保加利亞乳酸桿菌於膽鹽逆境下之蛋白質體研究

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Title:	保加利亞乳酸桿菌於膽鹽逆境下之蛋白質體研究 Proteomic research of Lactobacillus delbrueckii subsp. bulgaricus under bile salt stress environment
Authors:	林妤楹 Lin, Yu-Yin
Contributors:	生物科技研究所
Keywords:	益生菌逆境保加利亞乳酸桿菌蛋白質體二維電泳 Probiotics Stress Lactobacillus delbrueckii subsp. bulgaricus Proteomic 2-DE
Date:	2013-06
Issue Date:	2013-10-08 14:25:18 (UTC+8)
Abstract:	益生菌被定義為活的微生物，可改善宿主腸道微生物相的平衡，並對宿主帶來健康之效益，如抗炎、抗癌、降低膽固醇以及抗過敏等。乳酸菌已被證實是藉由碳水化合物進行發酵而產生乳酸且已普遍作為益生菌的根據。作為益生菌，必須通過消化道並能存活且能定殖於腸道中。因此，膽鹽耐受性為乳酸菌存活於消化道關鍵特性之一。本研究的目的為探討保加利亞乳酸菌生長於膽鹽逆境環境下之生長情形，並藉由二維電泳分析其蛋白質表現之差異。我們使用膠內水解、質譜儀分析以及使用資料庫搜尋進行蛋白質辨識。體外逆境耐受性試驗指出，保加利亞乳酸桿菌可在0.05%以及0.1%膽鹽環境下存活，但是無法存活於0.3%膽鹽環境。因此，本研究選擇了0.05%以及0.1% 之膽鹽作為逆境之條件。經由蛋白質體分析，本實驗鑑定出10個表現量具有顯著差異之蛋白質。經由逆境所誘導之蛋白質如小熱休克蛋白、輔胺醯基胺肽酶、菸醯胺酶、尿苷二磷酸-N-乙醯醇丙二酮葡萄醣胺還原酶及丙酮酸氧化酶，我們推測這些被誘導之蛋白質，可以避免蛋白質變性、使蛋白質進行正確之折疊、鞏固細胞壁以及細胞膜之品質、維持培養環境之pH值以滲透壓，並且提供足夠的能量來抵禦逆境環境。因此，保加利亞乳酸桿菌可以通過消化道之膽鹽逆境環境，並給予宿主帶來健康之效益。 Probiotics are defined as live microbes which could improve the balance of the microbes at host intestinal tract and give health benefit to the host, such as anti-inflammatory, anti-carcinogenic and cholesterol-lowering effects and prevention of allergies. Lactic acid bacteria (LAB) having the property of producing lactic acid from sugars by fermentation are commonly regarded as probiotics based on their putative or proven mechanism. Tolerance to bile salts is among the most crucial properties as it determines the ability of bacteria to survive in gastrointestinal tract and consequently their capacity to play their functional role as probiotics. In this context, the objective of this study is to investigate the growth of Lactobacillus delbrueckii subsp. bulgaricus and protein expression under bile salt stress environment, using 2-DE comparative proteomics. Analysis of differential protein expression by tryptic digestion, mass spectrometry analysis, and protein identification by using database search. In vitro stress-resistant experiment indicated Lactobacillus delbrueckii subsp. bulgaricus could survive under 0.05% and 0.1% bile salt environment, but dead under 0.3%. Therefore, 0.05 and 0.1% bile salt are selected as the stress condition in this study. Identity by proteomic analysis, 10 identified proteins exhibited significant expression level changing by bile salt induced. We speculated these stress-induced of proteins, such as small heat shock protein, prolyl aminopeptidase, nicotinamidase, UDP-N-acetylenolpyruvoylglucosamine reductase and pyruvate oxidase, which could prevent protein denaturation, correct folding of partially unfolded proteins, maintain cell wall and cell membrane of Lactobacillus quality, maintain pH value and osmotic pressure in the culture environment, and provide enough energy to resist stress environment. Therefore, Lactobacillus delbrueckii subsp. bulgaricus could survive through bile salt condition, and give health benefit to the host.
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