PCR-DGGE for the profiling of cheese bacterial communities: strengths and weaknesses of a poorly explored combined approach

Abstract

Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) represents a still valid molecular tool for the profiling of complex microbial ecosystems, including cheeses. In the present study, a double PCR-DGGE approach has been applied to the investigation of the bacterial diversity of seven cheese models to objectively assess strengths and weaknesses of such an approach. To that end, the bacterial DNA was extracted directly from both the cheese replicates and the bulks of colonies harvested from the serial dilution agar plates of selective solid media used for the enumeration of presumptive lactobacilli, lactococci and thermophilic cocci, respectively. The results overall collected allowed the main bacterial taxa to be identified and roughly quantified. Rough quantification of the main cultivable species represents a strength of the PCR-DGGE approach applied, whereas its main weaknesses were represented by the low degree of selectivity of the conventional growth media used for cultivation of lactic acid bacteria and the underestimation of the effective microbial diversity occurring in the seven cheese models.

Impact of thistle rennet from Carlina acanthifolia All. subsp. acanthifolia on bacterial diversity and dynamics of a specialty Italian raw ewes' milk cheese

“…Illumina sequencing allowed both the majority and minority taxa to be identified, whereas single bacterial species were unambiguously recognized by PCR-DGGE. When the latter technique was applied, discrepancies were seen in the fingerprints obtained by the bulk and the direct approach, in terms of both the number of taxa identified and their relative abundance; this finding was in agreement with a recent finding by the same authors on semi-hard cheese models (Aquilanti et al, 2016) In Sopravissana raw ewes' milk, PCR-DGGE allowed the sole species L. lactis to be identified among LAB. Illumina sequencing confirmed the occurrence of Lactococcus spp.…”

Characterization of Bacterial Microbiota in Tilapia Fillets Under Different Storage Temperatures

“…In recent years, a highly sensitive and quantitative technique, the high-throughput sequencing of 16S rDNA, has emerged. Nevertheless, the earlier polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method, which is much simpler, quicker, and cheaper than high-throughput sequencing, continues to be a valid tool in the analysis of microbial diversity (Aquilanti et al, 2016;Delgado et al, 2013;Ding, Wu, Huang, & Zhou, 2015;Garofalo et al 2015). This paper investigates the bacterial microbiota in tilapia fillets under different cold storage temperatures by employing the PCR-DGGE method, finding that lower storage temperatures remarkably change the microbiota.…”

“…In recent years, a highly sensitive and quantitative technique, the high‐throughput sequencing of 16S rDNA, has emerged. Nevertheless, the earlier polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) method, which is much simpler, quicker, and cheaper than high‐throughput sequencing, continues to be a valid tool in the analysis of microbial diversity (Aquilanti et al., ; Delgado et al., ; Ding, Wu, Huang, & Zhou, ; Garofalo et al. ).…”

Διερεύνηση Των Μικροβιολογικών Και Φυσικοχημικών Χαρακτηριστικών Παραδοσιακής Φέτας Από Διάφορες Περιοχές.

“…Το αποτέλεσμα αυτό υποδηλώνει ότι μερικές φορές τα αποτελέσματα της PCR-DGGE μπορεί να μην καθορίζουν επακριβώς τη μικροχλωρίδα ενός τυριού και θα πρέπει να ληφθούν υπόψη και εκείνα των παραδοσιακών καλλιεργητικών τεχνικών. Η αξία της από κοινού χρήσης καλλιεργητικών και μη-καλλιεργητικών τεχνικών στην ανάλυση ενός σύνθετου μικροβιακού οικοσυστήματος όπως είναι το τυρί, έχει καταγραφεί αρκετά συχνά τα τελευταία χρόνια (Aponte et al, 2008;Aquilanti et al, 2016;Dolci et al, 2008a;Dolci et al, 2008b;Mangia et al, 2016;Randazzo et al, 2006;Rantsiou et al, 2008;Rossi et al, 2012).…”