The Potential of a Polyphasic PCR-DGGEApproach in Evaluating Microbial Diversity of Natural Whey Cultures for Water-Buffalo Mozzarella Cheese Production: Bias of Culture-Dependent and Culture-Independent Analyses

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

“…thermophilus grew on M17 agar plates incubated at 22°C but not at 45°C (ca_p), whereas the mesophilic species L. lactis, known to clearly prefer incubation temperatures around 30°C, was stably detected in the M17 bulks harvested from plates incubated at 45°( pe_r) or even in the MRS bulks (ca_r1, pe_p, cp_p and ca_p). The latter is the medium of choice for the enumeration and isolation of lactobacilli, and the findings are also in agreement with those previously reported (Ercolini et al 2001). On the one hand, this evidence encourages ongoing research efforts to develop new selective media for the isolation and enumeration of starter and non-starter LAB from cheese and dairy products, while on the other, it also clearly demonstrates the need to use a series of more selective media for the analysis of the cultivable fraction of cheese bacterial communities by PCR-DGGE, including acidified MRS (pH 5.7), whey agar medium (WAM), cheese agar medium (CAM), etc.…”

“…In some cases, such as ca_r1, the analysis of the cultivable community allowed a higher biodiversity to be detected, in terms of number of taxa identified, while in others, such as cp_r, species not detected in the bulks were, by contrast, identified through the direct approach. This evidence is substantially in agreement with what was previously highlighted by Ercolini et al (2001), thus decisively confirming the importance of combining the sequencing of DGGE bands after direct DNA extraction with the cultivation of microorganisms.…”

“…Notwithstanding the great potential of a combined use of the direct and bulk approaches for the monitoring of bacterial community structure in milk-based products, as revealed by a pioneer study conducted by Ercolini et al (2001), to date this double approach has only sporadically been applied to dairy microbiology, where the "standard" is unquestionably represented by the culture-independent PCR-DGGE analysis.…”

“…In the early investigation by Ercolini et al (2001), the DNA was extracted directly from natural whey cultures for the production of water buffalo Mozzarella cheese and the bulks of colonies harvested from all the serial dilution agar plates (from −1 to the last) of a pool of selective solid media; the DNA was then subjected to PCR-DGGE analysis for the amplification and separation of variable portions of the 16S rRNA gene. In the following years, the double PCR-DGGE approach was applied in a few investigations aimed at identifying the microbial diversity of raw milk cheeses, namely, Stilton (Ercolini et al 2003), water buffalo Mozzarella ), Goudatype cheeses (Van Hoorde et al 2008, 2010, an artisanal fresh sheep cheese produced in a limited area of eastern Croatia (Pogačić et al 2011) and traditional Italian semihard cheeses (Aquilanti et al 2013;Ricciardi et al 2014).…”

“…In the current study, the potential of the direct and bulk PCR-DGGE approach originally proposed by Ercolini et al (2001) for the profiling of dairy microbial communities was explored. To this end, seven semi-hard raw and pasteurised milk cheeses manufactured according to different cheese-making technologies were used.…”