PCR-DGGE markers for qualitative profiling of microbiota in raw milk and ripened cheeses
Introduction
Ripened cheeses are fermented products in which milk undergoes changes during fermentation and ripening in the presence of microorganisms forming a complex and dynamic system. The metabolic processes in cells drive biochemical changes, thus influencing the formation of desirable attributes in the final product. The microbiota of ripened cheeses is composed of starter bacteria, including starter lactic acid bacteria (SLAB) and, optionally, propionic acid bacteria, non-starter lactic acid bacteria (NSLAB) as well as yeasts and molds (Fox, Guinee, Cogan, & McSweeney, 2000).
Starter bacteria involved in lactic acid fermentation (LAB) and propionic acid fermentation (PAB) are responsible for the acidification of the cheese slurry, eye formation, and the taste and aroma of cheese (Fox & Wallace, 1997). Non-starter bacteria can be divided into two groups: non-starter lactic acid bacteria (NSLAB), which also determine the sensory attributes of cheese (Beresford, Fitzsimmons, Brennam, & Cogan, 2001), and contaminating bacteria which can lead to cheese spoilage and blowing defects (Cocolin, Innocente, Biasutti, & Comi, 2004), and pose a risk to consumer health (Koch et al., 2010).
Cheese production is a long and complex process. Microbial cells in the cheese matrix are exposed to various stressors which can lead to the death of microbial cells or the formation of viable but nonculturable bacteria (VBNC) that cannot be cultured in synthetic media (Joux & Lebaron, 2000). For this reason, sublethally damaged microbial cells or cells from small microbial populations, dominated by starter bacteria, are not always identified when a product sample is directly cultured to determine its microbiological purity.
Molecular culture-independent methods offer an alternative to traditional techniques. One of such methods is the polymerase chain reaction with denaturing gradient gel electrophoresis (PCR-DGGE) which amplifies hypervariable regions (V1-V9) in the 16S rRNA gene isolated from metagenomic DNA (Ercolini, Moschetti, Blaiotta, & Coppola, 2001b). PCR-DGGE can be used to evaluate ecosystem biodiversity, develop comprehensive profiles of microbial communities and monitor changes in those communities. This technique has been successfully used to analyze dairy ingredients and products. Franciosi, Settani, Cavazza, and Poznanski (2009) relied on PCR-DGGE to evaluate raw milk microbiota. In the work of Aponte, Fusco, Andolfi, and Coppola (2008), this method was used to identify bacteria in different types of regional cheeses. PCR-DGGE was deployed by Temmerman, Scheirlinck, Huys, and Swings (2003) to identify bacterial species in probiotic products. PCR-DGGE is also applied to analyze non-dairy fermented products, including sourdough (Meroth, Walter, Hertel, Brandt, & Hammes, 2003), wine (Renouf, Claisse, Miot-Sertier, & Lonvaud-Funel, 2006) and fermented sausage (Cocolin, Manzano, Cantoni, & Comi, 2001). The use of ladders similar to molecular markers used in horizontal electrophoresis could facilitate PCR-DGGE analyses. Due to the absence of such ladders, band patterns in the gel cannot be compared. The existing shortcomings prompted the authors to design markers composed of known reference strains, which could be used for PCR-DGGE qualitative profiling of microbiota in raw milk, and Swiss-Dutch-type cheeses manufactured in north-eastern Poland.
Section snippets
Reference strains
A total of 70 reference bacterial strains of known origin were used in the study. The strains are listed in Table 1.
Samples of raw milk and ripened cheeses
Ten samples were used to validate the reference marker, including 2 samples of raw milk and 2 samples of Swiss-Dutch-type ripened cheeses that were directly subjected for DNA isolation. The latter 2 samples were cultured in 3 different media: MRS, M17 and ML (Table 1), and DNA was isolated. The samples were collected in a local dairy plant in the Region of Warmia and Mazury (NE
Analysis of the position of reference strain bands in DGGE
The marker was designed with the use of U968-GC and L1401-r primers which amplify a V6−V8 region of the 16S rRNA coding gene. The mutual band positions of the reference strains (Table 1) were determined in a wide range of denaturing gradient (30–60%), and the results were used to discriminate the analyzed bacterial strains at genus, species and subspecies level.
In a 30–60% denaturing gradient, band positions in DGGE profiles differed significantly between the analyzed genera, but overlapping
Discussion
DGGE markers have also been developed by other authors (Aponte et al., 2014, Cocolin et al., 2004, Florez and Mayo, 2006, Temmerman et al., 2003). The cited researchers also postulated the need to determine the position of bands formed by different bacterial species, which is influenced by the applied amplification primers and separation conditions in DGGE. Gala et al. (2008) developed a DGGE marker for evaluating Parmigiano Reggiano cheese. The designed marker was composed of 15 bacterial
Conclusions
The reference marker described in this study was developed based on 24 species of starter bacteria used in the production of Swiss-Dutch-type cheeses (P. freudenreichii, L. lactis, L. mesenteroides), and non-starter bacteria that remained in milk after pasteurization and/or were part of the cheese plant microbiota (P. thoenii, L. acidophilus, L. plantarum, L. brevis, L. casei, L. delbrueckii, L. fermentum, L. helveticus, S. thermophilus). The marker comprised also representatives of undesirable
Acknowledgements
This study was supported by research grant No. NN312484140 from the National Science Center in Poland.
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