Influence of pasteurization, brining conditions and production environment on the microbiota of artisan Gouda-type cheeses Koenraad Van Hoorde a, * , Marc Heyndrickx b , Peter Vandamme a , Geert Huys a a Laboratory of Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium b Unit Technology and Food, Institute for Agricultural and Fisheries Research (ILVO), Brusselsesteenweg 370, B-9090 Melle, Belgium article info Article history: Received 26 August 2009 Received in revised form 3 December 2009 Accepted 4 December 2009 Available online 11 December 2009 Keywords: DGGE Artisan Gouda cheese Diversity Dynamics Lactic acid bacteria abstract To monitor the effect of the indigenous milk microbiota and of technological and environmental parameters on the microbiota established in ripened cheese, the diversity and dynamics of the predominant microbial communities in artisan Gouda-type cheeses produced under different conditions was studied. A total of 22 cheese types differing in milk source, milk treatment, production environment and brining conditions were analyzed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) using total DNA extracts as well as DNA extracted from culturable fractions. Through band position analysis and band sequencing, the majority of DGGE bands could be attributed to lactic acid bacteria (LAB), although a few bands also belonged to staphylococci and gamma-Proteobacteria. Aided by principal component analysis (PCA) and multivariate analysis of variance (MANOVA), cheeses produced at different locations could clearly be differentiated. The same approach also allowed to distinguish raw and pasteurized milk cheeses, the former showing a more diverse microbiota in terms of a higher species richness and number of DGGE bands. No substantial differences were found between cheeses brined at two different locations. In conclusion, the combined PCR-DGGE approach relying on both total DNA extracts and culturable fractions proved its value for analyzing the effect of technological and environmental parameters on the diversity and dynamics of the microbiota in Gouda-type cheeses. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction The typical characteristics of any given type of cheese strongly depend on the composition, dynamics and interactions of the cheese microbiota (Poznanski et al., 2004). Several steps in the cheese making process are known to affect this microbiota. Compositional factors such as the type of milk and starter (and adjunct) culture used have been recognized as major determinants of the cheese microbiota (Grappin and Beuvier, 1997; Poznanski et al., 2004; Callon et al., 2005). In addition, several studies have also highlighted the possible effect of the cheese production envi- ronment (Antonsson et al., 2001) and the influence of technological factors of cheese making (Martín-Platero et al., 2009; Moatsou et al., 2001). A better understanding of this microbiota diversity, how it is influenced by various environmental and technological parameters and how it develops during production and ripening could contribute to the selection of specific organoleptic properties, the enhancement of flavour formation and the control of microbial pathogens and spoilers in artisan cheeses (Kieronczyk et al., 2003; Grattepanche et al., 2008). Both culture-dependent and -independent approaches have been used to study microbial populations during cheese manufacturing (Giraffa and Carminati, 2008). Culture-based tech- niques, however, are laborious and time-consuming, and the fact that only a portion of the community members can be recovered on laboratory media, usually relying on a semi-randomized way of colony picking, results in an incomplete taxonomic inventory of the cheese microbiota (Giraffa and Neviani, 2001; Hugenholtz et al., 1998). Hence, molecular culture-independent tools are nowadays commonly applied for detailed microbial analyses of cheese ecosystems, many of them being based on community finger- printing of the predominant cheese microbiota (Jany and Barbier, 2008). Next to single-strand conformation polymorphism-PCR (SSCP-PCR), terminal restriction fragment length polymorphism (T- RFLP) and length-heterogeneity-PCR (LH-PCR), denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electro- phoresis (TGGE) are by far the most extensively used fingerprinting methods for studying bacterial diversity and population dynamics during cheese manufacturing (Coppola et al., 2008). Essentially, DGGE and TGGE rely on the electrophoretic sepa- ration of equally sized PCR amplicons obtained from a community * Corresponding author. E-mail address: koenraad.vanhoorde@ugent.be (K. Van Hoorde). Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm 0740-0020/$ e see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2009.12.001 Food Microbiology 27 (2010) 425e433