Microbial diversity and dynamics during the production of May bryndza cheese Domenico Pangallo a, ⁎, Nikoleta Šaková a,c , Janka Koreňová b , Andrea Puškárová a , Lucia Kraková a , Lubomír Valík c , Tomáš Kuchta b a Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava, Slovakia b Department of Microbiology and Molecular Biology, Food Research Institute, Priemyselná 4, P. O. Box 25, 824 75 Bratislava 26, Slovakia c Department of Nutrition and Food Quality Assessment, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia abstract article info Article history: Received 18 March 2013 Received in revised form 27 August 2013 Accepted 23 October 2013 Available online 30 October 2013 Keywords: Ewes' cheese Microbial dynamic Bacteria Fungi Yeasts Denaturing gradient gel electrophoresis (DGGE) Diversity and dynamics of microbial cultures were studied during the production of May bryndza cheese, a tra- ditional Slovak cheese produced from unpasteurized ewes' milk. Quantitative culture-based data were obtained for lactobacilli, lactococci, total mesophilic aerobic counts, coliforms, E. coli, staphylococci, coagulase-positive staphylococci, yeasts, fungi and Geotrichum spp. in ewes' milk, curd produced from it and ripened for 0 – 10 days, and in bryndza cheese produced from the curd, in three consecutive batches. Diversity of prokaryotes and eukaryotes in selected stages of the production was studied by non-culture approach based on amplification of 16S rDNA and internal transcribed spacer region, coupled to denaturing gradient gel electrophoresis and se- quencing. The culture-based data demonstrated an overall trend of growth of the microbial population contrib- uting to lactic acid production and to ripening of the cheese, lactobacilli, lactococci and Geotrichum spp. growing up to densities of 10 8 CFU/g, 10 9 CFU/g and 10 5 CFU/g, respectively, in all three consecutive batches of bryndza cheese. The diversity of bacteria encompassed Acinetobacter calcoaceticus, Acinetobacter guillouiae, Acinetobacter sp., Acinetobacter johnsonii, Citrobacter braakii, Clostridium bartlettii, Corynebacterium callunae, Corynebacterium maris, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter hormaechei, Enterococcus faecium, Enterococcus pallens, Escherichia coli, Haemophilus haemolyticus, Hafnia alvei, Kluyvera cryocrescens, Lactobacillus helveticus, Lactococcus garvieae, Lc. lactis subsp. cremoris, Lc. lactis subsp. lactis, “Leuconostoc garlicum”, Mannheimia glucosida, Mannheimia haemolytica, Pseudomonas sp., Ps. fluorescens, “Ps. reactans”, Raoultella ornithinolytica, R. terrigena, “Rothia arfidiae”, Staphylococcus aureus, Staph. epidermidis, Staph. felis, Staph. pasteuri, Staph. sciuri, Staph. xylosus, Streptococ- cus parauberis, Str. thermophilus and Variovorax paradoxus. The diversity of yeasts and fungi encompassed Alternaria alternata, “Ascomycete sp.”, Aspergillus fumigatus, Beauveria brongniartii, Candida xylopsoci, C. inconspicua, Cladosporium cladosporioides, Debaromyces hansenii, Fomes fomentarius, Galactomyces candidus, Gymnoascus reesii, Chaetomium globosum, Kluyveromyces marxianus, Metarhizium anisopliae, Penicillium aurantiogriseum, P. camemberti, P. freii, P. polonicum, P. viridicatum, Pichia kudriavzevii, Sordaria alcina, Trichosporon lactis and Yarrowia lipolytica. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Bryndza cheese is a soft spreadable cheese, made from unpasteur- ized ewes' milk. It is a traditional food product produced in mountain regions of Slovakia. May bryndza cheese is a highly valued variant of bryndza, which is produced in the beginning of summer season, in May. The season of production is believed to positively influence the quality of the cheese, probably by the quality of ewes' milk as influenced by the spring pastures (Görner, 1980; Palo and Kaláb, 1984). Traditionally, May bryndza cheese is produced from ewes' lump cheeses that are produced as an intermediate product in mountain cottages (salaš) and the production process continues in specialized bryndza-producing dairies lower in the valley, without the use of any starter-cultures. Ewes' milk is processed in salaš immediately after milking by renetting at 29 – 31 °C for 30 min. The curd is drained at 18 – 22 °C for 24 h and then left to ripen for 3 days at 18 – 20 °C. After that, the curd is transferred to a colder location (15 °C) and transported to a bryndza-producing dairy for processing. Here the ripening continues at 12 – 15 °C during 9 – 10 days. Alternatively, ewes' curd is produced and processed directly in specialized bryndza- producing dairies with ripenening at 12 – 14 °C during 10 – 14 days. The ripened cheese is decrusted and milled with salt solution, resulting in bryndza (Palo and Kaláb, 1984; Valík, 2004). Because composition and activity of microflora are believed to be responsible for flavour and aroma of various types of bryndza cheese, several culture-based as well as non-culture based studies were carried out with this cheese. Data from older culture-based stud- ies, which identified Lactobacillus spp., Lactococcus spp., Streptococcus spp., Enterococcus spp., Kluyveromyces marxianus and Galactomyces geotrichum / Geotrichum candidum (now called Galactomyces candidus / International Journal of Food Microbiology 170 (2014) 38–43 ⁎ Corresponding author. 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.10.015 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro