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186 A. LEBO PAVUNC et al.: Starter cultures for fresh cheese production, Mljekarstvo 63(4), 185-194 (2013)

both probiotic and functional starter culture strains(D idi et al., 2008). In general, strains harbouringantibiotic resistance plasmids are considered unsuit-able for application as functional starter cultures andprobiotics (Salminen et al., 1998; Saarela et al.,

2000). On the other hand, intrinsically antibiotic-resistant probiotic strains may benefit patients whosenormal intestinal microbiota has become unbalancedor greatly reduced in numbers due to the administra-tion of antibiotics (Salminen et al., 1998).

The production of many traditional cheeses indifferent European countries as well as in Croatiarelies on spontaneous fermentation by LAB (Ro-setti et al., 2009; Lebo Pavunc et al., 2012;Beganovi et al., 2013). Fresh cheese, produced

from raw milk by activity of autochthonous startercultures, represents an environment in which LABoriginate mainly from milk. The antibiotic resistanceof these microorganisms can be affected by antibiotictreatment of the milk-producing animals. Therefore,the objective of the present study was to investi-gate the antibiotic susceptibility and antibacterialactivity against food pathogens, of autochthonousstarter cultures Lactobacillus fermentum A8 andEnterococcus faeciumA7, which are important traitsfrom safety aspects. These cultures were previously

defined by Lebo Pavu nc et al. (2012), from func-tional and technological aspects, as starter culturesfor fresh cheese production. Since dairy productshave been frequently reported as contaminatedwith Listeria monocytogenes, antilisterial activityof Lb. fermentumA8 and Ec. faeciumA7, but alsoantimicrobial activity towards Salmonella entericaserovar Typhimurium FP1, Escherichia coli 3014and Staphylococcus aureus3048 in order to assessthe potential of these LAB strains to inhibit the

growth of the most common pathogens detected infermented dairy products, wasinvestigated.

Materials and methods

Bacterial strains

All LAB strains (n=15) were isolated andcharacterised in the Laboratory of Antibiotics, En-zymes, Probiotics and Starter Cultures Technology,Faculty of Food Technology and Biotechnology Uni-

versity of Zagreb. Examined strains were stored at-80 C in the De Mann Rogosa Sharpe (MRS) broth

(Difco, Darmstadt, Germany) with 15 % (v/v)glycerol. Test-microorganisms L. monocytogenesATCC 11911, S. aureus 3048, E. coli 3014 andS. entericaserovar Typhimurium FP1 were stored onnutrient broth (Biolife, Milano, Italy) plus 20 % (v/v)

glycerol at -80 C. All cultures were subculturedtwice before use in appropriate medium.

SDS-PAGE of soluble cell proteins and computer-aided comparison of the electrophoreticprotein patterns

SDS-PAGE of soluble cell proteins was per-formed according to Beganovi (2008). ProteinSDS-PAGE gels were scanned (Scanjet 3800;Hewlett Packard, CA, USA) and stored as TIFF

files. Recording of the electrophoretic proteinpatterns and grouping of the bacterial strains byUnweighted Pair Group Method Using AverageLinkage (UPGMA) cluster analysis were performedby using Gel-Compar II (Applied Maths, St-Ma-terns-Latem, Belgium).

Plasmid DNA isolation

The minipreparation of plasmid DNA was per-formed from 50 ml of the overnight grownLb. fer-mentum A8 and Ec. faecium A7 cultures. PlasmidDNA was extracted using Midi-prep kits (Prome-ga, San Luis Obispo, CA) according to the manu-facturers instructions. Extracted plasmids wereseparated by horizontal agarose gel electrophoresis(0.8 % agarose gel in 1 Tris-Phosphate-EDTA buffer,100 V, 2 h). After ethidium bromide staining, DNAwas visualised and digitalized images captured un-der UV light transillumination (MiniBIS Pro, DNRBio-Imaging Systems Ltd.). For the estimation of

the molecular weight of each plasmid, Gene Ruler1kb DNA Ladder Fermentas marker was used toestimate the molecular mass of the plasmids.

Antibiotic disk diffusion tests

For antibiotic disk diffusion tests, overnightgrown culture ofLb. fermentumA8 andEc. faeciumA7, diluted to give viable cell count of approximate-ly 108CFU/mL, was added into 12 mL of meltedMRS agar cooled to 50 C, and then was poured

into petri dishes. After the MRS agar plates had beensurface dried, susceptibility test disks of each of the

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187A. LEBO PAVUNC et al.: Starter cultures for fresh cheese production, Mljekarstvo 63(4), 185-194 (2013)

antibiotics were placed aseptically on the MRS agar.The 16 different antibiotics were tested in order tocover all the known chemical and functional groups ofantibiotics: ampicillin, bacitracin, penicillin G, vanco-mycin, azithromycin, clindamycin, chloramphenicol,

clarithomycin, erythromycin, gentamicin, streptomy-cin, spiramycin, tetracycline, neomycin, rifampicinand novobiocin. All antibiotic discs were purchasedfrom Oxoid (Basingstoke, United Kingdom). Agarplates with antibiotic disks were then incubated for24 h. The diameters of the inhibition zones weremeasured using a ruler and an average of 3 readingswas calculated.

E test

Bacterial suspensions with a turbidity equiva-lent to McFarland standard 1 were inoculated intoMRS agar plates. After drying the surfaces of theplates, the E test strips, (M. I. C. E. Evaluators,Oxoid Ltd, Baksingstoke, UK) of the antibiotictested (ampicillin, bacitracin, benzyl penicillin, clin-damycin, chloramphenicol, erythromycin, tetra-cycline, rifampicin and vancomycin) were applied.The plates were incubated under the same condi-tion as for the antibiotic disk diffusion tests. Theminimal inhibitory concentration (MIC) is the low-

est antibiotic concentration that inhibits the visiblebacterial growth after overnight incubation at opti-mal growth temperature of the certain strain. MICswere read directly from the test strip according tothe manufacturers instructions and were expressedas micrograms per millilitre.

Antibacterial activity of functional starter cultures

Three different methods were used in order toinvestigate an antibacterial activity ofLb. fermentum

A8 andEc. faeciumA7. Overnight cultures (5L) ofLb. fermentumA8 andEc. faeciumA7 were spotted

onto the surface of MRS agar (1.2 % w/v) plates,which were then incubated at 37 C for 24 h. Test-microorganisms were inoculated into soft agar me-dium (nutrient broth containing 0.7 % w/v agar) toa final concentration of approximately 107CFU/mL.The soft media were poured on the plates whichwere incubated for 24h at the optimal growth forindicator strains. Inhibition was scored positive in thepresence of a detectable clear zone around the colo-

ny of the producer strain. The ratio of the inhibitiondiameter (ID) to the spot culture diameter (CD) was

calculated for each assay, to determine the effectiveinhibition ratio (EIR): ((ID-CD)/CD). Lb. fermen-tumA8 andEc. faeciumA7 were further tested fortheir antimicrobial activity againstL. monocytogenesATCC 19111, S. Typhimurium FP1, E. coli 3014

and S. aureus3048 using the well-diffusion methoddescribed by Kos et al. (2008) and by turbidimetricmethod described by Beganovi et al. (2011).

Results and discussion

Autochthonous LAB strains SDS-PAGE proteinanalysis and plasmid DNA isolation

In order to define which LAB genus is pre-dominant in the autochthonous fresh cheese bacte-rial population, SDS-PAGE analysis of the solublecell proteins was performed from the selected MRScolonies and their protein profiles were compared.Results obtained by SDS-PAGE of soluble bacte-rial cell proteins, indicated that these were actuallyonly two different bacterial strains, i.e. eleven cocci-shaped isolates showed identical whole-cell proteinprofiles and four rod-shaped isolates showed identi-cal whole-cell protein profiles, respectively (Figure1). This implies that lactobacilli, but also enterococci

represent a significant proportion of the Croatianfresh cheese microbiota, being in accordance withGi ra ff a (2003) and Bernadeau et al. (2008) whoreviewed that traditionally produced cheeses origi-nating from Southern and Eastern European regionscontain high percentage of Lactobacillus andEnte-rococcus strains among total autochthonous cheesemicrobiota. Accurate taxonomic identification ofthe microorganism intended for application as func-tional starter culture is essential for its safety assess-ment. Hence two representatives of autochthonous

strains were identified by DNA fingerprinting usingAFLP as Lactobacillus fermentumA8 andEntero-coccus faeciumA7, respectively (Lebo Pavunc etal., 2012).

Before the application of functional starterculture, especially those from Enterococcus genera,in the food or feed, the presence of transferableresistances genes must be excluded, especially ifthese genes are present on the plasmids which oftenserve as vehicles to shuttle DNA between bacteria

(DAimmo et al., 2007; Bernardeau et al., 2008).Hence, in this study Lb. fermentum A8 and Ec.

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faeciumA7 were tested for the presence of plasmids

to determine whether these strains might carry anyplasmid-encoded antibiotic resistance genes (Figure2). Plasmid DNA analysis showed that strain Lb.fermentum A8 possess a plasmid of approximately10.0 kb, while no plasmid was detected in case ofEc. faeciumA7 (Figure 2). Gfeller et al. (2003) re-ported thatLb. fermentumROT1, isolated from a rawmilk cheese, has 19.3-kb plasmid pLME300 with twoantibiotic-resistance determinants vat(E) for strepto-gramin A and erm(LF) for erythromycin. Therefore,further antibiotic susceptibility was investigated to

examine whether these strains maybe have potentialplasmids which may carry antibiotic resistance genes.

Antibiotic susceptibility of Lb. fermentum A8 andEc. faecium A7

Antibiotic susceptibilities of Lb. fermentumA8 and Ec. faecium A7 were tested against anti-biotics chosen in order to provide a diverse repre-sentation of antimicrobial agent classes. AccordingEFSA (2008), as a basic requirement, the minimal

inhibitory concentration (MIC) of the antibioticsexpressed as mg/L or g/mL should be determined.

The antibiotics used in this study were chosen in or-

der to maximise the probability to identify possiblypresent resistance genotypes to the most commonlyused antibiotics by assessing the resistance pheno-types of Lb. fermentumA8 and Ec. faeciumA7 inorder to cover main chemical and functional groupsof antibiotics: ampicillin, bacitracin, penicillin G,vancomycin, azithromycin, chloramphenicol, clari-thromycin, clindamycin, erythromycin, gentamicin,streptomycin, spiramycin, tetracycline, neomycin,rifampicin and novobiocin, with concentrations indi-cated at Table 1. The appearance of inhibition zone

is taken as criterion for the antibiotic susceptibility.Lb. fermentumA8 andEc. faeciumA7 were suscep-tible to all tested antibiotics, with exception of theabsence ofLb. fermentumA8 susceptibility to van-comycin (Table 1). Examined strains were shown tobe less sensitive to aminoglycoside antibiotics gen-tamicin, streptomycin and neomycin, what could bedue to the MRS mediums low pH because of lacticacid production. Namely, the activity of aminoglyco-sides is markedly reduced at acidic pH values (Klareet al., 2007; Le bo Pavunc et al., 2011).

Figure 2. Agarose gel electrophoresis showingplasmid profiles 1Ec. faeciumA7; 2Lb.fermentumA8; 3Lactococcus lactisLMG9450; 4 Gene Ruler DNA LadderFermentas marker

Figure 1. Dendrogram based upon fingerprintsobtained by SDS-PAGE of soluble bacte-rial cell proteins of 15 autochthonouslactic acid bacteria isolates (lines indicateautochthonous LAB isolates: 6,7,5,13-Lb.fermentum; the other lines-Ec. faecium).Clustering was performed by UPGMA

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Lb. fermentum A8 strain displayed phenotypicresistance to vancomycin assayed by antibiotic disk

diffusion tests and E-test. Among antibiotic resist-ances, vancomycin resistance is of the major concernbecause vancomycin is one of the last antibioticsbroadly efficacious against clinical infections causedby multidrug-resistant pathogens (Zhou et al., 2005).Coming back to detected vancomycin resistance ofLb. fermentumA8, it should be stated that some LABhowever, including strains ofLactobacillussp.,Pedio-coccus sp. and Leuconostoc sp., are resistant to van-comycin. Such resistance is intrinsic, meaning that ischromosomally encoded and not transmissible (Zh ouet al., 2005). This intrinsic resistance to vancomycinis explained by the presence of D-alanine:D-alanineligase related enzymes (Bernardeau et al., 2008).Vancomycin inhibits the peptidoglycan biosynthesisby binding to muramylpentapeptides that terminatein D-alanyl-D-alanine (D-Ala-D-Ala) and blocks theaddition of these precursors into the peptidoglycanbackbone. Many Lactobacillus strains incorporateD-alanyl-D-lactate (D-Ala-D-Lac) into their mu-ramylpentapeptides by aid of D-Ala-D-Ala ligase

(Ddl). This is the basis for their vancomycin resist-ance as peptides that terminate in D-Ala-D-Lac

Antibioticsg/disk

Ec. faeciumA7 Lb. fermentumA8

Inhibition zones (mm)

Ampicillin(AMP) 2 184 261Bacitracin(B) 10 130 131

Penicillin G(P) 1 211 273

Vancomycin(VA) 30 111 0

Azithromycin(AZM) 15 112 171

Chloramphenicol(C) 30 191 251

Clarithromycin(CLR) 5 181 232

Clindamycin(DA) 2 172 250

Erythromycin (E) 15 161 211

Gentamicin (CN) 10 80.5 100.5

Spiramycin(SP) 100 171 191

Streptomycin (S) 10 111 100.5

Tetracycline(TE) 10 121 182

Neomycin (N) 10 90 103

Rifampicin (RD) 5 161 252

Novobiocin(NV) 5 131 132

Table 1. Antibiotic susceptibility of functional starter culturesEc. faeciumA7 andLb. fermentumA8 forfresh cheese production

are bound 1000-fold less efficiently by vancomycincompared to peptides terminating in D-Ala-D-Ala

(van Pijkeren and Britton, 2012). While the transfer of vancomycin resistancefromLactobacillussp. to other bacteria has not beenobserved, in many Enterococcus sp. vancomycin re-sistance is often plasmid encoded and transmissible.Several investigations showed the occurrence of van-comycin resistant enterococci (VRE) isolated fromfood or of animal origin, hence the most importantfactor during the selection of enterococci, as potentialfunctional starter cultures, is to test their resistanceagainst glycopeptides like vancomycin. Antibioticsusceptibility results obtained for strain Ec. faeciumA7 are in accordance with Lopes et al. (2006) whodemonstrated that bacteria fromEnterococcusgenus,isolated from autochthonous dairy products, were in-trinsically susceptible to the majority of the antibioticsused to cope with common infections, namely, peni-cillins, aminoglycosides, macrolides and vancomycin.Moreover, literature concerning antibiotic resistancein enterococci is limited to clinical enterococci andconfuses general characteristics of the genusEntero-

coccus including strains from fermented food origin,with characteristics of clinical enterococcal isolates.

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Table 2 summarises the distribution of MICsto several antibiotics which mode of action is basedon inhibiting the protein synthesis, cell wall synthe-sis or DNA synthesis, determined by E-test assay.

According to the results both strains were suscepti-ble to the nine different antibiotics applied in E-test.Although MIC determined for vancomycin in caseofEc. faecium A7 is slightly higher than demandedaccording to EFSA (2008) guidelines, it should beemphasised that this value is much lower than inEnterococcus strains which possess vanA or vanBtransferable vancomycin resistance with MIC values>64 g/mL and up to 1000 g/mL, respectively.Some enterococci may possess intrinsic, but nottransferable, resistance against vancomycin, cod-ed by vanC (MIC 2-32 g/L) (Sahlstrm et al.,2009). Obtained results are in agreement with in-vestigations of Ro ssetti et al. (2009) who studiedthe antibiotic susceptibility of dominant LAB associ-ated with Grana Padano cheese whey starters. Asstated by EFSA, microbiological breakpoint valuesfor vancomycin are not required for Lb. fermentumsince obligate and facultatively heterofermentativelactobacilli are known to be intrinsically resistant toglycopeptide antibiotics such as vancomycin (Dan-

ie ls en and Wind , 2003; EFSA, 2008; Ros sett i etal., 2009).

Antibacterial activity of Lb. fermentum A8 andEc. faecium A7

When proved safe from the aspect of antibiot-ic resistance, another important feature of autoch-thonous LAB, isolated from artisanal fermenteddairy products, is a potential to improve the micro-biological safety of food, because these strains arewell adapted to the environmental conditions ofthe milk as substrate and their antimicrobial activ-ity is resulting at least from the lactic acid produc-tion (u k ov i et al., 2010; Kos et al., 2011). Dueto the availability of nutrients in milk, fresh rawmilk cheeses may host different pathogenic micro-organisms. Levels of L. monocytogenes, as high as107 CFU/g, have been found in some naturally con-

taminated cheeses (Settani et al., 2011). Besides,L. monocytogenes, E. coli is one of the foodbornepathogens of concern for the dairy industry. Rawmilk is often recognised as matrix for the transmis-sion of this pathogen, and milk and different dairyproducts, including cheeses, have been implicatedin E. coli O157:H7 outbreaks (Rodrigues et al.,2005). Settani et al. (2011) reported that besidesthese two mentioned pathogens, according to Euro-pean Commission Regulation, the presence of thetwo main other pathogens Staphylococcus aureus

and Salmonellamust be checked to assess micro-biological safety of the cheese. Hence, in this studyantimicrobial activity ofLb. fermentumA8 andEc.faeciumA7 against these four food pathogens wasinvestigated. The cell-free supernatants from thesestrains were sterilized by filtration, and tested bywell-diffusion assay against S. Typhimurium FP1,E. coli3014,L. monocytogenesATCC 11911 and S.aureus3048 (Table 3).

Apart from the best inhibitory activity against

L. monocytogenes ATCC 11911, culture superna-tants also inhibited S. Typhimurium FP1, E. coli3014 and S. aureus3048. These results are in ac-cordance with the results of Kos et al. (2008) whodemonstrated the antagonistic activity of probi-otic strain Lb. helveticusM92, isolated from dairyproduct, against S.Typhimurium FP1,E. coli3014,L. monocytogenes and S. aureus 3048 by in vitrocompetition test. The growth inhibition of fourdifferent pathogens by Lb. fermentumA8 and Ec.faeciumA7 cell free supernatants, analysed by tur-

bidimetric method, is presented in Figure 3.

Table 2. Minimum inhibitory concentration (MIC)of different antibiotics determined forEc. faeciumA7 andLb. fermentumA8,respectively

Antibiotics

Ec. faeciumA7

Lb. fermentumA8

MIC (g/mL)

Ampicillin 0.03 0.06

Bacitracin 1.50 3.00

Benzyl penicillin 0.032 0.064

Vancomycin 8.00 0

Chloramphenicol 1.00 1.00

Clindamycin 0.064 0.094

Erythromycin 1.00 0.75

Tetracycline 2.00 1.50

Rifampicin 0.064 0.094

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Table 3. Antagonistic activity ofEc. faeciumA7 i Lb. fermentumA8 againstE. coli3014, S.TyphimuriumFP1,L. monocytogenesATCC 11911 andS. aureus 3048, tested by a) agar-well diffusion assay andb) agar spot test

Test-microorganisms

Inhibition zones obtained with culture supernatants (mm)


E. coli3014 17.3 12.0

S. Typhimurium FP1 20.7 15.0

L. monocytogenesATCC 11911 25.0 18.0

S. aureus3048 19.0 16.3

a)

a) b)

b)

Test-microorganismsEIR*


E. coli3014 0.384 0.333

S. Typhimurium FP1 0.600 0.466

L. monocytogenesATCC 11911 0.787 0.680

S. aureus 3048 0.555 0.504

*Effective inhibition ratio achieved by culture supernatants

Figure 3. Antibacterial activity of cell-free supernatants (25 %, 50 %, 75 %, 100 % dilution in nutrient broth)from strains: a)Ec. faeciumA7, b)Lb. fermentumA8, againstE. coli3014, S.Typhimurium FP1,L. monocytogenesATCC 11911 and S. aureus3048 assayed by turbidimetric method, after 24 hof inhibition

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According to the results,Lb. fermentumA8 andEc. faeciumA7 reduced growth of the 4 examinedstrains within 24 h, although differences in the de-gree of inhibition were observed among the strains.Cell-free supernatants (100 %) significantly reduced

pathogen levels. However, at lower dilutions ofLb.fermentumA8 and Ec. faeciumA7 cell-free super-natants (75 %; 50 % and 25 %) inhibitory effectsdecreased although were still strong (Figure 3 a-b).The antimicrobial ability attributed to many LABagainst food and clinical pathogens is primarily dueto the production of organic acids, but also to otherantimicrobial metabolites such as bacteriocins. Pre-viously Lebo Pavunc et al. (2012) reported thatLb. fermentumA8 andEc. faeciumA7 produce highconcentrations of lactic acid after overnight growth,8.74 gL-1 and 7.94 gL-1, respectively. Alakomi etal., (2000) studied the antibacterial effect of lacticacid againstEscherichia coliO157:H7,Pseudomonasaeruginosa, and Salmonella enterica serovar Typh-imurium and reported that is largely, but not totally,assigned to its ability in the undissociated form topenetrate the cytoplasmic membrane, resulting inreduced intracellular pH and disruption of the trans-membrane proton motive force. Additionally highconcentrations of lactic acid may act as a potentia-

tor of the effects of other antimicrobial substancesproduced by LAB such as bacteriocins. Our resultssuggest that these strains, previously developed asfunctional starter culture, could offer an advantagedue to their antimicrobial activity towards commonfood pathogens,during fresh cheese production.

Conclusions

Strains Lb. fermentumA8 and Ec. faeciumA7,

originally isolated from autochthonous Croatian freshcheeses, were sensitive to all clinically effective anti-biotics applied in this study, with exception of theabsence of Lb. fermentum A8 susceptibility to van-comycin, which is referred to the specific structureof cell wall peptidoglycan, which is intrinsic and nottransferable resistance. Instability of aminoglycosideantibiotics, gentamicin, streptomycin and neomy-cin at lower pH values is a reason of obtained lowersensitivity of examined strains to those antibiotics.Lb. fermentumA8 reveals a presence of the plasmid

which is approximately of 10.0 kb in size, but its roleis yet to be determined. As Lb. fermentumA8 and

Ec. faeciumA7 are previously defined as functionalstarter cultures from the technological point of view,exhibited a strong inhibitory activity against foodpathogens and especially against L. monocytogenes,they may be useful in controlling the growth of these

pathogens during production of fresh cheese.

Acknowledgements

The authors are grateful for the financial sup-port provided by Ministry of Science, Educationand Sports of the Republic of Croatia (Project 058-0581990-2007 Probiotics, prebiotics and function-al starter cultures).

Osjetljivost na antibiotikei antimikrobna aktivnost autohtonihstarter kultura kao parametri sigurnosti

njihove primjene u proizvodnjisvjeeg sira

Saetak

U ovom radu istraena je osjetljivost na antibi-otike i antimikrobna aktivnost odabranih autohtonihsojeva bakterija mlijene kiseline, koji su u prethod-

nim istraivanjima zadovoljili tehnoloke kriterije zafunkcionalne starter kulture namijenjene proizvodnjisvjeeg sira. Prema profilu topljivih staninih prote-ina, dobivenom SDS-PAGE metodom, ustanovlje-no je da u tradicionalno proizvedenom svjeem siruprevladavaju dvije vrste bakterija mlijene kiseline,identificirane kaoLactobacillus fermentumA8 iEn-terococcus faeciumA7. Ovi bakterijski sojevi nisu bilirezistentni, nego su pokazali osjetljivost na testira-ne antibiotike: ampicilin, bacitracin, penicillin G,azitromicin, kloramfenikol, klaritromicin, klindami-

cin, spiramicin, tetraciklin, streptomicin, neomicin,gentamicin, eritromicin, rifampicin i novobiocin.Bakterijski sojLb. fermentumA8 posjeduje fenotip-sku rezistenciju na vankomicin, ali ta je rezistencijauroena, nije prenosiva na druge bakterije i stoga jeprihvatljiva s aspekta sigurnosti njegove primjenekao starter kulture. Ispitano je i antimikrobno djelo-vanje kulturaLb. fermentumA8 andEc. faeciumA7prema test-mikroorganizmima:Listeria monocytoge-nesATCC 11911,Escherichia coli 3014, Salmonellaenterica serovar Typhimurium FP1 i Staphylococcus

aureus3048. Prema dobivenim rezultatima,Lb. fer-mentumA8 iEc. faeciumA7 su sigurni s aspekta i-

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renja antibiotike rezistencije i mogu biti korisne kaozatitne kulture koje inhibiraju bakterije, najeekontaminante hrane, ukljuujui iL. monocytogenes.

Kljune rijei: antimikrobna aktivnost, antibi-otika rezistencija, autohtonestarter kulture

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