Next generation sequencing for quantitative measurements in food molecular microbiology

The microbiological composition plays a pivotal role in the production, final characteristics and safety of fermented foods. Such composition can be assessed by means of culture-based and molecular methods, where the latter are necessary to analyze non-culturable species. PCR-DGGE has been for several years the most applied technique in molecular microbiology: advantages include the relatively low cost and ease of use and the possibility of processing samples altogether; the method is however only semi-quantitative and the resolution power is limited to a few dominant species. Recent advancements in next generation sequencing technologies (NGS) are revolutionizing our comprehension and quantitative measurements of microbial communities. The initial approach is the same as for PCR-DGGE, i.e., the extraction of microbial DNA from food and the PCR amplification of 16s rRNA hypervariable regions. The PCR amplicons instead of being analyzed on a denaturating gel can be instead sequenced on NGS platforms such as Illumina, which provide multi-million reads of hundreds of base pairs. Here we present the validation of this approach on two case studies, one dealing with typical Italian fermented dry-sausages (salami) from different producers and ripening stages, the second with Grana Padano cheese produced with and without the use of lysozyme. A total of 722,196 high-quality reads where obtained for the salami, 1,240,592 for the cheese case study. More than 95% of these sequences could be classified up to the species level, and the number of species identified was very high: in the case of salami, 33 different species of lactobacilli where quantified, whereas on the same samples PCR-DGGE identified much less. Comparisons with RT-PCR measurements also showed that NGS has the potential for a quantitative assessment of hundreds of species altogether: further studies will be useful in order to validate and standardize the method for metrological measurements in food science