Precise Populations' Description in Dairy Ecosystems Using Digital Droplet PCR: The Case of Group in Starters.

group (composed of the and subspecies, recently reassigned as two distinct species) plays a major role in dairy fermentations. Usually present in starter cultures, the two species enable efficient acidification and improve the organoleptic qualities of the final product. Biovar diacetylactis strains produce diacetyl and acetoin, aromas from the citrate metabolization. As these populations have distinct genomic and phenotypic characteristics, the proportions of each other will affect the final product. Today, there is no quantitative test able to distinguish between the two species and the biovar in dairy ecosystems. In this study, we developed a specific, reliable, and accurate strategy to quantify these populations using, species-, and diacetylactis-specific fluorescent probes in digital droplet PCR assays (ddPCR). Species were distinguished based on three single nucleotide polymorphisms in the glutamate decarboxylase gene, and the gene involved in citrate metabolism was used to target the biovar. Used in duplex or singleplex, these probes made it possible to measure the proportion of each population. At 59°C, the probes showed target specificity and responded negatively to the non-target species usually found in dairy environments. Depending on the probe, limit of detection values in milk matrix ranged from 3.6 × 10 to 1.8 × 10 copies/ml. The test was applied to quantify sub-populations in the group during milk fermentation with a commercial starter. The effect of temperature and pH on the balance of the different populations was pointed out. At the initial state, and species represent, respectively, 75% and 28% of the total group and biovar diacetylactis strains represent 21% of the species strains. These ratios varied as a function of temperature (22°C or 35°C) and acidity (pH 4.5 or 4.3) with species promoted at 22°C and pH4.5 compared to at 35°C. The biovar diacetylactis strains were less sensitive to acid stress at 35°C. This methodology proved to be useful for quantifying and species and biovar diacetylactis, and could complete 16S metagenomics studies for the deeply description of group in complex ecosystems.