Iranian wheat flours from rural and industrial mills: Exploitation of the chemical and technology features, and selection of autochthonous sourdough starters for making breads

Accepted Manuscript Iranian wheat flours from rural and industrial mills: exploitation of the chemical and technology features, and selection of autochthonous sourdough starters for making breads Erica Pontonio , Luana Nionelli , José Antonio Curiel , Alireza Sadeghi , Raffaella Di Cagno , Marco Gobbetti , Carlo Giuseppe Rizzello PII:

S0740-0020(14)00291-3

DOI:

10.1016/j.fm.2014.10.011

Reference:

YFMIC 2303

To appear in:

Food Microbiology

Received Date: 19 June 2014 Revised Date:

2 October 2014

Accepted Date: 24 October 2014

Please cite this article as: Pontonio, E., Nionelli, L., Curiel, J.A., Sadeghi, A., Di Cagno, R., Gobbetti, M., Rizzello, C.G., Iranian wheat flours from rural and industrial mills: exploitation of the chemical and technology features, and selection of autochthonous sourdough starters for making breads, Food Microbiology (2015), doi: 10.1016/j.fm.2014.10.011. 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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Iranian wheat flours from rural and industrial mills: exploitation of the

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chemical and technology features, and selection of autochthonous sourdough

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starters for making breads

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Erica Pontonio1, Luana Nionelli1, José Antonio Curiel1, Alireza Sadeghi2, Raffaella Di Cagno1,

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Marco Gobbetti1, Carlo Giuseppe Rizzello1*

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Department of Soil, Plant and Food Science, University of Bari Aldo Moro, 70126 Bari, Italy Department of Food Science and Technology, Gorgan University of Agricultural Sciences and

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Running title: sourdough fermentation of Iranian wheat flours

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Abbreviations

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2-DE, Two-dimensional electrophoresis; Ban, Bandar Torkaman/Jorgan; Bar, Barbari; UD,

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Unfermented Dough; DY, Dough yield; FAA, Free amino acids; GI, Gluten index; HMW, High

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molecular weight; IEF, Isoelectric focusing; Kas, Kashmar; Lav, Lavash; LMW, Low molecular

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weight; ME, Methanolic extract; Ney, Neyshabur; OPA, o-phtaldialdehyde; PCA, Principal

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Component Analysis; QF, Quotient of fermentation; San, Sangak; SB, Sourdough bread; SLS,

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Selected sourdough starter; SS, Spontaneous sourdoughs; Taf, Taftoon; Tor, Toroujen; TPA,

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Texture Profile Analysis; TTA, Total titratable acidity; WSE, Water/salt-soluble extract.

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*Corresponding author. Tel.: +39 0805442948; Fax: +390805442911.

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E-mail address: [email protected] 1

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Abstract

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This study aimed at describing the main chemical and technology features of eight Iranian wheat

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flours collected from industrial and artisanal mills. Their suitability for bread making was

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investigated using autochthonous sourdough starters. Chemical analyses showed high

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concentration of fibers and ash, and technology aptitude for making breads. As shown through 2-

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DE analyses, gliadin and glutenin subunits were abundant and varied among the flours. According

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to the back slopping procedure, type I sourdoughs were prepared from Iranian flours, and lactic

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acid bacteria were typed and identified. Strains of Pediococcus pentosaceus, Weissella cibaria,

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Weissella confusa, and Leuconostoc citreum were the most abundant. Based on the kinetics of

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growth and acidification, quotient of fermentation and concentration of total free amino acids,

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lactic acid bacteria were selected and used as sourdough mixed starters for bread making.

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Compared to spontaneous fermentation, sourdoughs fermented with selected and mixed starters

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favored the increase of the concentrations of organic acids and total free amino acids, the most

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suitable quotient of fermentation, and the most intense phytase and antioxidant activities.

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Although the high concentration of fibers, selected and mixed starters improved the textural

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features of the breads. This study might had contribute to the exploitation of the potential of

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Iranian wheat flours and to extend the use of sourdough, showing positive technology, nutritional

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and, probably, economic repercussions.

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1. Introduction

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Since centuries soft wheat (Triticum aestivum) and durum wheat (Triticum durum) are cultivated

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in Iran. It seemed that these species and their main genetic diversity originated from this country

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(Moghaddam et al., 1997). Nowadays, wheat is the most important crop cultivated in Iran

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(Moghaddam et al., 1997). Because of the geographical position, Iran is considered to be as a dry

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or semy-dry region, where wheat flowering and grain filling are often subjected to environmental

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stresses and water deficit (Abdoli et al., 2012). The composition of the wheat flour is consequently

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affected. The wheat cultivated in Iran mainly includes primitive cultivars and wheat landraces,

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consisting of a mixture of genotypes, which evolved under peculiar environmental conditions and

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natural selection (Moghaddam et al., 1997).

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Bread is the most popular staple foods consumed in Iran. Traditional Iranian breads were already

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described, and appreciated for taste and overall quality (Fazeli et al., 2004). In particular, typical

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products such as Lavash, Taftoon, Barbari and Sangak breads are manufactured through

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traditional protocols, which include variable parameters of fermentation and baking, and lead to

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different and typical sensory and rheology features (Khaniki, 2005). Rural bread making is only or

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mainly based on spontaneous fermentation. On the contrary, bread making in the urban areas is

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recently subjected to the expansion of semi-automatic and fast equipment, and chemical

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leavening. The use of sodium bicarbonate almost replaced sourdough (Fazeli et al., 2004). As the

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consequence of chemical leavening, the shelf-life of baked goods markedly decreased, which

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leads to a huge waste of bread (Fazeli et al., 2004). A further nutritional consequence of chemical

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leavening concerns the high content of phytic acid into the breads, also due to the high flour

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extraction rates, which markedly decreases the mineral bioavailability (Didar, 2011).

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Cereal fermentation processes depend on specific determinants, which have to be strictly

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controlled to get standardized and agreeable products (Hammes and Gänzle, 1998). Among these

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determinants, the type of flour is one of the most important. It affects the technology features and

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the nutritional value of the baked goods and, more in general, the microbial fermentation through

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ACCEPTED MANUSCRIPT the level and type of fermentable carbohydrates, nitrogen sources and growth factors (Hammes et

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al., 2005). Overall, the use of industrial starter cultures for cereal fermentations is limited, and,

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when used, starter cultures often lack of biochemical properties to differentiate the products and to

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exploit the potential of the various flour matrices (Coda et al., 2014). Mainly based on the above

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considerations, the manufacture of bakery products with local flours and tailor made starter

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cultures for specific raw ingredients is deserving a marked interest to get new niche products

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(Coda et al., 2014). Nowadays, the chemical and technology characterization of Iranian flours, and

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the selection of lactic acid bacteria suitable for industrial or artisanal bread making would

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represent an useful tool to better address the biotechnology choices of the Iranian bakery

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industries.

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This study aimed at characterizing the chemical and technology features of eight Iranian wheat (T.

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aestivum and T. durum) flours. Suitable autochthonous lactic acid bacteria strains were selected

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for sourdough fermentation. A comparison between spontaneous and selected sourdough

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fermentations was made, and the main features of related breads were determined.

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2. Materials and methods

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2.1 Flours

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Eight wheat (Triticum aestivum and Triticum durum) flours collected in Iran were used in this

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study (Table 1). Four flours were purchased from industrial mills, which were located in the

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Mashhad area (North-East Iran). These corresponded to blends of national wheat varieties, usually

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designed for making local flat breads such as Lavash (Lav), Sangak (San), Barbari (Bar) and

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Taftoon (Taf). The other flours were collected from artisanal mills, which were located in rural

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areas close to the cities of Neyshabur (Ney), Toroujen (Tor), Kashmar (Kas) and Bandar

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Torkaman/Jorgan (Ban). Also in this case, flours consisted of blends of varieties and/or species of

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wheat. The wheat was cultivated during the season 2011-2012. Moisture, protein, ash, starch, fat,

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fibers, falling number, and gluten index/dry gluten were determined according to AACC standard

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methods: 44-15.02, 46-12.01, 08-01.01, 76-13.01, 30-10.01, 32-05.01, 56-81.03, and 38-12.02

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(AACC, 2003).

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2.2 Two-dimensional electrophoresis (2-DE)

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Proteins were selectively extracted from wheat flours, according to the method of Osborne (1907),

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further modified by Weiss et al. (1993). The concentration of proteins was determined by the

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Bradford method (Bradford, 1976). Two-dimensional electrophoresis (2-DE) was carried out with

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the immobiline-polyacrilamide system, as described by Di Cagno et al. (2002). Aliquots of

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proteins (30 µg) were used for the electrophoretic run. Isoelectric focusing (IEF) was carried out

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on immobiline strips, using the IPG phore at 20°C. A non-linear pH gradient from 3.0 to 10.0

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(IPG strips; Amersham Pharmacia Biotech, Uppsala, Sweden) was provided for the glutenin

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fraction. A linear pH gradient 6-11 was used for the gliadin fraction. The second dimension was

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carried out in a Laemmli system on 12% polyacrilamide gels (13 cm by 20 cm by 1.5 mm), at a

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constant current of 40 mA/gel and at 15°C for approximately 5 h, until the dye front reached the

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bottom of the gel. Two-DE protein standards (Bio-Rad Laboratories, USA) were used for IP and

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molecular mass estimation. Gels were silver stained (Bini et al., 1997). Image analysis of the gels,

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which was acquired by a gel scanner (Amersham Pharmacia Biotech, Uppsala, Sweden), was

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carried out with the UTHSCSA ImageTool software (Version 2.0, University of Texas Health

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Science Centre, San Antonio, Texas, available by anonymous FTP from maxrad6.uthscsa.edu). In

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details, grey scale (0-255) images of the gels, at 600 dots per inch, were obtained and processed.

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The intensity of the spots was calculated as the black pixel area, using a threshold method

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(Gámbaro et al., 2004).

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2.3 Type I sourdoughs

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using starter cultures or baker’s yeast. Flours were mixed with tap water at 60 x g for 5 min, with a

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IM 5-8 high-speed mixer (Mecnosud, Flumeri, Italy), and the doughs were incubated at 25°C for

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24 h. Dough yield (DY, dough weight x 100/flour weight) was 160. After this first fermentation,

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ten back-slopping steps (refreshments) were further carried out, mixing 25% of the previously

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fermented dough with flour and water (DY of 160), and incubating for 8 h at 25°C. After each

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fermentation, doughs were stored at 4˚C until the next refreshment. The pH value of doughs was

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determined by a pHmeter (Model 507, Crison, Milan, Italy) with a food penetration probe. Total

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titratable acidity (TTA) was determined after homogenization of 10 g of dough with 90 ml of

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distilled water, and expressed as the amount (ml) of 0.1 M NaOH needed to reach the value of pH

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of 8.3. The rate of volume increase of doughs was determined as described by Minervini et al.

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(2011). After six refreshments, the acidification rate and volume increase were stable, and

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spontaneous sourdoughs (SS) were characterized. Further refreshments did not modify the

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acidification rate and the volume of SS. All the analyses were carried out in triplicate.

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Not incubated and not fermented doughs (UD, unfermented doughs) were made by mixing flour

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and water (DY 160) in the same conditions and characterized.

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2.4 Determination of organic acids, quotient of fermentation, and free amino acids

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The water/salt-soluble extracts (WSE) of wheat flours and doughs, which were prepared according

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to Weiss et al. (1993), was used to analyze organic acids, peptides, and free amino acids (FAA).

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Organic acids were determined by High Performance Liquid Chromatography (HPLC), using an

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ÄKTA Purifier system (GE Healthcare, Buckinghmshire, UK) equipped with an Aminex HPX-

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87H column (ion exclusion, Biorad, Richmond, CA), and an UV detector operating at 210 nm.

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Elution was at 60°C, with a flow rate of 0.6 ml/min, using 10 mM H2SO4 as mobile phase (Coda

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et al., 2011). The quotient of fermentation (QF) was determined as the molar ratio between lactic

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and acetic acids. The peptide concentration was determined by the o-phtaldialdehyde (OPA)

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method (Church et al., 1983). A standard curve prepared using tryptone (0.25 to 1.5 mg/ml) was

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used as the reference. FAA were analyzed by a Biochrom 30 series Amino Acid Analyzer

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(Biochrom Ltd., Cambridge Science Park, England) with a Na-cation-exchange column (20 by

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0.46 cm internal diameter), as described by Rizzello et al. (2010a).

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2.5 Microbiological analysis and isolation of lactic acid bacteria. Ten grams of sourdough were

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homogenized with 90 ml of sterile peptone water (1% [wt/vol] of peptone and 0.9% [wt/vol] of

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NaCl) solution. Presumptive lactic acid bacteria were enumerated using modified MRS (mMRS)

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(containing 1% [wt/vol] maltose, 5% [vol/vol] fresh yeast extract, pH 5.6) agar medium (Oxoid,

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Basingstoke, Hampshire, United Kingdom). The medium was supplemented with cycloheximide

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(0.1 g liter). Plates were incubated at 30°C for 48 h, under anaerobiosis (AnaeroGen and

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AnaeroJar, Oxoid). At least ten colonies of presumptive lactic acid bacteria were randomly

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selected from the plates containing the two highest sample dilutions. Gram-positive, catalase-

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negative, non-motile rods and cocci isolates were cultivated into mMRS at 30°C for 24 h and re-

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streaked onto the same agar medium. All isolates considered for further analyses were able to

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acidify the culture medium.

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The cell density of yeasts was estimated on Sabouraud Dextrose Agar (SDA) (Oxoid) medium,

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supplemented with chloramphenicol (0.1 g liter) at 30°C for 48 h.

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2.6 Genotypic characterization by Randomly Amplified Polymorphic DNA-Polymerase

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Chain Reaction (RAPD-PCR) analysis. Genomic DNA of lactic acid bacteria was extracted

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according to De los Reyes-Gavilán et al. (1992). Three oligonucleotides, P4 5’-CCGCAGCGTT-

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3’, P7 5’-AGCAGCGTGG-3’ (Corsetti et al., 2003) and M13 5’-GAGGGTGGCGGTTCT-3’

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(Stendid et al., 1994), with arbitrarily chosen sequences, were used for bio-typing of lactic acid

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bacteria isolates. Reaction mixture and PCR conditions for primers P4 and P7 were those

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described by Corsetti et al. (2003), whereas those reported by Zapparoli et al. (1998) were used for 7

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System and compared using the Fingerprinting II InformatixTM Software (Bio-Rad Laboratories).

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Dice coefficients of similarity and UPGMA algorithm were used to estimate the similarity of the

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electrophoretic profiles. Since RAPD profiles of the isolates from one batch of each type of

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sourdough were confirmed by analyzing isolates from two other batches, strains isolated from a

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single batch were further analyzed.

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2.7 Genotypic identification of lactic acid bacteria and yeasts

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To identify presumptive lactic acid bacteria, two primer pairs (Invitrogen Life Technologies,

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Milan, Italy), LacbF/LacbR and LpCoF/LpCoR, were used for amplifying the 16S rDNA (De

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Angelis et al., 2006). PheS primers were used to identify at the species level within the genera

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Leuconostoc and Weissella (Naser et al., 2005). Electrophoresis was carried out on agarose gel at

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1.5% (wt/vol) (Gellyphor, EuroClone) and amplicons were purified with GFXTM PCR DNA and

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Gel Band Purification Kit (GE Healthcare).

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Sequencing electrophoregrams data were processed with Geneious (http://www.geneious.com).

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rDNA sequences alignments were carried out using the multiple sequence alignment method

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(Edgar, 2004) and identification queries were fulfilled by a BLAST search in GenBank

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(http://www.ncbi.nlm.nih.gov/genbank/).

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2.8 Selection of autochthonous lactic acid bacteria and preparation of selected sourdough

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starter

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Sixteen autochthonous lactic acid bacteria strains were cultivated into mMRS broth at 30°C for

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24 h. Cells were harvested by centrifugation (10,000 x g, 10 min, 4°C), washed twice in 50 mM

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sterile potassium phosphate buffer (pH 7.0) and re-suspended in tap water at the cell density of ca.

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8.0 log cfu/ml. Sixty-two grams of Ban flour (chosen as the common matrix to select strains) and

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37.5 ml of tap water, containing the above cellular suspension of each lactic acid bacterium (cell 8

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Mixing was done manually for 5 min. Sourdoughs were fermented at 30°C for 16 h, according to

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the optimal growth temperature of the selected lactic acid bacteria and the fermentation time

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allowing the obtaining of the proper biochemical properties (Coda et al., 2009; Nionelli et al.,

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2014).

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Kinetics of growth and acidification were determined and modelled in agreement with the

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Gompertz equation, as modified by Zwietering et al. (1990),: y= k + A exp{- exp[(µmax or Vmax

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e/A)(λ-t) + 1]}; where y is the growth expressed as log cfu/g/h or the acidification rate expressed

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as dpH/dt (units of pH/h) at the time t; k is the initial level of the dependent variable to be

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modelled (log cfu/g or pH units); A is the cell density or pH (units) variation (between inoculation

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and the stationary phase); µmax or Vmax is the maximum growth rate expressed as ∆log cfu/g/h or

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the maximum acidification rate expressed as dpH/h, respectively; λ is the length of the lag phase

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measured in hours. The experimental data were modelled by the non-linear regression procedure

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of the Statistica 8.0 software (Statsoft, Tulsa, USA).

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A pool of autochthonous lactic acid bacteria, consisting of Leuconostoc citreum San9,

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Pediococcus pentosaceus Bar4, and Weissella confusa Ney6, was used as selected sourdough

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starter (SLS) to ferment all the Iranian wheat flours. Cell suspensions were prepared as described

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elsewhere, the DY was 160 and the initial cell density of lactic acid bacteria was 7.0 log cfu/g.

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Fermentation with SLS was at 30°C for 16 h.

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2.9 Total phenols and antioxidant activity

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Total phenols concentration and antioxidant activity were determined in UD, SS and SLS. The

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1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was determined on the

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methanolic extract (ME) of the dough. Five grams of each dough were mixed with 50 ml of 80%

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methanol to get ME. The mixture was purged with nitrogen stream for 30 min, under stirring

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condition, and centrifuged at 4,600 × g for 20 min. ME were transferred into test tubes, purged 9

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determined as described by Slinkard and Singleton (1997), and expressed as gallic acid equivalent.

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The free radical scavenging capacity was determined using the stable 2,2-diphenyl-1-

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picrylhydrazyl radical (DPPH˙), as reported by Yu et al. (2003). The scavenging activity was

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expressed as follows: DPPH scavenging activity (%) = [(blank absorbance – sample absorbance) /

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blank absorbance] x 100. The value of absorbance was compared with 75 ppm butylated

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hydroxytoluene (BHT), which was used as the antioxidant reference.

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2.10 Phytase activity

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Phytase activity was determined on the WSE of UD, SS and SLS, by monitoring the rate of

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hydrolysis of p-nitrophenyl phosphate (p-NPP) (Sigma, 104-0). The assay mixture contained 200

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µL of 1.5 mM p-NPP (final concentration) in 0.2 M Na-acetate, pH 5.2, and 400 µL of WSE. The

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mixture was incubated at 45ºC and the reaction was stopped by adding 600 µL of 0.1 M NaOH.

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The p-nitrophenol released was determined by measuring the absorbance at 405 nm (De Angelis et

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al., 2003). One unit (U) of activity was defined as the amount of enzyme required to liberate 1

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µmol/min of p-nitrophenol under the assay conditions.

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2.11 Bread making

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Sourdough breads (SB, DY of 160) were manufactured at the pilot plant of the Department of

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Soil, Plant and Food Science of the University of Bari (Italy), according to the two-stage

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protocol including the production of sourdough (fermentation for 16h at 30°C, step I) first, and

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the mixing with flour, water, and baker’s yeast (1.5 h at 30°C, step II), later (Plessas et al.,

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2011; Ravyts et al., 2011). SLS were used at the percentage of 25% (w/w) (Sadeghi et al.,

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2008) and baker’s yeast was added to all the doughs (2% w/w, corresponding to a final cell

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density of ca. 107 cfu/g). Baker’s yeast breads (YB), without addition of SLS, were

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manufactured and used as the controls. Doughs were mixed at 60 × g for 5 min with an IM 5-8

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high-speed mixer (Mecnosud, Flumeri, Italy) and fermentation was at 30°C for 1.5 h. All

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breads were baked at 220°C for 30 min (Combo 3, Zucchelli, Verona, Italy). Fermentations

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were carried out in triplicate and each bread was analyzed twice.

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2.12 Texture and image analyses

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Instrumental Texture Profile Analysis (TPA) was carried out with a TVT-300XP Texture

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Analyzer (TexVol Instruments, Viken, Sweden), equipped with a cylinder probe P-Cy25S. For the

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analysis, boule shaped loaves (300 g) were baked, packed in polypropylene micro perforated bags

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and stored for 24 h at room temperature. Crust was not removed. The selected settings were the

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following: test speed 1 mm/s, 30% deformation of the sample and one compression cycle. TPA

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was carried out (Gámbaro et al., 2004), using Texture Analyzer TVT-XP 3.8.0.5 software (TexVol

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Instruments). Height, width, depth, area, and specific volume of breads were measured by the

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BVM-test system (TexVol Instruments). The following textural parameters were obtained by the

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texturometer software: hardness (maximum peak force); fracturability (the first significant peak

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force during the probe compression of the bread); and resilience (ratio of the first decompression

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area to the first compression area).

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The chromaticity co-ordinates of the bread crust (obtained by a Minolta CR-10 camera) were also

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reported in the form of a color difference, dE*ab, as follows: dE*ab = ඥ(d‫)ܮ‬ଶ + (dܽ)ଶ + (dܾ)ଶ

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where dL, da, and db are the differences for L, a, and b values between sample and reference (a

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white ceramic plate having L = 93.4, a = –0.39, and b = 3.99).

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The crumb features of breads were evaluated after 24 h of storage using the image analysis

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technology with the UTHSCSA ImageTool as previously described by Rizzello et al. (2012).

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2.13 Statistical Analysis

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Data were subjected to one-way ANOVA; pair-comparison of treatment means was obtained by

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Tukey’s procedure at P<0.05, using the statistical software Statistica 8.0 (StatSoft Inc., Tulsa, 11

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USA). Chemical and rheology properties of flours were analyzed through Principal Component

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Analysis (PCA, Dijksterhuis 1997), using the software Statistica 8.0.

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3. Results

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3.1 Chemical and technology characterization of flours

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Four of the flours used in this study were provided by an industrial mill, which is located in the

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Mashaad area (North-east Iran). These flours were blends of national wheat varieties marketed for

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the manufacture of traditional Lavash (Lav), Sangak (San), Barbari (Bar), and Taftoon (Taf)

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breads. The other four flours were from artisanal mills, which are located in rural areas

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(Neyshabur - Ney, Toroujen - Tor, Kashmar - Kas, and Bandar - Ban). Ney and Kas flours were

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mixtures of grains from Triticum aestivum and Triticum durum cultivars. The other flours were

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made with mixture of varieties of soft wheat alone. Overall, the eight blends considered in this

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study were chosen as representative of the flours commonly used for the bread manufacture in Iran

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(Qarooni, 1996).

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The chemical and technology characteristics of the flours are shown in Table 1. Moisture ranged

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from 8.0 ± 0.3 to 13.6 ± 1.1%. Protein concentration was 10.9 ± 1.1 to 14.4 ± 1.3% of dry matter

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(d.m.). The only exception was San flour, which had the highest (P<0.05) concentration (16.4 ±

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1.3% of d.m.). All the flours had a content of ash higher than 0.62% of d.m. Ney and Kas flours,

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made with a mixture of soft and durum wheat, had a concentration of ash higher than 1.05 ±

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0.08% of d.m. These two flours also had concentrations of fat and fibers markedly (P<0.05) higher

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than the other ones. Nevertheless, the concentration of fibers was high (higher than 6.7 ± 0.43%)

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for almost all the flours. San and Ney flours showed the highest (P<0.05) values of dry gluten,

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whereas Lav, Taf and Tor flours had the highest values of gluten index (GI). Kas flour had the

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lowest values of both the indexes. The Falling number ranged from 400 ± 27 to 450 ± 32 sec. The

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only exceptions were Ney and Kas flours, which showed markedly lower values. Data collected

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from the chemical and technology characterization were subjected to Principal Component

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ACCEPTED MANUSCRIPT Analysis (PCA). The first and second factors explained 60.70 and 21.18% of the total variance

303

(Figure 1S). Bar, Taf, Tor and Lav flours were grouped in the same area of the plane because they

304

shared many features. Based on this result, Bar flour was chosen as the representative of this

305

group and only this was considered for further analyses.

306

Two-DE analysis of the gliadin fraction showed large variations of the total number of

307

polypeptides. San and Kas flours had the highest number of spots (74 and 68, respectively)

308

(Figure 1). The other flours showed numbers of gliadin polypeptides in the range of 37 to 48.

309

Within gliadins, the S-rich subunits (α-, β-, and γ-gliadins), having molecular masses of 30 to 45

310

kDa (Žilić et al., 2011), were the most abundant. In particular, 56 and 48 spots were found for San

311

and Kas flours, and 29-40 for the other ones. S-poor gliadin subunits (ω-gliadins), which are

312

usually located in the range of molecular masses of 46-74 kDa ( iliζ et al., 2011), were absent in

313

the Bar flour. A low number (1 to 5) of ω-gliadin spots were detected for the other flours. Bar and

314

Kas showed the highest number of spots (19 and 18, respectively) in the lower part (30 KDa) of

315

the electrophoresis gel, which usually included copious number of polypeptides (De Angelis et al.,

316

2008; Rizzello et al., 2014). Number and intensity of the spots (expressed as pixel area of the gel

317

image, and detected using a threshold method) were calculated for three different clusters of

318

molecular masses (I: >45 kDa; II: 30-45 kDa; and III: <30 kDa) by image analysis. Data were

319

subjected to PCA (Figure 1). The first and second factors explained 56.65 and 33.38% of the total

320

variance. Flours were clearly separated in the plane of the biplot. In particular, San and Bar flours

321

showed the highest and lowest areas for cluster I (aGli-I). San flour also showed the highest

322

number of spots and area for cluster II (aGli-II), followed by Ban and Bar flours. Bar flour had the

323

highest area and number of spots for cluster III (aGli-III). Overall, the other flours, which were

324

grouped with Bar flour (Fig. 1S), showed similar 2-DE profiles of gliadins and glutenins (data not

325

shown).

326

According to previous studies (D’Ovidio and Masci, 2004; Žilić et al., 2011), glutenins ranged

327

from 20.00 to 100.00 kDa, and pI 3.20 to 9.50 (Figure 2). The total number of polypeptides that

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ACCEPTED MANUSCRIPT were resolved through 2-DE ranged from 138 (San flour) to 168 (Kas flour). Spots were grouped

329

into three clusters: 70-140 kDa, corresponding to high molecular weight (HMW)-glutenins

330

(cluster I, D’Ovidio and Masci, 2004; Žilić et al., 2011), 30-70 kDa, referring to low molecular

331

weight (LMW)-glutenins (cluster II), and < 30kDa (cluster III). The highest number of HMW-

332

glutenins was found for Ban and Kas flours. Ney flour showed the highest number of LMW-

333

glutenins, followed by San flour. Number and area of the spots of each cluster were analyzed

334

through PCA (Figure 2). The first and second factors explained 55.75 and 23.43% of the total

335

variance. Kas flour showed the highest area of spots for the first cluster (aGlu-I), whereas San and

336

Bar flours had the highest area for cluster II (aGlu-II). Kas and Ney had the highest area for the

337

spots grouped into cluster III. Also considering glutenin polypeptides, flours were clearly

338

separated into the plane of the biplot.

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3.2 Spontaneous sourdough fermentation

341

UD had values of pH that ranged from 5.85 ± 0.03 to 6.28 ± 0.02, and corresponded to values of

342

TTA of 2.0 ± 0.3 – 3.3 ± 0.2 ml 0.1 N NaOH/10 g of dough. Lactic and acetic acids were not

343

detectable. Doughs made with Ney and Ban flours had the highest and lowest concentration of

344

total free amino acids (FAA), respectively. All UD showed appreciable concentrations of total

345

phenols (7.3 ± 0.2 –9.8 ± 0.21 mmol/kg of dough), especially, those made with Ban and Bar

346

flours. The number of presumptive lactic acid bacteria varied from 3.7 ± 0.3 to 4.1 ± 0.2 log cfu/g

347

of dough. Yeasts were found at 2.0 ± 0.2 to 2.8 ± 0.2 log cfu/g. After mature spontaneous

348

sourdoughs (SS) were obtained (Table 2), the values of pH significantly (P<0.05) decreased, and

349

ranged from 4.13 ± 0.02 to 4.51 ± 0.01. The only exceptions were Ney and Kas sourdoughs, which

350

showed slight but significantly (P<0.05) higher values. As expected, TTA increased and it was in

351

the range of 5.1 ± 0.3 – 7.9 ± 0.2. SS made with Ney flour showed the highest concentrations of

352

lactic and acetic acids (78 ± 2 and 18 ± 1 mmol/kg, respectively), and FAA (1851 ± 17 mg/kg).

353

The quotient of fermentation varied from 4 to 8, and the concentration of polyphenols ranged from

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ACCEPTED MANUSCRIPT 354

9.2 ± 0.2 to 10.9 ± 0.3 mmol/kg. Enumeration of presumptive lactic acid bacteria was in the

355

interval of 9.0 ± 0.1 – 9.7 ± 0.3 log cfu/g. Yeasts were found at densities lower than 4.0 ± 0.2 log

356

cfu/g.

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3.3 Isolation and identification of lactic acid bacteria from spontaneous sourdoughs

359

Gram-positive, catalase-negative, non-motile rods and cocci able to grow at 15°C and to acidify

360

the mMRS broth were subjected to RAPD-PCR analysis. Primers M13, P4, and P7 generated

361

different patterns (bands ranging from 5,000 to 100 bp), and were used for clusters analysis. The

362

reproducibility of the RAPD fingerprints was assessed by comparing the PCR products obtained

363

from three separate cultures of the same strain. The dendrogram for the 50 isolates is shown in

364

Figure 3. At the similarity level of 80%, almost all isolates were grouped into 11 clusters. With the

365

exception of clusters I and II, which included isolates from the same SS (San and Ban,

366

respectively), all the other clusters grouped isolates deriving from different SS.

367

Five strains that did not group into clusters and 11 strains, as the representatives of each cluster,

368

were identified by partial sequencing of the 16S rRNA. The following species were identified: Ln.

369

citreum (3 strains: 2 from SS made with San flour and 1 from SS made with Ban flour), P.

370

pentosaceus (6 strains: 4 from Ban, 1 from Bar and 1 from Ney), Weissella cibaria (4 strains: 2

371

from Ney, 1 from Bar and 1 from Ban), and W. confusa (3 strains: 1 from Nay, 1 from Ban and 1

372

from Kas). The identified strains, related clusters and source of isolation are shown in Figure 3.

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3.4 Selection of lactic acid bacteria

375

Since the number of isolates was rather elevated, the selection of the most performing strain was

376

carried out on the 16 strains chosen on the basis of the RAPD-PCR screening, as previously

377

reported by literature (Coda et al., 2009; Nionelli et al., 2014; Rizzello et al., 2010a);

378

acknowledging the possibility to loose some strains with interesting phenotype features. 15

ACCEPTED MANUSCRIPT Lactic acid bacteria strains were singly used to ferment Bar flour at 30°C for 16 h. Bar flour was

380

chosen as the matrix to assay the performance of lactic acid bacteria because characterized by

381

intermediate chemical features. Parameters of the kinetics of growth and acidification, QF and

382

FAA values of the 16 fermented doughs were taken into account for the selection. Strains showed

383

values of ∆log cfu/g and ∆pH, which ranged in the intervals 1.6 ± 0.2 – 2.5 ± 0.1 and 1.79 ± 0.05

384

– 2.38 ± 0.11, respectively (Table 3). The length of the lag phase (λ) varied from 0.69 ± 0.12 to

385

3.18 ± 0.09 h (growth) and 1.12 ± 0.10 to 4.41 ± 0.21 h (acidification). The values of the quotient

386

of fermentation were lower than 6.8 ± 0.2, and the concentration of FAA was 460 ± 5 – 853 ± 12.

387

The sourdough started with P. pentosaceus Bar4 showed the highest concentration of FAA, while

388

the lowest values of λ and quotient of fermentation were found for sourdoughs fermented with W.

389

confusa Ney6 and Ln. citreum San9, respectively. Mainly based on these features, the above

390

strains were selected and used as mixed starter.

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3.5 Sourdough fermentation with selected and mixed starter

393

Autochthonous P. pentosaceus Bar4, W. confusa Ney6 and Ln. citreum San9 were used in mixture

394

to ferment each of the Iranian flours, and selected sourdough starters (SLS) were obtained. Ney

395

and Kas sourdoughs showed the highest and the lowest values of A and λ (Table 4). The opposite

396

was found for Ban sourdough. The lowest and the highest values of µmax were found for Kas and

397

Bar sourdoughs, respectively. The kinetics of acidification showed that Bar sourdough also had

398

the highest values of Vmax (0.40 ± 0.03∆ph/h). In agreement with the latency phase of growth

399

(Table 4), Ney and Kas sourdoughs had the lowest values of latency phase of acidification (0.92 ±

400

0.10 and 0.93 ± 0.09 h, respectively). The values of pH were in the range 3.83 ± 0.03 – 4.14 ±

401

0.02, which corresponded to values of TTA of 6.8 ± 0.2 – 9.3 ± 0.3 ml 0.1 M NaOH/10 g of

402

dough. The final cell density of presumptive lactic acid bacteria did not significantly (P>0.05)

403

differ among sourdoughs (Table 5). Sourdoughs made with Ney and Kas flours were characterized

404

by the highest concentration of lactic acid and FAA. The lowest values were found for Bar and

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ACCEPTED MANUSCRIPT 405

Ban sourdoughs. The quotient of fermentation was 4 for all the sourdoughs. The concentration of

406

total phenols was the highest for San sourdough and it ranged from 11.6 ± 0.2 to 13.06 ± 0.3 for

407

the other ones.

408

3.6 Antioxidant and phytase activities

410

The antioxidant properties of UD, SS and SLS were determined based on the scavenging activity

411

towards DPPH radical. During assay, the colored stable DPPH radical is reduced to non-radical

412

DPPH-H, when in the presence of an antioxidant or a hydrogen donor. After 10 min of reaction,

413

the scavenging activity of DPPH was 60 ± 0.3% for BHT (75 ppm), and in the range 26.4 ± 1.0 –

414

53.2 ± 1.4% for the methanol extracts (ME) prepared from UD (Figure 4). UD made with Ney and

415

Kas flours had the highest antioxidant activity. The radical scavenging activity of SS was higher

416

than that found in the corresponding UD. The major increase was found for Bar flour. A further

417

increase of the antioxidant activity was found in the presence of SLS. This was particularly

418

evident when Ney and Kas flours were used.

419

The phytase activity of UD, SS and SLS is reported in Figure 4. The activity determined in the

420

water-soluble extracts (WSE) obtained from UD ranged from 0.35 ± 0.10 to 0.99 ± 0.02 U. It was

421

significantly (P<0.05) lower than that found in the corresponding SS. SLS allowed the highest

422

phytase activity. Compared to UD, increases of ca. 2-fold were found for SLS made with San, Bar,

423

and Ban flours. Activities ca. 5-fold higher were found for SLS made with Ney and Kas flours.

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3.7 Bread making

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According to the better results obtained for SLS compared with SS, experimental breads including

427

SLS as ingredient were produced and compared to baker’s yeast breads. Before baking, all the

428

doughs fermented with baker’s yeast alone had values of pH in the range 5.55 ± 0.45 – 5.85 ± 0.49

429

(Table 6). As expected, the use of sourdough affected the pH, which decreased to 4.61 ± 0.34 –

430

4.91 ± 0.39. Sourdough fermentation affected the specific volume of the breads. Compared to 17

ACCEPTED MANUSCRIPT baker’s yeast breads, it was significantly (P<0.05) higher. Bar and San sourdough breads had the

432

highest specific volume. The hardness was the highest when the fermentation was carried out with

433

baker’s yeast alone. Ney and Kas breads showed the highest values. The fracturability,

434

corresponding to the force at the first significant break during compression of the bread, followed

435

the same trend. Sourdough fermentation caused a slight but significant (P<0.05) decrease of the

436

resilience. The crumb structure of breads was evaluated by image analysis technology. Digital

437

images were pre-processed to estimate crumb cell-total area through a binary conversion (Table

438

6). Compared to baker’s yeast breads, the cell-total area (corresponding to the black pixel total

439

area) of sourdough breads were lower. Black pixel total area of sourdough breads were

440

significantly (P<0.05) higher than those observed for breads made with baker’s yeast alone. In

441

particular, San and Bar sourdough breads had the highest cell-total area. The visual inspection of

442

the crust of breads made with sourdoughs showed a more intense coloring compared to breads

443

made with baker’s yeast alone. Indeed, the crust lightness (L) of sourdough breads decreased.

444

Among sourdough breads, the lowest values of L were found for those made with Ney and Kas

445

flours. Almost the same differences were observed for the parameter dE*ab.

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4. Discussion

448

Iran produces more than 1,400,000 tons of wheat per year (Shahan et al., 2008). Bread has a

449

special status in the country and plays an important role in the Iranian diet. Indeed, it was

450

estimated that wheat bread provides 40-60% of national food requirements (Movahed et al., 2011).

451

Representative flours were collected from Iranian rural and urban areas. The exact percentages of

452

the different wheat varieties used for obtaining flour blends are unknown, since Iranian producers

453

use mixed seeds of local cultivars and landraces (Moghaddam et al., 1997). Protein concentration

454

and gluten index of the Iranian flours were typical of soft wheat flours, having good bread making

455

aptitude. The commercial blend marketed for Sangak bread and the flour from Neyshabur area

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ACCEPTED MANUSCRIPT may be classified as high-gluten flours. Only the flour from Bandar Torkaman had a low protein

457

concentration, which is typical of cake flours (Ionescu et al., 2010; Lafiandra and D’Egidio, 2010).

458

Roller-based milling is the most common process used in Iran for obtaining flours, where small

459

and medium plants are widespread. Mainly in the rural areas, Iranian baked goods are

460

manufactured with wheat flours obtained with high extraction rates (Tavajjoh et al., 2011). Indeed,

461

bran is substantially present into the flours, and high concentrations of fibers and ash were found.

462

Flours collected from the rural areas of Neyshabur and Kashmar showed high concentration of fat,

463

thus hypothesizing that wheat germ was not removed during milling. This presence leads to

464

oxidation and rancidity because of the fat content and the high lipase and lipoxygenase activities

465

(Rizzello et al, 2010a, 2010b). The above two flours also had low values of Falling number, which

466

corresponded to high α-amilase activity and extensive sprout damage (Lafiandra and D’Egidio,

467

2010).

468

The suitability of wheat flours for bread making is mainly influenced by gluten proteins (De

469

Angelis et al., 2008). Two-DE analysis of the gliadin fractions from Iranian wheat flours

470

demonstrated the abundant presence of α-, β-, and γ- gliadins, which are commonly related to

471

dough elasticity and leavening (Uthayakumaran et al., 1999). Each flour showed a peculiar

472

profiles. In particular, Sangak and Kashmar flours had the highest number of polypeptides. The

473

occurrence of ω-gliadins was lower. According to literature data (Žilić et al., 2011), the intensity

474

of α-, β- and γ- gliadin polypeptides was lower in the blends of durum wheat and soft wheat

475

(Neyshabur and Kashmar flours). The electrophoretic maps of the glutenins extracted from the

476

Iranian flours showed that LMW- were most abundant than HMW-polypeptides (Žilić et al.,

477

2011). LMW-glutenins influence the dough resistance and extensibility (D'Ovidio and Masci,

478

2004; Žilić et al., 2011). The industrial flours Sangak and Barbari showed a higher number and

479

intensity of LMW-glutenin spots compared to those collected from the rural areas. These

480

differences could be responsible for different technology aptitudes (Gupta and MacRitchie, 1994;

481

Wieser and Kieffer, 2001).

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ACCEPTED MANUSCRIPT Type I sourdoughs were prepared, and they stabilized, at least from a biochemical point of view,

483

after six refreshments (Minervini et al., 2012a, 2012b). Only ten isolates from the highest plate

484

dilutions of each spontaneous sourdoughs were subjected to typing and identification since the

485

main focus of the study was on the most adaptable and fast growing autochthonous strains and not

486

on the description of the microbiota diversity. Strains of P. pentosaceus were identified from three

487

spontaneous sourdoughs made with different Iranian flours. This species was isolated in

488

sourdoughs of different geographical origin (Coda et al., 2014) and it was usually found in

489

fermented vegetables (Corsetti et al., 2007). The other species found in Iranian spontaneous

490

sourdoughs were W. cibaria, W. confusa and Ln. citreum, which were previously isolated from

491

wheat flours and sourdoughs (Coda et al., 2014; De Vuyst et al., 2014). Species belonging to

492

Lactobacillus genus were not identified under the conditions of this study. This could be due to

493

the limited number of isolates subjected to analyses and/or to the use of only one medium

494

(mMRS) for isolation. Nevertheless, an abundant literature (Coda et al., 2010a, 2010b; Minervini

495

et al., 2012a; Nionelli et al., 2014; Ricciardi et al., 2005; Settani et al., 2013) showed that, when

496

lactobacilli are dominant in the sourdough biota, they are easily isolated using mMRS as well as

497

other specific media such as Sourdough Bacteria (SDB).

498

All the spontaneous sourdoughs harbored a population of yeasts, which, however, was markedly

499

lower than that commonly found in sourdoughs (Gobbetti, 1998; Hammes et al., 2005). The major

500

part of the advantages attributed to sourdough are due to lactic fermentation (Gobbetti, 1998).

501

Therefore, the selection of starters focused only on lactic acid bacteria strains. Autochthonous and

502

mixed strains were largely shown to be one of the best choice to get successful sourdough

503

fermentation (Coda et al., 2010a, 2010b). When selected and mixed starters were used, the cell

504

density of lactic acid bacteria was similar to that found after spontaneous fermentation. Almost all

505

the other features improved. The synthesis of lactic and acetic acids increased, and the quotient of

506

fermentation reached the optimal value for sensory attributes. The concentration of total free

507

amino acids also increased, thanks mainly to the proteolytic activity of the selected starters. It was

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ACCEPTED MANUSCRIPT shown that proteolysis improves nutritional and organoleptic features of baked goods (Rizzello et

509

al., 2014). Moreover, free amino acids are converted during baking into volatile compounds,

510

which are responsible for the typical flavor and odor of sourdough breads (Corsetti and Settanni,

511

2007).

512

Cereals contain phenolic acids and flavonoids in the free, soluble conjugated and insoluble bound

513

forms (Vaher et al., 2010). Since phenols are mainly located in the bran and germ fractions,

514

relevant concentrations were found in all the Iranian flours. As previously shown (Rizzello et al.,

515

2012), acidification with selected starters improves the extraction of total phenols. This would

516

explain the highest antioxidant activity, which was found in sourdoughs started with selected

517

strains. The anti-nutritional factor phytic acid is mainly located in the outer layers, pericarp and

518

germ of wheat kernel. Therefore, its concentration is particularly high in flours made with high

519

extraction rate (Rizzello et al., 2012). Phytase, which catalyzes the hydrolysis of phytic acid into

520

myo-inositol and phosphoric acid, makes available phosphate and leads to non-metal chelator

521

compound (Martinez et al., 1996). The changes of the traditional bread making processes and their

522

effect on the phytic acid degradation were largely debated with respect to the mineral availability

523

of Iranian breads (Didar, 2011; Sedaghati et al., 2011). It was estimated that 30% of Iranians

524

suffers iron or zinc deficiency (Didar, 2011). The value of pH reached with sourdough

525

fermentation is suitable to activate flour endogenous phytases. Besides, sourdough lactic acid

526

bacteria possess somewhat phytase activity (Di Cagno et al., 2008; Rizzello et al., 2010a, 2010b).

527

Compared to spontaneous fermentation, sourdoughs fermented with selected and mixed starters

528

showed an increase of the phytase activity up to 70%.

529

Usually, the high concentration of fibers is associated to the weak dough structure, decreased

530

bread volume and crumb elasticity, and increased loaf hardness (Rizzello et al., 2012). Under the

531

conditions of this study, the lowest specific volume and cell-total area of the crumb slices were

532

found for baker’s yeast breads made with Neyshabur and Kashmar flours. On the contrary, Sangak

533

and Barbari flours, which were characterized by an abundance of S-rich gliadins and LMW-

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ACCEPTED MANUSCRIPT glutenins, allowed the manufacture of breads with higher volume and cell-total area. Overall, the

535

use of sourdough improved both the above parameters. As previously reported for wholemeal

536

breads (Rizzello et al., 2012; 2010b), the hardness and fracturability of sourdough breads were

537

lower than those of breads fermented with baker’s yeast alone. The concentration of fibers

538

affected the bread color. Breads made with Neyshabur and Kashmar flours showed the darkest

539

crust. Sourdough fermentation increased the intensity of the crust color due to the proteolysis by

540

lactic acid bacteria, which released free amino acids available for the Maillard reaction during

541

baking (Rizzello et al., 2010b).

542

The analysis of the main chemical and technology characteristics of the Iranian flours

543

demonstrated a good suitability for bread making. Flours collected from rural areas contained

544

wheat germ and showed sprout damage. Selected lactic acid bacteria were suitable mixed starters

545

to exploit the technology and nutritional features of Iranian flours. The data from this study might

546

had helpful to show the potential of a biotechnology protocol to easily replace the largely used

547

chemical leavening, which was recognized as one of the main cause for nutritional problems,

548

waste of products, and losses of peculiar features of the traditional baked goods (Didar, 2011;

549

Fazeli et al., 2004).

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Acknowledgements

552

The authors thank the Fundación Alfonso Martín Escudero (Madrid, Spain) for the postdoctoral

553

fellowship of J.A. Curiel; Davide Minervini (Molini Tandoi, Corato, BA-IT) for the chemical

554

analyses.

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TE D

742 743

30

ACCEPTED MANUSCRIPT

Table 1. Chemical and technological characteristics of the Iranian wheat flours.

Type

Moisture (%)

Protein (% of d.m.)

Ash (% of d.m.)

Lav

Commercial blend designed for making Lavash bread, purchased from an industrial mill in Mashhad area, Iran

Triticum aestivum

12.7±0.8b

12.3±1.0

0.83±0.10c

San

Commercial blend designed for making Sangak bread, purchased from an industrial mill in Mashhad area, Iran

Triticum aestivum

11.7±0.6c

16.4±1.3a

Bar

Commercial blend designed for making Barbari bread, purchased from an industrial mill in Mashhad area, Iran

Triticum aestivum

9.3±0.5e

12.2±1.2

Taf

Commercial blend designed for making Taftoon bread, purchased from an industrial mill in Mashhad area, Iran

Triticum aestivum

12.2±1.2b

12.4±1.2

Ney

Blend purchased from an artisanal mill in the rural area of Neyshabur, Iran

Tor

Blend purchased from an artisanal mill in the rural area of Toroujen, Iran

Kas

Blend purchased from an artisanal mill in the rural area of Kashmar, Iran

Ban 746 747 748

Blend purchased from an artisanal mill in the rural area of Bandar Torkaman/Jorgan, Iran

d

Fat (% of d.m.)

Fibers (% of d.m.)

Dry gluten (% of d.m.)

Gluten Index

Falling number (sec)

1.45±0.10c

6.70±0.43e

11.0±1.3 b

100±9a

450±32a

67±6b

1.45±0.09c

2.65±0.20f

12.2±1.0a

96±7b

410±24b

0.77±0.03d

69±6a

1.31±0.11c

7.43±0.32d

11.5±0.9

96±9b

417±30d

0.78±0.2d

64±4c

1.31±0.14c

9.30±0.65c

11.3±1.5 b

99±3a

400±27b

SC

66±5b

0.82±0.07c

M AN U d

TE D

Triticum aestivum and Triticum durum

d

Starch (% of d.m.)

RI PT

Origin

b

10.2±0.9d

14.4±1.3 b

1.99±0.21a

57±6e

4.70±0.24a

11.71±1.09a

12.4±1.7a

96±6b

85±9c

12.2±0.9b

12±0.8d

1.05±0.08b

64±7c

1.70±0.15b

9.04±0.47c

11.1±0.8 b

98±5a

446±22a

Triticum aestivum and Triticum durum

8.0±0.3e

13.9±0.9c

1.94±0.11a

61±4d

4.7±0.31a

10.46±1.79b

8.6±0.5d

78±5c

72±6c

Triticum aestivum

13.6±1.1a

10.9±1.1e

0.62±0.09e

66±5b

1.24±0.08d

7.60±0.52d

9.9±0.8c

97±8b

408±29b

Triticum aestivum

EP

Flours

AC C

744 745

The data are the means of three independent experiments ± standard deviations (n = 3). a-e Values in the same column with different superscript letters differ significantly (P<0.05). 31

ACCEPTED MANUSCRIPT

Flours

pH

(mL of 0.1 N NaOH/10g)

Acetic acid

(mmol/kg)

(mmol/kg)

QF

FAA

Total phenols

(mg/ kg)

(mmol/kg)

Lactic acid bacteria (log cfu/g)

Yeast (log cfu/g)

4.51±0.01b

5.1±0.3c

55±1c

7±2b

8a

857±11c

9.7±0.1c

9.2±0.1b

3.0±0.1c

Bar

4.39±0.03c

5.3±0.2c

39±2d

9±2b

4b

821±10d

10.7±0.2a

9.7±0.3a

4.0±0.2a

Ney

4.71±0.01a

7.9±0.2a

78±2a

18±1a

4b

1851±17a

10.1±0.1b

9.7±0.2a

3.0±0.2c

Kas

4.78±0.02a

6.7±0.1b

68±1b

16±3a

4b

1208±14b

9.2±0.2d

9.4±0.1b

3.7±0.3a

Ban

4.13±0.02d

5.5±0.2c

60±3c

8±1b

8a

629±12e

10.9±0.3a

9.0±0.1c

3.4±0.3b

TE D

M AN U

San

The data are the means of three independent experiments ± standard deviations (n = 3). a-d Values in the same column with different superscript letters differ significantly (P<0.05). See materials and methods for further information on flours, including the significance of abbreviations.

EP

753 754 755 756 757 758

Lactic acid

SC

TTA

RI PT

Table 2. Chemical and microbiological characteristics of spontaneous sourdoughs (SS) from five Iranian flours.

AC C

749 750 751 752

32

ACCEPTED MANUSCRIPT

RI PT

FAA**

A

λ

µmax

∆pH

(log cfu/g)

(h)

(∆log cfu/g/h)

(pH units)

m 1.6±0.2 M 2.5±0.1

m 0.69±0.12

m 0.78±0.11

m 1.79±0.21

m 1.12±0.10

m 0.24±0.05

m 1.5±0.1

m 460±5

M 3.18±0.09

M 1.51±0.08

M 2.38±0.14

M 4.41±0.21

M 1.83±0.09

M 6.8±0.2

M 853±12

Leuconostoc citreum San9

2.1±0.1b

0.92±0.12b

0.93±0.09a

1.85±0.22b

3.56±0.21a

1.83±0.09a

1.5±0.1c

466±3c

Pediococcus pentosaceus Bar4 Weissella confusa Ney6

1.6±0.2c

2.26±0.09a

0.81±0.13b

2.19±0.11a

2.65±0.23b

0.48±0.02b

5.0±0.3a

853±12a

2.25±0.1a

0.78±0.21b

0.78±0.11b

2.04±0.12b

1.12±0.10c

0.24±0.03c

2.31±0.3b

615±9b

Vmax

(h)

(∆pH/ h)

M AN U

SC

λ

QF

*

(mg/kg)

TE D

Bar flour was used as common matrix for sourdough fermentation. The data are the means of three independent experiments ± standard deviations (n = 3). a-c Values in the same column with different superscript letters differ significantly (P<0.05).

EP

765 766 767 768 769

Table 3. Parameters of the kinetics of growth and acidification, quotient of fermentation (molar ratio between lactic and acetic acids, QF) and concentration of total free amino acids (FAA, mg/kg) of sourdoughs started with single lactic acid bacteria strains isolated from the five Iranian spontaneous sourdoughs and fermented at 30°C for 16 h. The minimum (m) and maximum (M) refer to whole number of isolates. Values for individual lactic acid bacteria, which were further used as selected starters for sourdough fermentation, are also included.

AC C

759 760 761 762 763 764

33

ACCEPTED MANUSCRIPT

A

λ

µmax

(log cfu/g)

(h)

San

1.70±0.12b

Bar

RI PT

Selected

λ

Vmax

(∆log cfu/g/h)

(pH units)

(h)

(∆pH/h)

1.92±0.06a

0.83±0.10b

1.80±0.09b

2.26±0.12b

0.34±0.11b

2.05±0.09a

1.21±0.10b

1.27±0.08a

1.81±0.02b

1.77±0.07c

0.40±0.03a

Ney

2.15±0.10a

0.82±0.10c

0.93±0.12b

1.82±0.12b

0.92±0.05d

0.28±0.04b

Kas

2.10±0.20a

0.83±0.09c

0.44±0.14d

1.96±0.15a

0.93±0.09d

0.23±0.02c

Ban

1.50±0.18b

2.03±0.12a

0.76±0.12c

1.61±0.07c

2.43±0.06a

0.30±0.03b

M AN U

starter (SLS)

SC

∆pH

sourdough

TE D

Data are the mean of three independent fermentations twice analyzed. a-d Values in the same column with different superscript letters differ significantly (P<0.05). See materials and methods for further information on flours, including the significance of abbreviations.

EP

789 790 791

Table 4. Parameters of the kinetics of growth and acidification of the sourdoughs made with the Iranian San, Bar, Ney, Kas, and Ban flours, and fermented with Leuconostoc citreum San9, Pediococcus pentosaceus Bar4 and Weissella confusa Ney6 at 30°C for 16 h.

AC C

770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788

34

ACCEPTED MANUSCRIPT

sourdough

pH

starter (SLS)

(mmol/ kg)

(mmol/ kg)

QF

San

3.83±0.03e

6.8±0.2d

62±4c

16±3c

Bar

3.92±0.01c

8.7±0.1b

53±2d

Ney

4.14±0.02a

9.3±0.3a

99±2a

Kas

4.08±0.02b

9.1±0.2a

Ban

3.88±0.01d

7.2±0.2c

M AN U

NaOH/10g)

Acetic acid

4

LAB

FAA

Polyphenols

(mg/kg)

(mmol/kg)

1059±11c

14.1±0.1a

9.0±0.1

(log cfu/g)

15±2c

4

1048±19c

11.6±0.2c

9.3±0.2

24±3a

4

2287±24a

13.1±0.2b

9.3±0.1

86±3b

20±4b

4

1433±11b

13.6±0.3b

9.1±0.1

55±1d

15±2c

4

871±14d

11.9±0.3c

8.7±0.3

TE D

Data are the mean of three independent fermentations twice analyzed. a-e Values in the same column with different superscript letters differ significantly (P<0.05). See materials and methods for further information on flours, including the significance of abbreviations.

EP

797 798 799 800

(mL of 0.1 N

Lactic acid

SC

TTA

Selected

RI PT

Table 5. Main microbiological and chemical features of sourdoughs produced with the Iranian San, Bar, Ney, Kas, and Ban flours, and fermented with Leuconostoc citreum San9, Pediococcus pentosaceus Bar4 and Weissella confusa Ney6 at 30°C for 16 h.

AC C

792 793 794 795 796

35

ACCEPTED MANUSCRIPT

San

Bar

Ney

YB

SB

YB

SB

YB

5.61±0.43b

4.67±0.34d

5.55±0.45b

4.61±0.34d

5.85±0.49a

2.01±0.16c

2.43±0.22b

2.18±0.22c

2.62±0.21a

1.64±0.15e

Hardness (g)

8819±376d

4437±287g

6674±532e

3670±354h

Fracturability (g)

7403±564d

2999±249f

5432±465e

Resilience

0.73±0.05a

0.66±0.04c

37.6±0.7c

L

Kas

Ban

SB

YB

SB

YB

SB

4.91±0.39c

5.79±0.38a

4.84±0.42c

5.73±0.33a

4.69±0.47d

1.73±0.12d

1.65±0.17e

1.75±0.09d

1.68±0.13e

2.00±0.18c

13200±1242a

10100±932b

14230±1262a

10702±953b

9888±882c

5931±562f

2634±254f

9543±884a

8003±749c

9784±854a

8624±724b

8625±728b

4703±376e

0.75±0.06a

0.65±0.07c

0.78±0.05a

0.71±0.08b

0.70±0.04b

0.68±0.06c

0.73±0.07a

0.67±0.06c

51.7± 1.1b

38.2±0.9c

55.1±1.3a

27.9±2.3e

35.6±1.3c

27.4±1.0e

33.4±2.1d

28.4±1.7e

36.9±2.1d

68.25±0.27a

64.36±0.21c

69.35±0.17a

66.78±2.03b

63.14±0.18c

56.51±1.36e

63.33±0.18c

61.97±0.3d

71.15±0.21a

63.60±1.17c

a

5.48±0.13d

6.44±0.19c

5.23±0.11d

4.59±0.82e

6.01±0.13c

10.01±0.49a

6.35±0.23c

8.54±0.11b

5.31±0.32d

6.03±0.11c

b

21.73±0.27c

22.17±0.43b

20.71±0.17d

21.08±2.46c

20.73±0.09d

21.75±0.45c

21.44±0.18c

22.4±0.09b

22.53±0.71b

23.04±0.81a

∆E

31.33±0.25e

35.11±0.22c

29.68±0.17f

33.08±1.60d

35.17±1.03c

42.24±1.18a

35.41±1.21c

37.50±0.59b

29.52±0.85e

35.95±1.01c

pH Specific volume 3

(cm /g)

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Structural characteristics

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Black pixel ratio (%)

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Image analysis

805 806 807 808 809

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Table 6. Characteristics of breads made with the five Iranian San, Bar, Ney, Kas, and Ban flours. Sourdough breads (SB) were made with the addition of 25% (w/w) of selected sourdoughs (SLS) previously fermented with Leuconostoc citreum San9, Pediococcus pentosaceus Bar4 and Weissella confusa Ney6 at 30°C for 16 h. Baker’s yeast breads (YB) were made without addition of SLS and used as the controls. Baker’s yeast was added to all the doughs (2% w/w); fermentation was at 30°C for 1.5 h.

SC

801 802 803 804

Data are the mean of three independent fermentations twice analyzed. a-h Values in the same row with different superscript letters differ significantly (P<0.05). See materials and methods for further information on flours, including the significance of abbreviations.

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Legends to figures

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Figure 1. Two-dimensional gel electrophoresis (2-DE) analysis of gliadins extracted from Iranian San (A), Bar (B), Ney (C), Kas (D) and Ban (E) wheat flours. See materials and methods for further information on flours, including the significance of abbreviations. The principal component biplot (Dijksterhuis, 1997), based on number (n) and intensity of polypeptides, calculated as pixel area (a) by image analysis of the gels, is shown. Number and area of the spots were calculated for three clusters of molecular masses, separated by dashed lines: cluster I, >45 kDa (nGli-I and aGli-I, respectively); cluster II, 30-45 kDa (nGli-II and aGli-II, respectively); and cluster III, <30kDa (nGli-III and aGli-III, respectively).

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Figure 2. Two-dimensional gel electrophoresis (2-DE) analysis of glutenins extracted from Iranian San (A), Bar (B), Ney (C), Kas (D) and Ban (E) wheat flours. See materials and methods for further information on flours, including the significance of abbreviations. The principal component biplot (Dijksterhuis, 1997) based on number (n) and intensity of polypeptides, calculated as pixel area (a) by image analysis of the gels, is shown. Number and area of the spots were calculated for three clusters of molecular masses, separated by dashed lines: cluster I, >45 kDa (nGli-I and aGli-I, respectively); cluster II, 30-45 kDa (nGli-II and aGli-II, respectively); and cluster III, <30kDa (nGli-III and aGli-III, respectively). Figure 3. Dendrogram obtained by combined random amplification of polymorphic DNA patterns for the isolates from spontaneous sourdoughs (SS) using primers M13, P4 and P7. The codes used to identify isolates refer to the source of isolation (San, Bar, Ney, Kas, and Ban spontaneous sourdoughs). See materials and methods for further information on flours, including the significance of abbreviations. Cluster analysis was based on the simple matching coefficient and unweighted pair group with arithmetic average.

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Figure 4. Antioxidant (A) and phytase (B) activities of doughs before fermentation (UD, unfermented doughs), spontaneous sourdoughs (SS), and selected sourdough starters (SLS) made with Iranian San, Bar, Ney, Kas, and Ban flours. See materials and methods for further information on flours, including the significance of abbreviations. Antioxidant activity, calculated as scavenging activity on DPPH radical, was expressed as follows: DPPH scavenging activity (%) = [(blank absorbance – sample absorbance) / blank absorbance] x 100. One unit (U) of phytase activity was defined as the amount of enzyme required to liberate 1 µmol/min of p- nitrophenol under the assay conditions.

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Figure 1S. Principal component analysis (PCA) based on the main chemical characteristics of the eight Iran wheat flours: Lav, San, Bar, Taf, Ney, Tor, Kas, and Ban. As: Ash, Dg: Dry gluten, Fa: Fat, Fi: Fibers, Fn: Falling number, Hu: Humidity, Pr: Protein, St: Starch.

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Highlights •

Iranian wheat flours were characterized on the basis of chemical characteristics.

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Type I sourdoughs were prepared through back slopping procedure and characterized. Microbiota was investigated and a pool of strains was selected as starters for

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fermentation.

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making were demonstrated.

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