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?（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?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.
