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International Journal of Food Micro

Characterization of microflora in homemade semi-hard white Zlatar cheese

Amarela Terzic-Vidojevic a,⁎, Maja Vukasinovic a,b, Katarina Veljovic a,Mihailo Ostojic c, Ljubisa Topisirovic a

a Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, P.O.Box 23, 11010 Belgrade, Serbia and Montenegrob Faculty of Technology and Metallurgy, Karnegieva 4, 11000 Belgrade, Serbia and Montenegro

c Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11080 Zemun, Serbia and Montenegro

Received 11 November 2005; received in revised form 12 September 2006; accepted 26 October 2006

Abstract

The Zlatar cheese belongs to the group of traditionally homemade cheeses, which are produced from nonpasteurized cow's milk, withoutadding of any bacterial starter culture. Changes were followed in lactic acid bacteria population and chemical composition during the ripeningperiod of cheese up to 60 days. Results showed that the percentage of lactic acid cocci was higher in raw milk and one day old cheese and theirpercentage was gradually decreasing, whereas the number of lactobacilli was increasing. After 30 days of cheese ripening the number of cocciincreased again, reaching the number of lactobacilli. The results of API 50 CH system and rep-PCR analysis showed that Lactobacillus paracaseisubsp. paracasei, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcuus faecium and Enterococcus faecalis were the main groupspresent during the ripening of Zlatar cheese. Results revealed that in older cheeses (45 and 60 days old) enterococci were the main group present.It was also demonstrated that 57 isolates showed antimicrobial activity. The number of bacteria showing antimicrobial activity slowly decreasedduring the ripening period and in samples of 60 days old cheese producers of antimicrobial activities were not detected.© 2006 Elsevier B.V. All rights reserved.

Keywords: Homemade cheese; Non-starter lactic acid bacteria (NSLAB); API 50 CH test; rep-PCR; 16S rRNA; Antimicrobial activity

1. Introduction

Large quantities of different cheeses have been producedtraditionally for centuries in Serbia from cow's, sheep's andgoat's milk. One of them is a white semi-hard cheese which isproduced on the highlands of the mountain Zlatar, and given thename Zlatar cheese. This artisan cheese is manufactured inhouseholds from raw cow's milk without the addition of anyknown starter culture.

Complexity and varied sensory properties of traditionalcheeses manufactured from raw milk depend on their microbialcommunity (Cogan et al., 1997). Non-starter lactic acid bacteria(NSLAB) constitute complex microbial associations that arecharacterized by the occurrence of various species and manybiotypes as a result of a number of selective conditions persistingduring the manufacturing process and different ecological niches(McSweeney et al., 1993). The NSLAB community is composed

⁎ Corresponding author. Tel.: +381 11 3975960; fax: +381 11 3975808.E-mail address: lab6@eunet.yu (A. Terzic-Vidojevic).

0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.ijfoodmicro.2006.10.038

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mostly ofmesophilic lactobacilli such asLactobacillus paracasei,Lactobacillus plantarum, and Lactobacillus curvatus (Mannuet al., 2000; Beresford et al., 2001). Nevertheless, pediococci,micrococci and leuconostoc could also be present (Fitzsimonset al., 1999; Manolopoulou et al., 2003; Callon et al., 2004). TheNSLAB dominate the microflora of long-ripened cheese for mostof its ripening period and they certainly have the potential to affectand contribute to cheese maturation (Fox et al., 1998).

Phenotypic, biochemical and physiological tests have nor-mally been used to identify taxonomically the microbial flora incheese. The development of PCR-based molecular techniquesfor the identification of bacterial species offers new perspectivesin microbial taxonomic studies (Mannu et al., 2002; Berthier andEhrlich, 1998; Ward and Timmins, 1999; Drake et al., 1996).

Mountain Zlatar represents a specific ecological region inWestern Balkans with high plant biodiversity. Such specificdiversity must have an impact on the formation of the specificmicroflora found in milk. We have selected an isolatedhousehold in which the traditional cheese production has along history. To date no published information exists on either

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chemical or microbiological characteristics of Zlatar cheese.Therefore, the aim of this work was to study the changes in thegross chemical composition and lactic acid bacteria populationof Zlatar cheese during the ripening process. Characterization ofisolated lactic acid bacteria to the strain level would help inselecting those that could be used for the preparation of startercultures for production of the standardized Zlatar cheese, havingorganoleptic qualities as good as the artisan cheese.

2. Materials and methods

2.1. Cheese manufacturing and sampling

Lactic acid bacteria (LAB) were isolated from white brineZlatar cheeses at different periods of ripening. This cheese ismadeby adding the rennet to raw nonpasteurized milk immediatelyafter milking and formation of a curd (coagulation) takes 1 to 2 h.Afterwards the curd is cut into smaller pieces and salted. Thethickness of the slices is between 0.5 and 1.0 cm and no holesinside the cheese body were present. For longer ripening periodcheese slices are being transferred into a small wooden barreleveryday and this process continues until the barrel is full —when the cheese is poured over with the brine, covered up withwooden cover and left on for the ripening. The ripening time isbetween 1 and 60 days at 10 to 15 °C and is performed in its ownbrine made of whey created by self-pressing of cheese.

A sample of raw cow's milk (500 ml) used for cheeseproduction and one sample (500 g) of cheese 1, 10, 20, 30, 45and 60 days old were collected from October to December2003. These samples were taken from farmhouses in a sterileplastic container and transported to the laboratory underrefrigeration. Chemical and microbiological analyses of thesesamples were performed within the following 24 to 48 h.

2.2. Chemical analysis

All samples were subjected to chemical analysis comprisingdry matter, pH, total fat, protein and NaCl content. Dry matterwas determined by weighing before and after drying in an ovenat 102±1 °C according to the IDF Standard 4 (1958). The pH ofcheese was measured by placing electrodes in direct contactwith grated cheese. Microprocessor pH Meter HI 9311 (HannaInstruments, Lisbon, Portugal) was used for these measure-ments. Total fat content was evaluated by acido-butyrometricmethod of van Gulik (ISO-3433, 1975). Kjeldahl procedure wasused to determine the nitrogen (N) content in cheese, whichpermitted calculation of the protein content (N×6.38) (IDFStandard 25, 1964). The NaCl contents in curd and cheeseduring ripening were determined by the Mohr method (IDFStandard 12 B, 1988). All analyses were performed in triplicate.

2.3. Microbiological analyses — isolation, characterizationand tentative identification of lactic acid bacteria

For microbiological analysis 20 g of each sample was takenfrom the cheese interior and homogenized with pastille in sterilemortar and transferred to 180 ml sterile 2% (w/v) sodium citrate

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solution in a sterile conical flask. Decimal dilutions of thehomogenates were prepared with sterile 0.85% (w/v) sodiumchloride and were plated on media most suitable for isolation ofLAB: a) for presumptive lactobacilli, on MRS agar pH 5.7(Merck GmbH, Darmstadt, Germany) at 30 °C and 45 °C for72 h in aerobic conditions, and in anaerobic conditions inanaerobic jars with Anaerocult A (Merck, GmbH, Darmstadt,Germany) for 5 days; b) for presumptive lactococci on M17agar pH 7.2 (Merck, GmbH, Darmstadt, Germany) at 30 °C for72 h (Mannu et al., 2002).

The LAB were isolated from one sample of cow's milk andsix samples of cheese of different ripening time. Samples aredesignated as ZLM—raw milk, ZLS1—cheese sample imme-diately after it was made, and samples ZLS10, ZLS20, ZLS30,ZLS45 and ZLS60 after a ripening period of 10, 20, 30, 45 and60 days, respectively.

Thirty to fifty colonies per sample were randomly taken fromboth MRS (30 °C and 45 °C) and M17 (30 °C) agar platescorresponding to the highest dilution at which growth occurred.The cell morphology of all strains of LAB was determined bymicroscopy (Olympus U-RFL-T, BX51, GmbH, Hamburg, Ger-many). After microscopic observations, the colonies were sub-cultured to purity on MRS or M17 medium. Gram-positive andcatalase-negative isolates were stored in milk at +4 °C and alsofrozen at −20 °C and −80 °C in M17 (for cocci) and in MRS (forrods) broth containing 15% of glycerol (v/v) (Mannu et al., 2002).Overall 306 isolates were isolated and after catalase test, Gramstaining and microscopy, 253 were chosen for further analyses.

Gram-positive and catalase-negative isolates of LAB wereidentified to genus level, by tests as follows: (a) colony mor-phology and pigmentation, (b) growth at 30 and 45 °C in MRSand M17 broth, (c) growth at 4.0 and 6.5% (w/w) NaCl in MRSand M17 broth, (d) production of carbon dioxide from glu-cose by sub-culturing the isolates in tubes with MRS brothDurham's tubes, (e) L-arginine and esculin hydrolysis, (f) citrate-utilization, (g) diacetyl production — only for LAB whichcoagulated casein, (h) activity in milk and (i) test in litmus milk(Bergey's Manual, 1984; Mundt, 1986a,b; Sneath et al., 1986;Prescot et al., 1996). According to tests cited above 25 isolates ofLAB were chosen for preliminary identification using API 50CH tests (BioMérieux, Montalien-Vercien, France).

In general, for the identification of isolates reference strainsof LAB were used (Table 1).

2.4. DNA and PCR analysis

Total DNA from 25 isolates preliminary identified with API50 CH tests plus 2 isolates of presumptive enterococci waspurified by the method given by Hopwood et al. (1985). Forrep-PCR analysis total DNA from different isolates of lacticacid bacteria was used as template for PCR amplifications withBOXA1R (5′-CTACGGCAAGGCGACGCTGACG-3′) and(GTG)5 (5′-GTGGT GGTGGTGGTG-3′) oligonucleotide pri-mers, each with its optimal PCR program (Versalovic et al.,1994), using Taq DNA polymerase (Fermentas UAB, Vilnius,Lithuania). Reactions were carried out in a thermal cycler GeneAmpR PCR System 2700 (Applied Biosystems, Foster City,

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Table 1The list of used reference strains

Bacterial strains Source or reference

Lactococcus lactis subsp. lactis biovar.diacetylactis S50

Kojic et al. (1991)

Lactococcus lactis subsp. cremoris NS1 Strahinic et al. (2005)Lactobacillus plantarum A112 Vujicic and Topisirovic (1993)Lactobacillus rhamnosus BGT10 Pastar et al. (2003)Lactobacillus casei ATCC 393 American Type Culture

Collection, Rockville, Md., USALactobacillus paraplantarum BGKP-15 a Laboratory collectionLactobacillus paraplantarum BGDA-17a a Laboratory collectionLactobacillus fermentum BGHI-14a a Laboratory collectionLactobacillus brevis BGHI-3a a Laboratory collectionLactococcus lactis subsp. lactis

BGSM1-19 aLaboratory collection

Enterococcus durans BGZLS20-35b a Laboratory collectionEnterococcus faecalis BGZLS45-14 a Laboratory collectionEnterococcus faecium BGGJ8-3 a Laboratory collectionLactobacillus paracasei subsp. paracasei

BGBUK 2-16 aLaboratory collection

a These strains were identified by molecular methods, AFLP, SDS-PAGE andrep-PCR with (GTG)5 primer in the Laboratorium voor Microbiologie,Univesitet Gent, Gent, Belgium.

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California, USA). The PCR products were separated by elec-trophoresis on 1.5% agarose gel (15×20 cm) containing 0.5 μgml− 1 ethidium bromide, for 16 h in 1×TAE buffer and 55 V(constant voltage) at +4 °C (Versalovic et al., 1994). Elec-trophoresis was performed using an Electrophoresis PowerSupply-EPS 301 (Amersham Biosciences, San Francisco,USA). The buffer 50×TAE prepared from 242 g TRISBASE, 57.1 ml glacial acetic acid, 100 ml 0.5 M EDTA pH8.0 and demineralized water to 1000 ml. The rep-PCR profileswere visualized under ultraviolet light, followed by digitalimage capturing using a CCD camera Biometra BDR2/5/6 (BioDoc Analyze GmbH, Göttingen, Germany).

For the sequencing of the 16S rRNA region total DNA fromisolate BGZLS30-44 was used as a template for PCR amplifica-tions with U968-GC (5′-CGCCGGGGGCGCGCCCCGGGC-GGGCGG GGCACGGGGGGAACGCGAAGAACCTTAC-3′) and L1401 (5′-GCGTGTGTACAAGACCC-3′) primers(Zoetendal et al., 1998; Randazzo et al., 2002), using TaqDNA polymerase (Fermentas UAB, Vilnius, Lithuania). Reac-tions were carried out in thermal cycler as cited above. Theobtained PCR product was purified by QIAquick PCRPurification KIT/250 (QIAGEN GmbH, Hilden, Germany),and sequenced by CRIBI-BMR servizio sequenziamento DNA,

Table 2Changes of physicochemical parameters of Zlatar cheese throughout ripening

Parameters Days of ripening

1 10 20

Dry matter (%) 37.5±1.28 38.8±1.33 41Fat (%) 19.5±1.71 19.5±1.54 19Proteins (%) 11.2±0.80 12.9±0.69 14Fat in dry matter (%) 50.0±2.89 52.2±3.22 47NaCl (%) 2.1±0.48 2.3±0.47 2pH 5.2±0.16 4.6±0.43 4

Mean±standard deviation of 3 independent measurements.

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Univesita di Padova, Italy. The sequence was aligned in theNCBI database using the standard nucleotide–nucletide ho-mology search BLAST (http://www.ncbi.nlm.nih.gov/BLAST).

2.5. Detection of antimicrobial activity

Antimicrobial activity of isolated lactic acid bacteria wasscreened by agar-well diffusion method (Tagg and Mc Given,1971) usingLactococcus lactis subsp. cremorisNS1,L. plantarumA112 and L. lactis subsp. lactis BGMN1-596 as indicator strains.Soft GM17 andMRS agar (0.7%, w/v), containingLactococcus orLactobacillus indicator strains, was overlaid ontoGM17 andMRSplates, respectively. Wells were made in the lawn of hardened softagars. Aliquots (50 μl) of supernatant of overnight cultures (16 h)were placed in the wells. To confirm the production ofproteinaceous substance, a crystal of proteolytic enzyme pronaseE (Sigma Chemie GmbH, Deisenhofen, Germany) was placedclose to the edge of supernatant containing well. The plates wereincubated overnight at 30 °C. A clear zone of inhibition around thewell, but not in the vicinity of pronase E crystal, was taken as anindication of proteinaceous nature of produced antimicrobialsubstance i.e. a potential bacteriocin-like compound.

3. Results

3.1. Results of chemical analysis

The chemical composition of Zlatar cheese was determinedduring 60 days of ripening. Results showed that tested parameterswere within the range accepted for white cheeses (Table 2). Drymatter was minimum 40% (w/w) and salt in dry matter wasmaximum 10% (w/w). During the ripening process, dry matter incheeses increased mostly as a result of syneresis and osmosis inbrine and after 60 days it reached a final value of 50.0%. Thecontent of NaCl increased during first 20 days of ripening from2.1% (w/w) to 2.7% (w/w) and afterwards a gradual decrease ofNaCl content occurred. After 60 days of cheese ripening it was2.4% (w/w). The pH value was 5.2 in 1 day old cheese and duringthe ripening time pHvalue was constantly decreasing and reachedthe value of 3.5 in 60 days old cheese.

3.2. Isolation ofmicroorganisms and phenotypic characterization

Analysis of LAB population changes during the cheeseripening was performed. For this purpose, the population of

30 45 60

.0±1.50 45.0±0.92 50.0±1.09 50.0±1.17

.5±1.56 22.5±1.83 27.0±2.04 27.6±2.04

.9±0.72 16.0±0.56 17.0±0.81 16.6±0.71

.5±2.58 50.0±3.02 54.0±3.24 54.9±3.39

.7±0.52 2.6±0.55 2.4±0.58 2.4±0.66

.3±0.28 4.0±0.31 3.7±0.28 3.5±0.22

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LAB was analysed starting from the milk of which cheesewas manufactured and cheese samples of different ripeningperiods.

From all samples of milk and cheese used in the study, 306randomly chosen isolates were taken for the analysis and results

Fig. 1. rep-PCR patterns obtained with BOXA1R primer. (A) and (B) Line 1, GenLactobacillus casei ATCC 393; line 3, Lactobacillus paracasei subsp. paracaseparaplantarum BGKP-15; line 6, Lactobacillus paraplantarum BGDA17-a; line 7,line 9, Lactobacillus brevis BGHI3-a; line 10, Gene Ruler™ 100 bp DNA Ladder (Fe2; line 13, BGZLM1-4; line 14, BGZLS1-1; line 15, BGZLS10-9; line 16, BGZLS10DNA Ladder (Fermentas UAB, Vilnius, Lithuania). (B) Line 11, BGZLS20-8; liBGZLS45-26; line 16, BGZLS45-27; line 17, BGZLS45-29; line 18, BGZLS60-39; lBOXA1R primer. Line 1, Ready-Load 1 kb plus DNA Ladder (Gibco/BRL); line 2, LBGBUK2-16; line 4, Lactobacillus plantarum A112; line 5, Lactobacillus paraplantaLoad 1 kb plus DNA Ladder (Gibco/BRL); line 8, Lactobacillus fermentum BGHBGHI3-a; line 11, BGZLS10-17; line 12, BGZLS30-44; line 13, BGZLS60-39; linobtained with (GTG)5 primer of lactococci (lines 5–9) and enterococci (lines 14–16).line 2, Lactococcus lactis subsp. lactis BGSM1-19; line 3, Lactococcus lactis subsp.line 5, BGZLM1-24; line 6, BGZLS1-8; line 7, BGZLS10-28; line 8, BGZLS20-34;UAB, Vilnius, Lithuania); line 11, Enterococcus faecium BGGJ3-8; line 12, Enteroc14, BGZLM1-9; line 15, BGZLS10-4; line 16, BGZLS60-27; line 18, Gene Ruler™

Please cite this article as: Terzic-Vidojevic, A. et al. Characterization of microflorMicrobiology (2006), doi:10.1016/j.ijfoodmicro.2006.10.038

showed that 253 isolates were Gram-positive and catalase-negative and taken for further study.

Results showed that the percentage of cocci and rods insamples of milk and cheeses of different ripening period wasfluctuating. Rawmilk and one day old cheese had a considerably

e Ruler™ 100 bp DNA Ladder (Fermentas UAB, Vilnius, Lithuania); line 2,i BGBUK2-16; line 4, Lactobacillus plantarum A112; line 5, LactobacillusLactobacillus fermentum BGHI14-a; line 8, Lactobacillus rhamnosus BGT10;rmentas UAB, Vilnius, Lithuania). (A) Line 11, BGZLM1-1; line 12, BGZLM1--10; line 17, BGZLS10-15; line 18, BGZLS20-3; line 19, Gene Ruler™ 100 bpne 12, BGZLS20-13; line 13, BGZLS30-44; line 14, BGZLS45-25; line 15,ine 19, BGZLS60-46; line 20, BGZLS60-50. (C) rep-PCR patterns obtained withactobacillus casei ATCC 393; line 3, Lactobacillus paracasei subsp. paracaseirum BGKP-15; line 6, Lactobacillus paraplantarum BGDA17-a; line 7, Ready-I14-a; line 9, Lactobacillus rhamnosus BGT10; line 10, Lactobacillus brevise 14, Ready-Load 1 kb plus DNA Ladder (Gibco/BRL). (D) rep-PCR patternsLine 1, Gene Ruler™ 100 bp DNA Ladder (Fermentas UAB, Vilnius, Lithuania);cremoris NS1; line 4, Lactococcus lactis subsp. lactis biovar. diacetylactis S50;line 9, BGZLS30-17/1; line 10, Gene Ruler™ 100 bp DNA Ladder (Fermentasoccus faecalis BGZLS45-14; line 13, Enterococcus durans BGZLS20-35b; line100 bp DNA Ladder (Fermentas UAB, Vilnius, Lithuania).

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greater number of cocci than rods (results not shown). However,during the ripening period the number of lactic acid cocci wasgradually decreasing whereas the number of lactic acid rodsbacteria was increasing. This trend continued until 30 days ofcheese ripening (sample ZLS30), when the number of cocciincreased again and the percentage of cocci and rods equalized in60 days ripened cheese (sample ZLS60).

Further analysis of isolated lactic acid cocci was performedby examination of their ability to grow at different temperaturesas well as in the presence of different salt concentrations.Preliminary characterization included analysis of their ability togrow at 45 °C and the ability to grow in bouillon with 6.5% (w/w) NaCl. In addition, some of their physiological features werealso investigated such as their ability of arginine and esculinhydrolysis, production of diacetyl as well as citrate utilization.Results showed that a number of thermophilic lactic acid cocciin Zlatar cheese, which grew at 45 °C and in media with 6.5%(w/w) NaCl was increasing during the ripening period and theyalso hydrolyzed esculin. These two characteristics are typicalfor enterococcal species. In addition, it was found that 92 out of115 isolated cocci hydrolyzed arginine followed by theproduction of ammonia (results not shown). Accordingly, itcould be inferred that in cheeses older then 30 days enterococciare the most abundant species.

Previous reports dealing with the analysis of microflora inartisanal cheeses showed that enterococci are numerous species(Mas et al., 2002).

A large number (66%) of mesophilic lactobacilli from Zlatarcheese produced diacetyl (results not shown), particularly inisolates from cheeses at 45 and 60 days of ripening. Althoughthe number of cocci which produced diacetyl was smaller (only21%), this number also increased during the ripening period.

Test in litmus milk showed that over 90% of isolates pro-duced acid and changed the color of the indicator from purple tored, created curd and reduced litmus and changed the color ofthe indicator from purple to white by hydrogen ions. This testalso showed that most of the isolates coagulated skimmed milkwithin 24 h. From this group, 8 isolates exhibited good acidi-fication activity in milk, and after 5 to 7 h of incubation the pHreached a value of 4.8 (results not shown).

3.2.1. API 50 CH testsPreliminary characterization of all 253 LAB isolates has been

done based on their morphological, physiological and biochem-

Table 3Identification of 27 isolates of lactic acid bacteria comprising Lactobacillus spp. (Lhomemade Zlatar cheese by different methods

Methods Lb. spp. Lb. paracas. subsp.parac.

Lb. brevis Lc. spp. Lc. laclactis

Traditionalmethods a

19 isolates 5 isolates

API b 18 isolates 1 isolate 5 isolarep-PCR 18 isolates 1 isolate 5 isolates16S rRNAc 1 isolatea Morphological, biochemical and physiological tests.b Fermentation of carbohydrates by API 50 CH (BioMérieux).c Sequence is compared by BLAST search on NCBI site.

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ical characteristics. They have been divided in 25 groups accord-ing to their observed features such as microscopy appearance, theability to grow under similar temperature and salt conditions, etc.(results not shown). From each group one isolate has been chosenfor further examinations. Twenty-five isolates of selected LABwere characterized for sugar fermentation by using theAPI 50CHsystem (results not shown). The results have shown that 17isolates belonged to L. paracasei subsp. paracasei, 2 isolatesbelonged to Lactobacillus brevis, 5 to species L. lactis subsp.lactis, and one isolate belonged to genus Enterococcus spp.

The results referring to physiological characteristics haveshown that several subgroups of species L. paracasei subsp.paracasei existed. According to their ability of arginine hydro-lysis, growth at 45 °C and growth in bouillon with 6.5% (w/w)NaCl, four subgroups within L. paracasei subsp. paracaseiisolates could be distinguished. The isolates BGZLM1-1,BGZLM1-2, BGZLM1-4, BGZLS1-1, BGZLS10-9 andBGZLS20-8 belong to the first subgroup and they are typicalrepresentatives of the species L. paracasei subsp. paracasei. Incontrast, the second subgroup contains isolates that did notgrow at 45 °C, but could grow in media with 6.5% (w/w) NaCl(isolates BGZLS20-3, BGZLS20-13, BGZLS45-25,BGZLS45-26, BGZLS45-27, BGZLS45-29 and BGZLS60-46). The isolates BGZLS60-39 and BGZLS60-50 belong to thethird subgroup and they grew at 45 °C and in media with 6.5%(w/w) NaCl. The members of all three groups were arginine-negative. However, two isolated L. paracasei subsp. paracasei(BGZLS10-10 and BGZLS10-15) were arginine positive, couldgrow at 45 °C and in media with 6.5% (w/w) NaCl.

According to the API test, isolates BGZLS10-17 andBGZLS30-44 belonged to the species L. brevis, albeit thesetwo isolates did not exhibit typical physiological characteristicsof this species. They were arginine-negative and did not grow inmedia with 6.5% (w/w) NaCl.

3.3. rep-PCR identification and 16S rRNA sequence analysis

The results obtained by rep-PCR from 27 isolates of lactic acidbacteria (25 of them have been analysed by API 50 CH test andtwo presumptive enterococci) with BOXA1R and (GTG)5primers are shown in Fig. 1A, B, C and D. These two enterococciwere taken for molecular determination since the results obtainedin biochemical and physiological testings showed some dis-crepancies in relation to other tested enterococci.

b.), Lactococcus spp. (Lc.) and Enterococcus spp. (Ec.) isolated from milk and

tis subsp. Ec. spp. Ec.faecalis

Ec.faecium

Ec. avium Unidentified

3 isolates

tes 1 isolate1 isolate 1 isolate 1 isolate

1 isolate

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Table 4Antimicrobial activity among isolates of lactic acid bacteria tested in agar-diffusionassay

Genus Isolates Indicator strains

Lactococcuslactis subsp.lactisBGMN1-596

Lactococcuslactis subsp.cremorisNS1

LactobacillusplantarumA112

Lactobacillus spp. 6 isolates c5 isolates c3 isolates c2 isolates c t1 isolate t t

Lactococcus spp. 8 isolates c c t5 isolates c c+ t t

Enterococcus spp. 6 isolates c c+ t t4 isolates c c3 isolates c+ t c+ t c+ t3 isolates c+ t c+ t2 isolates c2 isolates t1 isolate c t t1 isolate c c t1 isolate c c+ t1 isolate c t1 isolate t c

c: clear zone of inhibition; t: turbid zone.

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Species-specific rep-PCR identification showed certaindifferences in band organization on gels within the samespecies (L. paracasei subsp. paracasei) indicating the presenceof different strains of the same species (Fig. 1A and B). Theisolates BGZLS10-17 and BGZL30-44 were identified by rep-PCR method as species L. brevis (Fig. 1C).

Regarding lactococci, it was shown that rep-PCR methodcan be useful only for the identification to the level of genusLactococcus spp. (Fig. 1D).

Table 3 gives a brief view of identification results for 27LAB isolated from homemade semi-hard white Zlatar cheese bydifferent methods.

3.4. Detection of antimicrobial activity

It was confirmed that 57 of 253 isolates examined producedantimicrobial compounds that were sensitive to pronase E(Table 4). Most of the producers of antimicrobial compoundswere isolated from samples of a one day old cheese (100%), andfrom a milk sample (63%). In 10 days old cheese 32% ofantimicrobial compounds producers have been determined, in20 days old cheese 16%, in 30 days old cheese 24%, and in45 days old cheese only 4% of antimicrobial compoundsproducers have been determined. From 60 days old cheese noantimicrobial compounds producer has been isolated (resultsnot shown).

4. Discussion

A better understanding of the role which natural lactic acidbacteria have in the production of traditional cheeses requires a

Please cite this article as: Terzic-Vidojevic, A. et al. Characterization of microflorMicrobiology (2006), doi:10.1016/j.ijfoodmicro.2006.10.038

deeper study of the microbial community involved in theirproduction and ripening. The strains dominating the first stagesare not necessarily present in the later phases of ripening.Recent studies have shown that artisan cheeses have differentand typical microbial population dynamics related to the localproduction technology and geographic area of origin (Gobbettiet al., 2002; Mannu et al., 2002).

During the ripening dry matter in cheeses increases mostlyas a result of syneresis and osmosis during the ripening inbrine and after 60 days reaching the final value of 50.0%.The NaCl content increases during first 20 days of ripeningas a result of moving of salt from the brine into the cheese.The observed decrease of cheese's pH value during ripeningappears to be due to the activity of LAB. The evolution ofthe pH value and NaCl content during cheese ripeningwas also described previously (Mas et al., 2002; Kasimogluet al., 2004). During the ripening period the number oflactobacilli was increasing most probably because of theirhigher ability to grow under the low pH conditions (Caridiet al., 2003).

Mesophilic lactobacilli are important in the maturation ofcheeses as they are able to ferment citrate and could be involvedin proteolysis as well as in other enzymatic processes thatoccurred during cheese ripening (Crow et al., 2001). Thepresence of mesophilic lactobacilli in the 60 days old cheesesample may explain why older cheeses have better taste andaroma for consumers (Palles et al., 1998).

The results of the examination (Fitzsimons et al., 1999,2001) showed that there are differences in band pattern onthe gel among some representatives inside of the species L.paracasei subsp. paracasei. We also had difficulties in iden-tifying one isolate, BGZLS30-44 which according to API50 CH method belonged to L. brevis, but its band pattern fromrep-PCR differed significantly from the referent strain. Finalsequencing of 16S rRNA region confirmed that it was L.brevis.

Lactic acid bacteria have the ability to produce antimicro-bial substances like hydrogen peroxide, diacetyl and bacter-iocins which are antagonistic to spoilage and pathogenicorganisms. Because of increased public interest in food safety,including demands for less artificial additives, researchattention is focused on the use of naturally occurring meta-bolites produced by food grade bacteria. Bacteriocin producedby lactic acid bacteria may be a very promising source to beused as biological food preservatives (Daeschel, 1989; Bolmand Morvedt, 1991; Piard and Desmazeaud, 1992). A largenumber of LAB isolated from the Zlatar cheese showed anti-microbial activity. As they inhibited 2 or 3 different indicatorstrains, one could speculate that more than one principle isinvolved (Messi et al., 2001; Atanassova et al., 2003; Lozoet al., 2004).

Acknowledgement

This work was supported by the Ministry of Science andEnvironmental Protection of the Republic of Serbia (MSEPRS)grant No.: 143036 and partially by grant BTN-351010.

a in homemade semi-hard white Zlatar cheese. International Journal of Food

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a in homemade semi-hard white Zlatar cheese. International Journal of Food