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.

Easy DNA extraction method and optimisation of PCR-Temporal Temperature Gel Electrophoresis to identify the predominant high and low GC-content bacteria from dairy products.

Molecular fingerprinting of bacterial ecosystems has recently increased in food microbiology. The aim of this work was to develop a rapid and easy method to extract DNA from various cheeses, and to optimize the separation of low and high GC-content bacteria by PCR-Temporal Temperature Gel Electrophoresis (PCR-TTGE). Seventy six strains belonging to 50 of the most frequently encountered bacterial species in dairy products were used to construct a database. Specific PCR-TTGE ladders containing 17 species forming a regular scale were created. Amplicons of these species were sequenced and the GC-content plotted against the migration distance: the correlation coefficients obtained were r(2)=0.97 and r(2)=0.99, respectively for high and low GC-contents. The extraction method developed did not use any harmful solvent such as phenol/chloroform. The concentrations of DNA extracted from hard cooked and pressed cheeses, quantified by picogreen molecular probes, were between 0.7 and 6 microg/g for core samples and 8 to 30 microg/g for rind samples. Experimental as well as commercial dairy products were analysed using the developed method and the reproducibility of the profiles was 89%. The method appears to be particularly efficient in the characterization of the ecosystem of cheese rinds.