International Journal of Food Microbiology 170 (2014) 38–43 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro 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 article info abstract Article history: Diversity and dynamics of microbial cultures were studied during the production of May bryndza cheese, a tra- Received 18 March 2013 ditional Slovak cheese produced from unpasteurized ewes' milk. Quantitative culture-based data were obtained Received in revised form 27 August 2013 for lactobacilli, lactococci, total mesophilic aerobic counts, coliforms, E. coli, staphylococci, coagulase-positive Accepted 23 October 2013 staphylococci, yeasts, fungi and Geotrichum spp. in ewes' milk, curd produced from it and ripened for 0 – Available online 30 October 2013 10 days, and in bryndza cheese produced from the curd, in three consecutive batches. Diversity of prokaryotes fi Keywords: and eukaryotes in selected stages of the production was studied by non-culture approach based on ampli cation Ewes' cheese of 16S rDNA and internal transcribed spacer region, coupled to denaturing gradient gel electrophoresis and se- Microbial dynamic quencing. The culture-based data demonstrated an overall trend of growth of the microbial population contrib- Bacteria uting to lactic acid production and to ripening of the cheese, lactobacilli, lactococci and Geotrichum spp. growing Fungi up to densities of 108 CFU/g, 109 CFU/g and 105 CFU/g, respectively, in all three consecutive batches of bryndza Yeasts cheese. The diversity of bacteria encompassed Acinetobacter calcoaceticus, Acinetobacter guillouiae, Acinetobacter Denaturing gradient gel electrophoresis sp., Acinetobacter johnsonii, Citrobacter braakii, Clostridium bartlettii, Corynebacterium callunae, Corynebacterium (DGGE) 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 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. Bryndza cheese is a soft spreadable cheese, made from unpasteur- After that, the curd is transferred to a colder location (15 °C) and ized ewes' milk. It is a traditional food product produced in mountain transported to a bryndza-producing dairy for processing. Here the regions of Slovakia. May bryndza cheese is a highly valued variant of ripening continues at 12 – 15 °C during 9 – 10 days. Alternatively, bryndza, which is produced in the beginning of summer season, in ewes' curd is produced and processed directly in specialized bryndza- May. The season of production is believed to positively influence the producing dairies with ripenening at 12 – 14 °C during 10 – 14 days. quality of the cheese, probably by the quality of ewes' milk as influenced The ripened cheese is decrusted and milled with salt solution, resulting by the spring pastures (Görner, 1980; Palo and Kaláb, 1984). in bryndza (Palo and Kaláb, 1984; Valík, 2004). Traditionally, May bryndza cheese is produced from ewes' lump Because composition and activity of microflora are believed to cheeses that are produced as an intermediate product in mountain be responsible for flavour and aroma of various types of bryndza cottages (salaš) and the production process continues in specialized cheese, several culture-based as well as non-culture based studies bryndza-producing dairies lower in the valley, without the use of any were carried out with this cheese. Data from older culture-based stud- starter-cultures. Ewes' milk is processed in salaš immediately after ies, which identified Lactobacillus spp., Lactococcus spp., Streptococcus spp., Enterococcus spp., Kluyveromyces marxianus and Galactomyces ⁎ Corresponding author. geotrichum / Geotrichum candidum (now called Galactomyces candidus / 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.10.015 D. Pangallo et al. / International Journal of Food Microbiology 170 (2014) 38–43 39 Geotrichum candidum) as main components of the microflora of bryndza 1999) oriented to 16S rRNA gene. For amplification of ITS region of cheese (Görner, 1980; Palo and Kaláb, 1984; Valík, 2004), were updated yeasts and fungi, primers ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) by a study of Berta et al. (2009), in which a range of Lactobacillus spp. and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′)wereused(White isolates were identified by 16S rDNA sequencing. Non-culture-based et al., 1990). The PCR mixture (25 μl) contained 50 pmol of each primer, study of Chebeňová-Turcovská et al. (2011) revealed the presence of 200 μmol l−1 of dNTP, 1.5 U SuperHot-Taq DNA polymerase (Bioron, Lb. delbrueckii, Lb. brevis, Lc. lactis, Lc. raffinolactis, Str. macedonicus, Str. Ludwigshafen, Germany) and 1 X PCR buffer. Three μl of extracted thermophilus, Leuconostoc pseudomesenteroides, Debaromyces hansenii, DNA was used as a template in the first amplification. The temperature Mucor fragilis, Yarrowia lipolytica and Galactomyces geotrichum / program consisted of initial denaturation at 94 °C for 5 min, 30 cycles Geotrichum candidum (now called Galactomyces candidus / Geotrichum (94 °C for 30 s, 54 °C for 45 s, 72 °C for 1 min) and a final polymeriza- candidum) in bryndza cheese. Another research team cultivated and tion step at 72 °C for 10 min. For each DNA target (16S rDNA and ITS), identified fungal species (Laurenčík et al., 2008), including a novel four reactions of 25 μl(100μl altogether) were produced. The four species Geotrichum bryndzae (Sulo et al., 2009). reactions of each DNA target were mixed together and the specificity In the current study, we attempted to provide a detailed insight in of amplification was checked by agarose gel electrophoresis. the microflora of May bryndza cheese, as the most valued variant of bryndza cheese, and also to describe the dynamics of the microflora 2.4. DGGE fingerprint analysis during the ripening of ewes' curd as an intermediate product in the pro- duction of May bryndza cheese. In order to describe the dynamics of the The PCR product of the first step (2 μl)wasusedastemplateinthe microbial cultures during the production of the cheese, analyses were second amplification, a semi-nested PCR for each DNA target. The 16S performed at various stages of the production (ewes' milk, curd pro- rDNA was re-amplified with primers 341f-GC (5′-CGC CCG CCG CGC duced from it and ripened for 0 – 10 days, bryndza produced from the GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG curd). In order to describe also variability or trend during time of the CAG-3′; Muyzer et al., 1993)and518r(5′-ATT ACC GCG GCT GCT GG- year, three consecutive batches (end of April, May, beginning of June) 3′; Neefs et al., 1990) and primers ITS1f-GC (5′-CGC CCG CCG CGC were analysed. GCG GCG GGC GGG GCG GGG GCA CGG GGG GTC CGT AGG TGA ACC TGC GG-3′) and ITS2 (5′-GCT GCG TTC TTC ATC GAT GC-3′)wereused 2. Materials and methods for the semi-nested amplification of ITS fragment (White et al., 1990). The PCR conditions were the same as above. 2.1. Bryndza cheese samples Four semi-nested PCR products (4 reactions) for each DNA target were pooled, checked on agarose gel, and precipitated with 96% ethanol, Samples of ewes' milk, ewes' curd and bryndza were obtained from resuspended in 20 μlH2O and the precipitate (10 μl) was analysed by Farma Oľga Apoleníková, Pružina, Slovakia. Each batch of samples denaturing gradient gel electrophoresis [DGGE; 8% polyacrylamide gel contained samples of milk, curd produced from the milk on Day 0, 1, (acrylamide-bisacrylamide, 37.5:1); denaturation gradient 25% – 55% 2, 3, 4, 5, 6, 7, 8, 9 and 10 of ripening, and bryndza cheese produced for separation of 16S rDNA amplicons or 20% – 50% for separation of from the curd ripened for 10 days.