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.

Puglisi, E., Bassi, D., Polka, J. U., Rebecchi, A., Cocconcelli, P. S., Morelli, L., Next generation sequencing for quantitative measurements in food molecular microbiology, Relazione, in 1st IMEKOFOODS Conference “Promoting objective and Measurable Food Quality & Safety”, (Roma, 12-15 October 2014), IMEKFOODS, Roma 2014: 1-1 [http://hdl.handle.net/10807/64804]

Next generation sequencing for quantitative measurements in food molecular microbiology

Puglisi, Edoardo;Bassi, Daniela;Polka, Justyna Urszula;Rebecchi, Annalisa;Cocconcelli, Pier Sandro;Morelli, Lorenzo
2014

Abstract

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.
Inglese
1st IMEKOFOODS Conference “Promoting objective and Measurable Food Quality & Safety”
1st IMEKOFOODS Conference “Promoting objective and Measurable Food Quality & Safety”
Roma
Relazione
12-ott-2014
15-ott-2014
Puglisi, E., Bassi, D., Polka, J. U., Rebecchi, A., Cocconcelli, P. S., Morelli, L., Next generation sequencing for quantitative measurements in food molecular microbiology, Relazione, in 1st IMEKOFOODS Conference “Promoting objective and Measurable Food Quality & Safety”, (Roma, 12-15 October 2014), IMEKFOODS, Roma 2014: 1-1 [http://hdl.handle.net/10807/64804]
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10807/64804
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