使用groESLgene鑑定乳酸菌並探討與吸附特性之相互關係

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题名:	使用groESLgene鑑定乳酸菌並探討與吸附特性之相互關係
其它题名:	Studies on the identification and correlation on adhesion properties of lactic acid bacteria using groESL gene
作者:	黃宇男 Huang, Yu-Nan
贡献者:	曾浩洋;盧錫祺 Tsen, Hau-Yang;Lu, Hsi-Chi 東海大學食品科學系
关键词:	乳酸菌;種專一性核酸擴增引子組;熱休克蛋白10-60系統;吸附特性 Lactic acid bacteria;Species-specific primer;groESL system;adhesion properties
日期:	2008
上传时间:	2011-03-11T06:07:40Z (UTC)
摘要:	乳酸菌於發酵乳製品、肉類和蔬菜中扮演重要的角色。許多乳酸菌能做為益生菌或動物飼料添加物使用。品質管制有賴可靠的方法鑑定益生菌與發酵產品中的乳酸菌種。本研究從熱休克蛋白10-60系統中設計專一性核酸擴增引子組和探針偵測發酵乳製品中各種乳酸桿菌與嗜熱鏈球菌。其靈敏度與專一性之聚合?鏈反應足以有效偵測到人工添加乳酸菌與市售乳酸菌發酵產品中的乳酸菌種。我們進而以groESL系統開發多套式聚合?鏈反應、同步擴增鏈反應和點墨雜交法鑑定乳酸菌種。兩組多套式聚合?鏈反應能分別同步偵測Lactobacillus acidophilus、L. delbrueckii subsp. bulgaricus和Streptococcus thermophilus (group-1)以及 L. casei、L. rhamnosus 和 L. zasei (group-2)。同步定量偵測之靈敏度可達103 CFU/mL。點墨雜交法能專一性的偵測到所有目標乳酸菌種，未來將可應用於商業發酵產品的品質管制。就我們所知，這是第一篇研究從熱休克蛋白10-60系統中設計專一性引子組和探針來鑑定商業產品中的乳酸菌種之報告。相關文獻顯示乳酸菌的熱休克蛋白60能釋放出胞膜外，並且與乳酸菌吸附至腸道黏膜或腸道上皮細胞有關聯。另一方面，有研究指出鈣離子能促進乳酸菌吸附至腸道上皮細胞。因此在本研究的第二部份，我們探討七株乳酸菌種在鈣離子存在下與腸道癌上皮細胞株的吸附特性，以及和血清型沙門氏菌的競爭排除試驗。結果顯示不論鈣離子存在與否，相對於控制組，七株乳酸菌種皆能顯著地(P<0.01)降低沙門氏菌吸附至腸道上皮細胞的菌量達兩倍以上。以反轉錄聚合?鏈反應分析乳酸菌於培養期間中熱休克蛋白基因的表現量，相對於控制組，乳酸菌於10 mM濃度的鈣離子存在下，能顯著地(P<0.01)增加乳酸菌吸附至腸道上皮細胞上，並能促進熱休克蛋白60基因的表現量。我們的結果顯示鈣離子能促進熱休克蛋白60基因的表現量進而增加乳酸菌的吸附能力。未來可應用改善弱吸附能力但具有其他益生特性之乳酸菌實用價值上。 Lactic acid bacteria (LAB) play an important role in fermentation of dairy products, meats, cheeses and vegetables. Certain LAB strains could be used as probiotics for human and feed supplements for animals. Reliable methods for the detection or identification of these LAB strains are demanded for quality assurance of these probiotics and fermented products. In this study, heat shock protein 10-60 system sequence (hsp10-60; groESL) was used for the designing of PCR primers specific or probe for the detection of various Lactobacillus spp. and Streptococcus thermophilus strains in fermented diary products. The sensitiveity and specificity of PCR amplication were verified both in intentionally inoculated and commercial fermentation products. Moreover, based on groESL sequence, we further developed multiplex PCR, real-time PCR as well as dot blot hybridization for LAB identification. Two sets of multiplex PCR have been used for the simultaneory detection of Lactobacillus acidophilus, L. delbrueckii subsp. bulgaricus, Streptococcus thermophilus (group-1), L. casei, L. rhamnosus and L. zasei (group-2). Detection limits of these LAB were lower than 103 CFU/mL without non-specific amplification, as demonstrated in SYBR Green I based real-time PCR assay. Dot blot hybridization revealed unbiased detection of all tested strains, and showed the potential in commercial use. To our knowledge, this is the first report of a species-specific identification of LAB by using groESL system.Previous studies have showed the relationship of the localization of LAB GroEL and the adhesion of LAB to mucus and epitheial cells. Independent reports indicated that calcium ions greatly promoted the attachment of LAB to culture cells. In the second part of this study, we investigated the adhesive features and competitive exclusion of 7 LAB strains including Lactobacillus acidophilus, L. fermentum (2 strains), L. fermentum, L. plantarum, L. brevis , Pediococcus acidilactici and Enterococcus faecium to Salmonella enterica serovar Typhimurium, in the presence of calcium ions and Caco-2 cells. Protection assays performed with pathogenic S. enterica showed that all tested Lactic acid bacteria strains have significantly reduced (P<0.01) the attachment of S. enterica to Caco-2 cell lines by more than 2-fold both in the presence and the absence of calcium ions. In vitro adhesion assays and reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that 10mM calcium ions significantly enhaned (P<0.01) both the binding of Lactic acid bacteria to Caco-2 cell lines and expression of groEL gene. As the first attempt ever to correlate the expression of groEL with the addition of calcium ions, our data have provided some basic supporting evidences. A direct link of the functions of groEL and calcium ions to adhesion of LAB to cells awaits more efforts, and would a key to a better understanding and control of probiotic properties.
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