Grattepanche F, Lacroix C, Audet P, Lapointe G
STELA Dairy Research Centre, Pavillon Paul Comtois, Université Laval, Québec, QC, G1K 7P4, Canada.
Appl Microbiol Biotechnol. 2005 Jan;66(4):414-21. doi: 10.1007/s00253-004-1705-4. Epub 2004 Jul 23.
During cheese making, interactions between different strains of lactic acid bacteria play an important role. However, few methods are available to specifically determine each bacterial population in mixed cultures, in particular for strains of the same species. The aim of this study was to develop a real-time PCR quantification method to monitor the population of Lactococcus cremoris ATCC 19257 in mixed culture with Lactobacillus rhamnosus RW-9595M and the bacteriocin-producing microorganism Lc. diacetylactis UL719. The specificity of the two primers 68FCa33 and 16SR308 used to amplify a 240-bp fragment of DNA from Lc. cremoris was demonstrated by conventional PCR. Using these primers for real-time PCR, the detection limit was 2 cfu/reaction or 200 cfu of Lc. cremoris ATCC 19257 per millilitre of mixed culture in milk. In pure culture batch fermentation, good correlation was obtained between real-time PCR and the conventional plating method for monitoring Lc. cremoris growth. In mixed culture batch fermentation, Lb. rhamnosus and Lc. cremoris decreased due to nisin Z production by Lc. diacetylactis. The decrease of the Lc. cremoris cell population detected by real-time PCR was not possible to observe by the plate count method in the presence of a Lc. diacetylactis population that was 1 log higher.
在奶酪制作过程中，不同乳酸菌菌株之间的相互作用起着重要作用。然而，很少有方法能够特异性地确定混合培养物中每种细菌群体，特别是同一物种的菌株。本研究的目的是开发一种实时PCR定量方法，以监测在与鼠李糖乳杆菌RW - 9595M和产细菌素的微生物双乙酰乳酸乳球菌UL719混合培养时，乳脂乳球菌ATCC 19257的群体数量。用于从乳脂乳球菌扩增240 bp DNA片段的两种引物68FCa33和16SR308的特异性通过常规PCR得到证实。使用这些引物进行实时PCR，检测限为每反应2 cfu或每毫升牛奶混合培养物中200 cfu的乳脂乳球菌ATCC 19257。在纯培养分批发酵中，实时PCR与监测乳脂乳球菌生长的常规平板计数法之间具有良好的相关性。在混合培养分批发酵中，由于双乙酰乳酸乳球菌产生乳酸链球菌素Z，鼠李糖乳杆菌和乳脂乳球菌数量减少。在双乙酰乳酸乳球菌群体数量高1个对数的情况下，通过平板计数法无法观察到实时PCR检测到的乳脂乳球菌细胞群体数量的减少。
