Abstract
Simultaneous quantitative detection of Lactococcus (Lc.) lactis and Leuconostoc species bacteriophages (phages) has not been reported in dairies using undefined mixed-strain DL-starters, probably due to the lack of applicable methods. We optimized a high-throughput qPCR system that allows simultaneous quantitative detection of Lc. lactis 936 (now SK1virus), P335, c2 (now C2virus) and Leuconostoc phage groups. Component assays are designed to have high efficiencies and nearly the same dynamic detection ranges, i.e., from ~1.1 x 105 to ~1.1 x 101 phage genomes per reaction, which corresponds to ~9 x 107 to ~9 x 103 phage particles mL-1 without any additional up-concentrating steps. The amplification efficiencies of the corresponding assays were 100.1±2.6, 98.7±2.3, 101.0±2.3 and 96.2±6.2. The qPCR system was tested on samples obtained from a dairy plant that employed traditional mother-bulk-cheese vat system. High levels of 936 and P335 phages were detected in the mother culture and the bulk starter, but also in the whey samples. Low levels of phages were detected in the cheese milk samples.
Highlights
Strains of Lactococcus (Lc.) lactis are extensively used in the manufacture of most fermented dairy products, and Leuconostoc species are widely used as well
We introduced a high-throughput quantitative PCR (qPCR) assay based on the BioMark HD System (Fluidigm, USA) for simultaneous quantitative detection of dairy Lc. lactis and Leuconostoc phages
The main objective of this study was to develop a system for simultaneous quantification of 936, P335, c2, and Leuconostoc phage species
Summary
Strains of Lactococcus (Lc.) lactis are extensively used in the manufacture of most fermented dairy products, and Leuconostoc species are widely used as well. In the manufacture of cheese, Lc. lactis strains cause rapid acidification of milk through the production of lactic acid. The presence of Lc. lactis enhances the survival of Leuconostoc strains, which grow poorly in milk on their own [2,3]. Leuconostoc strains were shown to influence multiple organoleptic properties of fresh and semi-hard cheese varieties (such as Tilsitter, Edam and Gouda) and unripened dairy products [2,4,5,6]. Industrial cheese fermentation vats are ideal environments for the multiplication of virulent bacteriophages (phages), since they are not inactivated by the heat treatment that is commonly used in the dairy industry [7,8,9]. Infection by phages of Lc. lactis results in partial or complete
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