International Journal of Food Microbiology 170 (2014) 38–43

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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

a r t i c l e i n f o a b s t r a c t

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
Keywords:
and eukaryotes in selected stages of the production was studied by non-culture approach based on amplification
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

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
⁎ Corresponding author.
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 /

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
Geotrichum candidum) as main components of the microflora of bryndza
cheese (Görner, 1980; Palo and Kaláb, 1984; Valík, 2004), were updated
by a study of Berta et al. (2009), in which a range of Lactobacillus spp.
isolates were identified by 16S rDNA sequencing. Non-culture-based
study of Chebeňová-Turcovská et al. (2011) revealed the presence of
Lb. delbrueckii, Lb. brevis, Lc. lactis, Lc. raffinolactis, Str. macedonicus, Str.
thermophilus, Leuconostoc pseudomesenteroides, Debaromyces hansenii,
Mucor fragilis, Yarrowia lipolytica and Galactomyces geotrichum /
Geotrichum candidum (now called Galactomyces candidus / Geotrichum
candidum) in bryndza cheese. Another research team cultivated and
identified fungal species (Laurenčík et al., 2008), including a novel
species Geotrichum bryndzae (Sulo et al., 2009).
In the current study, we attempted to provide a detailed insight in
the microflora of May bryndza cheese, as the most valued variant of
bryndza cheese, and also to describe the dynamics of the microflora
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
microbial cultures during the production of the cheese, analyses were
performed at various stages of the production (ewes' milk, curd pro-
duced from it and ripened for 0 – 10 days, bryndza produced from the
curd). In order to describe also variability or trend during time of the
year, three consecutive batches (end of April, May, beginning of June)
were analysed.
2. Materials and methods
2.1. Bryndza cheese samples
Samples of ewes' milk, ewes' curd and bryndza were obtained from
Farma Oľga Apoleníková, Pružina, Slovakia. Each batch of samples
contained samples of milk, curd produced from the milk on Day 0, 1,
2, 3, 4, 5, 6, 7, 8, 9 and 10 of ripening, and bryndza cheese produced
from the curd ripened for 10 days. Three consecutive batches from the
end of April 2012, from May 2012 and from the beginning of June
2012 were analysed. Fresh samples were analysed by culture, and
samples frozen at −20 °C for a maximum of 3 weeks were analysed
by non-culture DNA-based methods.
2.2. Culture-based analysis
Bacteria were grown and quantified on the following media:
lactobacilli on de Man – Rogosa – Sharpe agar (Merck, Darmstadt,
Germany) at 30 °C during 72 h; lactococci on M17 agar (Merck) at
30 °C during 72 h; total mesophilic aerobes on glucose-tryptone-yeast
extract agar (Merck) at 30 °C during 48 h; coliforms and E. coli on
Chromocult C medium (Merck) at 37 °C during 24 h; Staphylococcus
spp. on Baird–Parker agar (Merck) at 37 °C during 48 h. Coagulase ac-
tivity of staphylococci was determined by rabbit plasma tube coagulase
test (Bio-Rad, Marnes-la-Coquette, France) at 37 °C during 24 h. Yeasts
(including Geotrichum spp.) and molds were grown and quantified on
yeast extract-glucose-chloramphenicol agar (Merck) at 25 °C during
5 days with colony morphology evaluated by microscopy.
2.3. DNA extraction and first PCR amplification
DNA was isolated from 1 ml milk, or 1 g ewes' curd or bryndza
cheese by shaking at 45 °C during 30 min in 20 ml of 2% sodium citrate
solution with glass beads, with subsequent removal of the fat layer and
chaotropic solid phase extraction using DNeasy Tissue kit (Qiagen,
Hilden, Germany; Chebeňová-Turcovská et al., 2011). Bacterial 16S
rDNA and eukaryotic ITS fragment were amplified in two steps; part
of the PCR product of the first step was used for the construction of
the clone libraries and part in the second amplification step, a semi-
nested PCR which was used for DGGE fingerprint analysis. The first
step involved primers 341f (5′-CCT ACG GGA GGC AGC AG-3′; Muyzer
et al., 1993) and 985r (5′-GTA AGG TTC TTC GCG TT-3′; Heuer et al.,
39
1999) oriented to 16S rRNA gene. For amplification of ITS region of
yeasts and fungi, primers ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′)
and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) were used (White
et al., 1990). The PCR mixture (25 μl) contained 50 pmol of each primer,
200 μmol l−1 of dNTP, 1.5 U SuperHot-Taq DNA polymerase (Bioron,
Ludwigshafen, Germany) and 1 X PCR buffer. Three μl of extracted
DNA was used as a template in the first amplification. The temperature
program consisted of initial denaturation at 94 °C for 5 min, 30 cycles
(94 °C for 30 s, 54 °C for 45 s, 72 °C for 1 min) and a final polymeriza-
tion step at 72 °C for 10 min. For each DNA target (16S rDNA and ITS),
four reactions of 25 μl (100 μl altogether) were produced. The four
reactions of each DNA target were mixed together and the specificity
of amplification was checked by agarose gel electrophoresis.
2.4. DGGE fingerprint analysis
The PCR product of the first step (2 μl) was used as template in the
second amplification, a semi-nested PCR for each DNA target. The 16S
rDNA was re-amplified with primers 341f-GC (5′-CGC CCG CCG CGC
GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG
CAG-3′; Muyzer et al., 1993) and 518r (5′-ATT ACC GCG GCT GCT GG-
3′; Neefs et al., 1990) and primers ITS1f-GC (5′-CGC CCG CCG CGC
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′) were used
for the semi-nested amplification of ITS fragment (White et al., 1990).
The PCR conditions were the same as above.
Four semi-nested PCR products (4 reactions) for each DNA target
were pooled, checked on agarose gel, and precipitated with 96% ethanol,
resuspended in 20 μl H2O and the precipitate (10 μl) was analysed by
denaturing gradient gel electrophoresis [DGGE; 8% polyacrylamide gel
(acrylamide-bisacrylamide, 37.5:1); denaturation gradient 25% – 55%
for separation of 16S rDNA amplicons or 20% – 50% for separation of
ITS amplicons, 100% denaturant containing 7 M urea and 40% (v/v)
formamide]. DGGE was run on DCode System (Bio-Rad) in × 0.5 TAE
(20 mM Tris, 10 mM acetate, 0.5 mM Na2 EDTA; pH 8.0) at 200 V at
60 °C for 3 h for bacteria or for 5 h.
2.5. Construction of clone libraries
The rest of the PCR products from the first amplifications were used
for the construction of the bacterial 16S rDNA and eukaryotic ITS clone
libraries. Briefly, the PCR products were purified by QIAquick PCR puri-
fication kit (Qiagen), ligated to pGEM-T Easy vector (Promega, Madison,
Wisconsin, USA), transformed to E.coli XLI-Blue, and spread to LB plates
with ampicillin (100 μg/ml), X-Gal (0.1 mM) and IPTG (0.2 mM). A
number of about 50 white colonies from each clone library were
checked by vector-specific PCR with primers SP6 (5′-ATT TAG GTG
ACA CTA TAG AAT AC-3′) and T7 (5′-TAA TAC GAC TCA CTA TAG GG-
3′). Positive clones of each library were analysed by DGGE in conditions
described above, with bacterial primers 341f-GC and 518, and ITS
primers ITS1f-GC and ITS2. Profiles of individual clones were compared
with each other and with the profile of the whole community. Clones
with different profiles were sequenced by primers SP6 and T7. The ob-
tained sequences were compared with those present in the GenBank
database using a BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
In addition, the sequences were deposited in the GenBank database
under the accession numbers KC797504–KC797582.
3. Results and discussion
Groups of microorganisms were quantified during the production of
May bryndza cheese, collecting culture-based data on lactobacilli,
lactococci, total mesophilic aerobic counts, coliforms, E. coli, staphylo-
cocci, coagulase-positive staphylococci, yeasts, fungi and Geotrichum
spp. from analyses at various stages of the production (ewes' milk,
curd produced from it and ripened for 0 – 10 days, bryndza produced
40 D. Pangallo et al. / International Journal of Food Microbiology 170 (2014) 38–43

from the curd) in three consecutive batches. Data for selected stages of
production are presented in Table 1 and time course of densities of se-
lected groups of microorganisms is illustrated in Fig. 1. These culture-
based data demonstrated the extent of variability but also an overall
trend of growth of the microbial population contributing to lactic acid
production and to ripening of the cheese. The great microbial variability
in milk and on day 0 should be connected to different conditions in pro-
cessing environment; this aspect and its influence on fermentation will
be studied when more data from different farms are available. Cultures
of lactobacilli grew to the final density of 108 CFU/g, lactococci grew to
the final density of 109 CFU/g and Geotrichum spp. grew to the final
density of 105 CFU/g in bryndza cheese. High counts of contaminating
and potentially pathogenic microflora were observed, which might be
important from the hygienic point of view if insufficient competition
takes place. Our data correspond with the fact that the cheese was
made from unpasteurized ewes' milk and the development of the
populations was led solely by competition (Valík, 2004).
In order to confirm the group identity of lactococci grown on M17
agar from ewes' curd samples (batch 1, batch 2), which was found
confusing in a previous study (Berta et al., 2009), selected colonies
were identified by rDNA amplification and sequencing, and found to
be Lactococcus lactis subsp. lactis (data not shown). Analogically, select-
ed colonies grown on MRS agar from ewes' curd or bryndza cheese
(batch 1, batch 2) were identified by rDNA amplification and sequenc-
ing, and found to be Lb. casei or Lb. plantarum (data not shown).
Diversity of prokaryotes and eukaryotes in selected stages of
bryndza cheese production was also studied by DGGE fingerprint
coupled to the construction of clone libraries for bacteria and eukaryotic
microorganisms. This kind of strategy permits the screening of the
various stages and the consequent selection of those stages which
were fully analysed by the culture-independent strategy. A common
approach to studying the microbial diversity in cheese environments
is based on the use of DGGE in combination with excision of the short
DNA band from the gel (Quigley et al., 2011). An alternative approach,
which uses semi-nested PCR coupled to the construction of clone librar-
ies, allows the inclusion of bigger 16S rDNA and ITS sequences, which fa-
cilitates a more reliable phylogenetic identification of microorganisms
than that obtained by sequence analysis of excised and directly se-
quenced DGGE bands. In addition, the obtained clones can be easily
screened by DGGE and those different can be sequenced (Michaelsen
et al., 2010).
The results produced by the culture-independent strategy are
presented in Tables 2 and 3. It is evident how the microflora diversity
decreased from batch 1 to batch 3. The presence of filamentous fungi
belonging to Cladosporium cladosporioides, Beauveria brongniartii and
several Penicillium species detected in different stages of the first and
the second batch perhaps represented mould spoilage due to secondary
contamination during cheese production. This aspect was already treat-
ed by other authors (Kure et al., 2001; Delavenne et al., 2011). Filamen-
tous fungi were detected also in the final product (bryndza cheese),
mainly in the first batch. To our knowledge, Penicillium species such as
Fig. 1. Time course of culture densities of lactobacilli (filled circles), lactococci (open
circles), Geotrichum spp. (filled triangles) and coliforms (open triangles) during ripening
of ewes' curd, Batch 1 (Day 0 – 10) and in bryndza cheese produced from it (the final
time in the graph).
P. aurantiogriseum, P. camemberti, P. freii, P. polonicum, and P. viridicatum,
were never detected in bryndza cheese or in other ewes' cheeses, but
the mycotoxin production of some of these species in food was demon-
strated (Frisvad et al., 2004). Another particularity of bryndza from the
first batch was the presence of Trichosporon lactis.
Microorganisms belonging to the species Galactomyces candidus
(formerly G. geotrichum) and Yarrowia lipolitica were detected in each
production stage and generally in each batch confirming that these
yeasts are typical for bryndza cheese, which is in accordance with the
results of previous investigations (Laurenčík et al., 2008; Chebeňová-
Turcovská et al., 2011). The species Gymnoascus reesii was characteristic
for batch 2 and was detected in the ewes' curd from day 0 to bryndza
cheese. To our knowledge, this fungus was never detected in the cheese
environment. Members of the genus Gymnoascus, which is characteris-
tic by keratinolytic properties, were associated with different animals
and also wool (Solé et al., 2002; Błyskal, 2009). Probably in our case,
they colonized the sheep and their presence in bryndza process was
only occasional.
The data obtained in this study provide evidence of the presence of a
rich eukaryotic microflora, which is still less investigated, compared to
the bacterial one. Our data contribute to the knowledge of fungal diver-
sity in ewes' cheese environment.
The bacterial microflora, similar to the eukaryotic one, exhibited the
highest diversity in the first two batches. Indeed, Gammaproteobacteria
belonging to the genera Acinetobacter and Pseudomonas were detected
largely in the milk sample of the first batch. The milk of the second
batch was characterized by other kinds of Gammaproteobacteria of
the genera Enterobacter, Kluyvera and Raoultella. The detection of
Gammaproteobacteria decreased drastically already in the ewes' curd
on day 0 of the first two batches and in the milk sample of batch 3.
Mannheimia glucosidica (11 clones) was the only Gammaproteobacteria
strain detected in bryndza cheese (second batch). Generally, members
of this genus are responsible of mastitis in small ruminants (Omaleki
et al., 2011) and the 16S rDNA sequences of our clones showed 100%

Table 1
Culture-based quantitative data on groups of microorganisms during the production of May bryndza cheese.

Group of microorganisms Density [CFU/ml] (milk) or [CFU/g] (rest) (Batch 1/Batch 2/Batch 3)

Milk Ewes' curd, Day 0 Ewes' curd, Day 5 Bryndza cheese

Lactobacilli 1.4 × 105/105/1.2 × 105 7.2 × 108/105/4.2 × 107 5.9 × 108/8.4 × 108/5.7 × 108 4.7 × 108/5.4 × 108/9.7 × 108
Lactococci 6.3 × 105/1.3 × 105/1.2 × 105 9.7 × 108/105/2.7 × 107 1.7 × 108/9.8 × 108/7.0 × 108 109/1.1 × 109/1.1 × 109
Total mesophilic aerobes 6.8 × 105/1.7 × 105/1.8 × 105 7.0 × 109/106/8.9 × 108 2.2 × 109/8.9 × 108/1.6 × 109 3.8 × 108/1011/9.0 × 1010
Coliforms 4.0 × 104/6.7 × 104/4.1 × 103 103/8.3 × 104/5.0 × 106 1.3 × 107/4.5 × 106/4.4 × 106 1.5 × 105/9.0 × 103/5.4 × 104
E. coli 5.9 × 102/4.5 × 101/2.3 × 102 103/4.5 × 102/1.8 × 104 9.8 × 104/105/4.5 × 104 103/5.0 × 102/1.3 × 103
Staphylococci 5.0 × 104/9.8 × 104/3.1 × 104 7.0 × 106/3.5 × 105/3.8 × 106 1.3 × 106/2.6 × 106/2.2 × 106 3.9 × 105/2.6 × 106/2.9 × 106
Coagulase-positive staphylococci 5.0 × 101/102/2.0 × 104 3.2 × 105/103/105 103/4.0 × 105/1.1 × 106 1.1 × 105/5.0 × 104/1.6 × 106
Yeasts 5.6 × 104/1.2 × 103/4.4 × 103 1.2 × 104/4.3 × 103/4.1 × 104 9.7 × 105/1.7 × 106/1.1 × 106 2.0 × 106/8.6 × 105/1.1 × 106
Fungi 101/2.9 × 104/3.8 × 102 2.7 × 101/2.1 × 105/3.3 × 102 3.1 × 105/103/8.2 × 104 9.6 × 104/102/2.0 × 104
Geotrichum spp. 4.2 × 102/101/1.8 × 101 6.6 × 103/3.3 × 102/1.3 × 102 4.0 × 105/1.1 × 106/5.2 × 105 1.7 × 105/4.6 × 105/1.3 × 105
 D. Pangallo et al. / International Journal of Food Microbiology 170 (2014) 38–43 41

Table 2
List of identified eukaryotic microorganisms.

Species identification on the basis of the highest ITS similarity score Number of clones (Batch 1/Batch 2/Batch 3)

Milk Ewes' curd, Day 0 Ewes' curd, Day 5 Bryndza cheese

Alternaria alternata JX045850 - 100% 0/0/0 0/0/0 0/0/0 0/9/0

“Ascomycete sp.” AY230245 - 99% 3/0/0 0/0/0 0/0/0 0/0/0
Aspergillus fumigatus JN850983 - 99% 0/0/0 0/0/0 0/0/0 1/0/0
Beauveria brongniartii JX110373 - 100%; JX110381 - 99% 0/1/0 30/0/1 8/0/0 15/1/0
Candida xylopsoci JF896574 - 99% 1/0/0 0/0/0 0/0/0 0/0/0
Candida inconspicua EU315757 - 99% 8/0/0 0/0/0 0/0/0 0/0/0
Cladosporium cladosporioides JQ936096 - 100%; DQ026006, JQ936197 - 99% 8/0/0 0/4/0 0/0/0 0/3/0
Debaryomyces hansenii HE681104 - 100% 0/0/0 0/3/0 0/3/0 0/0/0
Fomes fometarius JF927882 - 99% 0/0/0 9/0/0 0/0/0 2/0/0
Galactomyces candidus JN974290 - 100% 0/28/24 0/7/17 5/8/19 0/3/31
Galactomyces geotrichum AJ876893, JF262194 - 100% 8/12/14 0/3/15 6/8/19 1/0/2
Gymnoascus reesii HM991269 - 100% 0/0/0 0/3/0 0/4/0 0/4/0
Chaetomium globosum FJ426397, JN209920 - 99% 0/1/0 1/0/0 0/0/0 3/0/0
Kluyveromyces marxianus AB771424 - 100%; JQ425346 - 99% 18/0/1 0/0/2 0/0/0 0/2/0
Metarhizium anisopliae HM055426 - 99% 0/0/0 1/0/0 0/0/0 0/0/0
Penicillium aurantiogriseum JF311946 - 99% 0/0/0 2/0/0 1/0/0 4/0/0
Penicillium camemberti AF033474 - 100% 0/0/0 1/0/0 0/0/0 8/0/0
Penicillium freii JN942696 - 99% 0/0/0 0/0/0 5/0/0 2/0/0
Penicillium polonicum JN986766 - 100% 0/0/0 2/0/0 0/0/0 2/0/0
Penicillium viridicatum JN942697 - 100% 0/0/0 0/0/0 0/0/0 7/0/0
Pichia kudriavzevii JQ726607 - 100% 1/0/0 0/0/0 0/0/0 0/0/0
Sordaria alcina GQ996575 - 99% 0/0/0 1/0/0 0/0/0 0/0/0
Trichosporon lactis HE660076 - 99% 0/0/0 0/0/0 0/0/0 7/0/0
Yarrowia lipolytica JX171197, HM627146, AM279245 - 100% 0/10/3 0/1/4 2/0/4 0/2/7

Table 3
List of identified prokaryotic microorganisms.

Species identification on the basis of the highest 16S rRNA similarity score Number of clones (Batch 1/Batch 2/Batch 3)

Milk Ewes' curd, Day 0 Ewes' curd, Day 5 Bryndza cheese

Acinetobacter calcoaceticus JX164201 - 99% 0/0/0 0/0/1 0/0/0 0/0/0

Acinetobacter guillouiae JN092611 - 99% 2/0/0 0/0/0 0/0/0 0/0/0
Acinetobacter johnsonii JF915343 - 100%; JQ435689 - 99% 1/0/0 2/0/0 0/0/0 0/0/0
Acinetobacter sp. JX266367 - 99% 0/0/0 0/0/2 0/0/0 0/0/0
Citrobacter braakii HQ288930 - 100% 0/0/0 0/0/0 0/1/0 0/0/0
Clostridium bartlettii NR_027573 - 99% 0/0/1 0/0/0 0/0/0 0/0/0
Corynebacterium callunae NR_037036 - 99% 0/0/0 3/0/0 0/0/0 0/0/0
Corynebacterium maris FJ423600 - 99% 0/0/0 1/0/0 0/0/0 0/0/0
Enterobacter aerogenes JQ682638 - 99% 0/0/0 0/0/0 0/0/1 0/0/0
Enterobacter asburiae CP003026 - 100% 0/17/0 0/0/0 0/0/0 0/0/0
Enterobacter hormaechei JN645954 - 100% 0/0/0 0/0/2 0/2/0 0/0/0
Enterococcus faecium JX661714 - 99% 0/0/0 0/0/0 0/2/0 0/0/0
Enterococcus pallens NR_043794 - 100% 0/0/0 0/0/0 0/0/1 0/0/0
Escherichia coli AP012030 - 99% 0/0/1 0/0/0 0/0/0 0/0/0
Haemophilus haemolyticus JN227789 - 99% 0/0/0 0/1/0 0/0/0 0/0/0
Hafnia alvei JN014467 - 100% 0/0/0 1/0/0 0/0/0 0/0/0
Kluyvera cryocrescens JX294893 - 99% 0/3/0 0/0/0 0/0/0 0/0/0
Lactobacillus helveticus CP002429 - 100% 0/0/0 6/0/0 0/0/0 0/0/0
Lactococcus garvieae JX317635 - 100% 0/0/0 0/0/17 0/5/0 0/3/4
Lactococcus lactis subsp. cremoris JX402635 - 100% 0/7/18 0/0/21 0/29/38 0/26/32
Lactococcus lactis subsp. lactis JX119012, HE805077, HM638418 - 100%; HM218648, JQ795807 - 99% 7/0/0 23/2/0 46/3/8 33/0/0
“Leuconostoc garlicum” JQ805713 - 99% 0/0/0 2/0/0 0/0/0 0/0/0
Mannheimia glucosida AF053890 - 100% 0/0/0 0/3/0 0/0/0 0/11/0
Mannheimia haemolytica JQ975940 - 100% 0/0/0 0/1/0 0/3/0 0/0/0
Pseudomonas sp. JQ885953 - 100% 1/0/0 0/0/0 0/0/0 0/0/0
Pseudomonas fluorescens CP003041 - 100%; JX032808 - 99% 11/0/1 0/0/0 0/0/0 0/0/0
“Pseudomonas reactans” HM640280 - 100% 1/0/0 0/0/0 0/0/0 0/0/0
Raoultella ornithinolytica EU834264 - 100% 0/6/0 0/0/0 0/0/0 0/0/0
Raoultella terrigena HQ242728 - 100% 0/10/0 0/0/0 0/0/0 0/0/0
“Rothia arfidiae” DQ673322 - 99% 0/0/1 1/0/0 0/12/0 0/0/0
Staphylococcus aureus JN245970 - 100% 0/0/7 0/0/0 0/0/0 0/0/0
Staphylococcus epidermidis JN245969, JX534212 - 100%; JQ795860 - 99% 11/1/2 1/7/1 0/0/0 0/0/0
Staphylococcus felis NR_027215 - 99% 0/0/0 1/0/0 0/0/0 0/0/0
Staphylococcus pasteuri GQ901069 - 100% 0/0/0 0/2/0 0/0/0 0/0/0
Staphylococcus sciuri JX519590 - 100% 0/0/3 0/0/2 0/0/0 0/0/0
Staphylococcus xylosus JQ023729 - 99% 1/0/0 0/0/0 0/0/0 0/0/0
Streptococcus parauberis JQ945267 - 100% 0/0/0 1/0/0 0/0/0 2/0/0
Streptococcus thermophilus HE793101 - 99% 0/0/0 0/0/1 0/0/0 0/0/0
Variovorax paradoxus JX469400 - 99% 0/2/0 0/0/0 0/0/0 0/0/0
42 D. Pangallo et al. / International Journal of Food Microbiology 170 (2014) 38–43
similarity with Mannheimia strains of animal origin. The presence
of Gammaproteobacteria in milk and dairy products was already evi-
denced by other authors (Chaves-López et al., 2006; Hantsis-Zacharov
and Halpern, 2007); their extracellular enzymes, mainly proteases
and lipases, contribute to the spoilage of these foodstuffs and the forma-
tion of biogenic amines was studied in vitro (in a culture medium)
by Coton et al. (2012). The 16S rDNA sequences of detected
Gammaproteobacteria were similar to strains related to other environ-
ments than dairy samples and perhaps their high occurrence, mainly
on the early stages of cheese maturation of the first two batches, is relat-
ed to a possible contamination in the beginning of the production
season, due to the reusing of the equipment after a break in the produc-
tion during the winter season, or to insufficient sanitization of the whole
environment of the production facility.
The group of Actinobacteria represented by species Corynebacterium
callunae, C. maris and Rothia arfidiae presented 16S rDNA similarity with
strains isolated from other kinds of environments. Rothia arfidiae and
Corynebacterium maris are new recently identified species (Ben-Dov
et al., 2009; Ko et al., 2009).
The most abundant group was represented by the Firmicutes phy-
lum, which included members of the orders Bacillales (different kinds
of Staphylococcus spp.), Clostridiales (only one clone of Clostridium
bartlettii detected in milk of the third batch) and Lactobacillales
(Streptococcus thermophilus, Lactobacillus helveticus, “Leuconostoc
garlicum”, Enterococci and Lactococci). Our culture-independent
strategy evidenced the presence of Staphylococcus species mainly
in milk and in the ewes' curd on day 0; they were distributed in all
three batches. Lactobacillales were present during entire production,
from milk to bryndza cheese, and the dominant species was
Lactococcus lactis. Enterococcus faecium and E. pallens were detected
only in the ewes' curd on fifth day in batch 2 and batch 3. In ewes'
curd on day 0 in batch 1, Lactobacillus helveticus (6 clones),
“Leuconostoc garlicum” (2 clones) and Streptococcus parauberis (one
clone) were detected. Streptococcus thermophilus was exclusively de-
tected in ewes' curd on day 0 of the third batch. Except of the relative
new species E. pallens (Tyrrell et al., 2002), which was detected here
for the first time from a dairy environment, the other species had al-
ready been identified during ewes' cheese manufacturing (Casalta
et al., 2009; Kafili et al., 2009; Martín-Platero et al., 2009; Randazzo
et al., 2010).
Surprising was the low level of detection of lactobacilli strains, in
particular in the final product, bryndza cheese. Indeed, this was in
contrast with our previous findings, when Lactobacillus brevis and
Lb. delbrueckii were detected using a different culture-independent pro-
cedure (Chebeňová-Turcovská et al., 2011). In addition, DNA analysis-
based data disagree with data obtained in previous studies of our
group (Berta et al., 2009) and mainly with the microbial culture data
of this investigation. The impression is that PCR in the present study
was able to efficiently amplify DNA from the dominant species, namely
Lactococcus lactis (its high concentration in bryndza cheese was con-
firmed also by culture analysis), but PCR failed to amplify DNA from
lactobacilli (which reached a concentration of 108 CFU/g in bryndza
cheese, as determined by culture). On the other hand, PCR in some
cases allowed co-amplification of different lactic acid bacteria, such as
in ewes' curd on day 0 (Table 3), and detected a broad bacterial diversity
in the first stages of bryndza production. The strains detected in bryndza
cheese during this investigation, belonged to species Lc. lactis,
Lc. garvieae, Mannheimia glucosidica and Streptococcus parauberis.
The results obtained by culture-dependent and independent analyses
are not in a full concordance, but this is a common situation when
these two approaches are combined in investigation of cheese microflo-
ra (Rantsiou et al., 2008; Alegría et al., 2012). These data extend the
knowledge about the bacterial microflora of bryndza cheese, but can
be applied also to other ewes' cheeses from Central Europe.
The present study demonstrated the efficacy of exploitation of
the complementarity of culture-dependent and culture-independent
analyses. The high concentrations of different members of eukaryotic
and bacterial microflora were previously shown to concur, to reduce
and inhibit spoilage microorganisms in the final product, bryndza
cheese (Valík, 2004). The DNA analysis based on the combination of
DGGE and clone libraries construction facilitated the development of
a new useful strategy for investigation of the microbial diversity of
various food matrices.
The diversity and dynamics of microflora of ewes' cheese were ex-
tensively studied in Mediterranean countries (Abriouel et al., 2008;
Rantsiou et al., 2008; Casalta et al., 2009; Comunian et al., 2010, Fuka
et al., 2010; Randazzo et al., 2010; Feutry et al., 2012). The aspects of
ewes' cheeses from Central and Eastern Europe are less known, except
for some case (Alegría et al., 2012). The present study dealt, probably
for the first time, in parallel with microbial dynamics and diversity of
this type of ewes' cheese. May bryndza cheese, which was studied
here, is a variant of Slovenská bryndza (Slovakian bryndza cheese, a
PGI-status cheese) that has several similar properties with other ewes'
cheeses produced in Central and Eastern European countries, such as
Romania (brânză de burduf), Austrian (Liptauer-style cheese or
Brimsen) or Poland (Bryndza Podhalańska is a PDO-status ewes'
cheese). Therefore, our study provided data that can be useful also for
microbiological evaluation of various bryndza-like cheeses produced
in Central and Eastern Europe.
Acknowledgments
This work was supported by the project of the Slovak Research and
Development Agency APVV 0590-10. Authors thank Ing. Jadža Lejková
for technical assistance.
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