Purpose: A culture-dependent quantitative method (ABEV) and a culture-independent real-time PCR (qPCR) assays was developed to follow OHL population dynamic from milk samples to the end of cheese ripening.
Methods: A selective and specific ABEV medium was developed for direct enumeration of total OHL from cheese, and a specific qPCR assays was set up to target Lactobacillus fermentum and L. parabuchneri individual species. These tools were applied for OHL quantification in manufactured Emmental and Tomme cheeses.
Results: The ABEV medium was well adapted for specific enumeration and isolation of OHL species present in milk-derived samples, even in the presence of background microbiota. qPCR assays showed 100% specificity and could accurately quantify the targeted species in various types of cheese. Culture-dependent and -independent techniques evaluated in manufactured cheese samples generated similar bacterial counts. The behavior of L. fermentum and L. parabuchneri was characterized during cheese processes. PCR-TTGE was also used to confirm the presence of inoculated species and to analyze the composition of naturally present species.
Significance: Although the influence of OHL species during cheese ripening is recognized, information about their dynamic behaviour remains scarce due to the lack of simple analytical tools. This work illustrates how various culture-dependent and –independent methods can be implemented to study OHL, either as a population or as individual species. Both ABEV culture medium and qPCR tools present the advantage to be suitable for quantification of minor and dominant species and/or populations and are fully applicable to complex cheese ecosystems. A polyphasic approach combining these two quantitative methods with the more global PCR-TTGE technique showed their complementarity.