Department of Food Science, University of Parma, Parco Area delle Scienze 95/A, Parma, Italy.
Int J Food Microbiol. 2013 Jan 1;160(3):290-7. doi: 10.1016/j.ijfoodmicro.2012.10.011. Epub 2012 Oct 26.
A multiplex real time PCR (mRealT-PCR) useful to rapidly screen microbial composition of thermophilic starter cultures for hard cooked cheeses and to compare samples with potentially different technological properties was developed. Novel primers directed toward pheS gene were designed and optimized for multiple detection of Lactobacillus helveticus, Lactobacillus delbrueckii, Streptococcus thermophilus and Lactobacillus fermentum. The assay was based on SYBR Green chemistry followed by melting curves analysis. The method was then evaluated for applications in the specific detection of the 4 lactic acid bacteria (LAB) in 29 different natural whey starters for Parmigiano Reggiano cheese production. The results obtained by mRealT-PCR were also compared with those obtained on the same samples by Fluorescence in Situ Hybridization (FISH) and Length-Heterogeneity PCR (LH-PCR). The mRealT-PCR developed in this study, was found to be effective for analyzing species present in the samples with an average sensitivity down to less than 600 copies of DNA and therefore sensitive enough to detect even minor LAB community members of thermophilic starter cultures. The assay was able to describe the microbial population of all the different natural whey starter samples analyzed, despite their natural variability. A higher number of whey starter samples with S. thermophilus and L. fermentum present in their microbial community were revealed, suggesting that these species could be more frequent in Parmigiano Reggiano natural whey starter samples than previously shown. The method was more effective than LH-PCR and FISH and, considering that these two techniques have to be used in combination to detect the less abundant species, the mRealT-PCR was also faster. Providing a single step sensitive detection of L. helveticus, L. delbrueckii, S. thermophilus and L. fermentum, the developed mRealT-PCR could be used for screening thermophilic starter cultures and to follow the presence of those species during ripening of derived dairy products. A major increase in understanding the starter culture contribution to cheese ecosystem could be harnessed to control cheese ripening and flavor formation.
开发了一种多重实时 PCR（mRealT-PCR），可用于快速筛选高温发酵剂中微生物组成，并且可将具有潜在不同技术特性的样品进行比较。设计并优化了针对 pheS 基因的新型引物，以用于同时检测瑞士乳杆菌、德氏乳杆菌保加利亚亚种、嗜热链球菌和发酵乳杆菌。该方法基于 SYBR Green 化学，随后进行熔解曲线分析。然后将该方法应用于特定检测 Parmigiano Reggiano 奶酪生产中 29 种不同天然乳清发酵剂中 4 种乳酸菌（LAB）。通过 mRealT-PCR 获得的结果也与通过荧光原位杂交（FISH）和长度异质性 PCR（LH-PCR）获得的结果进行了比较。本研究开发的 mRealT-PCR 可用于分析样品中存在的物种，平均灵敏度低至少于 600 个 DNA 拷贝，因此足以检测高温发酵剂中微生物群落中的微量 LAB 成员。该检测法能够描述所有不同天然乳清发酵剂样品的微生物种群，尽管存在天然变异性。结果表明，乳清发酵剂中存在更多的嗜热链球菌和发酵乳杆菌，表明与之前的研究相比，这些物种在 Parmigiano Reggiano 天然乳清发酵剂样品中更为常见。该方法比 LH-PCR 和 FISH 更有效，而且考虑到这两种技术必须结合使用才能检测到较少的丰富物种，因此 mRealT-PCR 也更快。mRealT-PCR 可以一步灵敏地检测瑞士乳杆菌、德氏乳杆菌保加利亚亚种、嗜热链球菌和发酵乳杆菌，可用于筛选高温发酵剂，并在衍生乳制品成熟过程中跟踪这些物种的存在。这可以极大地提高对发酵剂对奶酪生态系统贡献的理解，从而控制奶酪成熟和风味形成。
