Characterisation of lactic acid bacteria from Turkish sourdough and determination of their exopolysaccharide (EPS) production characteristics

Accepted Manuscript Characterisation of lactic acid bacteria from Turkish sourdough and determination of their exopolysaccharide (EPS) production characteristics Enes Dertli, Emin Mercan, Muhammet Arıcı, Mustafa Tahsin Yılmaz, Osman Sağdıç PII:

S0023-6438(16)30166-9

DOI:

10.1016/j.lwt.2016.03.030

Reference:

YFSTL 5370

To appear in:

LWT - Food Science and Technology

Received Date: 13 November 2015 Revised Date:

20 January 2016

Accepted Date: 16 March 2016

Please cite this article as: Dertli, E., Mercan, E., Arıcı, M., Yılmaz, M.T., Sağdıç, O., Characterisation of lactic acid bacteria from Turkish sourdough and determination of their exopolysaccharide (EPS) production characteristics, LWT - Food Science and Technology (2016), doi: 10.1016/j.lwt.2016.03.030. 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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Characterisation of lactic acid bacteria from Turkish sourdough and determination of their exopolysaccharide (EPS) production characteristics

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Enes Dertli1*, Emin Mercan1, Muhammet Arıcı2, Mustafa Tahsin Yılmaz2, Osman

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Sağdıç2

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

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Department of Food Engineering, Faculty of Engineering, Yıldız Technical University, İstanbul, Turkey

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*Corresponding author: Enes Dertli, Department of Food Engineering, Faculty of

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Engineering, Bayburt University, Bayburt, 69000, Turkey. Tel: +90 (0) 458 2111153, Fax:

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+90 (0) 458 2111128, Email: [email protected]

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Abstract

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A total of 249 Lactic Acid Bacteria (LAB) isolates were found in traditional Turkish wheat

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sourdoughs from Eastern Black Sea region of Turkey. Genotypic characterization of these

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isolates revealed the presence of 47 distinct LAB strains belonging to 11 different species:

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Lactobacillus

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Lactobacillus rossiae, Lactobacillus sanfranciscensis, Lactobacillus brevis, Lactobacillus

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paralimentarius, Weissella paramesenteroides, Leuconostoc mesenteroides, Leuconostoc

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pseudomesenteroides and Weissella cibaria. The sourdough LAB microbiota differed

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depending on the sample origin and the collection period and heterofermentative LAB were

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dominant. The number of different species within a sourdough varied from 3 to 6 with the

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association of different hetero- and homofermentative LAB species. Exopolysaccharide

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(EPS) production characteristics of the isolates were determined and strain specific properties

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appeared to be important for the final EPS yields. Genes required for homopolysaccharide

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(gtf and lev) and heteropolysaccharide (epsA, epsB, p-gtf) production were PCR detected and

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several distribution patterns were observed. Results of this study confirmed the biodiversity

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of LAB species in traditional Turkish sourdough and highlighted the importance of EPS

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production in sourdough LAB strains.

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Keywords: Sourdough, Lactic acid bacteria (LAB), Microbial biodiversity,

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Exopolysaccharides (EPS)

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curvatus,

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Lactobacillus

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paraplantarum,

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Lactobacillus

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plantarum,

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1. Introduction

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There is an increasing demand for sourdough based bakery products due to several

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advantages of sourdough in comparison to use of baker’s yeast in cereal fermentations

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(Robert, Gabriel, & Fontagne-Faucher, 2009). Preparation of bread dough with sourdough

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improves technological properties of dough, enhances the nutritional and sensory properties

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of bread and increases the keeping properties of bread by retarding the staling process and

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preventing bacterial and mould spoilage (Arendt, Ryan, & Dal Bello, 2007; Hammes &

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Gänzle, 1997). Sourdough is a mixture of flour and water that is fermented with lactic acid

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bacteria (LAB) and yeasts (De Vuyst & Neysens, 2005; Gobbetti, 1998). The sourdough

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microflora contains metabolically active LAB and yeasts that form this intermediate product

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for dough preparation. The positive effects of sourdough generally rely on the metabolism of

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the LAB in sourdough that can be originated from flour and other dough ingredients (De

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Vuyst & Neysens, 2005). The main metabolic activities of sourdough LAB determining

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importance of sourdough are their proteolytic activity (Gobbetti et al., 1995), formation of

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volatile, antibacterial and antimould compounds (Corsetti & Settanni, 2007) as well as their

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exopolysaccharide (EPS) production characteristics (Galle & Arendt, 2014).

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Several types of traditional sourdoughs having cultural and geographical identities exist all

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over the world in which different types of flours and other ingredients as well as fermentation

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methodologies are used (De Vuyst & Neysens, 2005; Robert et al., 2009). These differences

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in sourdough production process determine the sourdough LAB microflora. In addition to the

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LAB in sourdoughs, natural yeasts also play important roles on fermentation process and in

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general the LAB: yeast ratio in sourdough is 100:1. Both homo- and hetero-fermentative

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LAB species are present in sourdoughs although unlike to the other food fermentations

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heterofermentative species generally dominate sourdoughs. Sourdough has a rich LAB

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microflora in which Lactobacillus strains are present more frequently than Leuconostoc,

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Pediococcus and Weissella species (De Vuyst & Neysens, 2005). The variety of sourdough

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LAB depends mainly on fermentation kinetics which determines the final characteristics of

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the bread. The most frequent Lactobacillus species in sourdoughs appeared to be

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Lactobacillus sanfranciscensis, L. brevis and L. plantarum although more than 30

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Lactobacillus species have been isolated from traditional sourdoughs. Additionally several

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studies also showed the importance of Leuconostoc and Weissella as less dominant species in

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sourdoughs (Wolter, Hager, Zannini, Galle, et al., 2014).

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Recent studies showed that one of the important properties of sourdough LAB is their EPS

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production characteristics (Galle & Arendt, 2014). EPS are natural biopolymers produced by

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several LAB species that can either encapsulate bacteria or be secreted in their environment

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(Dertli et al., 2013). EPS play critical roles in stress resistance at single cell level (Dertli,

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Mayer, & Narbad, 2015) and they have unique physicochemical effects improving the

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technological properties of sourdough and bread (Tieking, Korakli, Ehrmann, Gänzle, &

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Vogel, 2003). Structurally, EPS are divided into two groups as homopolysaccharides and

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heteropolysaccharides which are composed of only one type of sugar monomer and two or

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more types of sugar monomers, respectively. For the production of homopolymeric one, only

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one gene determined as gtf or ftf is required whereas for the heteropolymeric EPS production

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a complex eps gene cluster harbouring several genes is required (Dertli et al., 2013).

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Identification of the EPS production characteristics of sourdough LAB is crucial in order to

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reflect their overall role during fermentation process.

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Up to date, only few reports appeared on identification of LAB microflora of Turkish

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sourdoughs despite the great importance of sourdough technology in Turkish bakery industry

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and the presence of different Lactobacillus species as well as some Pedioccocus species were

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shown in Turkish sourdoughs (Gül, Özçelik, Sağdıç, & Certel, 2005; Şimşek, Çon, &

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Tulumogˇlu, 2006). In this study sourdough samples were collected from Eastern Black sea

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region of Turkey where famous ‘Vakfıkebir bread’ is produced with sourdough technology in

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order to identify the LAB microflora of traditional Turkish sourdoughs. Our results showed

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the presence of 11 different species of LAB in sourdoughs in which collection period

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determined the variety of the presented LAB species. Both homo- and heterofermentative

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LAB species were present although the later ones were dominant. PCR detection of the eps

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genes revealed that all selected strains were positive for genes required for the

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homopolysaccharide and heteropolysaccharide production although the level of EPS

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production varied among the tested strains. This study shows the wide diversity of LAB

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species present in sourdoughs collected from Vakfıkebir region and reveals the domination of

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the EPS producing LAB strains in sourdough samples.

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2. Materials and methods

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2.1. Sample collection

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In total 12 sourdough samples (A-L) were collected aseptically from small bakeries in

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Vakfıkebir, Trabzon in order to isolate and identify the LAB strains from traditional

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sourdoughs. Samples A-C, D-F and G-L were collected at one month intervals representing

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the three collection periods. All sourdough samples were produced from wheat flour with

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regular propagation by backslopping at 20-30 °C to keep microorganisms in an active state

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and all sourdoughs were at the final stage of fermentation stage before the inoculation to final

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dough for the bread production.

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2.2. Characterization of sourdough samples and microbiological analyses

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The pH value of sourdough samples was determined by a pH meter (WTW 720) with a

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suitable penetration probe. Total LAB and yeast counts of sourdough samples were

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determined by plating to corresponding agar plates. For the isolation of LAB from sourdough

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samples, serial dilutions were conducted up to 10-5 dilution factor with PBS and plated onto

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MRS5 agar containing 10 g of maltose, 5 g of fructose, 5 g of glucose, 10 g of tryptone, 5 g

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of meat extract, 5 g of yeast extract, 5 g of C2H3NaO2 · 3H2O, 3 g of ammonium chloride, 2.6

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g of K2HPO4 · 3H2O, 4 g of KH2PO4, 0.1 g of MgSO4 · 7H2O, 0.05 g of MnSO4 · 4H2O, 0.5 g

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of cysteine-HCl, 1 ml of Tween 80, and 1 ml of a vitamin mixture (pH 5.8) per liter and 0.1 g

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of cycloheximide for the inhibition of yeast growth (Meroth, Walter, Hertel, Brandt, &

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Hammes, 2003) and plates were incubated under anaerobic conditions at 30°C for 2 days. At

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the end of incubation period colonies with potential different morphologies and slimy

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characteristics were picked randomly from agar plates of all samples and propagated into

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MRS5 medium and incubated at 30 or 37°C for 2 days and then tested for Gram stain, cell

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morphology and catalase reaction.

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2.3. Bacterial growth conditions

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In total, 249 LAB isolates were selected for further analysis. All isolates were grown in

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MRS5 medium at 30°C anaerobically and stock solutions of isolates were prepared in 20%

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(v/v) glycerol and stored at -80oC. For the isolation of genomic DNA from bacterial cultures

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all strains were grown overnight at 30°C in MRS5 broth. For the isolation of EPS from

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sourdough LAB, selected strains were grown in 100 ml MRS5 culture at 30°C and 37°C for 2 d

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under anaerobic conditions.

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2.4. Genotypic characterization by rep-PCR, Box-PCR and RAPD-PCR analysis

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For the discrimination LAB strains isolated from sourdough at species level firstly rep-PCR

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analysis was performed as described elsewhere (Sagdic, Ozturk, Yapar, & Yetim, 2014). For

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the isolation of genomic DNA a commercial isolation kit was used and extractions were

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performed according to manufacturer’s protocol (Qiagen, Turkey). For the repetitive

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sequence based-PCR (rep-PCR) analysis primer (GTG)5 (5′-GTGGTGGTGGTGGTG-3′) was

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used and PCR reactions were prepared containing 1 µl DNA template, 10 µl 5×PCR buffer

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for Taq polymerase (Promega), 0.4 µl dNTPs (Bioline), 2 µl 20 mM primer (GTG)5, 0.25 µl

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5 U Taq polymerase and up to 50 µl of sterile H2O. PCR was performed using a thermal

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cycler (Benchmark, TC9639) with the following program: Initial denaturation for 10 min at

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95°C, 35 cycles at 94°C for 60 s, 40°C for 60 s, and 65°C for 8 min; and followed by a final

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elongation step of 65°C for 16 min. The rep-PCR products were separated on a 1% (wt/vol)

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agarose gel and visualised by ethidium bromide staining and photographed under UV

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illumination.

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The BOX-PCR repetitive element analysis as a second molecular identification method was

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performed with primer BOX A1R (5′-CTACGGCAAGGCGACGCTGACG-3′) using the

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following program: initial denaturation for 7 min at 95°C, 35 cycles at 94°C for 60 s, 53°C

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for 60 s, and 65°C for 8 min; and followed by a final elongation step of 65°C for 16 min and

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PCR products were separated using previously described methodology (Sagdic et al., 2014).

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In the final step of strain differentiation of all 249 sourdough isolates, RAPD-PCR analysis

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was conducted with primer M13. Bacterial genomic DNA was prepared as described above

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and was used as a template for PCR amplification. Each PCR mixture contained 5×PCR

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buffer, 2.5 mM of dNTPs, 1.5 U polymerase and 25 pMol of primer M13

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(GAGGGTGGCGGTTCT). PCR was performed with the following program: 35 cycles of

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94°C for 1 min, 40°C for 20 s, then final step of 72°C for 2 min. The PCR products were

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separated with electrophoresis on 1.6% (w/v) agarose gels at 90 V for 1.5 h and band patterns

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were visualised.

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2.5. Bacterial identification by 16S RNA gene sequencing

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After determination of different strains within the 249 isolates bacterial identification was

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performed with 16S rRNA gene sequencing. The 1.5 kb 16S rRNA genes of isolates were

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amplified with primers AMP_F and AMP_R (Baker, Smith, & Cowan, 2003). PCR reaction

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

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dNTPs, 1 µl of 20 mM primers AMP_F and AMP_R, 0.25 µl 5U polymerase and up to 50 µl

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

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of 95°C for 30 s, 55°C for 20 s, and 72°C for 30 s and 72°C for 5 min final extension. PCR

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products were run on a gel to check the amplication and amplicons were further purified

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using SureClean kit (Bioline). Sequencing reactions were prepared using primers AMP_F/

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AMP_R at 1.6 µM concentrations and the ABI Prism BigDye Terminator Sequence Kit

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(Applied Biosystems) according to the manufacturer’s protocol. Sequences obtained were

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interrogated by using Ribosomal Database Project II (Cole et al., 2005) and the identities of

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isolates were determined on basis of more than 0.98 matching score and aligned with the

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NCBI database using the BLAST algorithm with a similarity criterion of 97–100%. The 16S

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rRNA gene sequences for all of the LAB species isolated from sourdoughs were arranged in

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MEGA5. Phylogenetic trees were constructed using neighbor-joining (NJ) method with 1000

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bootstrap replicates (Saitou & Nei, 1987). All phylogenetic analyses were performed using

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MEGA5 (Tamura et al., 2011).

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2.6. Molecular detection of eps genes in sourdough isolates

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The EPS production characteristics of sourdough isolates at strain level of different species

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were determined with the previously described methodology (Dertli, 2015) and strains were

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selected for further screening of the eps genes. The target genes for the detection of the eps

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genes were gtf (glucansucrase), lev (levansucrase) and epsA (putative transcriptional

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regulator), epsB (putative polymerization and chain length determination protein gene) and p-

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gtf (putative priming glycosyltransferase gene) representing genes required for the production

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of homopolymeric and heteropolymeric EPS, respectively. Additionally L. rhamnosus GG, S.

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thermophilus ED1 (heteropolymeric genes) and EPS producer L. mesenteroides strain

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(homopolymeric genes) were used as positive controls. Table 1 shows the primers, target

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amplicon lengths and PCR conditions used in this study for detection of eps genes.

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2.7. Isolation of EPS and determination of EPS production levels

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For the isolation of EPS, all strains were grown in 100 ml MRS5 culture, inoculated at 1%

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(v/v) with an overnight culture then incubated at 30°C and 37°C for 2 d anaerobically. All

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strains reached similar OD values and cell numbers were determined and EPS were isolated

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from LAB strains as described previously (Dertli et al., 2013). The isolated EPS further were

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dissolved in distilled water and the EPS levels were quantified (Dubois, Gilles, Hamilton,

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Rebers, & Smith, 1956).

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2.8. Nucleotide accession number

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The 16S sequences of the identified strains in this study were deposited in GenBank under

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accession numbers KP903367, KR003289 – KR003317 (30 strains), KR422317 – KR422333

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(16 strains).

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3. Results

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The twelve sourdough samples were characterized in terms of pH, LAB and yeast counts and

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the results are shown in Table 2. The pH of the sourdough samples which were at the final

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stage of the fermentation period were ranged between 3.37 to 3.95 (Table 2). The LAB

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counts of these samples ranged between 8.35 and 8.91 log cfu/g and the yeast counts were

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approximately 100 fold lower than the LAB numbers and ranged between 6.70 and 6.96 cfu/g

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(Table 2).

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The genotyping identification of the 249 isolates by different methods enabled to discriminate

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47 distinct strains belonging to 11 species (Table 3). The distribution of the species within

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different sourdough samples can be seen in Table 3. Homofermentative LAB strains isolated

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from Turkish sourdoughs in this study were L. plantarum, L. paraplantarum and L. curvatus

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whereas L. rossiae, L. sanfranciscensis, L. brevis, L. paralimentarius, W. paramesenteroides,

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Leuc. mesenteroides, Leuc. pseudomesenteroides and W. cibaria were isolated as

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heterofermentative LAB strains. As can be seen in Table 3, heterofermentative LAB species

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dominated the Turkish traditional sourdoughs and the number of different species per

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sourdough varied from 3 (samples A to F) to 6 (samples G to L). Interestingly, sourdough

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samples in this study can be grouped into three groups depending of the variety of the LAB

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strains originating from the difference in collection period. Group 1 consisted samples A-C

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collected in the first month in which L. plantarum, L. rossiae, L. sanfranciscensis were

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presented and all sourdough samples contained all three LAB species. Sourdough samples

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collected in the second month were reflected as Group 2 (samples D-F) and consisted L.

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plantarum, L. brevis and L. paralimentarius as the LAB species and similar to the group 1

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samples all three sourdough samples consisted one of the strains of these three LAB species.

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Similarly samples G-L collected in the third month formed the Group 3 with the presence of

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L. paraplantarum (samples G, I, J), L. curvatus (samples G, H, K, L), W. paramesenteroides

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(samples K, G, L), Leuc. mesenteroides (samples I, G, J, H), Leuc. pseudomesenteroides and

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W. cibaria (all sourdough samples in this group). In the first two groups L. plantarum as a

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homofermentative lactobacilli were presented whereas L.paraplantarum and L. curvatus

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coexisted together as the homofermentative species in the third group sourdough samples

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(Table 3). Additionally obligate heterofermentative species presented in sourdoughs except L.

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paralimentarius as a facultative heterofermentative species and these species were well-

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distributed within the sourdoughs. Apart from these lactobacilli, no strains of pediococci and

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enterococci were present in sourdough samples.

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Figure 1 represents the MEGA5 alignments of the 16S rRNA genes of distinct sourdough

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strains showing their phylogenetic relationship with the formation of seven different groups.

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The cluster alignments analysis showed that 16S rDNA sequences for majority of the strains

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of L. sanfranciscensis, L. brevis, L. plantarum, L. paralimentarius and W. cibaria were

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similar and clustered as denoted in groups 1, 2, 3, 4 and 6 respectively. Similarly MEGA5

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alignments

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paramesenteroides strains in group 7 showing the phylogenetic similarities based on 16S

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rRNA genes. Finally Mega 5 alignments analysis placed L. curvatus and L. rossiae strains

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together as group 5 with a number of nucleotide substitution compared to the other groups

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(Figure 1).

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In order to determine the exopolysaccharide production characteristics of LAB strains from

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11 different LAB species isolated in this study, we have chosen one strain from one species

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based on the formation of slimy colonies on MRS5 agar and EPS production characteristics

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were further tested at two different incubation temperatures. EPS production was higher at

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37°C than at 30°C except W. cibaria N9 which showed similar EPS production levels at both

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temperatures (Figure 2). The highest EPS production levels were detected for L. rossiae ED1,

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L. plantarum ED10, L. brevis ED25 and L. sanfranciscensis ED5 at both 30 and 37°C and

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these strains produced 1410.3 ± 33.2, 1153.8 ± 62, 1389.2 ± 45.7 and 1286.9 ± 89.7 µg/107

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cells of EPS, respectively at 30°C (Figure 2). EPS production levels of L. paralimentarius

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ED106 and W. paramesenteroides N7 were detected to be 737.3 ± 70.7 and 753.46 ± 32.6

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µg/107 cells,

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pseudomesenteroides N13, W. cibaria N9, L. paraplantarum N15 and L. curvatus N19

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produced lower levels of EPS at 30°C compared to the other strains (Figure 2).

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Screening for EPS genes revealed that all identified LAB strains harboured at least one type

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of eps gene required for homopolymeric and heteropolymeric EPS biosynthesis (Table 4). All

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LAB isolates harboured a glucansucrase (gtf) gene responsible for homopolymeric type EPS

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production detected with primer Dexreu targeting the gtfA nucleotide sequence of L. reuteri

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LB 121 (Kralj et al., 2002). Additionally presence of levansucrase gene was PCR detected

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with primer LevV which was previously designed based on the levansucrase gene of L.

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sanfranciscensis (Tieking et al., 2003). The levansucrase gene was detected in L.

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sanfranciscensis ED5,

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paraplantarum N15, Leuc. pseudomesenteroides N13 and W. cibaria N9 (Table 4). The

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presence of the 800-bp PCR products with an epsA gene using previously designed primer set

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(Low et al., 1998) was detected in L. rossiae ED1, L. brevis ED25, W. paramesenteroides

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N7, Leuc. mesenteroides N6 and L. curvatus N19 (Table 4). Additionally only 3 out of 11

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strains harboured the epsB with a PCR product of 1150-bp detected with primer set designed

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for this gene (Vuyst & de Ven, 1998). All sourdough isolates harboured the priming

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glycosyltransferase gene in which three different primer sets were used for PCR amplification

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(Table 4). The detection of the p-gtf gene was observed with the previously designed hybrid

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primer set (epsD/E) suggesting the presence of the consensus region in p-gtf genes of the

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isolates but specific primer set (P1) designed for L. casei group (Provencher, LaPointe,

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Sirois, Van Calsteren, & Roy, 2003) was not able to amplify p-gtf genes from different strain

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groups (Table 4). Finally, with primer epsEFG only L. brevis E-25 presented the expected

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fragment size of c. 1600 bp.

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4. Discussion

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The quality of the sourdough bread mainly depends on the sourdough microflora in particular

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to the LAB strains and determination of the LAB species contributing to the formation of

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sourdough is indispensable. Knowledge on the microflora of European sourdoughs is

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expanding but there is not such that many studies conducted with Turkish sourdoughs

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especially from Trabzon region. This work, in fact, aimed to identify the main LAB species

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responsible for the formation of Turkish sourdough and to make these strains available for

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further laboratory scale fermentations to identify their technological roles.

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The pH of sourdough samples were in a similar range with the previous observations (Gül et

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al., 2005; Palomba et al., 2012; Şimşek et al., 2006). Both LAB and yeasts are involved in

paralimentarius E-106,

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sourdough fermentation process and similar to the previous knowledge, the LAB : yeast ratio

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in sourdough samples were found to be 100:1 (De Vuyst & Neysens, 2005; Gobbetti, 1998).

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Genotypic characterization of the 249 isolates obtained in this study revealed the presence of

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47 different strains belonging to 11 different species: L. plantarum, L. paraplantarum, L.

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curvatus,

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paramesenteroides, Leuc. mesenteroides, Leuc. pseudomesenteroides and W. cibaria. These

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strains were clearly grouped into 3 depending on the collection period showing the

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importance of sampling and origin to identify the LAB microflora of sourdoughs and this

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grouping was also in accordance with the previous findings showing the importance of the

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bakery environment, the origin of flours and flour type, differences in the technological

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applications for the determination of the microbial composition of sourdoughs (De Vuyst et

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al., 2002; Scheirlinck et al., 2007). All isolates could be designated to well-known sourdough

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species but to the best of our knowledge this is the first report presenting the presence of

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some LAB strains in Turkish sourdoughs including L. sanfranciscensis and Weissella species

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but no Enterococcus or Pediococcus strains were isolated in this study which might be related

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with the fact that sourdoughs were at the final stage of the fermentation process (De Vuyst &

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Neysens, 2005). L. sanfranciscensis has been reported to be one of the dominant bacterial

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species in traditional sourdoughs (De Vuyst & Neysens, 2005; Gobbetti & Corsetti, 1997;

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Lee et al., 2015) and we also found this species in sourdough samples although L.

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sanfranciscensis was obtained from only group 1 samples. The presence of L. plantarum and

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L. brevis strains were also reported in Turkish sourdoughs (Gül et al., 2005; Şimşek et al.,

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2006) but other lactobacilli isolated in this study were not reported previously. Importantly

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Group 3 samples consisted Leuconostoc and Weissella species as heterofermentative flora

330

similar to the previous observation in French sourdoughs. Additionally similar to the

331

traditional French sourdoughs these species were presented together with L. paraplantarum

332

and L. curvatus as homofermentative species (Robert et al., 2009).

333

Heterofermentative LAB species dominate the sourdough microflora in particularly when

334

sourdough fermentation occurs spontaneously (Corsetti et al., 2003; Corsetti et al., 2001; De

335

Vuyst & Neysens, 2005; De Vuyst et al., 2002). Similar to the previous knowledge, our

336

results also confirm the dominance of the heterofermentative LAB in traditional sourdoughs

337

and LAB species were well distributed within the different sourdough samples. More

338

importantly the coexistence of these species within the same sourdough samples at the end of

339

the fermentation period can be explained by their similar growth rates affected by

L.

sanfranciscensis,

L.

brevis,

L.

paralimentarius,

W.

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rossiae,

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L.

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fermentation conditions especially by fermentation temperature and sourdough pH (Ganzle,

341

Ehmann, & Hammes, 1998). Additionally the proportion of the obligately heterofermentative

342

species were also dominant in Turkish sourdoughs compared to the receding presence of

343

these species in French sourdoughs (Robert et al., 2009). These results revealed the complex

344

microflora of traditional Vakfıkebir sourdoughs formed with the well-known sourdough

345

isolates which is a prerequisite for final technological, nutritional and physicochemical

346

properties of sourdough (Nionelli et al., 2014). The biodiversity of the LAB strains in

347

sourdough environment can be corresponded to different biochemical and/or technological

348

functions to obtain an optimal final product (Coda, Di Cagno, Edema, Nionelli, & Gobbetti,

349

2010; Coda et al., 2010). Similarly it was shown that co-cultivation of sourdough lactobacilli

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results in expression of some genes/proteins (Di Cagno, De Angelis, Coda, Minervini, &

351

Gobbetti, 2009) which can be important for the final quality related with the functional

352

properties of sourdough. These examples reveal the importance of the complex sourdough

353

microflora for the final functional properties of sourdough.

354

Recent interest on sourdough LAB strains is determination of their EPS production

355

characteristics due to functional roles of in situ EPS formation on technological and

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physicochemical properties of sourdough and bread (Galle & Arendt, 2014; Galle, Schwab,

357

Arendt, & Ganzle, 2011; Palomba et al., 2012; Wolter, Hager, Zannini, Czerny, & Arendt,

358

2014). In LAB, biosynthesis mechanism of EPS production is well-described (Horn et al.,

359

2013) and some LAB strains produce only heteropolysaccharide (Lebeer et al., 2009) or

360

homopolysaccharide type EPS (Kralj et al., 2002) and both types of EPS (Dertli et al., 2013;

361

Van der Meulen et al., 2007). For the heteropolymeric EPS production an eps gene cluster

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harbouring epsA, epsB, epsD-epsE genes as well as other genes is required (Horn et al., 2013)

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whereas a single gene described as gtf or ftf is responsible for homopolymeric glucan or

364

fructan type, respectively (Palomba et al., 2012). The investigation of the presence of eps

365

genes in sourdough LAB strains revealed that all sourdough isolates were positive for both gtf

366

and p-gtf genes required for homopolymeric and heteropolymeric EPS production,

367

respectively. In general cereal-based LAB species have been associated with the formation of

368

homopolymeric EPS (Tieking & Gänzle, 2005) but recent studies also showed the production

369

of heteropolymeric EPS by sourdough isolates which were shown to alter the

370

physicochemical properties of sourdough (Galle, Schwab, Arendt, & Gänzle, 2011). Our

371

results also confirm both findings and reveal the importance of the EPS production in

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sourdough LAB strains. Additionally strains capable of producing two types of EPS can

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particularly be of interest for sourdough fermentation process due to their potential

374

synergistic effect on final technological properties of sourdough (Van der Meulen et al.,

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2007). Dextran type EPS production is well known for Weissella species (Wolter, Hager,

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Zannini, Galle, et al., 2014) although they were also shown to produce two types of EPS as

377

well as both capsular and ropy EPS (Malang, Maina, Schwab, Tenkanen, & Lacroix, 2015).

378

In this study heteropolymeric EPS genes were also detected in Weissella species in

379

accordance with previous observation (Van der Meulen et al., 2007).

380

Several factors including medium and fermentation conditions as well as genetic factors

381

affect the EPS production levels of LAB (De Vuyst & Degeest, 1999). Sourdough isolates

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showed different levels of EPS production characteristics depending on incubation

383

temperature although they were reached to the similar numbers at the end of incubation

384

period at both 30 and 37°C. The increment of the EPS production at 37°C without any

385

alteration in the growth rates might be related with the fact that optimum EPS production

386

temperature might not reflect the optimum growth rate as previously reported (Looijesteijn &

387

Hugenholtz, 1999). Nevertheless our findings revealed that EPS production characteristic

388

altered depending on the fermentation temperature suggesting the importance of sourdough

389

fermentation conditions on final in situ EPS production levels as previously suggested

390

(Tieking et al., 2003).

391

Similar to the previous observations this study revealed the dominant presence of the EPS

392

producing strains in traditional sourdoughs (Palomba et al., 2012; Tieking et al., 2003). EPS

393

production by LAB strains has both technological and functional roles such as their role in

394

sourdough process and probiotic action, respectively. For instance, the main technological

395

functions of the in situ EPS production during sourdough fermentation process by LAB

396

strains are the development of the viscoelastic properties of dough and the improvement of

397

the texture and shelf life of bread (Galle & Arendt, 2014). Similarly EPS production was

398

shown to affect the biofilm formation, adhesion, aggregation, immune responses and

399

protection under harsh conditions as well as pathogen exclusion related to the probiotic

400

functions (Dertli et al., 2015; Fanning et al., 2012). In addition to the technological roles of

401

EPS in the sourdough process, EPS production may have protective roles for LAB strains in

402

this complex environment related to the functions of EPS in probiotic action. Similarly it can

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be also suggested that the potential prebiotic effect of EPS might be an important reason for

404

LAB to produce EPS as fermentation occurs synergistically. More studies are definitely

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required in order to determine the prebiotic effect of EPS and its role at interspecies and

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intraspecies level during sourdough fermentation.

407

In conclusion, this study identified LAB strains responsible for fermentation of traditional

408

sourdoughs from Eastern Black Sea region of Turkey and determined their EPS production

409

characteristics. In total 11 different species were identified in which some were shown at first

410

time in Turkish sourdoughs. Results revealed that both homofermentative and

411

heterofermentative LAB species presented in sourdoughs although heterofermentative species

412

were dominant. The origin of sourdoughs reflected as collection period in this study seemed

413

to be important in LAB biodiversity. The PCR screening of eps genes revealed that all strains

414

harboured genes required in biosynthesis of homopolymeric and heteropolymeric EPS and

415

importantly the level of EPS production altered at strain level. These findings will help to

416

prepare standard sourdoughs with functional characteristics using these strains identified in

417

this study. Preparation of standard sourdoughs will improve the sourdough technology in

418

Turkish bakery industry. More studies identifying the functional roles of these strains such as

419

antifungal effects will also improve the current status of the sourdough technology in Turkish

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food industry.

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Acknowledgments

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This study was supported with the grant 114O695 by The Scientific and Technological

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Research Council of Turkey (TUBİTAK).

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References

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Arendt, E. K., Ryan, L. A. M., & Dal Bello, F. (2007). Impact of sourdough on the texture of bread. Food Microbiology, 24(2), 165-174. doi: http://dx.doi.org/10.1016/j.fm.2006.07.011 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 Coda, R., Di Cagno, R., Edema, M. O., Nionelli, L., & Gobbetti, M. (2010). Exploitation of Acha (Digitaria exiliis) and Iburu (Digitaria iburua) flours: Chemical characterization and their use for sourdough fermentation. Food microbiology, 27(8), 1043-1050. Coda, R., Nionelli, L., Rizzello, C. G., De Angelis, M., Tossut, P., & Gobbetti, M. (2010). Spelt and emmer flours: characterization of the lactic acid bacteria microbiota and selection of mixed starters for bread making. Journal of Applied Microbiology, 108(3), 925-935. doi: 10.1111/j.1365-2672.2009.04497.x Cole, J. R., Chai, B., Farris, R. J., Wang, Q., Kulam, S. A., McGarrell, D. M., Garrity G.M., Tiedje, J. M. (2005). The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis. Nucleic Acids Res, 33(Database issue), D294-296. doi: 10.1093/nar/gki038 Corsetti, A., De Angelis, M., Dellaglio, F., Paparella, A., Fox, P. F., Settanni, L., & Gobbetti, M. (2003). Characterization of sourdough lactic acid bacteria based on genotypic and cell-wall protein analyses. J Appl Microbiol, 94(4), 641-654. Corsetti, A., Lavermicocca, P., Morea, M., Baruzzi, F., Tosti, N., & Gobbetti, M. (2001). Phenotypic and molecular identification and clustering of lactic acid bacteria and yeasts from wheat (species Triticum durum and Triticum aestivum) sourdoughs of Southern Italy. Int J Food Microbiol, 64(1-2), 95-104. Corsetti, A., & Settanni, L. (2007). Lactobacilli in sourdough fermentation. Food Research International, 40(5), 539-558. Deveau, H., & Moineau, S. (2003). Technical note: use of RFLP to characterize Lactococcus lactis Strains producing exopolysaccharides. Journal of dairy science, 86(4), 1472-1475. De Vuyst, L., & Degeest, B. (1999). Heteropolysaccharides from lactic acid bacteria. FEMS microbiology reviews, 23(2), 153-177. De Vuyst, L., & Neysens, P. (2005). The sourdough microflora: biodiversity and metabolic interactions. Trends in Food Science & Technology, 16(1), 43-56. De Vuyst, L., Schrijvers, V., Paramithiotis, S., Hoste, B., Vancanneyt, M., Swings, J., Kalantzopoulos G., Tsakalidou E., Messens, W. (2002). The Biodiversity of Lactic Acid Bacteria in Greek Traditional Wheat Sourdoughs Is Reflected in Both Composition and Metabolite Formation. Applied and Environmental Microbiology, 68(12), 6059-6069. doi: 10.1128/aem.68.12.6059-6069.2002 Dertli, E. (2015). Isolation and Identification of an Exopolysaccharide Producer Streptococcus thermophilus Strain from Turkish Yogurt. Kafkas Univ Vet Fak Derg, 21(2), 229-232. Dertli, E., Colquhoun, I. J., Gunning, A. P., Bongaerts, R. J., Le Gall, G., Bonev, B. B., Mayer, M. J., & Narbad, A. (2013). Structure and biosynthesis of two exopolysaccharides produced by Lactobacillus johnsonii FI9785. Journal of Biological Chemistry, 288(44), 31938-31951. Dertli, E., Mayer, M. J., & Narbad, A. (2015). Impact of the exopolysaccharide layer on biofilms, adhesion and resistance to stress in Lactobacillus johnsonii FI9785. BMC microbiology, 15(1), 8. Di Cagno, R., De Angelis, M., Coda, R., Minervini, F., & Gobbetti, M. (2009). Molecular adaptation of sourdough Lactobacillus plantarum DC400 under co-cultivation with other lactobacilli. Research in Microbiology, 160(5), 358-366. doi: http://dx.doi.org/10.1016/j.resmic.2009.04.006 Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical chemistry, 28(3), 350-356.

AC C

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RI PT

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EP

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Fanning, S., Hall, L. J., Cronin, M., Zomer, A., MacSharry, J., Goulding, D., Motherway M.O., Shanahan F., Nally K., Dougan G., & van Sinderen, D. (2012). Bifidobacterial surfaceexopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protection. Proc Natl Acad Sci U S A, 109(6), 2108-2113. doi: 10.1073/pnas.1115621109 Felsenstein J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783-791. Galle, S., & Arendt, E. K. (2014). Exopolysaccharides from sourdough lactic acid bacteria. Crit Rev Food Sci Nutr, 54(7), 891-901. doi: 10.1080/10408398.2011.617474 Galle, S., Schwab, C., Arendt, E. K., & Ganzle, M. G. (2011). Structural and rheological characterisation of heteropolysaccharides produced by lactic acid bacteria in wheat and sorghum sourdough. Food Microbiol, 28(3), 547-553. doi: 10.1016/j.fm.2010.11.006 Ganzle, M. G., Ehmann, M., & Hammes, W. P. (1998). Modeling of Growth of Lactobacillus sanfranciscensis and Candida milleri in Response to Process Parameters of Sourdough Fermentation. Appl Environ Microbiol, 64(7), 2616-2623. Gobbetti, M. (1998). The sourdough microflora: interactions of lactic acid bacteria and yeasts. Trends in Food Science & Technology, 9(7), 267-274. Gobbetti, M., & Corsetti, A. (1997). Lactobacillus sanfranciscoa key sourdough lactic acid bacterium: a review. Food Microbiology, 14(2), 175-188. Gobbetti, M., Simonetti, M. S., Corsetti, A., Santinelli, F., Rossi, J., & Damiani, P. (1995). Volatile compound and organic acid productions by mixed wheat sour dough starters: influence of fermentation parameters and dynamics during baking. Food Microbiology, 12, 497-507. Gül, H., Özçelik, S., Sağdıç, O., & Certel, M. (2005). Sourdough bread production with lactobacilli and S. cerevisiae isolated from sourdoughs. Process Biochemistry, 40(2), 691-697. doi: http://dx.doi.org/10.1016/j.procbio.2004.01.044 Hammes, W., & Gänzle, M. (1997). Sourdough breads and related products Microbiology of fermented foods (pp. 199-216): Springer. Horn, N., Wegmann, U., Dertli, E., Mulholland, F., Collins, S. R., Waldron, K. W., Mayer, M. J., & Narbad, A. (2013). Spontaneous mutation reveals influence of exopolysaccharide on Lactobacillus johnsonii surface characteristics. PLoS One, 8(3), e59957. doi: 10.1371/journal.pone.0059957 Kralj, S., van Geel-Schutten, G. H., Rahaoui, H., Leer, R. J., Faber, E. J., van der Maarel, M. J. E. C., & Dijkhuizen, L. (2002). Molecular Characterization of a Novel Glucosyltransferase from Lactobacillus reuteri Strain 121 Synthesizing a Unique, Highly Branched Glucan with α(1→4) and α-(1→6) Glucosidic Bonds. Applied and Environmental Microbiology, 68(9), 4283-4291. doi: 10.1128/aem.68.9.4283-4291.2002 Lamothe, G., Jolly, L., Mollet, B., & Stingele, F. (2002). Genetic and biochemical characterization of exopolysaccharide biosynthesis by Lactobacillus delbrueckii subsp. bulgaricus. Archives of microbiology, 178(3), 218-228. Lebeer, S., Verhoeven, T. L., Francius, G., Schoofs, G., Lambrichts, I., Dufrene, Y., Vanderleyden J., De Keersmaecker, S. C. (2009). Identification of a Gene Cluster for the Biosynthesis of a Long, Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase. Appl Environ Microbiol, 75(11), 3554-3563. doi: 10.1128/AEM.02919-08 Lee, H., Baek, H., Lim, S. B., Hur, J. S., Shim, S., Shin, S. Y., Han N.S., Seo, J. H. (2015). Development of species-specific PCR primers and polyphasic characterization of Lactobacillus sanfranciscensis isolated from Korean sourdough. Int J Food Microbiol, 200, 80-86. doi: 10.1016/j.ijfoodmicro.2015.02.007 Looijesteijn, P. J., & Hugenholtz, J. (1999). Uncoupling of growth and exopolysaccharide production by Lactococcus lactis subsp. cremoris NIZO B40 and optimization of its

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synthesis. Journal of Bioscience and Bioengineering, 88(2), 178-182. doi: http://dx.doi.org/10.1016/S1389-1723(99)80198-4 Low, D., Ahlgren, J. A., Horne, D., McMahon, D. J., Oberg, C. J., & Broadbent, J. R. (1998). Role of Streptococcus thermophilus MR-1C capsular exopolysaccharide in cheese moisture retention. Applied and environmental microbiology, 64(6), 2147-2151. Malang, S. K., Maina, N. H., Schwab, C., Tenkanen, M., & Lacroix, C. (2015). Characterization of exopolysaccharide and ropy capsular polysaccharide formation by Weissella. Food Microbiology, 46(0), 418-427. doi: http://dx.doi.org/10.1016/j.fm.2014.08.022 Meroth, C. B., Walter, J., Hertel, C., Brandt, M. J., & Hammes, W. P. (2003). Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCRDenaturing Gradient Gel Electrophoresis. Applied and Environmental Microbiology, 69(1), 475-482. doi: 10.1128/aem.69.1.475-482.2003 Nei M. and Kumar S. (2000). Molecular Evolution and Phylogenetics. Oxford University Press, New York. Nionelli, L., Curri, N., Curiel, J. A., Di Cagno, R., Pontonio, E., Cavoski, I., Gobbetti M., Rizzello, C. G. (2014). Exploitation of Albanian wheat cultivars: characterization of the flours and lactic acid bacteria microbiota, and selection of starters for sourdough fermentation. Food Microbiol, 44, 96-107. doi: 10.1016/j.fm.2014.05.011 Palomba, S., Cavella, S., Torrieri, E., Piccolo, A., Mazzei, P., Blaiotta, G., Ventorino V., Pepe, O. (2012). Polyphasic screening, homopolysaccharide composition, and viscoelastic behavior of wheat Sourdough from a Leuconostoc lactis and Lactobacillus curvatus exopolysaccharide-producing starter culture. Appl Environ Microbiol, 78(8), 2737-2747. doi: 10.1128/AEM.07302-11 Provencher, C., LaPointe, G., Sirois, S., Van Calsteren, M. R., & Roy, D. (2003). Consensusdegenerate hybrid oligonucleotide primers for amplification of priming glycosyltransferase genes of the exopolysaccharide locus in strains of the Lactobacillus casei group. Appl Environ Microbiol, 69(6), 3299-3307. Robert, H., Gabriel, V., & Fontagne-Faucher, C. (2009). Biodiversity of lactic acid bacteria in French wheat sourdough as determined by molecular characterization using species-specific PCR. Int J Food Microbiol, 135(1), 53-59. doi: 10.1016/j.ijfoodmicro.2009.07.006 Sagdic, O., Ozturk, I., Yapar, N., & Yetim, H. (2014). Diversity and probiotic potentials of lactic acid bacteria isolated from gilaburu, a traditional Turkish fermented European cranberrybush (Viburnum opulus L.) fruit drink. Food Research International, 64, 537-545. Saitou, N., & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular biology and evolution, 4(4), 406-425. Scheirlinck, I., Van der Meulen, R., Van Schoor, A., Vancanneyt, M., De Vuyst, L., Vandamme, P., & Huys, G. (2007). Influence of Geographical Origin and Flour Type on Diversity of Lactic Acid Bacteria in Traditional Belgian Sourdoughs. Applied and Environmental Microbiology, 73(19), 6262-6269. doi: 10.1128/aem.00894-07 Şimşek, Ö., Çon, A. H., & Tulumogˇlu, Ş. (2006). Isolating lactic starter cultures with antimicrobial activity for sourdough processes. Food Control, 17(4), 263-270. doi: http://dx.doi.org/10.1016/j.foodcont.2004.10.011 Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular biology and evolution, 28(10), 2731-2739. Tieking, M., & Gänzle, M. G. (2005). Exopolysaccharides from cereal-associated lactobacilli. Trends in Food Science & Technology, 16(1–3), 79-84. doi: http://dx.doi.org/10.1016/j.tifs.2004.02.015 Tieking, M., Korakli, M., Ehrmann, M. A., Gänzle, M. G., & Vogel, R. F. (2003). In situ production of exopolysaccharides during sourdough fermentation by cereal and intestinal isolates of lactic acid bacteria. Applied and environmental microbiology, 69(2), 945-952.

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Van der Meulen, R., Grosu-Tudor, S., Mozzi, F., Vaningelgem, F., Zamfir, M., de Valdez, G. F., & De Vuyst, L. (2007). Screening of lactic acid bacteria isolates from dairy and cereal products for exopolysaccharide production and genes involved. Int J Food Microbiol, 118(3), 250258. doi: 10.1016/j.ijfoodmicro.2007.07.014 Vuyst, D., & de Ven, V. (1998). Production by and isolation of exopolysaccharides from Streptococcus thermophilus grown in a milk medium and evidence for their growth-associated biosynthesis. Journal of Applied Microbiology, 84(6), 1059-1068. Wolter, A., Hager, A. S., Zannini, E., Czerny, M., & Arendt, E. K. (2014). Influence of dextranproducing Weissella cibaria on baking properties and sensory profile of gluten-free and wheat breads. Int J Food Microbiol, 172, 83-91. doi: 10.1016/j.ijfoodmicro.2013.11.015 Wolter, A., Hager, A. S., Zannini, E., Galle, S., Gänzle, M. G., Waters, D. M., & Arendt, E. K. (2014). Evaluation of exopolysaccharide producing Weissella cibaria MG1 strain for the production of sourdough from various flours. Food Microbiology, 37(0), 44-50. doi: http://dx.doi.org/10.1016/j.fm.2013.06.009

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Tables

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Table 1. Primers used for the detection of eps genes.

619

Table 2. Characteristics of sourdough samples collected from different bakeries.

620 621

Table 3. Species distribution of sourdough samples (+ and - represent the presence and the abundance of each species within the corresponding sample).

622 623

Table 4. Screening of genes involved in homopolymeric EPS (gtf and lev) and heteropolymeric EPS (epsA, epsB, p-gtf) production.

624

Figures

625 626 627 628 629 630 631 632 633 634 635 636

Figure 1. Dendogram showing multiple sequence alignment of 16S rRNA gene sequences of sourdough strains. Pairwise phylogenetic distances were calculated based on 1400 nt of 16S rRNA gene. The evolutionary history was inferred using the Neighbor-Joining method (Saitou & Nei, 1987). The optimal tree with the sum of branch length = 0.25610000 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the pdistance method (Nei & Kumar, 2000) and are in the units of the number of transitional differences per site. All positions containing gaps and missing data were eliminated. There were a total of 625 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011).

637 638 639

Figure 2. The level of EPS production of selected sourdough LAB strains at 30 and 37°C. EPS were isolated from stationary-phase cultures of LAB strains grown in MRS5 medium (c. ~ 107 cells)

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Table 1

epsEFG-F

GAYGARYTNCCNCARYTNWKAAYGT

epsEFG-R

TGCAGCYTCWGCCACATG

TAGTGACAACGGTTGTACTG

epsA-R epsB fw

GATCATTATGGACTGTCAC CGTACGATTCGTACGACCAT

epsB rev

TGACCAGTGACACTTGAAGC

epsA

epsB

1,600

30 cycles of 94°C (30 s), 49°C (45 s), 72°C (1 min)

800

35 cycles of 94°C (15 s), 40°C (30 s), 72°C (1 min)

(Low et al., 1998)

1150

35 cycles of 94°C (45 s), 46°C (1 min), 72°C (1 min)

35 cycles of 95°C (30 s), 42°C (45 s), 72°C (1 min)

GAYGTNTGGGAYWSNTGGC

LevV-R

TCNTYYTCRTCNSWNRMCAT

Lev

800

epsD/E-F

TCATTTTATTCGTAAAACCTCAATTGAY GARYTNCC

p-gtf

189

35 cycles of 95°C (30s) 42°C (45s) 72°C (1 min)

p-gtf

276

35 cycles of 95°C (30s) 42°C (45s) 72°C (1 min)

600

35 cycles of 95°C (30 s), 42°C (45 s), 72°C (1 min)

TE D

LevV-F

epsD/E-R

AATATTATTACGACCTSWNAYYTGCCA ATGAGTTTGGTTGGACCAAGACCTCC

EP

G-Lr-Bacta-F-26

TTTAATAGGCTCCAGTTGGA

AC C

G-Lr-Bactb-R-20b

DexreuV

GTGAAGGTAACTATGTTG

DexreuR

ATCCGCATTAAAGAATGG

a

gtf

Y=C or T; R=A or G; W=A or T; K=G or T; S= C or G; M=A or C; V=A, C, or G; N=A, C, G, or T; I=

inosine; b P1.

Reference (Lamothe, Jolly, Mollet, & Stingele, 2002)

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epsA-F

p-gtf

PCR conditions

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Sequencea (5′-3′)

Expected amplicon (bp)

SC

Primer

Gene target

(Deveau and Moineau, 2003) (Vuyst & de Ven, 1998) (Tieking, Korakli, Ehrmann, Ganzle, & Vogel, 2003) (Provencher , LaPointe, Sirois, Van Calsteren, & Roy, 2003) (Provencher et al., 2003)

(Tieking et al., 2005)

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Table 2 Number of isolates

TE D EP AC C

16 17 23 20 18 17 21 27 22 21 23 24

RI PT

Yeast log CFU/g 6.96 ± 0.01 6.91 ± 0.02 6.90 ± 0.01 6.79 ± 0.03 6.70 ± 0.04 6.85 ± 0.02 6.95 ± 0.01 6.94 ± 0.03 6.71 ± 0.01 6.85 ± 0.02 6.91 ± 0.01 6.82 ± 0.01

SC

LAB log CFU/g 8.61 ± 0.01 8.54 ± 0.04 8.35 ± 0.01 8.72 ± 0.01 8.61 ± 0.02 8.63 ± 0.03 8.71 ± 0.01 8.96 ± 0.01 8.91 ± 0.02 8.50 ± 0.01 8.61 ± 0.04 8.85 ± 0.02

pH 3.82 3.76 3.58 3.81 3.91 3.95 3.61 3.38 3.42 3.65 3.48 3.37

M AN U

Sourdough A B C D E F G H K L M N

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Table 3 D-F

G-L

+ -

+ -

+ +

+ + -

+ + -

+ + + +

SC

RI PT

A-C

AC C

EP

TE D

M AN U

Sourdough Homofermentative L. plantarum L.paraplantarum L. curvatus Heterofermentative L. rossiae L. sanfranciscensis L. brevis L. paralimentarius W.paramesenteroides Leuc. mesenteroides Leuc. pseudomesenteroides W. cibaria

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Table 4

Gtf Dexreu

Lev LevV

epsA

p-gtf P1

epsB

+ -

+ + + + + + + + + + +

AC C

EP

TE D

M AN U

SC

RI PT

L. rossiae ED-1 +* + L. sanfranciscensis ED-5 + + L. plantarum ED-10 + L. brevis E-25 + + L. paralimentarius E-106 + + + W.paramesenteroides N7 + + + Leuc. mesenteroides N6 + + L.paraplantarum N15 + + L. curvatus N19 + + Leuc. pseudomesenteroides N13 + + + W. cibaria N9 + + + * + presence of the corresponding gene, - no detection of the corresponding gene

p-gtf p-gtf epsEFG epsD/E

AC C

EP

TE D

M AN U

SC

RI PT

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AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

-

AC C

EP

TE D

M AN U

SC

-

Heterofermentative LAB species dominated traditional Turkish sourdoughs from Eastern Black Sea region of Turkey. Several LAB species including L. sanfranciscensis were identified first time in Turkish sourdoughs. The origin of sourdoughs reflected as collection period in this study seemed to be important in LAB biodiversity. All tested strains harboured different eps genes required for homopolymeric and heteropolymeric exopolysaccharide (EPS) production.

RI PT

-