Accepted Manuscript Microbial communities involved in Kashar cheese ripening Oğuz Aydemir, Henning Harth, Stefan Weckx, Muhammet Dervişoğlu, Luc De Vuyst PII:

S0740-0020(14)00252-4

DOI:

10.1016/j.fm.2014.10.002

Reference:

YFMIC 2281

To appear in:

Food Microbiology

Received Date: 25 August 2014 Revised Date:

1 October 2014

Accepted Date: 7 October 2014

Please cite this article as: Aydemir, O., Harth, H., Weckx, S., Dervişoğlu, M., De Vuyst, L., Microbial communities involved in Kashar cheese ripening, Food Microbiology (2014), doi: 10.1016/ j.fm.2014.10.002. 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.

ACCEPTED MANUSCRIPT 1

Microbial communities involved in Kashar cheese ripening

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Oğuz Aydemir a,*, Henning Harth b, Stefan Weckx b, Muhammet Dervişoğlu c, Luc De

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Vuyst b

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a

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18100 Çankırı, Turkey

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Department of Food Engineering, Faculty of Engineering, Çankırı Karatekin University,

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b

Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Department

of Bioengineering Sciences, Faculty of Sciences and Bioengineering Sciences, Vrije

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Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium

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c

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Samsun, Turkey

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Department of Food Engineering, Faculty of Engineering, Ondokuz Mayıs University, 55139

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*Corresponding author:

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Asst. Prof. Dr. Oğuz Aydemir, Department of Food Engineering, Faculty of Engineering,

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Çankırı Karatekin University, 18100 Çankırı, Turkey

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Tel: +90 376 2189500–8353. E–mail: [email protected]

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ABSTRACT

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The microbiota of non–starter lactic acid bacteria (NSLAB) and their concomitant community

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dynamics during cheese ripening were investigated for traditional Turkish Kashar cheeses

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made from raw cows’ milk. Five batches of 15 Kashar cheeses produced in different dairy

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plants located in Kars were analyzed during their whole ripening phase up to 180 days.

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Lactobacilli and lactococci were determined as the prevailing microbial groups. The

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molecular classification and identification of 594 LAB isolates during Kashar cheese ripening

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were performed through (GTG)5–PCR fingerprinting of their genomic DNA followed by

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verification of the (GTG)5–PCR clusters obtained after numerical analysis through 16S rRNA

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gene sequencing of representative isolates. Lactobacillus casei (247 isolates, 41.6 %),

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Lactobacillus plantarum (77 isolates, 13.0 %), and Pediococcus acidilactici (58 isolates, 9.8

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%) were the prevailing NSLAB species in all Kashar cheeses of the different dairy plants

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investigated throughout cheese ripening. The data of the present study contribute to the

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inventory of unique cheese varieties to enable the prevention of losses of microbial

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biodiversity and the selection of starter cultures for controlled cheese manufacturing.

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Keywords Kashar cheese, Cheese ripening, Non–starter lactic acid bacteria

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1. Introduction

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Raw milk cheeses produced from unpasteurized milk and following traditional

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manufacturing procedures consist of a very diverse and rich microbial ecosystem, of which

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the composition highly influences the quality of the cheeses (McSweeney et al., 1993;

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Grappin and Beuvier, 1997; De Angelis et al., 2001; Marino et al., 2003). The coagulation

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temperature of the milk will facilitate the growth of most microorganisms; however, the

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temperature applied during curd processing has the potential to inhibit the growth of some of

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them (Steffen et al., 1993). While variations in manufacturing parameters such as cooking

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temperature and curd handling play an important role in determining the characteristics of

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cheeses, the cheese ripening microbiota plays a critical and crucial role in the development of

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the unique characteristics of each cheese variety (Beresford et al., 2001). Non–starter lactic

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acid bacteria (NSLAB) from the environment are a significant proportion of the microbial

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ACCEPTED MANUSCRIPT communities of most ripened cheeses. They do not contribute to acid production during

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cheese manufacture as the LAB starter culture does, but do have an impact on flavour

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development in the ripening cheese (Beresford and Williams, 2004). The primary

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environmental factors controlling the growth of microorganisms in the cheese matrix are

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water and salt content, pH, presence of organic acids and nitrate, redox potential, and ripening

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temperature (Beresford et al., 2001). Increasing information on the natural microbiota present

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in cheeses can help to prevent a loss of microbial biodiversity and consequently a loss of a

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wide range of cheeses produced by different methods, whose typical features depend on local

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and regional traditions and on the indigenous microbial communities present in raw milk and

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selected for by the cheesemaking environment (Fortina et al., 2003).

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Kashar cheese, a pasta–filata cheese made from raw ewes’ or cows’ milk or a mixture

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of them, is one of the most important Turkish cheese varieties and is similar to Caciocavallo,

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Provolone, Regusono, Kashkaval, and Mozzarella (Aran, 1998; Çetinkaya and Soyutemiz,

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2006). However, nowadays, it is generally made from raw cow’s milk (Kamber, 2008).

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Kashar cheese is typically produced in the city of Kars situated in the North–East of Turkey.

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Kashar cheeses have typically a cylindrical shape of around 30 cm in diameter and a height of

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20 cm, with an average weight of 12 kg.

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In general, Kashar cheeses are made in a traditional way, although their ripening phase

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is a laborious and expensive process. At farmhouse level, Kashar cheeses are produced from

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raw milk without the addition of any starter culture. According to traditional Kashar cheese

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manufacturing, the raw milk is filtered through a cloth and transferred into vats. Then, rennet

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is added to the milk at 30–33 °C. Coagulation of the milk is achieved in about 45 min,

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followed by cutting of the curd in small pieces (size of rice grains), which are poured into a

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cheese cloth. The remaining whey is drained off by placing heavy blocks on top of it. After

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pressing, the curd is cut into large parts and then grated. The grated curd is put into a metal

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basket with holes and cooked at 70–80 °C in water with 5–8 Baume degrees for about 2–3

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min. The cooked curd is then kneaded like bread dough. In some dairies, dry salting is

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performed during kneading. Then, the curd is placed into round metal moulds for 12–24 h.

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The cheeses are sprinkled with salt and placed on wooden shelves in a ripening room. The

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cheeses are periodically turned and stacked. After a first ripening period of 3-4 weeks at room

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temperature, 5–6 wheels of cheeses are stacked between round wooden plates and put into

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sacks for a second ripening in a cold room (3–4 °C) for at least one month to maximally two

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years.

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ACCEPTED MANUSCRIPT Although several studies have been performed on the characterisation of Kashar

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cheese, there is very little information available on the actual composition of its microbial

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ecosystem (Aran, 1998; Soyutemiz et al., 2000; Gülmez et al., 2004; Kamber, 2005;

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Çetinkaya and Soyutemiz, 2006; Özdemir and Demirci, 2006; Fırat, 2006; Var et al., 2006;

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Sert et al., 2007; Cıbık et al., 2010). No information at all is available on the molecular

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identification of the LAB communities involved in Kashar cheese production and their

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evolution during cheese ripening. Therefore, the aim of the present study was to characterise

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the LAB composition of Kashar cheeses during cheese ripening.

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2. Materials and methods

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2.1. Cheese manufacturing and sampling

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A total of 75 Kashar cheeses were manufactured from raw cows’ milk in five selected

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dairy plants (further referred to as A, B, C, D, and E) located in Kars. Each dairy plant

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produced 15 independent Kashar cheeses, each one weighing ca. 3 kg, according to their own

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traditional procedures (Table 1). None of the dairy plants performed pasteurization of the raw

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milk neither applied a starter culture or used CaCl2. All Kashar cheeses produced were kept in

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the same ripening room of dairy plant A (at room temperature and 60–70 % relative humidity)

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for 20 d. After this first ripening period, the Kashar cheeses were wrapped into baking paper

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and placed into a sack. Then, the cheeses were ripened in the same cold room of dairy plant A

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(at 3–4 °C and 70–80 % relative humidity) for 160 d. Hence, the Kashar cheeses were ripened

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for a total period of 180 d. Three Kashar cheeses of each 15–cheese batch were sampled at 5

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time points, namely after 3, 30, 60, 120, and 180 d following manufacture. These cheese

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samples were transported to the laboratory for microbiological analysis.

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2.2. Community dynamics

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Ten–g cheese samples were homogenised in 90 mL of a 2 % (w/v) solution of sodium

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citrate (Carlo Erba, Milano, Italy) for 1 min in a stomacher (AES Chemunex, Bruz, France).

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Decimal dilutions of the suspensions were prepared with Ringer’s solution (Merck,

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Darmstadt, Germany). Microbiological analyses were performed using the following agar

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media and incubation conditions: yeast–glucose–chloramphenicol agar (YGC agar; Merck)

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for yeasts and moulds at 30 °C for 72 h (Manolopoulou et al., 2003), kanamycin–aesculin–

ACCEPTED MANUSCRIPT azide agar (KAA agar; Merck) for enterococci at 30 °C for 24 h (Manolopoulou et al., 2003),

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Rogosa agar (RO agar; Merck) adjusted to pH 5.5 with acetic acid for lactobacilli at 30 °C for

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5 d under anaerobiosis (Ferrazza et al., 2004), M17 agar (Merck) for lactococci at 30 °C for

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72 h (Zarate et al., 1997), de Man–Rogosa–Sharpe (MRS) agar (Merck) supplemented with

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vancomycin (30 µg/mL; Sigma–Aldrich, Steinheim, Germany) for leuconostocs at 30 °C for

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72 h (Manolopoulou et al., 2003), violet–red–bile agar (Merck) for coliforms at 37 °C for 24 h

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(Gonzalez et al., 2003), chromocult–trytpone–bile–X–glucuronide agar (Merck) for

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Escherichia coli at 44 °C for 24 h (Gross et al., 2007), and Baird–Parker agar (Merck) for

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Staphylococcus aureus at 37 °C for 24 h, the presence of which was confirmed by a positive

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coagulase test (Turkish Standards, 2001). All microbial counts were expressed as colony

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forming units (cfu) per g of cheese sample.

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2.3. Isolation of lactic acid bacteria

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Three or four colonies were randomly isolated from each countable plate of the M17

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(presumptive lactococci), RO (presumptive lactobacilli) and MRS–vancomycin (presumptive

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leuconostocs) agar media for each cheese sample. In total, 594 colonies were isolated from 75

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cheese samples. The isolates were subcultured in MRS medium (lactobacilli and

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leuconostocs) or M17 medium (lactococci), supplemented with 15 % (v/v) glycerol as a

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cryoprotectant, and stored at –20 °C.

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2.4. (GTG)5-PCR fingerprinting of genomic DNA for classification and identification of the

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lactic acid bacteria isolates

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Isolates were grown overnight in MRS medium (lactobacilli and leuconostocs) or M17

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medium (lactococci) and 2–mL aliquots of the cultures were centrifuged (8000 × g, 15 min, 4

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°C). The cell pellets were subjected to genomic DNA extraction and purification using the

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NucleoSpin 96 Tissue kit (Macherey Nagel GmbH, Düren, Germany), following the

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manufacturer’s instructions, after first applying an enzymatic lysis of the cells using 20 µg/µL

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lysozyme (Sigma–Aldrich) and 0.5 U/µL mutanolysin (Sigma–Aldrich) in TET buffer (20

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mM Tris–HCl; 2 mM EDTA; 1 % Triton X–100; pH 8.0).

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Classification and identification of the isolates was performed by (GTG)5–PCR

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fingerprinting of their genomic DNA, numerical cluster analysis of these DNA fingerprints

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using the BioNumerics 5.1 software (Applied Maths, Sint–Martens–Latem, Belgium), and

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verification of the identity of the (GTG)5–PCR clusters through 16S rRNA gene sequencing

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of representative isolates, as described previously (Ravyts et al., 2008).

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2.5. Statistical analysis

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Statistical analysis of the data as to assess the effect of the ripening time and other

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processing factors on the size and nature of the microbial communities was performed by

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one–way and two–factor randomized complete block design, using SPSS 12.0 statistical

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software (SPSS Inc., Chicago, IL, USA). The mean differences were analyzed using Duncan’s

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multiple–range test.

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3. Results

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3.1. Effect of the ripening time on the microbial community dynamics

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Presumptive lactobacilli (MRS agar counts), lactococci (M17 agar counts),

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leuconostocs (RO agar counts), enterococci (KAA agar counts), and yeasts and moulds (YGC

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agar counts) were the different microbial groups present during the ripening of the Kashar

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cheeses investigated (Table 2). Coliforms, E. coli, and Staph. aureus were not found in any of

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the cheese samples analysed.

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In dairy plant A cheeses, the lactobacilli counts of the samples ranged from 4.35 to

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7.84 log cfu/g upon cheese ripening. At 30 d of ripening, there was a significant (p < 0.05)

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increase of the counts of the lactobacilli. Afterwards, there was no significant difference in

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their counts. The lactococci counts showed a slight increase on the 30th day of cheese

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ripening (p > 0.05). Thereafter, their counts showed a decreasing trend. The lactobacilli

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counts outnumbered the lactococci counts after 120 d. The counts of the leuconostocs

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increased from 4.08 log cfu/g after 3 d to 8.09 log cfu/g after 60 d of cheese ripening (p
0.05) and remained constant

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throughout the ripening phase.

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ACCEPTED MANUSCRIPT Concerning dairy plant B cheeses, the lactobacilli counts of the samples did not

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change significantly during cheese ripening (p > 0.05). The lactococci counts ranged from

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3.62 to 6.74 log cfu/g during cheese ripening. Only after 60 d of ripening, there was a

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significant (p < 0.05) decrease in the counts of the lactococci. Afterwards, there was no

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significant difference in their counts. The lactobacilli counts outnumbered the lactococci

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counts at all time points sampled during cheese ripening. The counts of the leuconostocs

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showed a very significant increase on the 30th day (p < 0.01). Thereafter, the leuconostoc

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counts remained more or less constant. The yeast and mould counts showed an irregular

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progress during cheese ripening and reached their highest counts after 30 d (p < 0.05).

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However, a very significant decrease of the yeast and mould counts was found after 60 d of

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ripening (p < 0.01). The enterococci counts decreased significantly after 30 d (p < 0.05) to

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remain constant thereafter. Enterococci were not found in the cheese samples of the last two

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sampled time points during the ripening phase.

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In dairy plant C cheeses, the lactobacilli counts ranged from 2.01 to 2.72 log cfu/g and

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there was no significant change during cheese ripening (p > 0.05). The counts of the

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lactococci exhibited a significant increase after 30 d of ripening (p < 0.05); afterwards, their

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counts did not change significantly (p > 0.05). The counts of the lactococci were higher than

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those of the lactobacilli at all time points sampled during the ripening phase. The leuconostoc

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counts did not show a significant change during cheese ripening (p > 0.05). The yeast and

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mould counts showed a slightly increasing trend during cheese ripening. The counts of the

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yeast and moulds were significantly higher after 180 d of cheese ripening than those at the 3rd

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and 60th day (p < 0.05). Enterococci, at a level of 1.03 log cfu/g, were only found after 3 d of

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cheese ripening, to disappear upon.

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Concerning dairy plant D cheeses, the counts of the lactobacilli showed a significant

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increase after 30 d of cheese ripening (p < 0.05), to remain more or less constant afterwards.

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In contrast with the lactobacilli counts, the lactococci counts remained constant till the 120th

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day of cheese ripening and decreased significantly (p < 0.05) at the end of the ripening phase.

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The counts of the lactococci were higher than those of the lactobacilli till the 120th day, but

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the lactobacilli counts outnumbered the lactococci counts after 180 d of ripening. The counts

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of the leuconostocs increased from 7.05 to 8.55 log cfu/g after 30 d of cheese ripening (p
0.05). The counts of the

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lactobacilli showed an increase till the 30th day of cheese ripening, to exhibit a decreasing

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trend afterwards. The counts of the lactococci showed a decreasing trend till the 60th day of

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cheese ripening and then remained constant. The counts of the lactococci were higher than

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those of the lactobacilli at all time points sampled during the cheese ripening phase. The

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counts of the leuconostocs ranged between 2.19 and 3.09 log cfu/g and there was no

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significant change during cheese ripening (p > 0.05). The yeast and mould counts tended to

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remain constant during cheese ripening. The enterococci counts decreased significantly after

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30 d of cheese ripening (p < 0.05) and disappeared after 60 d.

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3.2. Molecular identification of the lactic acid bacteria isolates and community dynamics at

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species level

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A total of 594 strains isolated from MRS, M17 and RO agar media were subjected to a

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molecular identification. The species and number of isolates identified are summarized in

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Table 3 and the community dynamics of these species present during cheese ripening for each

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dairy plant is represented in Fig. 1. Generally, the species variety was large on the 3rd day of

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cheese ripening and decreased as long as ripening progressed.

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In dairy plant A cheeses, Lactobacillus fermentum was the prevailing species (20 %)

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during the first cheese ripening period. In this period, other prevailing species were

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Lactobacillus delbrueckii subsp. bulgaricus and Pediococcus acidilactici. On the 30th day,

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Lactobacillus casei (58 %) and Lactobacillus plantarum (12 %) prevailed. Lactobacillus casei

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and Staphylococcus saprophyticus subsp. saprophyticus were the species most frequently

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isolated from cheese samples of 60 and 120 d of ripening. At the end of the cheese ripening

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phase, Lb. casei (73 %) was the prevailing species. Hence, in cheeses from dairy plant A, Lb.

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casei (48 %), Staph. saprophyticus subsp. saprophyticus (9 %), and Lb. plantarum (7 %) were

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the predominant species throughout cheese ripening. Enterococcus faecium was found with

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frequencies ranging from 5 to 7 % during the whole ripening phase. Only few isolates were

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identified as Streptococcus thermophilus, Enterococcus durans, Lactobacillus paracasei,

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Lactobacillus reuteri, Leuconostoc lactis, and Weissella halotolerans.

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ACCEPTED MANUSCRIPT In dairy plant B cheeses, S. thermophilus (25 %) and P. acidilactici (21 %) were the

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prevailing species on the 3rd day of cheese ripening. Staphylococcus saprophyticus subsp.

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saprophyticus (38 %) and Lb. plantarum (25 %) were the prevailing species on the 30th day.

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On the 60th day, Lb. casei (25 %), Lb. plantarum (25 %), and P. acidilactici (21%) prevailed.

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Lactobacillus casei (33 %), Lb. plantarum (20 %), and Lactobacillus coryniformis subsp.

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torquens (20 %) prevailed at 120 d of ripening. At the end of the ripening period, Lb. casei

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(77 %) and Lb. plantarum (12 %) were the prevailing species. Hence, in dairy plant B

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cheeses, the predominant species throughout cheese ripening were Lb. casei (32 %), Lb.

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plantarum (19 %), and P. acidilactici (13 %). Only few isolates were identified as E. faecium,

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E. durans, and W. halotolerans.

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In dairy plant C cheeses, P. acidilactici (42 %), Lb. casei (19 %), and Lb. reuteri (15

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%) were the prevailing species during the first cheese ripening period. On the 30th day of

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cheese ripening, Lb. plantarum (36 %) and P. acidilactici (29 %) prevailed. After 30 d of

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cheese ripening, Lb. casei prevailed. Hence, in cheeses from dairy plant C, Lb. casei (44 %),

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P. acidilactici (17 %), Lb. plantarum (9 %), and Lb. reuteri (9 %) were the predominant

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species throughout cheese ripening. Only few isolates were identified as Staph. saprophyticus

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subsp. saprophyticus, S. thermophilus, Lb. coryniformis subsp. torquens, Leuc. lactis, and E.

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durans.

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In dairy plant D cheeses, Lb. casei was prevailing during 120 d of cheese ripening.

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Afterwards, Lb. plantarum (42 %) became prevailing in the cheese samples. Hence, in dairy

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plant D cheeses, Lb. casei (53 %) and Lb. plantarum (18 %) were the predominant species

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during cheese ripening. The presence of E. durans (18 %) during the first cheese ripening

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period was remarkable. Moreover, among all dairy plants, E. durans and E. faecium were

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present at the highest rate (10 %) in cheeses of dairy plant D during cheese ripening. Only few

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isolates were identified as S. thermophilus, P. acidilactici, Lb. fermentum, Lb. reuteri, and

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Leuc. lactis.

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In dairy plant E cheeses, P. acidilactici (39 %), Lb. casei (22 %), and E. durans (17

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%) were the prevailing species during the first cheese ripening period. On the 30th and 60th

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day, Lb. casei and Lb. plantarum prevailed. Pediococcus acidilactici (75 %) and Lb. casei (25

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%) prevailed on the 120th day. At the end of the cheese ripening phase, Lb. coryniformis

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subsp. torquens (45 %), Lb. casei (20 %), and S. thermophilus (20 %) became prevailing.

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Hence, in the dairy plant E batch of cheeses, Lb. casei (25 %), P. acidilactici (15 %), and Lb.

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coryniformis subsp. torquens (10 %) were the predominant species during cheese ripening.

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Only few isolates were identified as E. durans, E. faecium, Lb. sakei, Lb. paracasei, W.

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halotolerans, Weissella paramesenteroides, and Leuconostoc mesenteroides.

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3.3. Effect of the dairy plant and ripening time on Kashar cheese ripening

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The dairy plant and cheese ripening time had a significant effect on the presumptive

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lactobacilli, leuconostoc, lactococci, and enterococci counts (p < 0.01 in each case). The

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lowest and highest lactobacilli counts were on the 3rd and 30th day of cheese ripening,

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respectively. On the 30th day, an increase of the lactobacilli, leuconostoc, and lactococci

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counts was found in all cheese samples, except for the lactococci counts of dairy plant E.

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Afterwards, the lactobacilli counts tended to remain constant, while the lactococci counts of

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the cheese samples decreased. On the 60th day of cheese ripening, small decreases in the

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leuconostoc counts were found in all cheese samples, except in those from dairy plant A.

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Thereafter, the leuconostoc counts of the cheese samples showed some decreases, except for

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dairy plant C. The highest average enterococci counts of the cheese samples during Kashar

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cheese ripening were on the 3rd day (p < 0.01). In Kashar cheeses of dairy plants B, C, and E,

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the enterococci counts showed a decreasing trend throughout cheese ripening and disappeared

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on the 120th, 30th, and 60th day of ripening, respectively.

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Kashar cheeses of dairy plants A and D displayed higher lactobacilli, leuconostoc,

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lactococci, and enterococci counts than the other batches of cheeses during cheese ripening (p

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< 0.05 in each case). There was no difference in lactococci counts of the Kashar cheeses

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between dairy plants A and D in terms of mean values (p > 0.05), while the other cheese

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batches were significantly different from these two batches and from each other (p < 0.01).

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The lactococci counts of the Kashar cheese samples of the different dairy plants were in

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increasing order C = E < B < A < D. Cheeses of dairy plant D showed the highest enterococci

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counts throughout cheese ripening (P < 0.05). All these data indicate an influence of the lower

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cooking temperature and time applied in the Kashar cheeses of dairy plants A and D.

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The Kashar cheeses of all dairy plants investigated displayed high yeast and mould

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counts. During cheese ripening, there was no difference between the cheeses of dairy plants

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A, B and C in terms of their mean values (p > 0.05). While the highest average values of yeast

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and mould counts were found in cheese samples from dairy plant D (p < 0.05), cheese

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samples from dairy plant E showed the lowest values during cheese ripening (p < 0.01). All

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cheese samples showed increases of the yeast and mould counts on the 30th day, but they all

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showed decreases on the 60th day in the cold room. Afterwards, yeast and mould counts in all

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cheese samples increased slightly upon further ripening in the cold room.

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4. Discussion

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Lactobacillus casei (41.6 %), Lb. plantarum (13.0 %), and P. acidilactici (9.8 %) were

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the most isolated LAB species from all Kashar cheeses of the different dairy plants

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investigated throughout cheese ripening. Whereas P. acidilactici was present at the beginning

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of the cheese ripening phase, Lb. casei and Lb. plantarum were present after a first period of

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cheese ripening. In each Kashar cheese, Lb. casei was most prevalent, with an increasing

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trend in the cheeses of dairy plants A, B, and C upon cheese ripening. Also, Aran (1998)

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found that Lb. casei subsp. casei (34.3 %) and Lb. plantarum (7.5 %) are the most frequently

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isolated LAB species during Kashar cheese ripening, with an increasing proportion of the

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former upon four months of cheese ripening. Lactobacillus casei and Lb. plantarum are

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commonly present in cheeses, being predominant in for instance Cheddar cheese (Peterson

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and Marshall, 1990; Jordan and Cogan, 1993). These mesophilic lactobacilli are often part of

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the NSLAB secondary microbiota as are Lb. paracasei, Lb. curvatus, and pediococci

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(Fitzsimons et al., 1999). Whereas Lb. coryniformis subsp. torquens was found during the late

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stages of cheese ripening in the Kashar cheese samples of dairy plants B and E, Lb. reuteri

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was found at the early stages of cheese ripening, especially in Kashar cheeses of dairy plant

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C. Lactobacillus coryniformis subsp. torquens is sometimes found in dairy environments and

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has been isolated from cheeses occasionally (Coppola et al., 2003; Martin et al., 2005; Dolci

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et al., 2008). Lactobacillus reuteri has been isolated from cheeses seldomly (Dellaglio et al.,

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1981). Pediococcus acidilactici was present in high numbers at early stages of the Kashar

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cheese ripening, in particular in cheeses from dairy plants B, C, and E. This LAB species is

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most frequently isolated from cheeses; also, pediococci are used as adjunct starter cultures in

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Feta and Cheddar cheese productions (Beresford and Williams, 2004). Streptococcus

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thermophilus was found in all cheese samples with similar frequencies (4.7–7.9 %)

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throughout cheese ripening as a minor component of the Kashar cheese LAB microbiota. This

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LAB species often accompanies mesophilic LAB species during cheese manufacturing

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(Champagne et al., 2009). In the present study, Leuconostoc and Lactococcus species were

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hardly found among the LAB species during Kashar cheese ripening. Leuconostoc lactis (1.2

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%) was the predominant leuconostoc. Only one isolate of Lc. lactis subsp. cremoris in Kashar

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cheeses of dairy plant A and two isolates of Lc. lactis subsp. lactis in cheeses of dairy plant E

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ACCEPTED MANUSCRIPT were identified during cheese ripening. Aran (1998) found 4.8 % and 8.0 % of leuconostocs

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among fresh and ripened Kashar cheese isolates, respectively. Further, they reported that Lc.

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lactis subsp. lactis was important only in the curd to acidify the milk and is inactivated during

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cheese texturing. Cıbık et al. (2010) identified 37 strains of Lc. lactis subsp. lactis and 5

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strains of Lc. lactis subsp. cremoris isolated from white pickled and Kashar cheeses,

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respectively. Remarkably, Staph. saprophyticus subsp. saprophyticus was found in cheeses

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from dairy plants A, B, and C. This species has already been isolated from different cheeses

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(Rea et al., 2007); it may originate from the udder skin (Braem et al., 2013). Staphylococcus

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saprophyticus is known to cause urinary tract infections in women (Kuroda et al., 2005).

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The LAB microbiota of Kashar cheese slightly differed from other pasta‒filata

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cheeses. For instance, in Caciocavallo cheese, Lb. brevis, Lb. coryneformis subsp.

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coryneformis, Lb. fermentum, Lb. parabuchneri, Lb. paracasei subsp. paracasei, Lb.

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pentosus, and/or Lb. plantarum have been determined as the predominant species (Corsetti et

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al, 2001; Gobbetti et al. 2002; Coppola et al., 2003; Piraino et al., 2005). In Mozzarella

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cheese, Lc. Lactis, Lactobacillus spp. such as Lb. fermentum and Lb. plantarum and S.

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thermophilus were found as the prevailing species (Coppola et al., 2001; De Angelis et al.,

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2006). In Provolone cheese, Lb. rhamnosus, S. macedonicus, and S. thermophilus were

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predominant (Aponte et al., 2008). Cultivation-independent methods might be valuable to

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complement the results of the present study.

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The presumptive lactobacilli counts of the Kashar cheese samples of the present study

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ranged from 1.69 to 8.74 log cfu/g and their average counts were 4.51 log cfu/g during cheese

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ripening. Whereas Sert et al. (2007) determined 5.70 log cfu/g of lactobacilli in raw–milk

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Kashar cheese and 5.59 log cfu/g of lactobacilli in Kashar cheese made with a starter culture,

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Aran (1998) reported MRS agar counts from 7.24 to 8.11 log cfu/g during Kashar cheese

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ripening. The present study unravelled a significant effect of the dairy plant (e.g., cooking

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temperature and time, as shown for dairy plants A and D) and cheese ripening time on the

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lactobacilli counts, which increased mainly after 30 d of ripening to remain constant upon.

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Özdemir and Demirci (2006) noticed a similar trend for the lactobacilli counts of Kashar

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cheeses. Sert et al. (2007) and Moatsou et al. (2001) determined an increase in lactobacilli

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counts at the beginning of the ripening phase followed by a decrease afterwards in the Kashar

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and Kasseri cheeses investigated, respectively. In general, mesophilic lactobacilli form a

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significant portion of the microbiota of most cheese varieties during ripening (Beresford et al.,

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2001). Despite the fact that mesophilic lactobacilli are inhabitants of raw milk and the dairy

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environment, they are frequently overgrown by strong acidifiers of the genus Lactococcus.

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ACCEPTED MANUSCRIPT Alternatively, they do gain access during the cheesemaking process, so that they are often

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found as a secondary microbiota during the ripening phase of different cheeses. This is

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especially true for raw-milk cheeses, but mesophilic lactobacilli are also common in cheeses

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manufactured with modern technologies (Wouters et al., 2002). The presumptive lactococci

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counts of the Kashar cheese samples investigated varied between 2.27 and 9.24 log cfu/g and

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their average counts were 5.71 log cfu/g during cheese ripening. Fırat (2006) reported an

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average of 6.36 log cfu/g of lactococci in Kashar cheese made without a starter culture during

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90 d of cheese ripening. Mesophilic lactococci are generally associated with the milk

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environment (Wouters et al., 2002). The evolution of the lactococci counts in the cheese

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samples of the present study is in agreement with the results of previous studies on Kashar

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cheeses (Çetinkaya and Soyutemiz, 2006; Fırat, 2006; Sert et al., 2007). In several artisan

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cheeses produced from raw milk, indigenous lactococci are found as the predominant LAB,

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while in other cheeses, they are just dominant during the first period of the ripening phase

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(Pogačić et al., 2011). In the present study, a significant effect of the dairy plant (e.g., cooking

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temperature and time, as shown for dairy plants A and D) and cheese ripening time on the

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lactococci counts was found. As leuconostocs were identified only sporadically in the Kashar

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cheese samples investigated, the RO agar counts most probably represented lactobacilli as

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well. The presumptive leuconostoc counts of the cheese samples ranged from 2.19 to 8.55 log

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cfu/g and their average was 4.71 log cfu/g. As for lactococci, the natural habitat of

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leuconostocs is plant material containing fermentable carbohydrates. They are introduced in

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the dairy environment through the green pasture and via fodder fed to the cows (Vedamuthu,

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2006).

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Enterococcus faecium (3.9 %) and E. durans (3.4 %) were isolated from all cheese

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samples during Kashar cheese ripening, except from cheeses of dairy plant C. Aran (1998)

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found that E. faecium (28.4 %) is the second predominating LAB species in Kashar cheese

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during cheese ripening. In general, enterococci are an important component of the microbiota

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of cheeses produced in Italy, Spain, Portugal, Greece, Turkey, and Egypt. Enterococcus

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faecalis, E. faecium and E. durans are the most frequently isolated enterococcal species, as

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they may be present in raw milk (Beresford and Williams, 2004; Zamfir et al., 2006). High

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enterococci counts in artisan cheeses are usually associated with poor hygienic practices

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(Franz et al., 1999). Indeed, enterococci may get into milk either directly via human or animal

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faeces or indirectly through contaminated water sources, milking equipment, or bulk storage

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tanks (Giraffa, 2003). However, they may contribute to functionalities such as antibiosis and

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flavour formation (Foulquié Moreno et al., 2006; De Vuyst et al., 2011). As they occur

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frequently in a wide range of dairy products, several strains show potential as functional

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starter cultures for food fermentations based on their biochemical properties (Wouters et al.,

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2002; Wessels et al., 2004; Foulquié Moreno et al., 2006; De Vuyst et al., 2011). The presumptive enterococci counts of the cheese samples ranged from

Microbial communities involved in Kaşar cheese ripening.

The microbiota of non-starter lactic acid bacteria (NSLAB) and their concomitant community dynamics during cheese ripening were investigated for tradi...
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