Bacterial Community Dynamics during Production of Registered Designation of Origin Salers Cheese as Evaluated by 16S rRNA Gene Single-Strand Conformation Polymorphism Analysis

Abstract

Microbial dynamics during processing and ripening of traditional cheeses such as registered designation of origin Salers cheese, an artisanal cheese produced in France, play an important role in the elaboration of sensory qualities. The aim of the present study was to obtain a picture of the dynamics of the microbial ecosystem of RDO Salers cheese by using culture-independent methods. This included DNA extraction, PCR, and single-strand conformation polymorphism (SSCP) analysis. Bacterial and high-GC% gram-positive bacterial primers were used to amplify V2 or V3 regions of the 16S rRNA gene. SSCP patterns revealed changes during the manufacturing of the cheese. Patterns of the ecosystems of cheeses that were provided by three farmers were also quite different. Cloning and sequencing of the 16S rRNA gene revealed sequences related to lactic acid bacteria (Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Lactobacillus plantarum, and Lactobacillus pentosus), which were predominant during manufacturing and ripening. Bacteria belonging to the high-GC% gram-positive group (essentially corynebacteria) were found by using specific primers. The present molecular approach can effectively describe the ecosystem of artisanal dairy products.The typical sensorial qualities of traditional cheese ultimately depend on several factors, including traditional cheese-making practices, feeding of dairy cows, and the dynamics of microbial communities. The qualitative and quantitative composition of the microbial flora, its evolution, and its activity during ripening play an important role in the development of hygienic and sensorial qualities. In order to better understand the functions of the microbial community, a full description of the microbial ecosystem is required. Classically, this has been addressed by enumerating members of certain microbial groups by using various culture media, followed by identification of a number of dominant isolates by phenotypic tests (16,18,33) or molecular techniques such as ribotyping (21), random amplified polymorphic DNA analyses, and sequencing (4, 27). However, cultivation-dependent approaches do not necessarily provide reliable information on the composition of entire microbial communities. It is therefore difficult to assess the significance of cultured microorganisms in microbial ecosystems because of the disparity between culturable and in situ diversity. Indeed, microbial communities may contain viable but nonculturable bacteria and also bacteria that would be culturable if appropriate medium had been developed. Thus, in order to study interactions between microorganisms, it is important to study the ecosystem without dissociating it. It has been shown that a dual approach, e.g., inventory by using a cultivation-dependent method and di-

Microbiota of Karakačanski skakutanac, an artisanal fresh sheep cheese studied by culture-independent PCR-ARDRA and PCR-DGGE

“…Taking into account this drawback of culture-independent PCR approach, identical ARDRA profiles could be explained although initial diverse profiles were expected due to the use of raw milk in the cheese production. It has been reported by other authors [3,4,11,14,15] that population structure of LAB community present in the traditional raw milk cheeses is diverse and complex. The dominance of only one species was also confirmed by PCR-DGGE.…”

“…The majority of the cheeses produced from raw sheep milk are hard and long ripened and their unique characteristics arise from specific indigenous microbiota which contributes to diversity of taste and flavour of artisanal cheeses [3,4]. Autochthonous microbiota of such cheeses represent a heritage which has to be protected and preserved, as it contributes to natural microbial biodiversity and typical characteristics of the traditional cheeses [11].…”

Development of a defined starter culture mixture for the fermentation of sato, a Thai rice-based alcoholic beverage

“…To assess the microbial community, cultivation-dependent techniques have been employed in the past. This type of approach however is limited not only by the long time it takes in obtaining the results, but also in that it may fail to give reliable information on the composition of the microbial communities since some microorganisms are not cultivatable in such environments 11 . Thus a polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) approach can be applied to solve the problem.…”

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products

“…The hypervariable regions are flanked by conserved sequences, which can serve for amplification with "universal" primers (Baker et al, 2003). The variable V1 Bonetta et al, 2008), V3 Ercolini et al, 2001;Ogier et al, 2002;Duthoit et al, 2003;Ercolini et al, 2003;Mauriello et al, 2003;Andrighetto et al, 2004;Ercolini et al, 2004;Feurer et al, 2004a;Feurer et al, 2004b;Lafarge et al, 2004;Ogier et al, 2004;Duthoit et al, 2005;Flórez and Mayo, 2006;Delbes et al, 2007;El-Baradei et al, 2007;Parayre et al, 2007;Abriouel et al, 2008;Ercolini et al, 2008;Gala et al, 2008;Van Hoorde et al, 2008;Alegría et al, 2009;Casalta et al, 2009;Dolci et al, 2009;Giannino et al, 2009;Mounier et al, 2009;Serhan et al, 2009;Dolci et al, 2010;Fontana et al, 2010;Van Hoorde et al, 2010;Masoud et al, 2011), V2 (Duthoit et al, 2003;Delbes and Montel, 2005;Saubusse et al, 2007), V4-V5 , V1-V3 (Randazzo et al, 2002), V4-V8 (Randazzo et al, 2006), V5-V6 (Le Bourhis et al, 2005;Le Bourhis et al, 2007) and V6-V8 …”

“…In direct DNA extraction procedures (McKillip et al, 2000;Duthoit et al, 2003;Feurer et al, 2004a;Feurer et al, 2004b;Callon et al, 2006;Monnet et al, 2006;Delbes et al, 2007;Masoud et al, 2011), the cheese samples are first homogenised in a liquid solution by a method involving bead-beating, a mortar and pestle or other mechanical treatments. Efficient treatments of casein degradation and cell lysis, followed by phenol/chloroform extractions, are then needed to remove most contaminating compounds.…”