Slovak „Bryndza“ is a natural white, gently spreadable, slightly moist fresh ripened cheese with curds and own texture, made in a traditional way from well-fermented ripened ewe's lump cheese.

A characteristic feature of the production of Slovak „Bryndza“ is the crushing and grinding of mature ewe's or a mixture of ewe's and cow's lump cheese and their mixing with salt or specially prepared saline solution to achieve the required composition, which distinguishes this production from the production of other ewe's cheese produced outside Slovakia. Its characteristic sensory attributes are due to the natural microflora contained in raw ewe's milk and ewe's lump cheese and to the characteristic production method. The basic raw material for the production of Slovak „Bryndza“ is ewe's lump cheese or a mixture of ewe's and cow's lump cheese, or a mixture of cured ewe's lump cheese and cow's lump cheese aged under specific conditions (Commission Regulation No. 676/2008).

Microorganisms represent without doubt the largest group of living organisms in the world, with only a small fraction of microbial species which have been identified until now. They can be highly diverse in their biochemistry, physiology and nutritional modes. Most of them are reproducing swiftly and the significant plasticity of their genome allows them to easily adapt to changing environmental conditions, as well as perform a variety of essential ecosystem functions, on which food production depends on. According to FAO (2009), the main functional groups for food processing are beneficial microorganisms (fermentation and probiotics). Microbial food cultures include bacterial food cultures, fungi and yeasts. These microorganisms determine the characteristics of the fermented food, e.g., acidity, flavour and texture, as well as health benefits that go beyond elementar nutrition (Vogel et al., 2011).

The aim of our study was to isolate and identificate the microorganisms from Slovak ewe's cheese „Bryndza“ obtained from different Slovak regions.

There were 60 samples of Slovak ewe's cheese „Bryndza“ from east, middle and west part of Slovakia evaluated for microbiological quality in our study. All samples were obtained in May 2019. These samples were placed in sterile sample containers and transported on ice to the laboratory for microbiological investigations. Samples were kept in a refrigerator (4 ±1 °C) until the testing began. The primary dilution of the ewe's cheese was made for preparing the samples for testing: a 5 mL of sample material was added to 45 mL of 0.87 % sterile saline. Then the serial dilutions (10^-2 to 10^-4) were done and a 100 μL of each dilution was plated out.

Different groups of microorganisms were isolated from the 60 ewe's cheese „Bryndza“ samples (Table 1). Total bacterial count in ewe's cheese ranged from 3.87 ±0.58 CFU.g^-1 from west Slovak producers to 4.32 ±0.17 CFU.g^-1 from middle Slovak producers. Generally, the coliform bacteria ranged from 3.46 ±0.26 CFU.g^-1 from east Slovak producers to 3.64 ±0.19 CFU.g^-1 for bryndza from middle Slovak producers. The number of lactic acid bacteria ranged from 3.14 ±0.09 CFU.g^-1 from east Slovak producers to 3.24 ±0.21 CFU.g^-1 in the bryndza cheeses from west Slovak producers. Table 2a and Table 2b showes isolated species of bacteria.

The eukaryotic microorganisms were represented largely by members of the genera of Dipodascus and Kluyveromyces, which were present at a level of 99 isolates and 60 isolates, and by other yeasts, which were present in Candida genera (Figure 3). The moulds were present generally at middle levels, with the most colonies of Rhizopus spp. With 21 isolates. All samples contained high numbers of lactic acid bacteria belonging to genera Lactobacillus, Lactococcus and Leuconostoc. In order to obtain a better view of the lactic acid bacteria isolates, the Enterococcus, Lactobacillus, Lactococcus, Leuconostoc and Pediococcus strains were identified by mass spectrometry assays. Representatives of the species Leuconostoc mesenteroides ssp. mesenteroides were isolated and identified in all bryndza cheese samples (Figure 2).

Different Lactobacillus species, such as Lb. brevis, Lb. delbrueckii, Lb. fermentum, Lb. helveticus, Lb. harbinensis, Lb. jonsonii, Lb. paracasei ssp. paracasei, Lb. plantarum, Lb. paraplantarum, Lb. rhamnosus and Lb. suebicus, were identified in the bryndza cheese samples from all producers (Figure 2). The most isolated species were Lb. brevis, Lb. fermentum and Lb. plantarum.

There were totally 1175 isolates identified by mass spectrometry include G^-, G⁺ and microscopic filamentous fungi in our study. Together 199 isolates were (Figure 1) isolated and identified from G^- and most frequently species was Escherichia coli, 599 isolates from G⁺ with most isolated species Leuconostoc mesenteroides ssp. mesenteroides (Figure 2) and 377 isolates of yeast and molds where the most frequently isolated species was Yarrowia lipolitica (Figure 3).

The distinctive flavour of bryndza cheese produced in month May is apparently composed from compounds contained in ewes’ milk and from the products of fermentation of the substrate by microflora. Principal volatile aroma-active compounds of May bryndza cheese have been characterized by Sádecká et al. (2014).

Due to composition and activity of microflora is estimated to have a great impact on the flavour of bryndza cheese, several culture-based as well as culture-independent microbiological studies were carried out in this regard. Data from older culture based studies, which identified Lactobacillus spp., Lactococcus spp., Streptococcus spp., Enterococcus spp., Kluyveromyces marxianus and Galactomyces geotrichum as main components of the microflora of bryndza cheese (Palo and Kalab, 1984; Görner and Valík, 2004; Görner, 1980) were updated by a study of Berta et al. (2009), in which a range of Lactobacillus spp. isolates were identified by 16S rDNA sequencing. Enterococci (Jurkovič et al., 2006), staphylococci (Mikulášová et al., 2014) and fungal species (Laurenčík et al., 2008) were cultured and identified in bryndza cheese. Culture-independent studies (Chebeňová-Turcovská et al., 2011; Pangallo et al., 2014) provided information on the diversity of bacteria and fungi and its dynamics during the production of bryndza cheese. In the production of bryndza cheese, also interactions between lactic acid bacteria and Galactomyces/Geotrichum group (Hudecová et al., 2011) and competition between lactic acid bacteria and coagulase-positive staphylococci (Medveďová and Valík, 2012) were studied.

Although basic information on May bryndza cheese is available regarding microbiological composition as well as aroma-active compounds, most of the previous experiments were done on a limited geographical basis, sometimes with products of just one factory. In order to obtain a more reliable and representative view, this study aimed to gain data for the products from the entire territory of Slovakia that is relevant to bryndza production, i.e. specified mountainous regions of Slovakia (Commission Regulation (EC) No. 676/2008). In Slovakia, the presence of Carpathian Mountains creates different climatic conditions that can have influence various characteristics of the produced bryndza cheese. These can relate, in particular, to the ewe's diet in terms of different plant species composition in the pasture and, therefore, to the quality of milk used for the production of bryndza (Ostrovský et al., 2009) and to different temperatures at which the lump cheese is produced, which can affect the microbial consortia in the beginning of the ripening process (Görner and Valík, 2004).

The aim of our study was to evaluate the microbiological quality of Slovak ewe's cheese bryndza from producers of east, middle and west Slovakia. The number of isolated group of microorganisms was accurate for the traditional cheese produced in Slovakia. Totally 1175 isolates of bacteria with score more than 2 were identified with MALDI TOF MS Biotyper.