Polyphasic approach for quantitative analysis of obligately heterofermentative Lactobacillus species in cheese

Obligately heterofermentative lactobacilli (OHL) present in cheese during ripening can influence the flavour and texture of the final product. In order to better evaluate, follow and control this population, there is a current need for easy-to-use tools. In this study, a culture-dependent quantitative method (ABEV medium) was set up for direct and selective enumeration of total OHL from cheese, and a culture-independent method based on specific real time PCR (qPCR) assays was developed to target Lactobacillus fermentum and Lactobacillus parabuchneri individual species. These tools were applied for OHL quantification in manufactured Emmental and Tomme cheeses. 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 behaviour of L. fermentum and L. parabuchneri was characterized from milk samples to the end of ripening. In addition, PCR-TTGE was used to confirm the presence of inoculated species and to globally analyze the composition of naturally present species. This polyphasic approach illustrates the complementarity of the different methods.