Instituto de Ciencias de la Vid y del Vino (Gobierno de La Rioja, Universidad de La Rioja, CSIC), C/ Madre de Dios 51, 26006, Logroño, La Rioja, Spain.
Appl Microbiol Biotechnol. 2013 Aug;97(15):6931-41. doi: 10.1007/s00253-013-4974-y. Epub 2013 May 18.
Lactic acid bacteria populations of red wine samples from industrial fermentations, including two different vinification methods were studied. For this investigation, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis was employed to supplement previous results that were obtained by culture-dependent methods. PCR-DGGE was aimed to study two targeted genes, 16S ribosomal DNA (rDNA) and rpoB, and the results were useful to evaluate the microbial populations in wine samples. Moreover, an improvement of a detection limit determined so far for DGGE analysis was obtained with the method described in this study, what made possible to identify lactic acid bacteria populations below 10(1) colony-forming unit/mL. The species Oenococcus oeni was the most frequently detected bacterium, but identifications close to species Oenococcus kitaharae and Lactococcus lactis that are not often found in wine were firstly identified in samples of this research. PCR-DGGE allowed to detect 9 out of 11 lactic acid bacteria species identified in this study (nine by PCR-16S rDNA/DGGE and four by PCR-rpoB/DGGE), while five species were detected using the modified de Man, Rogosa and Sharpe agar. Therefore, the two methods were demonstrated to be complementary. This finding suggests that analysis of the lactic acid bacteria population structure in wine should be carried out using both culture-dependent and culture-independent techniques with more than one primer pair.
对来自工业发酵的红酒样本中的乳酸菌种群进行了研究，包括两种不同的酿造方法。为了进行这项研究，采用聚合酶链反应-变性梯度凝胶电泳（PCR-DGGE）分析来补充以前通过依赖培养的方法获得的结果。PCR-DGGE 旨在研究两个靶向基因，16S 核糖体 DNA（rDNA）和 rpoB，并且结果有助于评估酒样中的微生物种群。此外，本研究中描述的方法提高了迄今为止确定的 DGGE 分析检测限，使得能够识别低于 10(1)菌落形成单位/mL 的乳酸菌种群。Oenococcus oeni 是最常检测到的细菌，但在本研究的样本中首次鉴定出接近 Oenococcus kitaharae 和 Lactococcus lactis 的菌种，这些菌种在葡萄酒中并不常见。PCR-DGGE 允许检测到本研究中鉴定出的 11 种乳酸菌中的 9 种（9 种通过 PCR-16S rDNA/DGGE 鉴定，4 种通过 PCR-rpoB/DGGE 鉴定），而通过改良的 Man，Rogosa 和 Sharpe 琼脂检测到 5 种。因此，这两种方法被证明是互补的。这一发现表明，应该使用两种以上的引物对，结合依赖培养和非依赖培养的技术，对葡萄酒中的乳酸菌种群结构进行分析。
