴散法（well-diffusion assay）測試乳酸桿菌 培養的上清液是否具有抑制細菌的效果。結果顯示，32 株（56.1%）的乳酸桿菌在 3.5 mg/ml 胃蛋白酶培養四個小時後，與對照組比減少數量少於 二個對數（log），但 19 株（33.3%）被完全抑制，無法存活；對 pH 3.0 的 酸性環境，有 56 株（98.2%）可以耐受，僅減少不到二個對數，而對 0.3%膽鹽的存活亦有 56 株（98.2%），減少不到二個對數。在抑菌試驗方 面對 Staphylococcus aureus ATCC 29213，有 45 株（78.9%）抑制圈大於 15 mm，對 Salmonella typhimurium BCRC 14193 則 54 株（94.7%）有大於 15 mm 抑制圈。

Probiotics are commonly found in the intestinal and genitourinary tracts of humans and animals. They can produce a variety of acidic metabolites such as lactic acid, acetic acid and other substances, making its living environment is not conducive to the growth of other microorganisms. And several antibacterial substances such as bacteriocin, hydrogen peroxide (H2O2) and reuterin can be produced. Because they have the effect of maintaining the health of the host but does not harm the host. Therefore, the US Food and Drug Administration (FDA) defines lactic acid bacteria (LAB) as generally recognized as safe (GRAS) microorganisms. Lactobacillus spp. is the most important genus. Lactic acid bacteria are also one of the most commonly used health foods. However, in addition to having a bacteriostatic capacity, good intestinal lactic acid bacteria must be able to pass through the harsh environment of the gastrointestinal tract. In this study, the main purpose is to use in vitro models to simulate the environmental challenges that lactic acid bacteria must face when passing through the gastrointestinal tract. And preliminary tests on the inhibition of Gram-positive bacteria and negative bacteria. In order to find useful probiotics that can be used in the human body in the future. In this study, the tolerance ability of 57 strains of 8 Lactobacillus strains was tested with 3.5 mg/ml pepsin in pH 2.5 environment. Then test whether the strain can tolerate gastric acid (pH 3). And 0.3% Oxgall was used to simulate the intestine environment. Finally, we used the well-diffusion assays to test whether the culture supernatant of Lactobacillus had the effect of inhibiting bacteria. The results showed that 32 strains (56.1%) of Lactobacillus were reduced by less than one log after incubation with 3.5 mg/ml pepsin for four hours. 19 strains (33.3%) were completely suppressed. For the pH 3 environment, 56 strains (98.2%) can tolerate and only less than one logarithm reduction. There were also 56 strains (98.2%) survived in 0.3% bile salts, reduced less than one log reduction. In the antibacterial test for Staphylococcus aureus ATCC 29213, there were 45 strains (78.9%) with inhibition zone greater than 15 mm. For Salmonella typhimurium BCRC 14193, 54 strains (94.7%) have a greater than 15 mm inhibition zone. The conclusion, in this study, we found that some strains have the potential to become probiotics in the gut. Follow-up animal experiments can be performed to verify the actual effect of living body.

第一章緒言........................................................................................................... 1
第一節前言........................................................................................................ 1
第二節文獻回顧................................................................................................ 3
一、乳酸菌..................................................................................................... 3
(一)乳酸菌的定義 .................................................................................... 3
(二)乳酸菌之分類 .................................................................................... 3
(1) 專 性 同 型 發 酵 乳 酸 菌 （ obligately homofermentation
LAB）.................................................................................................4
(2)兼性異型發酵乳酸菌（facultatively heterofermentation
LAB） .............................................................................................. 4
(3)專性異型發酵乳酸菌（obligately heterofermentation LAB）... 5
(三)乳酸菌之應用 .................................................................................... 5
(1)食品工業上之應用...................................................................... 5
1. 增加風味 ............................................................................. 5
2. 增加營養價值 ..................................................................... 6
3. 食品保存 ............................................................................. 6
(2)醫藥科技之應用.......................................................................... 6
1. 維持正常腸道菌相............................................................. 6
2. 調節免疫作用 ..................................................................... 6
3. 抗癌作用 ............................................................................. 6
4. 降血壓功能 ......................................................................... 6
(四)乳酸菌之抑菌機制............................................................................ 7
(1)競爭性抑制作用.......................................................................... 7
1. 競爭營養成分 ..................................................................... 7
2. 競爭附著位置 ..................................................................... 7
(2)化學性抑制作用.......................................................................... 7
1. 有機酸（organic acids）.................................................... 7
2. 過氧化氫（hydrogen peroxide，H2O2）........................... 7
3. 二氧化碳（carbon dioxide，CO2） .................................. 8
4. 細菌素（bacteriocin） ....................................................... 8
5. 洛德因（reuterin）.............................................................8
第三節研究目的................................................................................................ 9
第二章材料與方法.............................................................................................10
第一節實驗材料與儀器設備.......................................................................... 10
一、菌株....................................................................................................... 10
二、培養基...................................................................................................11
三、實驗藥品............................................................................................... 11
四、實驗儀器............................................................................................... 11
第二節實驗流程..............................................................................................10
第三節實驗方法..............................................................................................10
一、菌株培養............................................................................................... 15
二、乳酸桿菌抗生素敏感性分析............................................................... 15
三、乳酸桿菌耐胃蛋白酶（pepsin）存活試驗........................................18
四、乳酸桿菌耐酸存活試驗....................................................................... 20
五、乳酸桿菌耐膽鹽存活試驗...................................................................22
六、乳酸桿菌瓊脂挖洞擴散法抑菌試驗 ..................................................24
第三章結果與討論.............................................................................................26
第一節乳酸桿菌抗生素敏感性......................................................................26
第二節乳酸桿菌耐胃蛋白酶存活率..............................................................26
第三節乳酸桿菌耐酸存活率.......................................................................... 27
第四節乳酸桿菌耐膽鹽存活率......................................................................28
第五節乳酸桿菌瓊脂挖洞擴散法抑菌結果..................................................28
第六節抗胃蛋白酶、酸及膽鹽且具良好抑菌效果之乳酸桿菌整理……..29
第四章結論.........................................................................................................31
參考文獻............................................................................................................. 32
表......................................................................................................................... 38
圖......................................................................................................................... 53

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