Characterization of functional properties of Enterococcus spp. isolated from Turkish white cheese

Accepted Manuscript Characterization of functional properties of Enterococcus spp. isolated from Turkish white cheese Hümeyra İspirli, Fatmanur Demirbaş, Enes Dertli PII:

S0023-6438(16)30572-2

DOI:

10.1016/j.lwt.2016.09.010

Reference:

YFSTL 5723

To appear in:

LWT - Food Science and Technology

Received Date: 28 May 2016 Revised Date:

4 September 2016

Accepted Date: 8 September 2016

Please cite this article as: İspirli, H., Demirbaş, F., Dertli, E., Characterization of functional properties of Enterococcus spp. isolated from Turkish white cheese, LWT - Food Science and Technology (2016), doi: 10.1016/j.lwt.2016.09.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Characterization of functional properties of Enterococcus spp. isolated from Turkish White cheese

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Hümeyra İspirli, Fatmanur Demirbaş and Enes Dertli *

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Department of Food Engineering, Faculty of Engineering, Bayburt University, Bayburt, Turkey

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* Enes Dertli, Department of Food Engineering, Faculty of Engineering, Bayburt University, Bayburt, 69000, Turkey Tel: +90 (0) 458 2111153, Fax: +90 (0) 458 2111172, Email: [email protected]

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ACCEPTED MANUSCRIPT Abstract

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Enterococcus species was isolated from Turkish white cheese and genotypic characterisation of

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these isolates revealed the presence of 12 distinct strains belonging to 5 different species:

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Enterococcus faecium, E. faecalis, E. durans, E. gallinarum and E. italicus. All strains showed

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good survival ability under bile salt conditions. The antibiotic resistance of these strains was

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low but two strains showed high levels of resistance including resistance to vancomycin. PCR

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detection of virulence determinant in these isolates revealed that cheese isolate enterococci

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contained virulence genes common in gut and food originated enterococci and importantly two

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strains harboured cylB gene related to cytolysin metabolism. No complete hemolytic activity

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was observed for Enterococcus strains but partial hemolytic activity was observed 8 out of 12

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strains. Additionally, all strains showed important levels of antimicrobial activity against food-

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borne pathogens and PCR screening of genes encoding enterocin A and B indicated the

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presence of enterocin B gene in all tested strains. The antimicrobial activities of the tested

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strains were lost after proteolytic enzyme treatments but no alteration was observed at different

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pHs and after heat treatments. In summary, this study reflected characteristics of Enterococcus

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strains presented in Turkish white cheese in terms of functional and safety perspectives.

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Keywords: Enterococcus species, Turkish white cheese, virulence determinants, antibiotic

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resistance, enterocins

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1. Introduction Recent interests in dairy technology constitute the none starter lactic acid bacteria (NSLAB)

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that can be critical for the development of desirable physicochemical and technological

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properties of different fermented food products including cheeses (Giraffa, 2003). One of the

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main species as NSLAB in cheese production are Enterococci species that have important roles

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during ripening period and they can also be part of cheese starter cultures (Giraffa, 2003). In

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the first place, Enterococci present in GI (gastrointestinal) tract of humans and animals and

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once they spread to the environment especially raw material such as milk, their high

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fermentation and survival ability in different food products place these species as one of the

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key species for the fermentation process (Nueno-Palop & Narbad, 2011; Giraffa, 2003).

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Additionally, Enterococci can be presented in cheese samples produced from both raw and

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pasteurised milk due to their high thermal resistance (Giraffa, 2003). Turkish white cheese is

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the most important cheese variety in Turkey (Dertli, Sert, & Akin, 2012) and can be produced

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under industrial scale as well as artisanal conditions and Enterococci are also important species

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present in Turkish white cheese that can affect the final quality of this cheese type (Çitak,

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Yucel, & Orhan, 2004).

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In addition to the technological functions of Enterococci, production of enterocins that are part

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of bacteriocins (C. M. Franz, Van Belkum, Holzapfel, Abriouel, & Gálvez, 2007), is important

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characteristics of these species result in inhibition of certain pathogens during the fermentation

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and ripening period of cheese and other food products. This characteristic increases their

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potential as both NSLAB as well as probiotic organisms. But as they can present in different

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niches including GI tract and fermented food products, they may show different levels of

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antibiotic resistance which is one of the major concern for food and gut isolates (Nueno-Palop

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& Narbad, 2011). Additionally, some Enterococcal species can be clinical and both food and

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gut isolate Enterococci should be assessed for the presence of virulence determinant genes

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(Eaton & Gasson, 2001). These virulence determinants include cylA and cylB which are

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associated with cytolysin mechanism that can be toxic to both eukaryotic and prokaryotic cells,

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the aggregation and immune evasion factors agg and esp, adhesins efaAfs and efaAfm and cob

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and gelE that may play roles on conjugation and hydrolysation of gelatine, collagen and other

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bioactive compounds, respectively (Eaton & Gasson, 2001; Nueno-Palop & Narbad, 2011).

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These characteristics make Enterococcal species present in food samples such as Turkish white

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cheese important and interesting species to be investigated for their technofunctional properties

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as well as from safety perspective of view (Rehaiem et al., 2014). In this respect the aims of

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ACCEPTED MANUSCRIPT this study are; to isolate and identify Enterococci species from traditional Turkish white cheese,

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to characterize the technofunctional properties of these species such as production of enterocins

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and to determine the safety status of Enterococci species in terms of antibiotic resistance,

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presence of virulence determinants and hemolyis reaction. Results of this study revealed that

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several distinct Enterococcal strains with different characteristics present in Turkish white

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cheese.

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2. Materials and Methods

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2.1. Isolation of Enterococci from Turkish white cheese

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In total seven different traditional white cheese samples were collected from different

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households in Northern Black Sea region of Turkey. Traditional white cheese samples were

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semi-hard in texture and were packed individually in plastic boxes containing brine solution in

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households. For the isolation of Enterococci, 10 g of representative cheese samples prepared by

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cutting out a sector were suspended in 90 ml Phosphate Buffer Saline (PBS) and after

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homogenisation serial dilutions were performed from these suspensions and potential

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Enterococci was isolated by plating to de Man, Rogosa, Sharpe (MRS) and Brain Heart

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Infusion (BHI) (Merck, Germany) agars and incubation of the plates were conducted at 37oC

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under anaerobic conditions for 48 h. From these plates potential different isolates were selected

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and further subcultured in BHI broth containing 6.5% NaCl in order to differentiate the

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Enterococci and these isolates were then stored at − 80 °C in glycerol (40% v/v).

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2.2. Genotypic characterization by RAPD-PCR analysis and bacterial identification by

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16S RNA gene sequencing

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For the discrimination potential Enterococci from cheese samples, in total 100 colonies

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(different isolates) were further tested for genotypic discrimination by RAPD-PCR analysis as

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described elsewhere (Dertli, Mercan, Arıcı, Yılmaz, & Sağdıç, 2016). For the isolation of

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genomic DNA a commercial isolation kit was used and extractions were performed according

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to manufacturer's protocol (Qiagen, Turkey). RAPD-PCR analysis was conducted with primer

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M13. Bacterial genomic DNA was prepared as described above and was used as a template for

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PCR amplification. Each PCR mixture contained 5 × PCR buffer (Promega), 2.5 mM of dNTPs

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(Bioline),

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(GAGGGTGGCGGTTCT). PCR was performed using a thermal cycler (Benchmark, TC9639)

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with the following program: 35 cycles of 94 °C for 1 min, 40 °C for 20 s, then final step of 72

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polymerase

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°C for 2 min. The PCR products were separated with electrophoresis on 1.6% (w/v) agarose

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gels at 90 V for 1.5 h and band patterns were visualised.

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Following genotypic discrimination selected strains were identified by 16S rRNA gene

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sequencing. The c.1.5 kb 16S rRNA gene of the selected strains were amplified with primers

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AMP_F

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AAGGAGGTGATCCARCCGCA-3’) (Baker, Smith, & Cowan, 2003). PCR reaction mixtures

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contained 1 µl DNA template from Genomic DNA, 10 µl 5× PCR buffer, 0.4 µl dNTPs, 1 µl of

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20 mM primers AMP_F and AMP_R, 0.25 µl 5U Taq polymerase and up to 50 µl of sterile

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H2O. PCR was performed with the following programme: 95°C for 2 min, 20 cycles of 95°C

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for 30 s, 55°C for 20 s, and 72°C for 30 s and 72°C for 5 min final extension. PCR products

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were run on a gel to check the amplication and amplicons were sent to Gen Plaza (Turkey) for

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sequencing. Sequences obtained were interrogated with the NCBI database using the BLAST

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algorithm with a similarity criterion of 97–100%. The 16S rRNA gene sequences for

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Enterococci strains were arranged in MEGA7. Phylogenetic trees were constructed using

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neighbour-joining (NJ) method with 1000 bootstrap replicates (Saitou & Nei, 1987). All

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phylogenetic analyses were performed using MEGA7 (Tamura et al., 2011).

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2.3. Resistance to low pH and bile salts

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To assess resistance to low pH and bile salts, strains were grown overnight aerobically in MRS

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medium and then a 1% inoculum subcultured into MRS medium as a control and MRS

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medium either adjusted to pH 4 using 1M HCl or containing bile salts (Bovine bile, Sigma) at

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concentration of 0.3% (v/v) to obtain an OD600 of 0.1. All samples in 20 ml bottles were

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incubated at 37°C unshaken and the growth of Enterococci strains were measured at OD600

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over 24 h using a Spectrophotometer (PG Instruments, T60).

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2.4. Antibiotic susceptibility

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Resistance of Enterococci strains against ampicillin (Amp, 10 µg), chloramphenicol (C, 30 µg),

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erythromycin (E, 15 µg), kanamycin (K, 30 µg), tetracycline hydrochloride (TE, 30 µg),

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vancomycin (VA, 30 µg), Gentamicin (Cn, 10 µg), Rifampicin (Rd, 5 µg), Carbenicillin (CAR,

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100 µg), Amoxicillin (Aml, 25 µg), Oxacillin (Ox, 1 µg) and Streptomycin (S, 10 µg), (Oxoid,

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UK) was determined using antibiotic disks. Each strain was activated in MRS broth and 1%

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inoculum added to MRS agar at 45–50 °C and poured into plates. Then, antibiotic disks were

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inhibition zones around the disks if presented were measured and expressed as centimetre (cm).

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2.5. Screening of Enterococci for virulence determinants

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The PCR screening of the Enterococcal strains for the virulence determinants was conducted

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targeting specific virulence factors (Agg, gelE, cylA, cylB, esp, efaAfs, efaAfm and cob) using

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the primer sets described previously (Eaton & Gasson, 2001). Genomic DNA was isolated

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from overnight cultures of Enterococci using a commercial DNA isolation kit (Qiagen,

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Turkey). The Tm of each primer set was determined and PCR was performed as described

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previously (Eaton & Gasson, 2001).

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2.6. Determination of hemolytic activity

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The haemolytic activity of Enterococcus strains was determined using previously described

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methodology (De Vuyst et al. 2003). Basically strains were grown overnight in MRS medium

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at 37 °C, and then transferred onto BHI and Blood Agar Base (Merck) plates containing 7% of

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human blood (Bayburt State Hospital, Bayburt, Turkey). The plates were incubated overnight

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under aerobic and anaerobic conditions at 37 °C and formation of a clear zone of hydrolysis, a

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partial hydrolysis or no reaction around colonies reflecting β hemolysis, α hemolysis and γ

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hemolysis, respectively was observed for the determination of the hemolytic activity (De Vuyst

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et al. 2003).

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2.7. Bacteriocin activity and detection of bacteriocin coding genes

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For the detection of antagonistic activity with regards to bacteriocin production of Enterococci,

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cultures were grown overnight with 1% inoculation in 10 ml MRS broth. Cells were removed

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by centrifugation at 14,000 g for 5 min. The supernatant was filtered through a sterile 0.22 µm

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syringe filter in order to remove all bacterial cells that may remain in the supernatant. The pH

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of the filtered supernatant was adjusted to pH 6.0 with NaOH for the elimination of possible

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inhibition effects organic acids following the inhibition of H2O2 with catalase (Merck)

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application at 25°C for 30 min. A final filtration step was applied and the 20 µl supernatants

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were applied to the TSB agar plates in which the target pathogen strains were previously spread

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and the antimicrobial activity was observed after 24h incubation at 37°C by measurement of

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the formed inhibition zones in each strain and expressed as diameters of the inhibition zone in

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mm. Three pathogen strains that can be present in white cheese; Salmonella typhimurium

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RSSK 95091, Escherichia coli BC 1402 and Staphylococcus aureus ATCC 25923 were used in

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this study. All pathogens were grown with Tryptic Soy Broth (TSB) medium under aerobic

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conditions at 37°C.

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The presence of the genes encoding enterocin A and B was PCR detected for E. faecium strains

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with

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(GCACTTCCCTGGAATTGCTC)

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entB_R (GTTGCATTTAGAGTATACATTTG), respectively (Toit, Franz, Dicks, & Holzapfel,

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2000). DNA was isolated from each strain and PCR conditions were performed as described

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previously (Fontana, Cocconcelli, Vignolo, & Saavedra, 2015). PCR products were run on a

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gel to check the amplicon size of 126 bp and 162 bp for the presence of enterocin A and B,

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respectively.

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2.8. Effect of enzymes, detergents, pH and temperature on the bacteriocin activity

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The effects of different conditions on bacteriocinogenic activity of Enterococci was tested as

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described previously (İspirli, Demirbaş, & Dertli, 2015). Briefly, the supernatants of

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Enterococcal strains were obtained as described above and proteinase K (Sigma), pepsin

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(Sigma) and catalase (Merck) were added to the supernatants at final concentration of 1 mg/ml

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and following 2 h incubation the antimicrobial activity of the supernatants was tested against

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all pathogens as described above. Similarly, for the determination of the effect of the detergents

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on bacteriocin activity of Enterococci, sodium dodecyl sulphate (SDS), Tween 20 and Triton

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X-100 were added to the supernatants at 1% final concentration and after 5 h incubation at

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37°C the antimicrobial activities were observed as described above. The effect of pH on

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bacteriocin activity was determined by adjusting the supernatants between pH 3.5 and 9.5 with

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sterile 1 N HCl or 1 N NaOH. Finally effect of high temperature on bacteriocin activity was

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determined by heating the supernatants at 80, 90 and 100°C for 30 min. Untreated supernatants

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of Enterococci were applied as control in all experiments.

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3. Results

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3.1. Isolation and Characterization of Enterococus strains

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In this study, the presence of Enterococci in traditional Turkish white cheese was tested and

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different Enterococcal strains were isolated and their functional roles including resistance to

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low pH and bile salts, inhibition of pathogens and presence of bacteriocin coding genes and

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production of bacteriocin(s) were determined. Additionally, from a safety perspective of view

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the antibiotic susceptibility and the presence of the virulence determinant genes within these

entA_F

(AAATATTATGGAAATGGAGTGTAT), and

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ACCEPTED MANUSCRIPT strains and the hemolytic activity of these strains were tested. After genotyping characterisation

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followed by the 16S rRNA gene sequencing distinct strains were identified in which 5

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Enterococcus durans, 4 Enterococcus faecalis, one of each Enterococcus faecium,

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Enterococcus gallinarum and Enterococcus italicus strains were presented in Turkish white

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cheese samples. Figure 1 represents the MEGA5 alignments of the 16S rRNA genes of distinct

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Enterococcal strains showing their phylogenetic relationship with the formation of different

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subgroups. The cluster alignments analysis showed that 16S rRNA sequences of the strains of

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Enterococcus durans and Enterococcus faecium were close and Enterococcus gallinarum

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strain placed separately from these strains with a number of nucleotide substitution. Similarly,

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Enterococcus faecalis strains formed a different subgroup compared to the other strains and

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this alignment analysis placed Enterococcus italicus strain separately from the other strains

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(Figure 1).

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3.2. Resistance to low pH and bile salts conditions

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The resistance of Enterococcal strains to low pH and bile salts conditions were tested and in

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general all strains except Enterococcus faecium BP8, Enterococcus durans BP10 and BP126

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showed similar growth rates compared to the control group under pH 4 conditions and the final

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concentrations of these three strains in medium environment were around 4 log10 cfu ml-1

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whereas their numbers in the control samples of these strains which were grown in standard

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MRS medium were around 8 log10 cfu ml-1 at the end of incubation period which reveals the

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40-50% reduction in the growth of these three strains under pH 4 conditions. Additionally, all

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Enterococcal strains showed similar growth rates under 0.3% (v/v) bile salt conditions

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compared to the control group as standard MRS medium (data not shown).

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3.3. Antibiotic susceptibility of Enterococcal strains

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There is a growing concern for the antibiotic resistance of food originated bacterial strains and

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in this respect the antibiotic resistance of Enterococcal strains isolated from Turkish white

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chese was determined (Table 1). Several patterns were observed for the antibiotic resistance of

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Enterococcal strains and three strains Enterococcus italicus BP45, Enterococcus faecalis PY44

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and Enterococcus durans PY113 showed no antibiotic resistance and strains Enterococcus

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durans BP2, Enterococcus faecium BP8, Enterococcus durans BP10 and Enterococcus

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gallinarum FN6 were only found to be resistant to Oxacillin and strain Enterococcus faecalis

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BP21 was both resistant to Oxacillin and Streptomycin. Similarly, strain Enterococcus faecalis

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PY99 was found to be resistant to Rifampicin, Oxacillin and Vancomycin. The highest

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ACCEPTED MANUSCRIPT antibiotic resintance among these strains was observed for strains Enterococcus durans PY126

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and Enterococcus durans PY146 and these strains were only sensitive to Kanamycin,

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Streptomycin and Gentamicin and Oxacillin for Enterococcus durans PY126 showing highest

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incidence of antibiotic resistance in these strains (Table 1).

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3.4. Distribution of virulence determinants in Enterococcal strains

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Another important concern for the Enterococcal strains is the presence of several virulence

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factors within these strains. In this respect, Enterococcal strains were PCR screened for the

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presence of the putative genes coding for the known virulent factors (Table 2). Enterococcus

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durans BP2 and Enterococcus italicus BP45 harboured only one virulence factor and these

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were gelE and efaAfm, respectively. The highest incidence of the presence of virulence

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determinants was observed for strains Enterococcus faecalis PY99 and Enterococcus durans

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PY113 and five virulence determinants including cylB were presented in these strains (Table

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2). The other tested strains were positive for two to three virulence determinants and in general

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gelE, efaAfs and efaAfm were presented in Enterococcal strains.

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3.5. Detection of hemolysis in Enterococcal strains

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The hemolytic activity of Enterococcal strains is also an important concern for their selection

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as starter cultures as well as probiotic organisms. In this study, Enterococcus durans BP2,

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Enterococcus faecalis BP4 and PY44 and Enterococcus gallinarum FN6 showed no hemolysis

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of human blood (γ hemolysis) whereas the other tested strains showed partial hydrolysis (α

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hemolysis) of human blood (Table 3).

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3.6. Inhibition of pathogens by Enterococcal strains

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Selection of starter cultures with antimicrobial properties generally originating from

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bacteriocin(s) production against certain pathogens has gained special interest and in this

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respect, the antagonistic activity of Enterococcal strains against food-borne pathogen strains

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was tested in this study (Table 4). E. gallinarum FN6 was the only strain showed no inhibition

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to the tested pathogens. Apart from this strain all strains showed antimicrobial activity against

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E. coli with different levels. Together with E. gallinarum FN6, E. faecalis PY99 did not show

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antimicrobial activity against S. typhimurium. Additionally, 4 out of 12 Enterococcal strains did

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not show any antimicrobial activity to Staph. aureus strain. This antimicrobial activity of

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Enterococcal strains depends on the production of bacteriocins and/or bacteriocin like

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substances by these species. We tested the presence of the genes coding for enterocins A and B

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ACCEPTED MANUSCRIPT genes in order to understand the origin of their antimicrobial activity. The PCR evaluation

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revealed that all strains were positive for the presence of enterocin A gene whereas only E.

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durans BP2 was found to be positive for the enterocin B gene suggesting production of

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enterocins by these cheese isolates. We then tested the effects of enzymes, detergents, different

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pH and high temperatures on the bacteriogenic effects of these isolates. As bacteriocins are

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proteinous substances, treatment of the cell-free supernatants of these Enterococcal strains with

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Proteinase K and pepsin resulted in complete loss of antimicrobial activity against tested

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pathogens. But no alteration of the antimicrobial activity observed after catalase treatment,

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setting the pH of the supernatant between 3.5-9.5 as well as high temperature application as 80,

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90 and 100°C 30 min and after SDS treatment. These results suggest that the antimicrobial

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activity of Enterococcal strains originates from production of bacteriocins.

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4. Discussion

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Enterococci species are important type of microorganisms that can be present in variety of

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cheeses produced by traditional ways as well as produced from pasteurised milk. The main

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roles of Enterococci species in cheese samples are related with their functions as nonstarter

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Lactic Acid Bacteria (NSLAB). Their presence and growth during the ripening period as

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NSLAB strains results in the development of flavour and physicochemical properties of semi-

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hard and hard cheese varieties due to their proteolytic and lipolytic activities (Giraffa, 2003).

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The presence of enterococci in Turkish white cheese might have important technological

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functions during its production as the manufacturing process of Turkish white cheese also

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includes the ripening period and these functions can be related with their biochemical

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properties as well as with their interactions with other desirable strains such as lactobacilli

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(Öner et al., 2006). Similarly, it was reported that E. faecium strains positively affected the

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technological functions of Feta cheese which have similar production process with white

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cheese supporting the role of enterococci in white cheese (Sarantinopoulos et al., 2002). In

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addition to their role as starter and adjunct cultures, several Enterococci species were also

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shown to act as probiotics (Eaton & Gasson, 2001). Despite their functional roles as starter

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cultures and probiotics, some Enterococcal strains can be detrimental to human and animal

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health due to presence of some virulence factors within these strains and also like other food or

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animal and human originated non-pathogenic strains they may possess important levels of

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antibiotic resistance genes which limits their potential usage as starter cultures and probiotics

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(Carlos, Semedo-Lemsaddek, Barreto-Crespo, & Tenreiro, 2010; Nueno-Palop & Narbad,

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ACCEPTED MANUSCRIPT 2011). In this respect, this study aimed to investigate the potential of different Enterococcal

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strains isolated from Turkish white cheese for their technofunctional roles.

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The isolation and identification of Enterococcal strains revealed the presence of E. faecium, E.

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faecalis, E. durans, E. gallinarum and E. italicus strains in Turkish white cheese. Previous

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reports showed the presence of these species except E. gallinarum and E. italicus strains in

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Turkish white cheese samples (Çitak et al., 2004; Hajikhani, Beyatli, & Aslim, 2007).

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Similarly, all strains except E. italicus were reported to be present in Feta cheese samples

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(Manolopoulou et al., 2003). E. italicus is also a strain associated with artisanal Italian cheeses

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(Fortina, Ricci, Mora, & Manachini, 2004) and presence of this strain in Turkish white cheese

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suggests its importance for variety of cheese samples. There can be several sources of

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enterococci presenting in traditional cheese samples such as milk, personnel involved in cheese

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making, milking equipment, bulk and cheese tanks (Gelsomino, Vancanneyt, Cogan, Condon,

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& Swings, 2002). In a comprehensive study reported the source of enterococci in traditional

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milk samples, the main contamination source of enterococci was suggested to be the process

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equipment in which the original source of these species in these instruments was reported to be

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unclear (Gelsomino et al., 2002). Similarly, Giraffa (2003) pointed out the main reason for the

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prevalence of enterococci in dairy products as the poor hygienic conditions during processing

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of milk. We also suggest that the poor processing conditions in terms of sanitization may be the

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main reason for the presence of these species in Turkish white cheese as these samples were

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collected from different households where a traditional way of production in terms of

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processing is still the case. Another source can be the brine solution as some species of

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enterococci can grow at 6.5% NaCl conditions but this probability is also related with the

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quality of tap water, pasteurisation conditions and processing tanks as well. It was reported to

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be very low in brine solutions used for the production of Feta cheese (Bintsis et al., 2000).

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Nevertheless, these results showed that Turkish white cheese samples have an important

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Enterococcal microflora and these strains can be crucial for the physicochemical development

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of white cheese as NSLAB and these strains may act potential probiotic strains.

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The resistance to low pH is crucial for the technofunctional properties of Enterococcal strains

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(Nueno-Palop & Narbad, 2011) and generally Enterococcal strains have good ability to grow

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under bile salt conditions (İspirli, Demirbaş, & Dertli, 2015). Our results were in agreement

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with previous observations that only three strains showed growth reduction under low pH

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conditions and no alteration in the growth of cheese isolate E. faecium, E. faecalis, E. durans,

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E. gallinarum and E. italicus strains was observed (Banwo, Sanni, & Tan, 2013; İspirli,

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ACCEPTED MANUSCRIPT Demirbaş, & Dertli, 2015; Pieniz, Andreazza, Anghinoni, Camargo, & Brandelli, 2014). The

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ability of these strains to grow under bile salt conditions can be related with their bile salt

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hydrolase activities as previously suggested (C. M. A. P. Franz, Specht, Haberer, & Holzapfel,

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2001). Both tests revealed the potential of these isolates as starter cultures as well as probiotic

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organisms.

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The antibiotic resistance of food and gut isolate strains including Enterococci is a major

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concern for selecting these strains as starter cultures or probiotic organisms due to the potential

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risk of transmittance of this resistance to non-resistant microorganisms (Hasman, Villadsen, &

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Aarestrup, 2005). Our results revealed that in general Enterococcal strains isolated from white

351

cheese revealed low level of resistance to the tested antibiotics but several trends were also

352

observed. For instance, two strains E. durans PY126 and E. durans PY146 showed high level

353

of resistance to the tested antibiotics including vancomycin which represents a major health

354

problem worldwide (Cetinkaya, Falk, & Mayhall, 2000). Apart from these two strains the other

355

tested strains showed lower level of incidence of antibiotic resistance compared to the other

356

studies conducted with Enterococci isolated from different sources including Turkish white

357

cheese (Abriouel et al., 2008; Çitak et al., 2004). A similar observation was reported on the low

358

antibiotic resistance of Enterococci isolated from goat’s milk showing that not only the food

359

origin but also other factors such as animal health or hygienic conditions may affect the

360

transmission of resistance (Schirru et al., 2012). Consequently, although generally low level of

361

antibiotic resistance among Enterococci was observed in this study, the presence of

362

vancomycin-resistant Enterococci in cheese samples should be noted.

363

Similar to the antibiotic resistance, Enterococci may possess several virulence factors and the

364

presence of putative virulence genes should be detected for safety considerations. The highest

365

number of the virulence determinants presented in cheese isolates was 5 detected for two

366

strains and generally these numbers were altered between 2 to 3. Previous reports revealed that

367

Enterococci especially clinical isolates harbours 6 to 11 virulence genes (Nueno-Palop &

368

Narbad, 2011) and these numbers can be lower for the non-clinical isolates (Eaton and Gasson

369

2001) and non-present for the food isolates (Santos et al., 2015; Zheng et al., 2015). The

370

presence of adhesion factors efaAfm and efaAfs genes was high in cheese isolate Enterococci

371

which was also the fact for other food and gut isolate strains reported previously and these two

372

virulence factor do not represent a high risk value (Eaton & Gasson, 2001; Ruiz-Moyano et al.,

373

2009) although the latter was shown to influence pathogenicity in animal models (Singh,

374

Coque, Weinstock, & Murray, 1998). Similarly, 2 out of 12 strains were positive for the other

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ACCEPTED MANUSCRIPT adhesion determinant (esp) supporting low level of occurrence of this indicator in non-medical

376

isolates as previously suggested (Eaton & Gasson, 2001). None of our tested isolates were

377

positive for the sex pheromone determinants (cob and agg) that can be related with both

378

virulence and antibiotic resistance gene transfer mechanisms (Ruiz-Moyano et al., 2009)

379

although a these virulence genes were reported to be at high occurrence level in Enterococci

380

(Eaton & Gasson, 2001). The presence of gelE which is associated with gelatinase activity as a

381

pathogenicity factor was at high levels in our study which was in accordance with previous

382

observations for both food isolates (Hammad, Hassan, & Shimamoto, 2015) as well as gut

383

isolates (Eaton & Gasson, 2001; İspirli et al., 2015). Importantly, two strains harboured the

384

cylB gene which is related with cytolysin metabolism that is the most important virulence trait

385

which lyses the eukaryotic cells (Kayser, 2003) and previous reports revealed the non-presence

386

of this virulence factor in non-clinical isolates including food originated Enterococci (Abriouel

387

et al. 2008; Nueno-Palop & Narbad 2011) but a recent report also showed presence of cyl gene

388

in cheese isolate (Hammad et al., 2015) Enterococci suggesting that the incidence of the

389

presence of this virulence factor may increase in future if positive strains will not be eliminated

390

from the food environment with related applications.

391

Similar to the other safety concerns, the hemolysis activity of Enterococcus strains should be

392

determined and in this study 8 out 12 strains showed α-hemolysis and rest of the strains showed

393

no hemolytic reaction and importantly no β-hemolysis was observed with the tested strains.

394

Previously several food and non-clinical isolates were reported to show both α-hemolysis and

395

β-hemolysis with the latter was observed to be at very low levels (Barbosa, Gibbs, & Teixeira,

396

2010; Eaton & Gasson, 2001). Although no β-hemolysis observed in our study, the high level

397

of α-hemolysis among Enterococci can be an important concern for the potential usage of this

398

strain as functional organisms.

399

Production of artisanal cheeses generally depend on use of unpasteurised milk and if other

400

cautions such as long term ripening are not applied and several pathogenic strains such as E.

401

coli, S. aureus and S. typhimurium that can cause serious health problems can be presented in

402

white-brined cheeses (Bintsis & Papademas, 2002). One of the expected properties of both

403

starter and NSLAB strains is production bacteriocins that inhibit the growth of certain

404

pathogens. Our results revealed that in general, all tested Enterococcus strains showed varying

405

levels of antimicrobial effects to selected Gram + and Gram – pathogens suggesting the

406

production of bacteriocins and some strains for instance E. durans BP2 showed high level of

407

inhibitory effect to the tested pathogens. Previous reports also showed that Enterococcal strains

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pathogens (De Kwaadsteniet, Todorov, Knoetze, & Dicks, 2005; Line et al., 2008). We also

410

tested the presence of the genes required for the production of enterocins A and B and all

411

strains were found to be positive for the enterocin B gene supporting that this antimicrobial

412

activity was depended on the production of enterocins as antimicrobial peptides (Fontana et al.,

413

2015). Importantly, when the supernatants of the Enterococcal strains treated with the

414

proteolytic enzymes all inhibitory activity was lost due to the proteinaceous nature of

415

bacteriocins as previously reported for other strains (Campos, Rodríguez, Calo-Mata, Prado, &

416

Barros-Velázquez, 2006; De Kwaadsteniet et al., 2005) whereas catalase treatment resulted in

417

no alteration in antimicrobial activity supporting the role of enterocins on inhibitory activity.

418

Additionally, after treatment with detergents, heat applications and at low and high pH values

419

between 3.5 and 9.5, no difference in the bacteriocinogenic activity of the Enterococcal strains

420

was observed and similar results were recorded previously (Campos et al., 2006; De

421

Kwaadsteniet et al., 2005; İspirli et al., 2015). These findings support the technofunctional

422

properties of the white cheese isolate Enterococcus strains for further applications as NSLAB

423

as well as potential probiotic strains.

424

5. Conclusion

425

In conclusion, this study showed the presence of distinct Enterococcal strains belonged to E.

426

durans, E. faecalis, E. faecium, E. italicus and E. gallinarum in Turkish white cheese which

427

might have significant technological functions during its production. These strains were further

428

tested for their low pH and bile salt conditions and promising results were obtained in terms of

429

their survival under these conditions. From safety perspective, the antibiotic resistance,

430

presence of the virulence factors as well as hemolytic reaction were tested in these strains and

431

some undesirable characteristics were observed for food isolate Enterococci. Nevertheless, all

432

strains were found to be effective for the inhibition of food-borne pathogens and this

433

antimicrobial role was suggested to be depending on enterocin production due to the PCR

434

detection of enterocin gene as well as proteinaceous nature of the antimicrobial component.

435

Further work will characterise the role of selected Enterococci strains as NSLAB in white

436

cheese environment as well as their probiotic potential.

437

Acknowledgments

438

This research was supported by Bayburt University through an internal fund.

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Abriouel, H., Omar, N. B., Molinos, A. C., López, R. L., Grande, M. J., Martínez-Viedma, P., Ortega, E., Cañamero, M.M. & Galvez, A.. (2008). Comparative analysis of genetic diversity and incidence of virulence factors and antibiotic resistance among enterococcal populations from raw fruit and vegetable foods, water and soil, and clinical samples. International journal of food microbiology, 123(1), 38-49. Baker, G. C., Smith, J. J., & Cowan, D. A. (2003). Review and re-analysis of domain-specific 16S primers. Journal of Microbiological Methods, 55(3), 541-555. doi: http://dx.doi.org/10.1016/j.mimet.2003.08.009 Banwo, K., Sanni, A., & Tan, H. (2013). Technological properties and probiotic potential of Enterococcus faecium strains isolated from cow milk. Journal of Applied Microbiology, 114(1), 229-241. doi: 10.1111/jam.12031 Barbosa, J., Gibbs, P. A., & Teixeira, P. (2010). Virulence factors among enterococci isolated from traditional fermented meat products produced in the North of Portugal. Food Control, 21(5), 651-656. doi: http://dx.doi.org/10.1016/j.foodcont.2009.10.002 Bintsis, T., & Papademas, P. (2002). Microbiological quality of white-brined cheeses: a review. International Journal of Dairy Technology, 55(3), 113-120. Campos, C. A., Rodríguez, Ó., Calo-Mata, P., Prado, M., & Barros-Velázquez, J. (2006). Preliminary characterization of bacteriocins from Lactococcus lactis, Enterococcus faecium and Enterococcus mundtii strains isolated from turbot (Psetta maxima). Food Research International, 39(3), 356-364. doi: http://dx.doi.org/10.1016/j.foodres.2005.08.008 Carlos, A. R., Semedo-Lemsaddek, T., Barreto-Crespo, M. T., & Tenreiro, R. (2010). Transcriptional analysis of virulence-related genes in enterococci from distinct origins. Journal of Applied Microbiology, 108(5), 1563-1575. doi: 10.1111/j.1365-2672.2009.04551.x Cetinkaya, Y., Falk, P., & Mayhall, C. G. (2000). Vancomycin-resistant enterococci. Clinical microbiology reviews, 13(4), 686-707. Çitak, S., Yucel, N., & Orhan, S. (2004). Antibiotic resistance and incidence of Enterococcus species in Turkish white cheese. International Journal of Dairy Technology, 57(1), 27-31. doi: 10.1111/j.1471-0307.2004.00122.x De Kwaadsteniet, M., Todorov, S. D., Knoetze, H., & Dicks, L. M. T. (2005). Characterization of a 3944 Da bacteriocin, produced by Enterococcus mundtii ST15, with activity against Gram-positive and Gram-negative bacteria. International Journal of Food Microbiology, 105(3), 433-444. doi: http://dx.doi.org/10.1016/j.ijfoodmicro.2005.03.021 Dertli, E., Mercan, E., Arıcı, M., Yılmaz, M. T., & Sağdıç, O. (2016). Characterisation of lactic acid bacteria from Turkish sourdough and determination of their exopolysaccharide (EPS) production characteristics. LWT - Food Science and Technology, 71, 116-124. doi: http://dx.doi.org/10.1016/j.lwt.2016.03.030 Dertli, E., Sert, D., & Akin, N. (2012). The effects of carbon dioxide addition to cheese milk on the microbiological properties of Turkish White brined cheese. International Journal of Dairy Technology, 65(3), 387-392. doi: 10.1111/j.1471-0307.2012.00843.x Eaton, T. J., & Gasson, M. J. (2001). Molecular Screening of EnterococcusVirulence Determinants and Potential for Genetic Exchange between Food and Medical Isolates. Applied and environmental microbiology, 67(4), 1628-1635. Fontana, C., Cocconcelli, P. S., Vignolo, G., & Saavedra, L. (2015). Occurrence of antilisterial structural bacteriocins genes in meat borne lactic acid bacteria. Food Control, 47(0), 53-59. doi: http://dx.doi.org/10.1016/j.foodcont.2014.06.021 Fortina, M. G., Ricci, G., Mora, D., & Manachini, P. (2004). Molecular analysis of artisanal Italian cheeses reveals Enterococcus italicus sp. nov. International journal of systematic and evolutionary microbiology, 54(5), 1717-1721.

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Sarantinopoulos, P., Kalantzopoulos, G., & Tsakalidou, E. (2002). Effect of Enterococcus faecium on microbiological, physicochemical and sensory characteristics of Greek Feta cheese. International Journal of Food Microbiology, 76 (1-2) 93-105. Schirru, S., Todorov, S. D., Favaro, L., Mangia, N. P., Basaglia, M., Casella, S., Comunian, R., de Melo Franco, B.D.G., & Deiana, P. (2012). Sardinian goat’s milk as source of bacteriocinogenic potential protective cultures. Food Control, 25(1), 309-320. doi: http://dx.doi.org/10.1016/j.foodcont.2011.10.060 Singh, K. V., Coque, T. M., Weinstock, G. M., & Murray, B. E. (1998). In vivo testing of an Enterococcus faecalis efaA mutant and use of efaA homologs for species identification. FEMS Immunol Med Microbiol, 21(4), 323-331. Toit, M. D., Franz, C. M. A. P., Dicks, L. M. T., & Holzapfel, W. H. (2000). Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces. Journal of Applied Microbiology, 88(3), 482-494. doi: 10.1046/j.13652672.2000.00986.x Zheng, W., Zhang, Y., Lu, H. M., Li, D. T., Zhang, Z. L., Tang, Z. X., & Shi, L. E. (2015). Antimicrobial activity and safety evaluation of Enterococcus faecium KQ 2.6 isolated from peacock feces. BMC Biotechnol, 15, 30. doi: 10.1186/s12896-015-0151-y

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537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553

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554 555 556 557

561 562 563 564 565 566

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560

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567 568 569 570 571 17

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Table 1. Resistance of Enterococcus strains against antibiotics (inhibition zone, cm).

574 575

Table 2. Detection of some virulence determinant genes in Enterococcus strains tested in this study.

576

Table 3. Hemolytic activity of Enterococcus strains

577

Table 4. Antibacterial activity of Enterococcus strains against selected pathogens.

578 579 580

Figure 1. Dendrogram showing multiple sequence alignment of 16S rRNA gene sequences of Enterococcus strains. The phylogenetic analysis was conducted as described previously (Dertli et al., 2016)

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b

-

C

CAR

CN

E

K

OX

1.0±0.1 1.5±0.1 0.8±0.1 1.0±0.1 0.8±0.1 1.5±0.1 1.1±0.1 0.9±0.2 0.8±0.1 1.0±0.1

1.2±0.1 2.4±0.1 0.9±0.1 1.0±0.1 1.0±0.1 2.2±0.2 1.0±0.1 0.4±0.2 0.8±0.1 1.0±0.1

1.1±0.1 1.2±0.1 1.0±0.1 1.1±0.1 0.8±0.02 1.6±0.1 1.0±0.1 0.8±0.02 0.2±0.02 1.0±0.1

-

-

0.6±0.1 0.2±0.1 0.5±0.1 0.2±0.1 1.1±0.1 0.5±0.1 0.5±0.1 0.2±0.1 0.5±0.1 0.6±0.1 0.5±0.1

0.4±0.2 0.7±0.1

-

0.6±0.01 0.4±0.01 0.5±0.01 0.2±0.02 0.6±0.01 0.3±0.02 0.4±0.01 0.3±0.01 0.4±0.01 0.4±0.01 0.4±0.1

-

RD

S

1.2±0.1 0.9±0.1 1.1±0.1 0.6±0.2 0.6±0.1 1.4±0.1 0.6±0.1 0.2±0.2 0.2±0.2

0.2±0.02 1.5±0.1 1.5±0.1 0.7±0.02 0.2±0.02 0.7±0.02 0.1±0.02 0.2±0.1 0.8±0.2 0.1±0.01 0.6±0.1 0.4±0.1 0.3±0.1

TE

VA

1.1±0.2 1.25±0.1 1.1±0.2 1.1±0.2 0.1±0.02 1.5±0.1 1.1±0.1 0.8±0.02 0.1±0.02 1.0±0.1

0.7±0.2 1.1±0.1 0.6±0.2 0.7±0.2 0.5±0.2 0.9±0.1 0.6±0.2 0.6±0.2

-

-

Table 1. Resistance of Enterococcus strains against antibiotics (inhibition zone, cm). a

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Codes represent the strain code of each strain given in Figure 1.

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b

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Aml: Amoxicillin, Amp: Ampicillin, CAR: Carbenicillin, C: Chloramphenicol, Cn: Gentamicin, E: Erythromycin, K: Kanamycin, Ox: Oxacillin, Rd: Rifampicin, S: Streptomycin, TE: Tetracycline, Va: Vancomycin, - No inhibition zone.

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PY126 PY146

AMP 1.1±0.2 1.3±0.1 0.8±0.01 1.0±0.1 0.9±0.01 1.9±0.1 1.0±0.1 0.4±0.02 0.5±0.02 1.1±0.1

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1.1±0.1 BP2 1.5±0.1 BP4 0.9±0.1 BP8 BP10 1.0±0.1 BP21 0.9±0.1 BP45 2.2±0.1 1.1±0.1 FN6 PY44 0.6±0.2 PY99 0.6±0.1 PY113 1.0±0.1

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AMLa

-

0.6±0.2

ACCEPTED MANUSCRIPT Table 2. Detection of some virulence determinant genes in Enterococcus strains tested in this study.

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Strains gelE agg cob cylA cylB esp efaAfs efaAfm Enterococcus durans (BP2) + Enterococcus faecalis (BP4) + + + Enterococcus faecium (BP8) + + Enterococcus durans (BP10) + + + Enterococcus faecalis (BP21) + + + Enterococcus italicus (BP45) + Enterococcus gallinarum (FN6) + + Enterococcus faecalis (PY44) + + Enterococcus faecalis (PY99) + + + + + Enterococcus durans (PY113) + + + + + Enterococcus durans (PY126) + + Enterococcus durans (PY146) + + + Results reveals the PCR detection of related gene; + and – represent the presence and absence of the corresponding gene.

ACCEPTED MANUSCRIPT Table 3. Hemolytic activity of Enterococcus strains

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Enterococcus durans (BP2) Enterococcus faecalis (BP4) Enterococcus faecium (BP8) Enterococcus durans (BP10) Enterococcus faecalis (BP21) Enterococcus italicus (BP45) Enterococcus gallinarum (FN6) Enterococcus faecalis (PY44) Enterococcus faecalis (PY99) Enterococcus durans (PY113) Enterococcus durans (PY126) Enterococcus durans (PY146)

α γ hemolysis hemolysis + + + + + + + + + + + + -

ACCEPTED MANUSCRIPT Table 4. Antibacterial activity of Enterococcus strains against selected pathogens. S. typhimurium ++ ++ ++ ++ ++ ++ + + + +

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S. aureus ++ ++ ++ ++ ++ + + +

SC

Enterococcus durans (BP2) Enterococcus faecalis (BP4) Enterococcus faecium (BP8) Enterococcus durans (BP10) Enterococcus faecalis (BP21) Enterococcus italicus (BP45) Enterococcus gallinarum (FN6) Enterococcus faecalis (PY44) Enterococcus faecalis (PY99) Enterococcus durans (PY113) Enterococcus durans (PY126) Enterococcus durans (PY146)

E. coli ++ ++ ++ ++ ++ ++ + + + + +

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Results are expressed as diameters of the inhibition zone and standard deviations in mm. ++ inhibition zone 5-10 mm, + inhibition zone 1-5 mm, -: no inhibiton zone

ACCEPTED MANUSCRIPT

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Figure 1. Dendrogram showing multiple sequence alignment of 16S rRNA gene sequences of Enterococcus strains. The phylogenetic analysis was conducted as described previously (Dertli et al., 2016)

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The genotypic characterisation revealed the presence of 12 distinct Enterococcus strains belonging to 5 different species in Turkish white cheese. Enterococcus faecium, E. faecalis, E. durans, E. gallinarum and E. italicus.presented in white cheese. Two strains showed resistance to vancomycin and the cylB gene realated to cytolysin metabolism presented in another two Enterococcus strains. All strains showed important levels of antimicrobial effects against food-borne pathogens with the presence of genes related to enterocin production.

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