Bryndza Cheese Research Articles

Microbial diversity and dynamics during the production of May bryndza cheese

Diversity and dynamics of microbial cultures were studied during the production of May bryndza cheese, a traditional 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 – 10days, 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 sequencing. The culture-based data demonstrated an overall trend of growth of the microbial population contributing to lactic acid production and to ripening of the cheese, lactobacilli, lactococci and Geotrichum spp. growing up to densities of 108CFU/g, 109CFU/g and 105CFU/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, Streptococcus 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.

Multiresistance of Staphylococcus xylosus and Staphylococcus equorum from Slovak Bryndza cheese

Staphylococcus xylosus, Staphylococcus equorum, and Staphylococcus epidermidis strains were isolated from Bryndza cheese and identified using PCR method. The antimicrobial susceptibility of these strains was assessed using disc diffusion method and broth microdilution method. The highest percentage of resistance was detected for ampicillin and oxacillin, and in contrary, isolates were susceptible or intermediate resistant to ciprofloxacin and chloramphenicol. Fourteen of the S. xylosus isolates (45%) and eleven of the S. equorum isolates (41%) exhibited multidrug resistance. None of the S. epidermidis isolate was multiresistant. The phenotypic resistance to oxacillin was verified by PCR amplification of the gene mecA.

Probiotic Potential and Safety Properties ofLactobacillus plantarumfrom Slovak Bryndza Cheese

One hundred and twenty-five acid-resistant presumptive lactobacilli were isolated from Slovak Bryndza cheese and screened for their antimicrobial activity against eight bacterial pathogens using spot agar assay. Out of twenty-six Lactobacillus strains with strong inhibition activity, twenty were identified as Lactobacillus plantarum and six as Lactobacillus fermentum. The most active eleven L. plantarum isolates were further characterized in vitro for some probiotic and safety properties. Only three isolates K10, K21, and ZS07 showed the ability to grow over 50% in the presence of 0.3% bile. Strong deconjugation efficiency was determined for CK06 and K21. The highest β-galactosidase activity was shown in isolates ZS11, B01, CK06, and ZS07. Only three of the strains had the ability to produce tyramine: CK06, LM1, and ZS11. Strains K09, K21, ZS11, and ZS15 were susceptible to all tested antibiotics. Analysis of the results confirmed the L. plantarum isolates ZS07 and K21 as the most suitable for probiotic use, due to their desirable probiotic and safety characteristics.

Application of quantitative approach focused on the competition of lactic acid bacteria culture with coagulase-positive staphylococci under the conditions related to artisanal cheese fermentation

A quantitative analysis of Staphylococcus aureus growth was carried out in milk fermented with the mesophilic mixed-strain culture of lactic acid bacteria (LAB), in relation to the conditions prevailing during artisanal ewes’ lump cheese production. Both the temperature and initial volume of the culture had a dramatic effect on the behaviour of the S. aureus strain under study. Depending on the conditions, the growth, reaching maximal population density (N<sub>max</sub>) and a strong inhibition of S. aureus were observed. Regression analysis of the results enabled us to explain the relationships between the parameters such as the growth or inhibition rate, lag phase and N<sub>max</sub>–N<sub>0</sub> and the temperature and initial culture density on the other hand. The surface methodology enabled to show that the rate of S. aureus inhibition (r<sub>inh</sub>) was affected by the temperature and volume of LAB inoculum following the equation: r<sub>inh </sub>=–0.1302 + 0.02325 × T – 0.000975 × T <sup>2 </sup>– 0.00001 × T <sup>2 </sup>× V <sub>0</sub><sup>2</sup> (R = 0.965). The fact that S. aureus increased in number mostly only by about 1 log and did not reach 10<sup>6</sup> CFU/ml during our model experiments was the most useful for good manufacturing practice. The knowledge obtained in this study concerned with the quantitative behaviour of S. aureus including the information on the inhibition at the selected environmental factors (temperature, inoculum size, and pH) is essential for the improvement of the fermentation process, quality and safety of artisanal ewes’ cheese production used as a raw material for industrial Bryndza cheese production.

Antibiotic resistance of Enterococcus species isolated from raw foods of animal origin in South West part of Slovakia

We determined the prevalence and antibiotic resistance of enterococci isolated from raw foods of animal origin. All samples were positive for enterococci. The lowest count of enterococci was found in pork (2.00 log CFU/cm<sup>2</sup>), while bryndza cheese contained the highest count (4.99 log CFU/g). Among the 349 Enterococcus isolates, 49% were E. faecalis, 29% E. faecium, and 13% Enterococcus spp. Tetracycline and gentamicin resistance was the most common. We found the highest tetracycline resistance levels (91%) in isolates from poultry samples. The isolates from the poultry samples also displayed multidrug resistance to all antibiotics tested. The most common vancomycin-resistant species in poultry and milk was E. faecalis. In contrast, the pork samples contained vancomycin-resistant E. faecium isolates. It is interesting to note that vancomycin resistance in the pork and poultry samples was found only in combination with either four (28%) or all five (14%) of the tested antibiotics. Our results suggest that raw products of animal origin are possible reservoirs of multi-antibiotic resistant enterococci in the food chain.

Food-borne enterococci and their resistance to oxidative stress

Enterococci are important food-borne pathogens that cause serious infections. Several virulence factors have been described including aggregation substance, gelatinase, cytolysin, and enterococcal surface protein. The ability to cause infections is mainly dependent on the response to oxidative stress due to the production of reactive oxygen species by immune cells. The aim of our study was to analyze the resistance of enterococcal strains from food to clinically relevant antiseptic agents with regard to the presence of selected virulence factors, and to uncover potential mechanisms of the antioxidative resistance. Eighty-two enterococcal isolates from Bryndza cheese were tested using in vitro growth assays to study the ability of these isolates to survive exposure to antiseptic agents - hydrogen peroxide, hypochlorite, and Chlorhexidine. Virulence genotypes of the isolates were determined by PCR, and RT real time PCR was used for gene expression under oxidative stress. Resistance against antiseptic agents depends on the concentration of applied chemicals, on the time of exposure, but also on virulence factors of the enterococcal strains. Oxidative stress induces the expression of antioxidative enzymes and down-regulates the expression of prooxidative enzymes. These effects are dependent on the virulence genotype of the enterococcal strains. These findings are important for future research, especially concerning the role of enterococci in oral diseases.

Culture-independent detection of microorganisms in traditional Slovakian bryndza cheese

In order to investigate the microflora of Slovakian bryndza cheese (a cheese containing unpasteurized or pasteurized ewes' milk component) by a culture-independent method, DNA was extracted directly from 7 bryndza samples and analysed by an innovative method. Using the universal prokaryotic and fungal primers, ribosomal DNA internal transcribed spacer (ITS) regions with variable length were amplified. The standard universal reverse primer L1 aligning to bacterial 23s rDNA was found unsuitable for some lactic acid bacteria and other species based on in silico analysis. Therefore, L1 primer was replaced by a combination of novel primers GplusR and GminusR aligning to the adjacent, more conserved DNA region. The amplification profiles were visualised by both standard electrophoresis and by fluorescent capillary gel electrophoresis. From representative samples, major amplicons were excised from the gel, cloned and sequenced. Sequencing revealed that the samples contained Lactobacillus delbrueckii, Lactobacillus brevis, Streptococcus thermophilus, Lactococcus lactis, Lactococcus raffinolactis, Streptococcus macedonicus, Leuconostoc pseudomesenteroides, Debaromyces hansenii, Mucor fragilis, Yarrowia lipolytica and Galactomyces geotrichum. These results represent an extension of the knowledge on the microflora of Slovakian bryndza cheese. The introduced automated ribosomal DNA intergenic spacer analysis of the bacterial and fungal genomes proved to be very effective in the application of studying microflora of cheese.

Randomly-amplified microsatellite polymorphism for preliminary typing of lactic acid bacteria from Bryndza Cheese

A high-throughput, medium-discrimination method for preliminary typing and selecting non-identical isolates of lactic acid bacteria in cheeses was developed. RAMP, a PCR with one microsatellite-targeted and one random primer, was used for preliminary typing of 1119 isolates of lactic acid bacteria from Slovak Bryndza cheese. A total of 59 genotypes were identified based on RAMP profiles consisting of 12-23 DNA fragments of 150-3000 bp. For example, 18, 17, 13 and 7 different RAMP-types were identified in Lactobacillus brevis, L. plantarum, L. paracasei and L. fermentum, respectively. The method facilitated well reproducible, medium-discrimination typing of Lactobacillus spp. and Pediococcus spp. at a subspecies level and proved to be suitable for preliminary typing of lactic acid bacteria isolated from cheese.

Pheno- and Genotyping of Staphylococcus aureus Isolates of Sheep Origin

The aim of the study was to determine the prevalence of genes encoding virulence factors in Staphylococcus aureus strains isolated from raw sheep milk, sheep cheese and Bryndza cheese. Genes encoding staphylococcal enterotoxin (sea, seb, sec, sed and see), toxic shock syndrome toxin-1 (tst), exfoliative toxins (eta and etb) and collagen-binding protein (cna) were detected. In a total of 79 S. aureus isolates all assessed toxins encoding genes were found, except for see, eta and etb. Overall, 75.9% of S. aureus isolates were found to be positive for one or more toxin genes. The sec gene was found most frequently (24.1%), followed by tst (22.8%), seb (13.9%), sed (10.1%) and sea (5.1%). The cna gene was detected in 55.7% of S. aureus isolates. Based on tandem repeats in coa gene, five coa types were observed, further divided into 16 subtypes based on their RFLP pattern. Similarly tandem repeats in spa gene divided S. aureus isolates into 7 types. In the parallel antibiotic resistance study, 69.6% isolates were resistant to at least one of the 11 tested antibiotics. The pheno- and genotyping of S. aureus isolates of sheep origin presented in this work update the epidemiological data in Slovakia.

Correlation and distribution of elemental markers of origin in the production of Bryndza sheep cheese

The successful classification of Bryndza, a typical Slovak national sheep cheese, from nine different Slovak producing regions, was based on canonical discriminant analysis. Elemental markers of origin (Ba, Cr, Cu, Hg, Mg, Mn, Ni and V) were determined by graphite furnace (GF) and flame atomic absorption spectrometry (AAS). High correlations of elemental markers were found between pasture soil, grass, and Bryndza cheese originated from different regions. The concentration of these markers in pasture soils, grasses, as well as in all the milk and Bryndza cheese-making process was monitored. Their content in sheep milk, lumpy cheese, whey, boiled sheep whey and final Bryndza cheese was significantly correlated with the soil elemental markers. The final product was only slightly contaminated by the individual elements during the Bryndza production, without any significant influence on the Bryndza origin classification and/or traceability.

Antimicrobial susceptibility ofEnterococcus species isolated from slovak bryndza cheese

Three hundred and ten enterococcal isolates (178 Enterococcus faecium, 68 E. durans, 49 E. faecalis, 8 E. italicus, 3 E. gallinarum, 3 E. casseliflavus, and 1 E. hirae) from Slovak Bryndza cheese were evaluated for susceptibility to nine antimicrobial agents (vancomycin, teicoplanin, ampicillin, streptomycin, gentamicin, erythromycin, rifampicin, nitrofurantoin, and ciprofloxacin). All enterococcal isolates from Bryndza cheese were susceptible to ampicillin, streptomycin, gentamicin, vancomycin, and teicoplanin as determined by the disk diffusion method. Vancomycin resistance genes vanA and vanB were not detected. Resistance rates of enterococcal isolates to rifampicin, erythromycin, ciprofloxacin, and nitrofurantoin were 24, 26, 2, and 1 %, respectively. Thirty-six % of E. faecium isolates and 22 % of the E. faecalis isolates were resistant to erythromycin. Resistance to rifampicin was similar in E. faecium (31 %) and E. faecalis (29 %). Both E. faecium and E. faecalis strains showed the same resistance to ciprofloxacin (2 %). E. durans isolates showed low levels of resistance to rifampicin, erythromycin, ciprofloxacin, and nitrofurantoin (1-4 %). Forty-eight (30 %) of the E. faecium isolates, two (3 %) of the E. durans isolates, and six (12 %) of the E. faecalis isolates exhibited multidrug resistance. The highest frequency of resistant enterococci was observed in Bryndza produced in winter season.

Genetic diversity of Enterococcus faecium isolated from Bryndza cheese

One hundred and seventy-six Enterococcus faecium isolates from Slovak dairy product Bryndza were tested for the presence of plasmid DNA. Eighty-two isolates were positive and their plasmid DNA was isolated and digested by EcoRI and HindIII restriction endonucleases. The patterns obtained were compared with those obtained after pulsed-field gel electrophoresis of macrorestriction fragments (PFGE), (GTG) 5–PCR and ERIC–PCR. All these molecular approaches were applied for the study of genetic variability and determination of strain relatednesses among plasmid-positive isolates of E. faecium. In general, all methods revealed a considerable genetic diversity of E. faecium isolates. Plasmid profiling and ERIC–PCR have offered a higher resolution than PFGE and (GTG) 5–PCR.

Identification of Slovakian, Polish, and Romanian bryndza cheeses origin by factor analysis of some elemental data

Study of traceability of typical sheep cheese bryndza made in Slovakia, Poland, and Romania was realized by means of some special elemental markers determined by atomic absorption spectrometry. Principal component factoring was used for elements variability determination, differentiation, and visualization of used data. Elemental markers Ba, Cu, Cr, Mg, and Ni enable effective identification of majority of Slovak bryndza cheese producing areas. As the best elemental markers of origin, enabling successful multiregional differentiation of Slovakian, Polish, and Romanian bryndza cheeses, Cr, Hg, Mn, and V were used.

Identification and characterization of enterococci from bryndza cheese

To identify enterococci isolated from sheep milk cheese--bryndza, and to compare differences in the composition of enterococcal microflora affected by the season, and to evaluate the potential presence of vancomycin resistance and virulence determinants. Bacterial strains were isolated during analysis of bryndza cheese and identified on the genus and species level by phenotypic methods and with commercial biochemical sets. The identification of the species, Enterococcus faecium, Ent. durans and Ent. faecalis, was confirmed by PCR using species-specific primers for ddl genes. PCR was also used for assessment of presence of vanA and vanB genes and virulence determinants gelE, agg and cytolysin genes namely: cylL(L), cylL(S), cylM, cylB and cylA. Among 308 Enterococcus sp. strains, 177 isolates were proved to be Ent. faecium, 59 to be Ent. durans and 41 to be Ent. faecalis. Vancomycin resistance genes vanA and vanB were not detected. Agar plate testing confirmed their absence. Gene gelE, however, was found in 20 Ent. faecalis isolates, but only 13 of them showed gelatinase-positive phenotype. Seven isolates had five cytolysin genes, but none of the isolates exhibited a positive haemolytic phenotype. Four isolates possessed the agg gene. The prevalence of Ent. faecium species was highest in samples from the winter season harvest. Ent. faecium is the dominant enterococcal species in bryndza cheese and the most prevalent in the winter season product. None of the Enterococcus sp. strains was proved to have vanA or vanB genes and the vancomycin resistance. To our knowledge, this is the first report of enterococcal microflora in bryndza cheese and its evaluation for the presence of vanA and vanB genes as well as virulence determinants.

Molecular identification and diversity of enterococci isolated from Slovak Bryndza cheese

Three hundred and eight presumed enterococcal isolates were recovered from Bryndza, a soft sheep milk cheese. The cheese samples were obtained from five different commercial distributors in Slovakia and were taken at three different seasonal intervals. All isolates were identified to the species level using genotypic tools. Species-specific PCR using ddl genes highlighted the predominance of Enterococcus faecium (176 isolates) and assigned 50 isolates to the species Enterococcus faecalis. The remaining 82 isolates were classified using repetitive element sequence-based polymerase chain reaction (PCR) with primer (GTG)(5)-(GTG)(5)-PCR, in combination with phenylalanyl-tRNA synthase gene (pheS) sequence analysis and by whole-cell protein analysis (SDS-PAGE). These strains were identified as Enterococcus durans (59 strains), Enterococcus italicus (8 strains), Enterococcus casseliflavus (3 strains), Enterococcus gallinarum (3 strains), Enterococcus hirae (1 strain), and 8 strains were members of the species Lactococcus lactis. Of the seven enterococcal species isolated, three of them, E. durans, E. faecalis and E. faecium were present in all samples studied, with E. faecium as the predominant one. The precise identification of enterococci in Bryndza cheese is an essential step in the process of evaluation of their functional properties which will be further studied and assessed.

Evaluation of (GTG) 5-PCR for identification of Enterococcus spp.

A set of reference strains and a group of previously unidentified enterococci were analysed by rep-PCR with the (GTG) 5 primer to evaluate the discriminatory power and suitability of this method for typing and identification of enterococcal species. A total of 49 strains representing all validly described species were obtained from bacterial collections. For more extensive evaluation of this identification approach 112 well-defined and identified enterococci isolated from bryndza cheese were tested. The (GTG) 5-PCR fingerprinting assigned all strains into well-differentiated clusters representing individual species. Subsequently, a group including 44 unidentified enterococci isolated from surface waters was analysed to evaluate this method for identification of unknown isolates. Obtained band patterns allowed us to identify all the strains clearly to the species level. This study proved that rep-PCR with (GTG) 5 primer is a reliable and fast method for species identification of enterococci.