International Journal of Food Microbiology 142 (2010) 19–24

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International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r.com/ locate / i j foodmicro

Bacterial communities associated with the production of artisanal Istrian cheese

Mirna Mrkonjić Fuka a,⁎, Marion Engel b, Andrea Skelin a, Sulejman Redžepović a, Michael Schloter b

a University of Zagreb Faculty of Agriculture, Department of Microbiology, Svetosimunska 25, 10000 Zagreb, Croatiab Helmholtz Zentrum München, Department for Terrestrial Ecogenetics, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany

⁎ Corresponding author. Tel.: +385 12394034; fax: +E-mail address: mirna_fuka@yahoo.de (M.M. Fuka).

0168-1605/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2010.05.008

a b s t r a c t

a r t i c l e i n f o

Article history:Received 28 October 2009Received in revised form 5 May 2010Accepted 12 May 2010

Keywords:Istrian cheese16S rRNA geneBacterial diversityLactococcus lactis subsp. lactis

In this work we report on the main bacterial microflora typical for fermentation and ripening of traditionalIstrian cheese. Samples from milk as well as Istrian cheese were analyzed during the ripening process byusing culture independent molecular fingerprinting methods as well as culture based approaches. Our resultsindicate changes in bacterial diversity pattern during the ripening process. Differences in bacterial diversityat the same ripening stage among different farms investigated were comparably low. Sequence analysis ofthe most prominent bands of denaturing gradient gel electrophoresis fingerprints revealed dominance ofLactococcus lactis subs. lactis in all samples and a strong presence of Enterococcus spp. which was alsoconfirmed by plate count analysis.

385 12393881.

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Traditional Istrian cheese is one of the most important Croatianproductswith a longhistory ofmanufacturing. It ismadeby farmers on asmall scale across the entire Istria region, located in west-south Croatia.Production of Istrian cheese includes usage of raw ewes' milk that isgently heated up to 35 °C prior the addition of natural rennet for milkcoagulation. The milk is not pasteurized and no starter cultures areapplied. Themilk coagulumis left for up to 1 h at roomtemperature (21–24 °C) until curd formation, which is cut into pieces and cooked at 42 °Cfor around 5 min before moulding. After 2 h of whey drainage with selfpressing, the surface of the fresh cheese is covered in coarse salt andheldat roomtemperature (21–24 °C) for 2 days. Thedry cheese is left to ripenfor 90–120 days at a constant temperature (14–19 °C) and humidity(65–98%). The resulting cheese has hard texture and strong flavour.

The use of specific starter cultures ensures sensorial propertiessimilar to thatof theartisanal cheese, greater safety anduniformityof thefinal products (Katechaki et al., 2008; Ross et al., 2000). Consequently ifno starter cultures are applied, the cheese quality is highly variable.Therefore, in order to preserve the specificity of Istrian cheese and tominimize variations in quality, it is necessary to investigate thecomposition of bacterial communities during the production process.However nothing is known so far about the bacterial populationsinvolved in the different stages of Istrian cheese ripening.

Therefore itwas the aimof this study to investigate the dynamic of theautochthonous bacterial communities present in Istrian cheese and toidentify the dominant species during the ripening process. To achieve this

aimmilk and cheese samples from four different farms located indifferentareas of Istria were collected. The raw milk, fresh cheese and cheesesamples after 30, 60, 90 and 120 days of ripening were analyzed usingculture independent molecular fingerprinting methods and plate countapproach.

2. Materials and methods

2.1. Sampling

Milk and cheese samples were collected from four farms (F1, F2, F3and F4) located at different areas in the Istrian region of Croatia duringthe year 2007. Due to the earlier beginning of the production of Istriancheese at F1 and F2 farms, the collection of samples started at F1 andF2 farms in May 2007 and at F3 and F4 farms in June 2007. All farmsproviding samples were part of a consortium for the preservation ofthe certified typical product “Istrian cheese PDO”. Milk (M) and freshcheeses (Ch0) were sampled at day zero; cheese samples werecollected after 30 (Ch30), 60 (Ch60), 90 (Ch90) and 120 (Ch120) days.Milk and mature cheese samples were analyzed in duplicates. Allsamples were transported to the laboratory at 4 °C and frozen at−80 °C for culture independent PCR-denaturing gradient gel electro-phoresis analysis or directly used for enumeration of bacterialpopulations. Total water content of the cheese samples rangedbetween 20 and 41%, pH between 4.8 and 5.2, salt content between2.3 and 4.6% and fat content between 38 and 53%.

2.2. Isolation of different bacterial groups

Enterococci, lactococci, mesophilic and thermophilic lactobacilli,and Enterobacteriaceae were analyzed from raw milk (M), fresh

20 M.M. Fuka et al. / International Journal of Food Microbiology 142 (2010) 19–24

cheese (Ch0) and ripened cheese (Ch120). The samples werehomogenized with a Stomacher (BagMixer® 400, Interscience, StNom, France) for 3 min and serially diluted in sterile physiologicalsolution (0.9% NaCl). By using the pour plate technique, aliquots of thesamples were inoculated on selective agar plates and incubated at theappropriate temperature (Table 1) for 48 h. Enterococci wereenumerated on citrate azide tween carbonate agar (CATC, Merck,Darmstadt, Germany) as described by Canzek Majhenic et al. (2005),mesophilic and thermophilic lactobacilli on MRS agar (Oxoid,Basingstoke, UK), lactococci were enumerated on M17 agar (Oxoid,Basingstoke, UK) and Enterobacteria were analyzed on violet red bileagar (VRBA, Oxoid, Basingstoke, UK) as described by Randazzo et al.(2006).

2.3. DNA extraction from milk and cheese samples

10 g of each cheese sample was taken from the cheese interior andaseptically blended with 90 ml of a sterile Ringer's solution in aBagMixer® 400 (Interscience, St Nom, France) for 3 min. 10 ml of eachcheese homogenate as well as 10 ml of each milk sample werecentrifuged at 3500×g for 10 min. After discarding the supernatant,the pellet was resuspended in prelysis buffer containing 500 μl of TEbuffer (10 mM Tris, 1 mM EDTA, pH 8.0) and 20 mg/ml of Lysosim,and incubated at 37 °C for 2 h. Total DNA was extracted and purifiedusing the Maxwell Tissue DNA Purification Kit (Promega, Wisconsin,USA) as recommended by the manufacturer.

2.4. Amplification of 16S rRNA genes

A part of the 16S rRNA genes was amplified using a primer setdesigned by Nubel et al. (1996) F968 primer (5′ - AACGCGAA-GAACCTTAC - 3′) and R1401 primer (5′ - CGGTGT GTACAAGACCC - 3′)containing a GC clamp in the forward primer. The PCR mixture andamplification programwere previously described (Nubel et al., 1996).PCR products were visualized on agarose gel after staining withethidium bromide and purified by Qiaqick PCR Purification Kit(Qiagen, Hilden, Germany) as recommended by the manufacturer.

2.5. Denaturating gradient gel electrophoresis (DGGE)

DGGE was performed with the D-Code system (Bio-Rad Labora-tories, Munich, Germany) as described byMuyzer et al. (1993). The 6%(w/v) polyacrylamide gels (ratio of acrylamide and bisacrylamide37:1) with a denaturant gradient from 40% to 60% were used foranalyzing the amplicons. The gels were run at 60 °C and 60 V for 16 h.DNA bands were stained as described by Cocolin et al. (2001). The

Table 1Bacterial counts (log cfu/g cheese or mlmilk) of the different bacterial groups inmilk (M), fre

Farm Sample Bacterial counts (log cfu/g cheese or ml milk)

VRBA, A, 37 °C(Enterobacteriaceae)

CATC, A, 37 °C(enterococci)

M(

F1 M 2.63 4.82 5Ch0 2.48 6.11 6Ch120 nd 7.40 7

F2 M 4.60 3.86 6Ch0 3.18 5.43 5Ch120 nd 6.45 6

F3 M 3.62 3.49 5Ch0 5.34 4.64 5Ch120 nd 7.04 6

F4 M nd 4.73 5Ch0 3.41 6.90 6Ch120 nd 6.18 7

nd=not detectable (under detection limit).A=incubation under aerobic conditions.An=incubation under anaerobic conditions.

DGGE profiles obtained were analyzed by GelCompar II Software(Applied Maths, Kortrijk, Belgium) as described by Giannino et al.(2009). Selected DGGE bands were extracted from the gels asdescribed by Cocolin et al. (2007) and reamplified by using theabove applying primers without GC clamp. Reamplified PCR productswere purified by MinElute PCR Purification Kit (Qiagen, Hilden,Germany) as recommended by the manufacturer and quantified by aNanodrop spectrophotometer (NanoDrop Technologies, Wilmington,USA).

2.6. Sequence analysis of 16S rRNA genes

Reamplified PCR products were sequenced on an ABI PRISM® 3730DNA Sequencer (Applied Biosystems, Foster City, USA) using a BigDyeTerminator v3.1 Cycle Sequencing Kit (Applied Biosystems, FosterCity, USA). Reaction conditions and thermal cycle program were aspreviously described by Mrkonjic Fuka et al. (2009). Unincorporateddye terminators were removed by ethanol precipitation.

Reamplified PCR products that gave mixed signals after sequenc-ing were cloned into pCR®2.1 vector of TA Cloning Kit (Invitrogen,Karlsruhe, Germany) following the manufacturer's instructions(Marzorati et al., 2008). Isolation of plasmids was done using theNucleoSpin Plasmid Mini Kit (Clontech Laboratories, Montain View,USA). Purified plasmids were tested for inserts by EcoRI digestion(MBI Fermentas, Heidelberg, Germany) at 37 °C for 1 h and sequencedlike described above.

The obtained sequences were aligned to sequences deposited inGenBank databases using NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) as described by Giannino et al. (2009). The presence ofchimeric sequences was checked using the chimera detectionprogram of the Ribosomal Database Project (http://rdp8.cme.msu.edu/cgis/chimera.cgi?su=SSU).

2.7. Nucleotide sequence accession numbers

The sequences determined in this study have been submitted toGenBank under accession numbers FJ770412–FJ770456.

3. Results

3.1. Bacterial communities

The viable bacterial counts of Istrian milk and cheese samplesestimated on different selective media are shown in Table 1. Thepopulation of lactococci and enterococci ranged from 5.30 to 7.94 and3.49 to 7.40 log cfu/g, respectively. These results reflect the strong

sh (Ch0) and ripened Istrian cheese (Ch120) samples from four different farms (F1–F4).

17, A, 30 °Clactococci)

MRS, An, 42 °C(thermophilic lactobacilli)

MRS, An, 30 °C(mesophilic lactobacilli)

.30 nd nd

.41 3.04 5.99

.94 5.93 4.75

.30 nd nd

.80 4.62 5.38

.72 5.94 5.04

.62 nd nd

.38 nd 4.98

.30 nd 5.34

.82 nd 3.22

.89 4.38 4.78

.40 5.00 5.01

21M.M. Fuka et al. / International Journal of Food Microbiology 142 (2010) 19–24

dominance of these groups in Istrian cheese samples. The bacterialcounts of mesophilic and thermophilic lactobacilli were either low oreven not detectable in some samples. The abundance of mesophiliclactobacilli was slightly higher (up to 5.99 log cfu/g) compared to thethermophilic microflora (0.00 to 5.94 log cfu/g). The number ofEnterobacteria varied between 0.00 and 5.34 log cfu/g. However noEnterobacteriaceae were detected in ripened cheese samples.

3.2. PCR-DGGE analysis of 16S rRNA gene fragments amplified from DNAextracted from cheese and milk samples

Total microbial DNA extracted from milk and cheese samplesranging from raw milk to 120 day ripened cheeses was analyzed byPCR-DGGE. Figs. 1a and 2a show DGGE profiles of the PCR productsbelonging to V6–8 regions of the bacterial 16S rRNA gene. Noticeable

Fig. 1. a) DGGE fingerprinting profiles of amplified 16S rRNA genes obtained from extracted(Ch30, Ch60, Ch90 and Ch120) collected from Farm 1 (F1) and Farm 2 (F2). b) Cluster analysisraw cheese (Ch0) and cheese after 30, 60, 90 and 120 days (Ch30, Ch60, Ch90 and Ch120) frospp. (band d) are present as double bands under the used DGGE conditions indicating that

bacterial diversity was found among the 24 samples analyzed. Allinvestigated samples displayed a complex profile with lowest averagenumber of bands from F2 cheeses and highest from F4 cheeses.Although, DGGE profiles of milk and cheese samples were differentand typical for each farm, similar patterns were also observed.

In general higher numbers of bands were found in fresh cheesesamples and in raw milk compared to samples obtained from cheeseduring the ripening. Whereas fingerprint patterns were highlydynamic during the initial ripening phase, after 60 days of ripeningoverall bacterial community composition was very stable. Nodifferences in the fingerprints were obtained when replicatesoriginated from milk and mature cheeses were analyzed (data notshown).

The comparison of DGGE profiles based on cluster analysis andDice correlation showed four distinct groups from F1 and F2 farms

DNA frommilk samples (M), raw cheese (Ch0) and cheese after 30, 60, 90 and 120 daysaccording to Dice coefficient showing the degree of similarity amongmilk samples (M),m Farm 1 (F1) and Farm 2 (F2). Lactococcus lactis subsp. lactis (band g) and Enterococcusthese bacteria harbor at least two different types of operons (operon heterogeneity).

Fig. 2. a) DGGE fingerprinting profiles of amplified 16S rRNA genes obtained from extracted DNA from milk (M), raw cheese (Ch0) and cheese after 30, 60, 90 and 120 days (Ch30,Ch60, Ch90 and Ch120) collected from Farm 3 (F3) and Farm 4 (F4). b) Cluster analysis according to Dice coefficient showing the degree of similarity among raw milk samples (M),raw cheese (Ch0) and cheese after 30, 60, 90 and 120 days (Ch30, Ch60, Ch90 and Ch120) from Farm 3 (F3) and Farm 4 (F4). Lactococcus lactis subsp. lactis (band g) is present asdouble band under the used DGGE conditions indicating that these bacteria harbor at least two different types of operons (operon heterogeneity).

22 M.M. Fuka et al. / International Journal of Food Microbiology 142 (2010) 19–24

(Fig. 1b); the first one included cheese samples from F1 farm and thesecond main cluster comprised the cheese samples from F2 farm.According to the diversity observed among samples from F1 and F2farms, the first group shared 53% of similarity and the second group84%. The third and fourth groups were monophyletic representingboth raw milk samples. The dendrograms for the F3 and F4 cheeses(Fig. 2b) showed more homogenous patterns with two main groups.One group contained milk and raw cheese samples and the secondgroup included only cheese samples from 30 to 120 days. Thesimilarity within both groups was 68%.

3.3. Phylogenetic identification of selected bands from DGGE fingerprints

After excision from DGGE gels, bands a to u were reamplified withprimers F978 and R1401 and sequenced. Bands b, f, k, n, o, p, r, s and uprovidedmixed signals andwere therefore sequenced after subcloning.

The results obtained are shown in Table 2. Although considerabledifferences were noticed among different farms or ripening stagessimilar patterns and recurrent bands were also observed. The majorbands identified in milk and cheese samples were phylogeneticallysimilar to Lactococcus lactis subs. lactis (Table 2; band g). They werepresent in highest intensities and frequencies throughout the investi-gated milk and cheese samples at all four farms, indicating the mainrole of this bacterial group before and during the fermentation process.Other dominant bands were identified as phylogenetically similar toEnterococcus spp. (Table 2; band d) and were typical for milk andcheese samples from F1. A more detailed analysis of these phylotypesby sequence analysis was not possible. Both L. lactis subs. lactis as wellas Enterococcus spp. had obviously multiple ribosomal operons withdifferent sequences, resulting in more than one band in thecorresponding fingerprints. Some other frequent bands of moderateintensity were identified as nonfermentative Acinetobacter spp.

Table 2Phylogenetic identification of excised bands from DGGE gels of 16S rRNA geneamplicons of DNA obtained from different cheese samples.

Samples Bands1 Identification Acc. no.2 Maxidentity %

F1 M, F4 Ch0 b Enterobactercancerogenus

FJ770434 99

F1, F3, F4 b, c, p, r, s, t,u

Acinetobacter sp. FJ770412a 99

F1 M, F4 Ch0 b Staphylococcusequorum

FJ770435 99

F1 M, F4 Ch0 b Klebsiella terrigena FJ770436 99F1 d Enterococcus spp.⁎ FJ770414 99F1 e Enterococcus sp. FJ770415 95F1 Ch0 f Staphylococcus sciuri FJ770432 100F1 Ch0 f Enterococcus durans FJ770416 100F1 Ch0 f Macrococcus

caseolyticusFJ770438 97

F1 Ch30–120,F2 Ch0–120, F3, F4

g Lactococcus lactissubsp. lactis

FJ770417 99

F2 M h Pseudomonas fragi FJ770418 99F3 M, F4 M, F3 Ch0,F4 Ch0

i, k Acinetobactercalcoalceticus

FJ770419b 94

F3 Ch0 j Uncultured bacteriumclone

FJ770420 99

F3, F4 k, s Pantoea agglomerans FJ770439c 99F3 Ch30, F4 Ch30 l Lactobacillus curvatus FJ770421 99F3 Ch0, Ch30, F4 M,Ch0, Ch90, Ch120

m, n, t, o Lactococcus lactis FJ770422d 99

F3 Ch0 o Staphylococcusepidermis

FJ770443 100

F3 Ch0, F1 M o, a Staphylococcus sp. FJ770441e 95F3 Ch0, F4 M n Staphylococcus

gallinarumFJ770424 93

F4 M, F3 Ch0 p Leptotrichia-like orakclone

FJ770446 93

F4 M, Ch0, F4Ch90, Ch120

p, u Staphylococcussaprophyticus

FJ770447f 100

F3 Ch0, F4 M, Ch0 r Enterobacter sp. FJ770448 93F3 Ch0, F4 M, Ch0 r Klebsiella

pneumoniaeFJ770449 99

F3 Ch0 j Streptococcusparauberis

FJ770429 99

F3, F4 s Enterobacterhormaechei

FJ770450 99

F4 Ch90, Ch120 u Enterococcus spp.⁎⁎ FJ770431 99

Accession number of sequences with equally or lower percent of max identity asdisplayed: aFJ770427, FJ770428, FJ770451, FJ770454, FJ770456, bFJ770440, cFJ770452,FJ770453, dFJ770423, FJ770430, FJ770425, FJ770426, eFJ770433, and fFJ770455.

1 Bands identification according to Figs. 1a and 2a.2 Abbreviations: Acc. no., accession number in GenBank.⁎ E. faecium or E. faecalis.⁎⁎ E. italicus, E. sulfureus or E. saccharominimus.

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(Table 2; bands b, c, i, k, p, r, s, t and u) or as Enterobacteriaceae (e.g.Enterobacter cancerogenus, Klebsiella terrigena, Klebsiella pneumoniae,Pantoea agglomerans and Enterobacter hormaechei) (Table 2; bands b, r, kand s) and Staphylococcus spp. (e.g. S. equorum, S. sciuri, S. gallinarum andS. saprophyticus) (Table 2; bands a, b, f, n, o, p and u). They wereespecially frequent in raw milk and fresh cheese and were rapidlyeliminated during the cheese ripening. The exceptions are speciesidentified as Acinetobacter spp., P. agglomerans and E. hormaechei aftercloning and sequencing the band s (Table 2). Only phylotypes similar toS. saprophyticus were found at F4 farm in 3 and 4 month old cheeses(Table 2; band u). Other bacterial phylotypes, belonging to differentgenera of LAB e.g. Lactobacillus curvatus, Streptococcus parauberis andEnterococcus spp. as well as non LAB as Pseudomonas fragi andMacrococcus caseolyticus (Table 2; bands e, f, h, j, l and u),were identifiedas minor or sporadically present species in milk or cheese samples.

4. Discussion

The bacterial community of Istrian cheese samples was complex.Most of the bacterial species were detected at the beginning of

fermentation (fresh cheese) and in rawmilk. Such microbiota includedmembers of thermo tolerant LAB as Enterococcus spp. and mesophilicLAB as non motile, obligate homofermentative species belonging to L.lactis subsp. lactis. They persist throughout the cheese maturation asindicated by molecular analysis and plate counting. The presence ofenterococci and their dominance at F1 farm have been often noticed forother types of Mediterranean cheeses, indicating their positive role inthe development of typical cheese aroma and flavour (Poznanski et al.,2004; Ogier and Serror, 2008; Foulquié Moreno et al., 2006; Abriouel etal., 2008; Marino et al., 2003). The occurrence of L. lactis subsp. lactis aspredominant species before and during the fermentation of Istriancheese has been reported for a number of artisanal dairy products (El-Baradei et al., 2007, 2008; Randazzo et al., 2006). The proteinases oflactococci, generally referred to as “wild lactococci”, are known to beinvolved in the first step of casein breakdown and to have the majorimpact on aroma formation through the production of organic acidssuch as lactic and acetic acids (Axelsson, 2004), thus playing not only apassive (enzyme release by autolysis) but also an active role duringcheese ripening. The large diversity of other subdominant phylotypesfound inmilk and fresh cheese like Acinetobacter spp., Enterobacter spp.,Pantoea spp., Staphylococcus spp., Klebsiella spp. and Pseudomonas spp.were presumably connected with poor hygienic conditions during thecheese production. The higher number of food borne pathogens in rawmilk is mainly reported in countries with poorly controlled hygienicproduction (de Buyser et al., 2001; Guerra et al., 2001; Samaržija et al.,2003), conditions typical for Istrian cheesemanufacturing.However, thebacteriological quality of the fermented products obtained from suchrawmilk is mostly high as a result of rapid acidification and/or negativemicrobial interaction during the ripening (Donnelly, 2004; Nero et al.,2008; Samaržija et al., 2007). However, their extracellular enzymesmaypersist and may have important influences on the Istrian cheeseripening process, as suggested for other traditional cheeses (Hantsis-Zacharov and Halpern, 2007; Giannino et al., 2009). Although in lowfrequency and low intensity some of the spoilage microorganismshowever remained during the whole ripening period at 3 of 4 farmsinvestigated. These organisms belonging to Acinetobacter spp., P.agglomerans and E. hormaechei may have a secondary activity in theripeningprocessof Istriancheese. Someauthors suggested that differentspecies of Acinetobacter and Enterobacteriaceae contributed to the tasteflavour of traditional cheeses due to the citrate catabolism and lipolytic(Addis et al., 2001) or proteolytic activity (Chaves-López et al., 2006;Zago et al., 2007). Although Enterobacteriaceae have been often isolatedas subdominant orminor species indifferent typesof traditional cheeses(Chaves-López et al., 2006; Martín-Platero et al., 2009; El-Baradei et al.,2007), this phylotype could not be detected in mature Istrian cheese(only in milk and fresh cheese) when using plate count technique. Assuch, detection of the corresponding sequences in our study could beattributed to dead cells as postulated by El-Baradei et al. (2008) orEnterobacteriaceae from our samples belong to the group of microbesthat cannot be easily cultured. Most concern in food production isrelated to the genus Staphylococcus, especially S. aureus due to thepathogenic potential of some strains. Our results indicated the sporadicpresence of Staphylococcus species in milk and fresh cheesewithout theoccurrence of S. aureus. However, the sequences related to coagulasenegative S. saprophyticuswere alsodetected inmature cheese at F4 farm.It is not clear whether this contamination originated frommilk or it is aresult of cheese manipulation by cheese sellers during the latter stagesof ripening. In some cases S. saprophyticus has been recognized as acausative agent of urinary infection (Hovelius and Mardh, 1984). Theoccurrence of coagulase negative staphylococci is not rare in traditionalcheese production (Addis et al., 2001; Giannino et al., 2009; El-Baradeiet al., 2007, 2008; Resch et al., 2008) and it is frequently recognized asbeneficial through the lysostaphin (Faria et al., 2009; Robinson et al.,1979; Recsei et al., 1987) or protease and lipase production (Addis et al.,2001), such indicating their potential impact on cheese flavour andtexture.

24 M.M. Fuka et al. / International Journal of Food Microbiology 142 (2010) 19–24

The relative highbacterial biodiversity found in Istrian cheesemaybeexplained by its heterogeneous production among different farmfacilities. Especially variable are salt concentration, ripening temperatureand humidity or hygienic standards (Magdić et al., unpublished results).El-Baradei et al. (2008) also interpreted noticeable heterogeneity oftraditional Egyptian Domiati cheese by its highly variable productionconditions. The variability among samples may also be the result of thedifferences in geographical areas of pastures as postulated by Gianninoet al. (2009) and Ercolini et al. (2008). This may influence thecomposition of the adventitious microflora characterizing the farmslocated in different areas. Although Istrian cheese is prepared from rawewe's milk without the addition of starter cultures the dominantbacterial population identified in our study by culture independentmolecular fingerprinting methods and culture based approach was thatof LAB. As L. lactis subsp. lactis and Enterococcus spp. were predominantspecies before and during the fermentation process, their isolation andcharacterization will be valuable for further use as starter cultures inIstrian cheese production.

However it has to be taken into account that other microorgan-isms, mainly yeasts may have an important impact on cheese qualityand can make a significant contribution to the maturation process(Addis et al., 2001; Ferreira and Viljoen, 2003; Fleet, 1990). The yeastspecies are often included as a part of the starter cultures for theartisanal cheese production, enhancing flavour development duringthe maturation (Ferreira and Viljoen, 2003; Narhvus and Gadaga,2003). As such, further work is still needed to characterize theindigenous yeast population and to elucidate the interaction that maybe taken place between dominant LAB and yeasts in Istrian cheese.

Acknowledgments

This study was funded by the German Academic Exchange Service(DAAD) and theMinistry of Science, Education and Sport of theRepublicof Croatia (MZOŠ) grant 178-1782128-2123. The authors would like tothank Dr. Metka Suhadolc from the Biotechnical Faculty University ofLjubljana for her altruistic help regarding the GelCompare Software.

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