AUTHOR=Kamilari Eleni , Efthymiou Marina , Anagnostopoulos Dimitrios A. , Tsaltas Dimitrios 

TITLE=Cyprus Sausages’ Bacterial Community Identification Through Metataxonomic Sequencing: Evaluation of the Impact of Different DNA Extraction Protocols on the Sausages’ Microbial Diversity Representation

JOURNAL=Frontiers in Microbiology

VOLUME=Volume 12 - 2021

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.662957

DOI=10.3389/fmicb.2021.662957

ISSN=1664-302X

ABSTRACT=Cyprus traditional sausages from the Troodos mountainous region of Pitsilia gained recently the Protected Geographical Indication (PGI) designation from the European Committee (EU 2020/C 203/06). Still, we lack authentication protocols for the distinction of “Pitsilia” from industrially produced Cyprus sausages. Microbial activity is an essential contributor to traditional sausages sensorial characteristics, but whether the microbial patterns might associate with the area of production is unclear. In the present research, High-Throughput sequencing (HTS) was applied to provide a linkage between the area of production and bacterial diversity of Cyprus sausages. To strengthen our findings, we used three different DNA extraction methods: i) the DNeasy PowerFood Microbial kit (Qiagen); ii) the NucleoSpin Food kit (Macherey Nagel); and iii) the blackPREP Food DNA I Kit (Analytik Jena), in which we applied three different microbial cell wall lysis modifications. The modifications included heat treatment, bead beating and enzymatic treatment. Results regarding metagenomic sequencing were evaluated in terms of number of reads, alpha diversity indexes and taxonomic composition. The efficacy of each method of DNA isolation was assessed quantitively based on the extracted DNA yield and the obtained copy number of a) the 16S rRNA gene, b) the internal transcribed spacer (ITS) region and c) three Gram-positive bacteria that belong to the genera Lactobacillus, Bacillus and Enterococcus via absolute quantification using qPCR. Compared with some examined industrial sausages, “Pitsilia” sausages had significantly higher bacterial alpha-diversity (Shannon, Simpson indexes). Principal coordinates analysis separated the total bacterial community composition (beta-diversity) of the sausages from the three Pitsilia areas from the industrial sausages, in all but one industrial sausage. Although the eight sausages shared the main abundant bacterial taxa based on 16S rDNA HTS, differences associated with bacterial diversity representation and certain genera were observed. The findings indicate that the microbial communities may be used as an additional tool for the identification of the authenticity of Cypriot sausages.