Renouf Vincent, Claisse Olivier, Lonvaud-Funel Aline
UMR oenologie-ampélologie, INRA-Université Victor Segalen Bordeaux2, 351 cours de la libération, 33405 Talence cedex, France.
J Microbiol Methods. 2006 Oct;67(1):162-70. doi: 10.1016/j.mimet.2006.03.008. Epub 2006 Apr 19.
Lactic acid bacteria (LAB) are essential in the quality of many fermented beverages like beer, cider and wine. In the two later cases, they convert malic acid into lactic acid during the malolactic fermentation. After fermentation, microbial stabilization is needed to prevent the development of spoilage bacteria species. Among them, cocci lead to different alterations: Pediococcus sp., and some strains of Leuconostoc mesenteroides and Oenococcus oeni can produce exopolysaccharides which modify wine viscosity and lead to ropiness. They also can produce acetic acid, biogenic amine, ethyl carbamate and volatile phenols. Therefore detection and identification are crucial. Results of phenotypic tests and DNA-DNA probes are not accurate enough. 16S RNA gene which is currently used for bacterial species identification presents intraspecies heterogeneity. The rpoB gene is an alternative to this limitation. However previous PCR targeting partial sequence of rpoB gene could not delimit cocci species. Therefore we compared the rpoB gene sequence of the six main cocci species found in fermented beverages: P. damnosus, P. dextrinicus, P. parvulus, P. pentosaceus, L. mesenteroides and O. oeni. The most discriminating partial sequence of the rpoB gene was chosen for designing primers. By PCR-DGGE the reliability of these primers was verified. It was controlled in a mixture of several cocci and other lactic acid bacteria (Lactobacillus sp.). Then we adapted the primers and the PCR conditions in order to achieve the identification of cocci species by real time PCR program including the fluorescent dye SYBR Green I, which gives faster results. PCR melt curves were established and a specific T(m) was attributed to each species.
乳酸菌（LAB）对于许多发酵饮料如啤酒、苹果酒和葡萄酒的品质至关重要。在后两种情况下，它们在苹果酸-乳酸发酵过程中将苹果酸转化为乳酸。发酵后，需要进行微生物稳定处理以防止腐败细菌种类的滋生。其中，球菌会导致不同的变化：片球菌属，以及一些肠系膜明串珠菌和酒酒球菌菌株可产生胞外多糖，这些多糖会改变葡萄酒的粘度并导致发粘。它们还可产生乙酸、生物胺、氨基甲酸乙酯和挥发性酚类。因此，检测和鉴定至关重要。表型测试和DNA-DNA探针的结果不够准确。目前用于细菌物种鉴定的16S rRNA基因存在种内异质性。rpoB基因是克服这一局限性的一种选择。然而，先前针对rpoB基因部分序列的PCR无法区分球菌物种。因此，我们比较了在发酵饮料中发现的六种主要球菌物种的rpoB基因序列：有害片球菌、糊精片球菌、渺小片球菌、戊糖片球菌、肠系膜明串珠菌和酒酒球菌。选择rpoB基因最具鉴别力的部分序列来设计引物。通过PCR-DGGE验证了这些引物的可靠性。在几种球菌和其他乳酸菌（乳酸杆菌属）的混合物中进行了检验。然后我们调整了引物和PCR条件以通过包括荧光染料SYBR Green I的实时PCR程序实现球菌物种的鉴定，该程序能更快得出结果。建立了PCR熔解曲线，并为每个物种赋予了特定的熔解温度（Tm）。
