International Dairy Journal 16 (2006) 1205–1210 PCR–DGGE as a tool for characterizing dominant microbial populations in the Spanish blue-veined Cabrales cheese Ana Bele´n Flo´rez, Baltasar Mayo à Instituto de Productos La´cteos de Asturias (CSIC), Carretera de Infiesto s/n, 33300-Villaviciosa, Asturias, Spain Received 21 April 2005; accepted 18 November 2005 Abstract The microbial populations of cheese milk and rennet extracts used in the production of traditional, Spanish, blue-veined Cabrales cheese were identified by PCR–DGGE analysis of the V3 region of the bacterial 16S rRNA gene and of the D1 region of the eukaryotic 26S rRNA genes. Ripe cheeses (60 days old) were examined in the same way. The results obtained by this culture-independent technique were compared to others previously obtained by conventional culturing methods. Rennet extracts were dominated by a number of Lactobacillus species, including Lb. plantarum, a non-starter lactic acid bacterium dominant during ripening. Lactococcus lactis was only found in one rennet extract. The cheese milk was clearly dominated by Lactococcus-like bacteria, with Lc. lactis in the greatest number. This bacterium was also dominant in the cheese samples (on both the surface and in the interior), in agreement with results obtained by culturing. The sequences of several bacterial DGGE bands from all samples showed less than 97% homology to known, cultured species. This indicates that unknown species are present in the Cabrales cheese environment and that culture-independent methods are needed to fully characterize this ecosystem. r 2005 Elsevier Ltd. All rights reserved. Keywords: Traditional cheeses; Blue-veined cheese; Cabrales cheese; Penicillium roqueforti; DGGE; Culture-independent microbiology 1. Introduction Over the last 10 years it has been recognized that conventional microbiological techniques do not provide a complete picture of the microbial diversity of some complex environments (Godon, Zumstein, Dabert, Ha- bouzit, & Moletta, 1997; Lin & Stahl, 1995; Ward, Ferris, Nold, & Bateson, 1998). The same is true of simpler environments, such as those of traditional fermented foods and beverages (Ampe, ben Omar, Moizan, Wacher, & Guyot, 1999; Cocolin, Manzano, Cantoni, & Comi, 2001). To overcome culture-associated drawbacks (unknown growth factors or growth conditions, obligate interaction among microorganisms or with their niche), an array of molecular techniques has been developed for studying microbial ecology and diversity without the need for culturing (Amann, Ludwingg, & Schleifer, 1995; von Wintzingerode, Go¨bel, & Stackebrandt, 1997). Many of these methods are based on amplifying rRNA or rDNA sequences by polymerase chain reaction (PCR) techniques. Denaturing gradient gel electrophoresis (DGGE), and its relative, temperature gradient gel electrophoresis (TGGE), were developed to analyse microbial communities based on the sequence-specific distinction of 16S amplicons (Muyzer & Smalla, 1998). Separation is based on the decreased electrophoretic mobility of partially melted double- stranded DNA molecules in polyacrylamide gels with a linear gradient of denaturing agents (urea and formamide) or temperature. A GC clamp of around 50 bp is attached to the 5 0 end of one of the primers, preventing the two DNA strands from undergoing complete disassociation (Shief- field, Cox, & Myers, 1989). If the total DNA of a microbial community is used in PCR amplification, the DGGE technique can provide a profile of the genetic diversity of the dominant populations (Muyzer, de Waal, & Uitterlin- den, 1993). If total RNA is used, the profile indicates the ARTICLE IN PRESS www.elsevier.com/locate/idairyj 0958-6946/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2005.11.008 à Corresponding author. Tel.: +34 985 89 21 31; fax: +34 985 89 22 33. E-mail address: baltasar.mayo@ipla.csic.es (B. Mayo).