Calmin Gautier, Lefort Francois, Belbahri Lassaad
Plants and Pathogens Group, Research Institute Earth Nature and Landscape, School of Engineering of Lullier, University of Applied Sciences of Western Switzerland, 150 Route de Presinge, 1254 Jussy, Switzerland.
Mol Biotechnol. 2008 Oct;40(2):170-9. doi: 10.1007/s12033-008-9073-4. Epub 2008 Jun 10.
The control of wine microbial population during and beyond fermentation is of huge importance for wine quality. Lactic acid bacteria (LAB) in wine are responsible for malolactic fermentation (MLF) which can be desired in some cases and undesirable in others. Some LAB do not perform MLF and their uncontrolled growth could contribute to severe wine spoilage such as undesired flavours. Their identification and detection is considered crucial for numerous biotechnological applications in food fermentations, where, through acidification and secretion of bacteriocins, they contribute to reduce food spoilage and growth of pathogenic microorganisms. LAB have traditionally been classified using morphological or biochemical features. Primary isolation, biochemical identification and phenotypic analysis are laborious, time consuming and inaccurate and often lead to misidentification within some genera such as Pediococcus. Molecular identification based on suitable marker genes could be an attractive alternative to conventional morphological and biochemical methods. We assessed here the applicability of four housekeeping genes recA, rplB, pyrG and leuS in combination with the mle gene in multi-loci sequence typing (MLST) of Pediococcus parvulus and Pediococcus damnosus. Sequencing and comparative analysis of sequence data were performed on 19 strains collected during wine fermentation. A combination of these five marker genes allowed for a clear differentiation of the strains analysed, indicating their applicability in molecular typing. Analysis of the observed nucleotide polymorphisms allowed designing highly discriminative primers for a multi-loci sequence typing (MLST) method that proved successful in detecting a particular isolate or sequence type of P. parvulus when using either conventional PCR or Real Time PCR.
在发酵过程及之后对葡萄酒微生物群落的控制对于葡萄酒质量至关重要。葡萄酒中的乳酸菌（LAB）负责苹果酸-乳酸发酵（MLF），在某些情况下这是期望的，而在其他情况下则是不期望的。一些乳酸菌不进行MLF，其不受控制的生长可能导致严重的葡萄酒变质，如产生不良风味。它们的鉴定和检测对于食品发酵中的众多生物技术应用至关重要，在这些应用中，通过酸化和细菌素的分泌，它们有助于减少食品变质和致病微生物的生长。传统上，乳酸菌是根据形态或生化特征进行分类的。初次分离、生化鉴定和表型分析费力、耗时且不准确，并且常常导致在某些属（如片球菌属）内出现错误鉴定。基于合适标记基因的分子鉴定可能是传统形态学和生化方法的一种有吸引力的替代方法。我们在此评估了四个管家基因recA、rplB、pyrG和leuS与mle基因相结合在细小片球菌和有害片球菌多位点序列分型（MLST）中的适用性。对葡萄酒发酵过程中收集的19株菌株进行了测序和序列数据的比较分析。这五个标记基因的组合能够清晰地区分所分析的菌株，表明它们在分子分型中的适用性。对观察到的核苷酸多态性的分析允许设计用于多位点序列分型（MLST）方法的高鉴别力引物，当使用常规PCR或实时PCR时，该方法在检测细小片球菌的特定分离株或序列类型方面证明是成功的。
