Accepted Manuscript

Use of sourdough made with quinoa (Chenopodium quinoa) flour and autochthonous
selected lactic acid bacteria for enhancing the nutritional, textural and sensory
features of white bread

Carlo Giuseppe Rizzello, Anna Lorusso, Marco Montemurro, Marco Gobbetti

PII: S0740-0020(15)00253-1
DOI: 10.1016/j.fm.2015.11.018
Reference: YFMIC 2499

To appear in: Food Microbiology

Received Date: 16 August 2015

Revised Date: 6 November 2015
Accepted Date: 27 November 2015

Please cite this article as: Rizzello, C.G., Lorusso, A., Montemurro, M., Gobbetti, M., Use of sourdough
made with quinoa (Chenopodium quinoa) flour and autochthonous selected lactic acid bacteria for
enhancing the nutritional, textural and sensory features of white bread, Food Microbiology (2015), doi:
10.1016/j.fm.2015.11.018.

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 ACCEPTED MANUSCRIPT
1 Use of sourdough made with quinoa (Chenopodium quinoa) flour and

2 autochthonous selected lactic acid bacteria for enhancing the nutritional,

3 textural and sensory features of white bread

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5 Carlo Giuseppe Rizzello*, Anna Lorusso, Marco Montemurro, Marco Gobbetti

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7 Department of Soil, Plant and Food Science, University of Bari Aldo Moro, 70126 Bari, Italy

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8

9 *Corresponding author. Tel.: +39 0805442950; Fax: +390805442911.

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E-mail address: [email protected]
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12

13 Abbreviations
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14 IDF: insoluble dietary fiber; SDF: soluble dietary fiber; DY: dough yield; TTA: Total titratable
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15 acidity; WSE: Water/salt-soluble extract; FAA: free amino acids; QS: quinoa sourdough; QD:
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16 quinoa dough; FQ: fermentation quotient; ME: methanolic extract; IVPD: in vitro protein

17 digestibility; QSB: quinoa sourdough bread; WB: wheat bread; QB: quinoa bread; TPA: Texture
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18 Profile Analysis; CS: Chemical Score; EAA: essential amino acid; EAAI: Essential Amino Acids
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19 Index; PER: Protein Efficiency Ratio; NI:Nutritional Index; HI: hydrolysis index; GI: glycemic

20 index; QF: quinoa flour.

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21 Abstract

22 Lactic acid bacteria were isolated and identified from quinoa flour, spontaneously fermented

23 quinoa dough, and type I quinoa sourdough. Strains were further selected based on acidification

24 and proteolytic activities. Selected Lactobacillus plantarum T6B10 and Lactobacillus rossiae

25 T0A16 were used as mixed starter to get quinoa sourdough. Compared to non-fermented flour,

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26 organic acids, free amino acids, soluble fibers, total phenols, phytase and antioxidant activities,

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27 and in vitro protein digestibility markedly increased during fermentation. A wheat bread was made

28 using 20% (w/w) of quinoa sourdough, and compared to baker’s yeast wheat breads manufactured

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29 with or without quinoa flour. The use of quinoa sourdough improved the chemical, textural, and

30 sensory features of wheat bread, showing better performances compared to the use of quinoa flour.

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Protein digestibility and quality, and the rate of starch hydrolysis were also nutritional features that
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32 markedly improved using quinoa sourdough as an ingredient.

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33 This study exploited the potential of quinoa flour through sourdough fermentation. A number of

34 advantages encouraged the manufacture of novel and healthy leavened baked goods.
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35
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36
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37 Keywords

38 Quinoa, Lactic acid bacteria, Sourdough, Bread

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39 1. Introduction

40 Quinoa (Chenopodium quinoa Willd.) is a seed crop, which is traditionally cultivated in the

41 Andean region since thousands of years. Commonly, quinoa grains and flour are used for human

42 consumption and animal feeding. Quinoa has a very elevated genetic variability, which makes

43 possible to select, adapt and breed cultivars for a wide range of environmental conditions (Bertero

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44 et al., 2004). Indeed, quinoa has the capacity of adapting to a range of agro-ecological conditions,

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45 showing tolerance to frost, salinity and drought, and having the potential to grow on marginal

46 soils. These features, together with an undoubtedly nutritional value, determine a worldwide

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47 interest for this crop (Stikic et al., 2012). During the last years, the production of quinoa markedly

48 increased, thus emphasizing the opportunity to cultivate this crop in various regions. Diverse

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climatic regions of USA, Canada, India, England, Denmark, Greece, Italy and other European
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50 countries were shown to be suitable for an extended cultivation (Stikic et al., 2012). FAO selected
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51 the quinoa as one of the crops that are destined to offer food security in the 21st century (Jacobsen

52 et al., 2003).
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53 The high nutritional value of quinoa seeds is mainly due to high concentrations of proteins,
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54 minerals, and vitamins (Fleming and Galwey, 1995). Proteins are rich in amino acids like lysine,

55 threonine and methionine, which, on the contrary, are deficient in cereals. A special interest was
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56 deserved to the use of quinoa for people who are affected by celiac disease. Quinoa was
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57 considered an efficient nutritional alternative to gluten-containing wheat, rye and barley (Jacobsen
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58 et al., 2003), being largely used as an ingredient for making breads, biscuits, cookies, crepes,

59 muffins, pancakes and tortillas. While the nutritional value and the chemical composition of

60 quinoa were characterized, several aspects concerning the technological applications have

61 received less attention (Stikic et al., 2012). Overall, the baking quality is considered rather low due

62 to the absence of gluten, (Stikic et al., 2012) and flavor, texture and appearance of baked goods,

63 including quinoa in the recipes, were reported only as moderately acceptable (Stikic et al., 2012).

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64 Sourdough fermentation has the potential to exploit the technological, nutritional, functional and

65 sensory features of wheat and non-wheat flours. Besides the well-known advantages documented

66 for processing wheat and rye, an abundant literature showed how the sourdough may enhance

67 various features of milling byproducts (Coda et al., 2015a; Rizzello et al., 2010a; Rizzello et al.,

68 2010b), minor cereals (Coda et al., 2011a; Coda et al., 2010a), legumes (Curiel et al., 2015;

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69 Rizzello et al., 2014a), teff, and buckwheat (Coda et al., 2014). In particular, sourdough

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70 fermentation improves dough workability, bread structure and organoleptic and nutritional

71 properties of raw flours. Furthermore, it increases the content of biogenic compounds and the

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72 uptake of minerals, and decreases the level of anti-nutritional factors and the value of the

73 glycaemic response (Gobbetti et al., 2014).

74
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Nevertheless, fermentation processes depend on specific determinants, which have to be strictly
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75 controlled to get standardized and agreeable products (Coda et al., 2014). Among these
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76 determinants, the type of flour is one of the most important. It affects the technological features

77 and the nutritional value of the baked goods and, more in general, the microbial fermentation
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78 through the level and type of fermentable carbohydrates, nitrogen sources and growth factors
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79 (Coda et al., 2014). To exploit the potential of particular flour matrices, the selection of adequate

80 starter cultures is needed (Coda et al., 2014). Regarding this aspect, the literature shows very few
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81 information on the quinoa lactic acid bacteria microbiota and on the selection of suitable strains

for industrial or artisanal baking.

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82
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83 First, this study aimed at selecting autochthonous lactic acid bacteria strains to be used for quinoa

84 sourdough fermentation. The quinoa sourdough made with selected starters was characterized and

85 used as an ingredient to enrich white wheat bread. An integrated characterization, including

86 nutritional, texture and sensory features, was carried out to show the numerous advantages of the

87 process.

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89 2. Materials and methods

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90 2.1. Quinoa flour

91 Organic quinoa (Chenopodium quinoa) dehulled seeds imported from Argentina (Fundacion

92 Nuevagestion, San Ignacio de Loyola, Jujuy) were used in this study. Quinoa flour (QF) was

93 obtained from seeds by the laboratory mill Ika-Werke M20 (GMBH, and Co. KG, Staufen,

94 Germany). Protein (total nitrogen × 5.7), lipids, ash and moisture contents were determined

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95 according to the AACC approved methods 46-11A, 30-10.01, 08-01, and 44-15A, respectively

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96 (AACC, 2010). Total carbohydrates were calculated as the difference [100- (proteins + lipids +

97 ash + total dietary fibers )]. Proteins, lipids, carbohydrates and ash were expressed as % of dry

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98 matter (d.m.).

99 The determination of insoluble (IDF) and soluble (SDF) dietary fibers was carried out by AOAC

100
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approved methods 991.42 and 993.19, respectively (Horwitz and Latimer, 2006).
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101
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102 2.2. Microbiological analysis and isolation of lactic acid bacteria. Lactic acid bacteria were

103 isolated from: quinoa flour (T0), quinoa flour dough, having dough yield (DY, dough weight x
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104 100/flour weight) of 160, and subjected to incubation at 30°C for 16 h (T1), and quinoa type I
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105 sourdough (T6). Quinoa type I sourdough was made and propagated through the traditional

106 protocol commonly used for wheat flour fermentation, without using starter cultures or baker’s
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107 yeast. Quinoa flour was mixed with tap water at 60 rpm for 5 min, with a IM 5-8 high-speed mixer

(Mecnosud, Flumeri, Italy), and the dough (DY 160) was incubated at 30°C for 16 h (T1). After
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108
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109 this first fermentation, six back-slopping steps (refreshments) were further carried out, mixing

110 30% of the previously fermented dough with flour and water (DY of 160), and incubating for 16 h

111 at 30°C. After each fermentation, doughs were stored at 4˚C until the next refreshment. The pH

112 value of doughs was determined by a pHmeter (Model 507, Crison, Milan, Italy) with a food

113 penetration probe. Total titratable acidity (TTA) was determined after homogenization of 10 g of

114 dough with 90 ml of distilled water, and expressed as the amount (ml) of 0.1 M NaOH required to

115 neutralize the solution, using phenolphthalein as indicator (official AACC method 02-31.01). The
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116 rate of volume increase of doughs was determined as described by Minervini et al. (2011). After

117 six refreshments, the acidification rate and volume increase were stable, and quinoa sourdough

118 was used for microbiological analysis and isolation of lactic acid bacteria. Ten grams of quinoa

119 flour (F), dough (T1) or sourdough (T6) were homogenized with 90 ml of sterile peptone water

120 (1% [wt/vol] of peptone and 0.9% [wt/vol] of NaCl) solution. Presumptive lactic acid bacteria

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121 were enumerated using three different agar media: MRS (Oxoid, Basingstoke, Hampshire, United

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122 Kingdom), modified MRS (mMRS) (containing 1% [wt/vol] maltose, 5% [vol/vol] fresh yeast

123 extract, pH 5.6) (Oxoid, Basingstoke, Hampshire, United Kingdom), and SDB (sourdough

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124 bacteria, Kline and Sugihara, 1971). Media were supplemented with cycloheximide (0.1 g liter).

125 Plates were incubated at 30°C for 48 h, under anaerobiosis (AnaeroGen and AnaeroJar, Oxoid).

126
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Approximately ten colonies of presumptive lactic acid bacteria were randomly selected from the
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127 plates containing the two highest sample dilutions. Gram-positive, catalase-negative, non-motile
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128 rods and cocci isolates were cultivated into MRS, mMRS, or SDB broth at 30°C for 24 h and re-

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

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131

132 2.3. Genotypic characterization by Randomly Amplified Polymorphic DNA-Polymerase

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

according to De los Reyes-Gavilán et al. (1992). Three oligonucleotides, P1 5’- ACGCGCCCT-3’,

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135 P4 5’-CCGCAGCGTT-3’, and M13 5’-GAGGGTGGCGGTTCT-3’, with arbitrarily chosen

136 sequences, were used for bio-typing of lactic acid bacteria isolates. Reaction mixture and PCR

137 conditions for primers P1 and P4 were those described by Corsetti et al. (2003), whereas those

138 reported by Zapparoli et al. (1998) were used for primer M13. RAPD-PCR profiles were acquired

139 through the Gel Doc 2000 Documentation System and compared using the Fingerprinting II

140 InformatixTM Software (Bio-Rad Laboratories). Dice coefficients of similarity and UPGMA

141 algorithm were used to estimate the similarity of the electrophoretic profiles. Since RAPD profiles
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142 of the isolates from one batch of each type of sourdough were confirmed by analyzing isolates

143 from two other batches, strains isolated from a single batch were further analyzed.

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145 2.4 Genotypic identification of lactic acid bacteria

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

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

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148 (Pontonio et al., 2015). Electrophoresis was carried out on agarose gel at 1.5% (wt/vol)

149 (Gellyphor, EuroClone) and amplicons were purified with GFXTM PCR DNA and Gel Band

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150 Purification Kit (GE Healthcare).

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

152
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rDNA sequences alignments were carried out using the multiple sequence alignment method
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153 (Pontonio et al., 2015) and identification queries were fulfilled by a BLAST search in GenBank
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154 (http://www.ncbi.nlm.nih.gov/genbank/).

155
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156 2.5. Selection of autochthonous lactic acid bacteria and preparation of selected sourdough
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157 starter

158 Autochthonous lactic acid bacteria strains (n.123) were cultivated into MRS, mMRS, or SDB
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159 broth (depending on the isolation media) at 30°C for 24 h. Cells were harvested by centrifugation

(10,000 x g, 10 min, 4°C), washed twice in 50 mM sterile potassium phosphate buffer (pH 7.0)
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160
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161 and re-suspended in tap water at the cell density of ca. 8.0 log cfu/ml. Quinoa flour (62.5 g) and

162 37.5 ml of tap water, containing the above cellular suspension of each lactic acid bacterium (cell

163 density in the dough of ca. log 7.0 cfu/g), were used to prepare 100 g of dough (DY of 160).

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

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

166 the obtaining of the proper biochemical properties (Coda et al., 2010b; Nionelli et al., 2014).

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167 pH and TTA of doughs were determined as described above. Water/salt-soluble extracts (WSE) of

168 doughs were prepared according to Weiss et al. (1993) and used to analyze free amino acids

169 (FAA). FAA were analyzed by a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd.,

170 Cambridge Science Park, England) with a Na-cation-exchange column (20 by 0.46 cm internal

171 diameter), as described by Rizzello et al. (2010a).

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172 Lactobacillus plantarum T6B10 and Lactobacillus rossiae T0A16 were used together as mixed

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173 starter to obtain a selected quinoa sourdough (QS). Cell suspensions were prepared as described

174 by Rizzello et al. (2010a), the DY was 160 and the initial cell density of lactic acid bacteria was

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175 7.0 log cfu/g. Fermentation of QS was at 30°C for 16 h.

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177 2.6. Quinoa sourdough characterization

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178 Kinetics of growth and acidification were determined and modelled in agreement with the
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179 Gompertz equation, as modified by Zwietering et al. (1990): y= k + A exp{- exp[(µmax or Vmax

180 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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181 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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182 modelled (log cfu/g or pH units); A is the cell density or pH (units) variation (between inoculation

183 and the stationary phase); µmax or Vmax is the maximum growth rate expressed as ∆log cfu/g/h or
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184 the maximum acidification rate expressed as dpH/h, respectively; λ is the length of the lag phase
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185 measured in hours. The experimental data were modelled by the non-linear regression procedure
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186 of the Statistica 8.0 software (Statsoft, Tulsa, USA). Moreover, the growth curve parameters were

187 estimated also using the dynamic model described by Baranyi and Roberts (1994) by using the

188 Excel add-in DMFit tool (version 3_5) for curve fitting.

189 pH and TTA were determined as described above. Proximal composition of the sourdough

190 fermented quinoa flour was determined as described above for raw quinoa flour, after milling a

191 freeze-dried sourdough with the laboratory mill Ika-Werke M20 (GMBH). The WSE was used to

192 analyze organic acid, and free amino acids. Organic acids were determined by High Performance
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193 Liquid Chromatography (HPLC), using an ÄKTA Purifier system (GE Healthcare,

194 Buckinghmshire, UK) equipped with an Aminex HPX-87H column (ion exclusion, Biorad,

195 Richmond, CA), and an UV detector operating at 210 nm. Elution was at 60°C, with a flow rate of

196 0.6 ml/min, using H2SO4 10 mM as mobile phase (Coda et al., 2011b). The fermentation quotient

197 (FQ) was determined as the molar ratio between lactic and acetic acids. Free amino acids were

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198 analyzed by a Biochrom 30 series Amino Acid Analyzer as described above.

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199 A dough, made with quinoa flour (DY 160) and without the inoculum of starters was incubated in

200 the same conditions of QS, and used as control (QD).

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201

202 2.7. Total phenols and antioxidant activity

203
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The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was determined on the
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204 methanolic extract (ME) of quinoa flour and doughs. Five grams of each sample were mixed with
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205 50 ml of 80% methanol to get ME. The mixture was purged with nitrogen stream for 30 min,

206 under stirring condition, and centrifuged at 4,600 × g for 20 min. ME were transferred into test
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207 tubes, purged with nitrogen stream and stored at ca. 4°C before analysis. The concentration of
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208 total phenols was determined as described by Slinkard and Singleton (1997), and expressed as

209 gallic acid equivalent. The free radical scavenging capacity was determined using the stable
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210 radical DPPH˙ (Rizzello et al., 2010a). The scavenging activity was expressed as follows: DPPH

scavenging activity (%) = [(blank absorbance – sample absorbance) / blank absorbance] x 100.
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211
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212 The value of absorbance was compared with 75 ppm butylated hydroxytoluene (BHT), which was

213 used as the antioxidant reference.

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215 2.8. Phytase activity

216 Phytase activity was determined on the WSE of doughs, by monitoring the rate of hydrolysis of p-

217 nitrophenyl phosphate (p-NPP) (Sigma, 104-0). The assay mixture contained 200 µL of 1.5 mM p-

218 NPP (final concentration) in 0.2 M Na-acetate, pH 5.2, and 400 µL of WSE. The mixture was
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219 incubated at 45ºC and the reaction was stopped by adding 600 µL of 0.1 M NaOH. The p-

220 nitrophenol released was determined by measuring the absorbance at 405 nm (Rizzello et al.,

221 2010a). One unit (U) of activity was defined as the amount of enzyme required to liberate 1

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

223

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224 2.9. Condensed tannins

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225 Condensed tannins were determined using the vanillin assay, as described by Price et al. (1978).

226 Samples were extracted with HCl:methanol (1:100) for 2.5 h at room temperature and centrifuged

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227 at 4,000 rpm for 20 min. Extracts were covered from light and analysed promptly at 30°C.

228 Vanillin reagent (equal volumes of 1% vanillin in methanol and 8% concentrated hydrochloric

229
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acid in methanol) was added to extracts. Blanks were prepared by adding 4% concentrated
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230 hydrochloric acid in methanol to extracts. The calibration curve was obtained using catechin and
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231 the results were expressed as catechin equivalents.

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233 2.10. In vitro protein digestibility

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234 The in vitro protein digestibility (IVPD) of QD and QS was determined by the method of Akeson

235 and Stahman (1964). One gram of each sample was incubated with 1.5 mg of pepsin, in 15 ml of
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236 0.1 M HCl, at 37°C for 3h. After neutralization with 2 M NaOH and addition of 4 mg of

pancreatin, in 7.5 ml of phosphate buffer (pH 8.0), 1 ml of toluene was added to prevent microbial
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237
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238 growth, and the solution was incubated for 24 h at 37°C. After 24 h, the enzyme was inactivated

239 by addition of 10 ml of trichloroacetic acid (20%, wt/vol), and the undigested protein was

240 precipitated. The volume was made up to 100 ml with distilled water and centrifuged at 5000 rpm

241 for 20 min. The concentration of protein of the supernatant was determined by the Bradford

242 method (Bradford, 1976). The precipitate was subjected to protein extraction, according to Weiss

243 et al. (1993), and the concentration of protein was determined. The in vitro protein digestibility

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244 was expressed as the percentage of the total protein, which was solubilized after enzyme

245 hydrolysis.

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247 2.11. Bread making

248 A quinoa sourdough bread (QSB, DY of 160) was manufactured at the pilot plant of the

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249 Department of Soil, Plant and Food Science of the University of Bari (Italy), according to the

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250 two-stage protocol including the production of sourdough (fermentation for 16h at 30°C, step I)

251 first, and the mixing with flour, water, and baker’s yeast (1.5 h at 30°C, step II), later. QS were

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252 used at the percentage of 20% (w/w) (Nionelli et al., 2014; Pontonio et al., 2015). A baker’s

253 yeast bread containing the same percentage of quinoa flour (12.5%) of QSB, but without the

254
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use of lactic acid bacteria as starters (QB), and a baker’s yeast wheat bread (WB) produced
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255 without the addition of quinoa flour, were manufactured and used as the controls. Baker’s yeast
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256 was added at the percentage of 2% w/w, corresponding to a final cell density of ca. 7 log cfu/g

257 in all the breads. Doughs were mixed at 60 × g for 5 min with an IM 5-8 high-speed mixer
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258 (Mecnosud, Flumeri, Italy) and fermentation was at 30°C for 1.5 h. All breads were baked at
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259 220°C for 30 min (Combo 3, Zucchelli, Verona, Italy).

260 Water activity (aw) was determined at 25ºC by the Aqualab Dew Point 4TE water activity
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261 meter (Decagon Devices Inc., USA). Saturated fats and sugars were determined with the

AACC methods 58-18.01 and 80-04.01, respectively (AACC, 2010). Fermentations were
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262
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263 carried out in triplicate and each bread was analyzed twice.

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265 2.12. Texture, image and color analyses

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

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

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

269 and stored for 24 h at room temperature. Crust was not removed. The selected settings were the
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270 following: test speed 1 mm/s, 30% deformation of the sample and one compression cycle. TPA

271 was carried out (Rizzello et al., 2012), using Texture Analyzer TVT-XP 3.8.0.5 software (TexVol

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

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

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

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

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276 area to the first compression area).

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

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278 reported in the form of a color difference, dE*ab, as follows: dE*ab =
(d) + (d) + (d )

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

280
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white ceramic plate having L = 93.4, a = –0.39, and b = 3.99).
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281 The crumb features of breads were evaluated after 24 h of storage using the image analysis
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282 technology with the UTHSCSA ImageTool as previously described by Rizzello et al. (2012).

283
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284 2.13. Nutritional characterization

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285 Energy value was calculated as reported by USDA method (IOM, 2002). The in vitro digestibility

286 of breads, was determined as described by Rizzello et al. (2014b). The supernatant, which
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287 contained the digested protein, was freeze-dried and used for further analyses. The modified
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288 method AOAC 982.30a (Horwitz and Latimer, 2006) was used to determine the total amino acid
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289 profile. The digested protein fraction, which derived from 1 g of sample, was added of 5.7 M HCl

290 (1 ml/10 mg of proteins), under nitrogen stream, and incubated at 110°C for 24 h. Hydrolysis was

291 carried out under anaerobic conditions to prevent the oxidative degradation of amino acids. After

292 freeze-drying, the hydrolyzate was re-suspended (20 mg/ml) in sodium citrate buffer, pH 2.2, and

293 filtered through a Millex-HA 0.22 µm pore size filter (Millipore Co.). Amino acids were analyzed

294 by a Biochrom 30 series Amino Acid Analyzer as described above. Since the above procedure of

295 hydrolysis does not allow the determination of tryptophan, it was estimated by the method of
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296 Pintér-Szakács & Molnán-Perl (1990). One gram of sample was suspended in 10 ml of 75 mM

297 NaOH, and shaken for 30 min at room temperature. The sample was centrifuged (10,000 rpm for

298 10 min), and 0.5 ml of the supernatant were mixed with 5 ml of ninhydrin reagent (1 g of

299 ninhydrin in 100 ml of HCl 37% : formic acid 96%, at the ratio 2:3) and incubated for 2 h at 37°C.

300 The reaction mixture was cooled at room temperature and made up to 10 ml with the addition of

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301 diethyl ether. The absorbance at 380 nm was measured. A standard tryptophan curve was prepared

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302 using a tryptophan (Sigma Chemicals Co.) solution in the range 0-100 µg/ml.

303 Chemical Score (CS) estimates the amount of protein required to provide the minimal essential

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304 amino acid (EAA) pattern, which is present in the reference protein (hen’s egg). It was calculated

305 using the equation of Block and Mitchel (1946), which compares the content of EAA of the breads

306
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for the amount of the same amino acid of the reference. The sequence of limiting essential amino
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307 acids corresponds to the list of EAA, having the lowest chemical score (Block and Mitchel, 1946).
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308 The protein score indicates the chemical score of the most limiting EAA that is present in the test

309 protein (Block and Mitchel, 1946). Essential Amino Acids Index (EAAI) estimates the quality of
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310 the test protein, using its EAA content as the criterion. EAAI was calculated according to the
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311 procedure of Oser (1959). It considers the ratio between EAA of the test protein and EAA of the

312 reference protein, according to the following equation:

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(
 ∗ 100)(
 ∗ 100)(… )(
 ∗ 100) []

 = 
!

(
 ∗ 100)(
 ∗ 100)(… )(
 ∗ 100)[ ]
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313 The Biological Value (BV) indicates the utilizable fraction of the test protein. BV was calculated

314 using the equation of Oser (1959): BV = ([1,09*EAAI]-11,70). The Protein Efficiency Ratio

315 (PER) estimates the protein nutritional quality based on the amino acid profile after hydrolysis.

316 PER was determined using the model developed by Ihekoronye (1981): PER = –0,468 +

317 (0,454*[Leucine]) – (0,105*[Tyrosine]). The Nutritional Index (NI) normalizes the qualitative and

318 quantitative variations of the test protein compared to its nutritional status. NI was calculated

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319 using the equation of Crisan and Sands (1978), which considers all the factors with an equal

320 importance: NI = (EAAI*Protein(%)/100).

321

322 2.14. Starch hydrolysis index and predicted glycaemic index

323 The analysis of starch hydrolysis was carried out on breads. The procedure mimicked the in vivo

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324 digestion of starch (De Angelis et al., 2009). Portions of breads, containing 1 g of starch, were

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325 given in randomized order to 10 volunteers. The glucose content was measured with Enzy Plus D-

326 Glucose kit (DiffchambVästraFrölunda, Sweden). The degree of starch digestion was expressed as

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327 the percentage of potentially available starch hydrolyzed at different times (30, 60, 90, 120, 150,

328 and 180 min). A non-linear model (De Angelis et al., 2009) was applied to describe the kinetics of

329
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starch hydrolysis. The hydrolysis curves were obtained with the equation reported below, using
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330 the software Statistica 8.0. Hydrolysis curves follow a first order equation: C = C∞ (1-e-kt) where C
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331 is the concentration at t time, C∞ is the equilibrium concentration, k is the kinetic constant and t is

332 the chosen time. Wheat flour bread (WB) was used as the control to estimate the hydrolysis index
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333 (HI = 100). The predicted GI was calculated using the equation: GI = 0.549*HI + 39.71 (Capriles
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334 and Areas, 2013) with wheat bread as the reference (GI wheat bread = 100).

335
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336 2.15. Sensory analysis

Sensory analysis of breads was carried out by 10 panellists (5 male and 5 female, mean age: 35
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337
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338 years, range: 18-54 years), according to the method described by Haglund et al (Haglund et al.,

339 1998; Rizzello et al., 2010b). Elasticity, colour of crust and crumb, acid taste, acid flavour,

340 sweetness, dryness, and taste were considered as sensory attributes using a scale from 0 to10, with

341 10 the highest score. Salty taste, previously described as another wheat sourdough bread attribute,

342 was also included (Rizzello et al., 2010b). Friedman's nonparametric test was used for the

343 statistical treatment of the results.

344
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345 2.16. Statistical Analysis

346 Fermentations were carried out in triplicate and each analysis was repeated twice. Data were

347 subjected to one-way ANOVA; pair-comparison of treatment means was obtained by Tukey’s

348 procedure at P<0.05, using the statistical software Statistica 8.0 (StatSoft Inc., Tulsa, USA).

349 Sensory analysis results were processed by the Friedman’s ANOVA non parametric test using the

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350 statistical software Statistica 8.0 (StatSoft Inc.).

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351

352 3. Results

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353 3.1 Isolation and identification of lactic acid bacteria The proximal composition of the quinoa

flour used in this study is reported in Table 1. In particular, the flour was characterized for a

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354 AN
355 protein concentration of 14.1 ± 0.6%, while the total dietary fibers were 10.3 ± 1.5%.

356 A total of 123 isolates were obtained from the various quinoa matrices. In particular, 18 isolates
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357 were from flour (T0), 44 from dough (T1), and 61 from sourdough (T6). Fifty-one isolates were

358 obtained using MRS, and 38 and 34 from mMRS and SDB, respectively. Gram-positive, catalase-
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359 negative, non-motile rods and cocci able to grow at 15°C and to acidify the media were subjected
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360 to RAPD-PCR analysis. Primers M13, P1, and P4 generated different patterns (bands ranging

361 from 5,000 to 100 bp), and were used for clusters analysis. Comparing the PCR products obtained
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362 from three separate cultures of the same strain, the reproducibility of the RAPD fingerprints was
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363 assessed. The dendrogram for the 123 isolates is shown in Figure 1. At the similarity level of 75%,
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364 almost all isolates were grouped into 16 clusters. Several clusters heterogeneously harbored strains

365 deriving from T0, T1, and T6. Cluster I only included strains deriving from T0, while clusters VII

366 and XVI included only strains isolated from T1. Clusters IX, X, XIII, and XIV grouped only

367 strains deriving from T6. Isolates from the same culture medium grouped together in clusters I,

368 VII and VIII (isolates from MRS, labeled with letter A), XI and XII (isolates from mMRS, labeled

369 with letter B), and X, XIII, and XIV (isolates from SDB, labeled with letter C).

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370 Eleven isolates that did not group into clusters and 16 isolates, which were representatives of each

371 cluster, were identified by partial sequencing of the 16S rRNA. The following species were

372 identified: Lb. plantarum (23 isolates: 7 from T0, 7 from T1, and 9 from T6, respectively),

373 Pediococcus pentosaceus (3 isolates from T1), and Lb. rossiae (1 isolate from T0).

374

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375 3.2 Selection of autochthonous lactic acid bacteria

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376 The identified strains were singly used to ferment quinoa flour at 30°C for 16 h. Overall, all the

377 strains were able to growth and acidify quinoa doughs. The final cell densities of lactic acid

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378 bacteria in the fermented quinoa sourdoughs were in the range 9.3 ± 0.2 to 9.7 ± 0.2 log cfu/g. The

379 cell growth during incubation was ca. 2 log cycles. The values of pH of the doughs decreased from

380
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5.94 ± 0.22 (before fermentation) to 2.64 ± 0.10 - 3.26 ± 0.15. The values of ∆pH values obtained
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381 using lactic acid bacteria as single starters are reported in Figure 2A. In particular, doughs
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382 fermented with L. rossiae T0A16 and L. plantarum T6B10 had the lowest values. TTA

383 significantly (P<0.05) increased from 7 ± 2 ml 0.1 M NaOH/10 g (before fermentation) to values
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384 of 18 ± 3 – 27 ± 2 ml 0.1 M NaOH/10 g. The values of pH and TTA of the non-inoculated dough
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385 (QD) were 5.82 ± 0.20 and 7.4 ± 1 ml 0.1 M NaOH/10 g, respectively.

386 The concentration of FAA of the doughs before fermentation was 4502 ± 93 mg/kg. After
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387 fermentation, the values increased to 6176 ± 110 - 13856 ± 230 mg/kg (Figure 2B). The highest

concentration was found for the dough fermented with L. plantarum T6B10. When QD was
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388
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389 incubated under the same conditions, the concentration of FAA reached the level of 5403 ± 75

390 mg/kg. Based on the above results, L. plantarum T6B10 and L. rossiae T0A16 were selected and

391 further used as mixed starter to get the quinoa sourdough (QS).

392

393 3.3 Sourdough fermentation with selected starters

394 During sourdough fermentation, the cell density of the selected starters increased by ca. 2 log

395 cycles. The kinetics of growth and acidification, which were determined during QS fermentation,
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396 showed values of latency phases lasting 1.97 ± 0.12 and 3.32 ± 0.23 h, respectively. The values of

397 µ max and Vmax were 0.40 ± 0.08 ∆log cfu/g/h and 0.44 ± 0.05 ∆pH/h, respectively. A and ∆pH

398 were 1.95 ± 0.25 ∆log cfu/g and 2.07 ± 0.12, respectively. To confirm the fitness of the Gompertz

399 model, the growth parameters were also calculated with the Baranyi and Roberts (1994) model

400 (Ricciardi et al., 2009), founding similar results. In particular, the estimated growth rate and lag

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401 phase were 0.52 (h-1) and 1.95h, respectively (r2 0,997).

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402 The main differences between quinoa flour (QF) and QS were related to the concentration of

403 soluble and insoluble fibers (Table 1). A significant (P<0.05) increase of soluble fibers was found

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404 in QS. Compared to QF, the insoluble fibers decreased by 35% in QS (Table 1).

405 Compared to QD, the pH of QS decreased to 3.83 ± 0.2 (Table 2). The values of TTA of QS was

406
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ca 4 fold higher than QD (Table 2). QS contained lactic and acetic acids at concentrations of 171 ±
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407 12 and 12 ± 2, respectively. The value of FQ was 13.70. Compared to QD, the fermentation with
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408 selected starters caused a marked increase of FAA, attaining a concentration ca. 4 times higher. As

409 determined through methanolic extraction, the total phenols concentration doubled during
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410 sourdough fermentation. Antioxidant and phytase activities were also the highest in QS.
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411 Compared to QD, the phytase activity of QS was ca. 3 times higher. QS had a concentration of

412 condensed tannins significantly (P<0.05) lower. The IVPD markedly increased (ca. 25%) during
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413 sourdough fermentation.

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414
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415 3.4 Chemical and technological characterization of quinoa sourdough bread

416 An experimental bread made with wheat flour and including QS as ingredient was produced and

417 compared to baker’s yeast wheat breads produced with or without the addition of QF. Before

418 baking, the doughs fermented with baker’s yeast alone (WB and QB) showed a similar pH values

419 (Table 3). As expected, the use of sourdough affected the value of pH, which decreased to 4.48 ±

420 0.18. An opposite trend was found for TTA (Table 3). The rate of volume increase of doughs did

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421 not significantly (P>0.05) differ between WB and SQB, while it was ca. 20% lower in QB (data

422 not shown).

423 The use of quinoa flour as an ingredient caused a significant (P<0.05) increase of the

424 concentrations of FAA and total phenols, as determined in doughs before baking. Compared to

425 WB, phytase and antioxidant activities also increased. Further significant (P<0.05) increases were

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426 found for all these chemical characteristics when QS was added to bread instead of the

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427 unfermented quinoa flour (Table 3). Compared to WB, the addition of QF caused an increase of

428 the concentration of almost all the individual FAA (Figure 3). The sourdough fermentation further

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429 strengthened this trend. In particular, the highest increases were found for Arg, Glu, Leu, and Lys

430 (Figure 3).

431
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Moreover, QSB was characterized by conspicuous concentrations of lactic and acetic acid. The
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432 fermentation quotient of QSB was 10.71 ± 0.16.No significant (P>0.05) were found among the
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433 three breads regarding water activity and moisture (Table 3). Compared to WB, QB and SQB were

434 characterized by higher concentrations of proteins, lipids, saturated fats, ash, and total dietary
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435 fibers (Table 3). SQB had the highest concentration of soluble fibers and the lowest concentration
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436 of sugars.

437 The textural properties of breads were determined after baking. Quinoa flour affected the specific
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438 volume of QB, that was ca 10% lower than WB. When sourdough fermentation was used, the

specific volume did not decrease, and no significant (P>0.05) differences were found between
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439
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440 WB and SQB.

441 The hardness was the highest for QB. When QS was used, the value was significantly (P<0.05)

442 lower than WB. Sourdough fermentation caused a significant (P<0.05) decrease of the resilience

443 and a slight increase of the fracturability (Table 3). The crumb structure of breads was evaluated

444 by image analysis technology. Digital images were pre-processed to estimate crumb cell-total area

445 through a binary conversion (Table 3). Compared to WB and SQB, the cell-total area

446 (corresponding to the black pixel total area) of QB was lower. The visual inspection of the crust of
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447 bread made with QS showed a more intense coloring with respect to to breads made with baker’s

448 yeast alone. Indeed, the crust lightness (L) of SQB decreased compared to QB and WB. Almost

449 the same differences were observed for the parameter dE (Table 3).

450

451 3.5 Nutritional characteristics

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452 The energy values of the three breads were not significantly (P>0.05) different (Table 4). A multi-

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453 step protocol, which mimics the in vivo gastrointestinal digestion, was used to estimate the bread

454 in vitro protein digestibility. The digestibility of SQB was significantly (P<0.05) higher than that

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455 of QB. Nevertheless, it was slight lower than that found for WB (Table 4). The digestible protein

456 fraction was further characterized. In particular, the amino acid composition and the related

457
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chemical scores were calculated, using the egg essential amino acid (EAA) pattern as the FAO
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458 (Food and Agriculture Organization) reference. Compared to WB, almost all the chemical scores
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459 of QB were significantly (P<0.05) higher, while they were significantly (P<0.05) lower than those

460 found for SQB (with the only exception of Cys).

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461 Based on chemical scores, the sequence of limiting amino acids and the protein score were
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462 determined. All breads showed Lys and Met as the most limiting amino acids, although their

463 chemical scores where significantly (P<0.05) higher in SQB compared to WB. The third limiting
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464 essential amino acid was Trp for both the baker’s yeast breads WB and QB, while Thr was

limiting for SQB. The highest protein score was calculated for SQB.
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465
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466 EAA and Biological Value (BV) indexes, which are commonly used to estimate the quality of

467 food proteins, were significantly (P<0.05) higher for QB and SQB with respect to WB (Table 4).

468 No significant (P>0.05) differences were found between the Protein Efficiency Ratio (PER) of

469 WB and SQB, while QB had a significantly (P<0.05) lower value. Compared to WB and QB, the

470 Nutritional Index (NI) of SQB was markedly higher (Table 4).

471 Starch hydrolysis was determined mimicking the in vivo digestion. The analysis is a presumptive

472 measure of the glycemic index (GI) in healthy subjects (De Angelis et al., 2009) (Figure 3). After
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473 180 min, the percentage of hydrolyzed starch was 65.1, 65.0, and 36.8% for WB, QB, and SQB,

474 respectively (Figure 4). No significant (P>0.05) differences were found between the HI of WB,

475 used as the reference (HI = 100), and QB. The values of HI for SQB was 56.5 ± 1.3. The predicted

476 GI for QB and SQB was 94.5 and 70.7, respectively.

477

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478 3.7 Sensory characteristics

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479 The results for bread sensory analysis are shown in Figure 5. As showed by the Friedman

480 ANOVA analysis of the results, significant differences among the experimental breads were found

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481 by the panelists, especially on the crust color, crumb color, acid taste, sweetness, and salty

482 attributes (Table 5). In detail, compared to WB, the addition of QF caused a slight decrease of the

483
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perceived elasticity (7.5 ± 0.2 vs 8.8 ± 0.5). At the same time, the scores for color of the crust and
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484 the crumb, salty, and overall taste attributes increased. Compared to QB, the SQB mainly differed
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485 for an intense crust color (7.3 vs 5.3), a markedly higher acid taste (4.65 vs 1.9) and a lower

486 sweetness (2.4 vs 6.5). Also the salty taste was the highest for sourdough bread.
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487
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488 4. Discussion

489 Research and development on local, ethnic, and ancient grains have a worldwide renewed interest
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490 (Coda et al., 2014), mainly due to their nutritional value and chemical composition (e.g., dietary

491 fibers, resistant starch, minerals, vitamins and phenolic compounds), which reflects on baked good
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492 features. Mainly for these reasons, local or indigenous varieties of cereal and pseudo-cereals
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493 attract bakery industries and consumers all over the world as niche products (Coda et al., 2014).

494 Quinoa deserved a special attention because of the quality and nutritional value of proteins, which

495 are an important source of essential amino acids like, the good balance between protein and fat

496 (Nascimento et al., 2014), and the potential use as a gluten-free ingredient. For these reasons there

497 is an emergent market for new bakery products manufactured with quinoa. Nevertheless, the use

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498 of this alternative flour is restricted due to the low baking and sensory quality of the baked

499 products (Enriquez et al., 2003; Lorenz and Coulter, 1991; Stikic et al., 2012).

500 Fermentation has been already reported as a potential tool to improve technological and sensory

501 characteristics of quinoa flour (Gallagher et al., 2003). Nevertheless, very few information are

502 available in literature, and the optimization of the processes are still needed.

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503 Sourdough fermentation is one of the oldest food biotechnologies, which was recently

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504 rediscovered for the positive effects on sensory, structure, nutritional and shelf life features of

505 leavened baked goods (Gobbetti et al., 2014). The leavening and acidifying properties of

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506 sourdough are associated to yeasts and lactic acid bacteria, selected among the spontaneous

507 microbiota through the backslopping procedure. Saccharomyces cerevisiae is the yeast most

508
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frequently isolated from sourdoughs, or it is added to sourdoughs as baker’s yeast to emphasize
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509 the leavening power (Gobbetti, 1998). However, the major part of the advantages attributed to
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510 sourdough is due to lactic acid bacteria complex microbiota (Gobbetti, 1998). Therefore, only

511 lactic acid bacteria were isolated, selected, and used for quinoa fermentation. To select proper
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512 strains to be used as starter, isolation was carried out on quinoa flour, but also on a spontaneously
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513 fermented dough and on type I sourdoughs, prepared (back slopped) under laboratory conditions,

514 according to a traditional protocol (Minervini et al., 2012). The strains deriving from the different
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515 quinoa matrices were typed and clustered on the basis of genetic molecular characteristic, however

they were heterogeneously grouped. Among the strains identified, L. plantarum was the most
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516
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517 represented species, even though strains belonging to P. pentosaceous and L. rossiae species were

518 also identified. In particular, L. plantarum strains were equally present in quinoa flour and

519 spontaneously fermented dough, where they were dominant. L. plantarum is often encountered in

520 cereal sourdough ecosystems (De Vuyst and Neysens, 2005), and its dominance during

521 fermentation, is related to a versatile metabolism (Coda et al., 2010b). Also P. pentosaceus and L.

522 rossiae are heterofermentative lactic acid bacteria frequently isolated from wheat sourdough, as

523 well as from other fermented foods (Semjonovs and Zikmanis, 2008). Both showed promising
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524 functional and technological properties and their use as starters was already proposed (Semjonovs

525 and Zikmanis, 2008, Robert et al., 2009). Compared to allochthonous or commercial starters,

526 mainly tailored for wheat flour fermentation, the selection of strains within the autochthonous

527 microbiota is an important pre-requisite (Coda et al., 2010b) for rapid adaptation, intense

528 acidification and positive influence on the nutritional and technological properties (Coda et al.,

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529 2010b). Furthermore, the use of mixtures of strains, each one selected for a different biochemical

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530 and/or technological trait, is successful to get optimal sourdough fermentation (Coda et al., 2010a,

531 2010b). L. plantarum T6B10 and L. rossiae T0A16 were selected on the basis of the acidification

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532 capacity and the proteolytic activity, since related to the most important technological and

533 nutritional advantages of sourdough fermentation (Coda et al., 2014; Gobbetti et al., 2014). When

534
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selected and mixed starters were used, the cell density of lactic acid bacteria and the lactic acid
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535 synthesis was similar to that commonly found in a wheat sourdough (Gobbetti, 1998), but the
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536 fermentation quotient was slightly higher than the optimal range for sensory attributes of wheat

537 baked goods (Corsetti and Settanni, 2007).

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538 The concentration of total free amino acids markedly increased. Besides the nutritional
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539 advantages, during baking free amino acids are converted into volatile compounds, which are

540 responsible for typical flavor and odor of sourdough breads (Corsetti and Settanni, 2007). It was
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541 reported that quinoa flour contains high concentration of phenol compounds such as phenolic

acids and flavonoids (Chlopicka et al., 2012; Miranda et al., 2011). In particular, the main
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542
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543 polyphenols present in quinoa are kaempferol and quercetin glycosides (Valcárcel-Yamani and da

544 Silva Lannes, 2012). As expected, relevant concentrations of total phenols were found in the

545 quinoa flour used in this study. As previously shown (Rizzello et al., 2012; Nionelli et al., 2014),

546 lactic acidification improves the extraction of total phenols when selected starters were used.

547 Esterase activities, able to hydrolyze complex phenolic compounds and their glycosylated forms

548 into the corresponding phenolic acids during sourdough fermentation were largely described for

549 lactic acid bacteria (Nionelli et al., 2014). The increased solubilization of phenolics might be
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550 related to the highest antioxidant activity found in quinoa sourdough. Phytic acid is an anti-

551 nutritional factor mainly present in the wheat kernel (Rizzello et al., 2012) and it was also found in

552 quinoa seeds, located in the external layers as well in the endosperm, at concentration of ca.1.5-2

553 g/100 g (Jancurová et al., 2009). Phytase, which catalyzes the hydrolysis of phytic acid into myo-

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

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555 compound (Gobbetti et al., 2014). Phytases have been studied intensively in recent years due to

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556 functionality in reducing phytate content in food for human consumption and animal feed (Rosero

557 et al., 2013), and high phytase activity was already found in quinoa flours (Rosero et al., 2013).

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558 As previously demonstrated for cereal-based doughs, the value of pH reached with sourdough

559 fermentation is suitable to activate flour endogenous phytases during fermentation. Besides,

560
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sourdough lactic acid bacteria possess somewhat phytase activity (Di Cagno et al., 2008; Rizzello
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561 et al., 2010a). Results confirmed that quinoa fermented with selected starters had a phytase
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562 activity ca. 2.75-times higher than raw quinoa flour. Tannins are polyphenolic secondary plant

563 metabolites, which can be found in high concentrations in the hulls of cereals and legumes (Coda
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564 et al., 2015). Recently, concentrations up to 500 mg/100 g have been reported for quinoa
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565 (Valcárcel-Yamani and da Silva Lannes, 2012). Condensed tannins, mainly composed of

566 flavonoid units, also have a marked repercussion on the nutritional value, since they can interfere
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567 with the digestive process by binding enzymes, other proteins and/or minerals (Curiel et al., 2015).

The degradation of condensed tannins can follow different pathways involving several enzymes
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568
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569 such as decarboxylases, oxygenases and it can also be carried out by microbial activity (Coda et

570 al., 2015b). It can be hypothesized that a combined action of quinoa endogenous enzymes and

571 lactic acid bacteria enzymes was responsible for the condensed tannins degradation into smaller

572 molecules.

573 The daily consumption of foods rich in dietary fiber is recommended to prevent several diseases

574 (Nionelli et al., 2014b), to regulate energy intake and satiety (Curiel et al., 2015) and to help the

575 glycaemic control into diabetic patients. Quinoa flour was characterized for relevant concentration
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576 of dietary fiber. The content of total dietary fiber did not show significant variations during

577 sourdough fermentation but, as previously shown for cereal-based matrices, the ratio between

578 insoluble (lignin, cellulose and some hemicelluloses) and soluble (pectin, some hemicelluloses and

579 other non-starch polysaccharides) dietary fiber decreased during sourdough fermentation through

580 lactic acidification and enzyme hydrolysis (Nionelli et al., 2014).

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581 White wheat bread started with baker’s yeast alone shows some drawbacks such as high glycemic

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582 index, low-resistant starch and low level of dietary fibers, low nutritional indexes related to

583 protein quality (Rizzello et al., 2014). Indeed, the use of ingredients including high nutritional

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584 value proteins, dietary fibers, minerals and phytochemicals was variously proposed to enhance the

585 nutritional features of white wheat bread (Rizzello et al., 2014)

586
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In this study, quinoa sourdough was used as an ingredient for the manufacture of white wheat
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587 bread and the characteristics of the bread were compared to those of white breads made with or
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588 without the use of raw (non fermented) quinoa flour. Overall, the use of sourdough fermented

589 quinoa had a positive effect on the nutritional properties of the white bread.
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590 The concentration of total free amino acids increased with the addition of quinoa flour, especially
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591 when sourdough was used. In particular, Lys, the major limiting amino acid of wheat flour, was

592 found in sourdough quinoa bread at the concentration of ca. 300 mg/kg. This concentration was
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593 ca. 50-times higher than that found in wheat flour bread. Quinoa conferred to white bread a value

of in vitro protein digestibility (IPVD) lower than control, but, as previously reported for other
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594
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595 proteic sources (Coda et al., 2015b; Rizzello et al., 2010a), sourdough fermentation increased the

596 value of IPVD. The enhanced protein digestibility may be attributed to proteolysis by lactic acid

597 bacteria and, probably, to the inactivation of some anti-nutritional factors such as trypsin inhibitor

598 and condensed tannins (Coda et al., 2015b). Overall, the sourdough fermentation increased the

599 concentration of the phenolic compounds (Nionelli et al., 2014a; Rizzello et al., 2010a). Indeed,

600 the concentration of total phenols and the antioxidant activity of the bread produced with the

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601 quinoa sourdough were higher than those found in the other two. Phytase activity follows the same

602 trend.

603 Proteins are one of the most critical components, which contribute to the nutritional value of

604 foods. Besides the amount, the quality of proteins is another important attribute (Rizzello et al.,

605 2014b). Usually, the quality of proteins is estimated through the determination of their amino acid

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606 composition. The amino acid composition is not the only predictor of the nutritive value, but this

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607 feature has to be combined with protein digestibility (Rizzello et al., 2014b). The in vitro

608 digestibility gives information on the stability of protein hydrolysates, and on how they withstand

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609 to digestive processes. The digestible protein fraction was used for the determination of the protein

610 quality indexes. Indeed, it was shown that the analysis of the total protein content should hide the

611
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effect of the proteolysis degree, which results otherwise in similar values for samples that are
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612 instead characterized by different bioavailability and nutritional features of the protein (Rizzello et
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613 al., 2014b). It is well known that wheat flour lacks of lysine and sulphur-containing amino acids.

614 Compared to wheat bread, the chemical scores for tryptophan and lysine of the sourdough quinoa
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615 bread were almost double. The Essential Amino Acid (EAA) and Biological Value (BV) indexes
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616 were the highest for the bread made with quinoa sourdough. EAA indicates the ratio of essential

617 amino acids of the sample compared to the reference. BV estimates the nitrogen potentially
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618 retained by human body after consumption. The Protein Efficiency Ratio (PER), which reflects the

capacity of a protein to support the body weight gain, was similar between the white wheat and the
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619
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620 sourdough quinoa breads. Within the indexes that are used to evaluate the nutritional value of

621 foods, only the Nutritional Index (NI) combines qualitative and quantitative factors. Indeed, NI is

622 considered a global predictor of the protein quality. Since the increased protein bioavailability, the

623 value of NI of the bread made with quinoa sourdough flour was ca. 89 and 78% higher than those

624 of the bread made with or without raw quinoa flour, respectively. Bread containing quinoa

625 sourdough had a markedly lower value of HI compared to those of the other two experimental

626 breads. This effect could be attributed to biological acidification, which is one of the main factors
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627 that decreases starch hydrolysis rate and HI (Curiel et al., 2014), and to the higher concentration of

628 dietary fibers.

629 The use of the quinoa sourdough had positive effect also on the texture and sensory properties of

630 the white bread, if compared to the use of the raw quinoa flour. Under the experimental conditions

631 of this study, highest specific volume and cell-total area of the crumb slices were found. The

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632 values of hardness of breads containing quinoa sourdough was lower than that of the bread

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633 containing raw flour, while fracturability value indicated an higher elasticity. Bread colour and

634 aroma develop during baking, simultaneously with the crust formation, and mainly derive from

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635 Maillard reaction and sugar caramelization (Rizzello et al., 2010b). The use of quinoa flour leaded

636 to an intense and appreciated colour of the crust, especially when sourdough was used. Most of the

637
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sensory attributes differentiated the two breads containing quinoa, and both of them from the
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638 wheat bread. Sourdough quinoa bread had mainly an acid taste and flavor, due to either sourdough
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639 fermentation and also resulted more salty than the others, probably due to the effect of

640 acidification and proteolysis by selected lactic acid bacteria. This sensory attribute may assume
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641 particular importance for the manufacture of baked goods according to the new EC directive
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642 which aimed at decreasing the concentration of salt in bread (WHO, 2007).

643 Despite the gain in healthiness, good sensory and texture properties still remain an essential
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644 requisite for baked goods. This work demonstrated that sourdough fermentation improved the

characteristics of quinoa flour, making it suitable for food processing. Indeed, it was confirmed
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645
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646 that the use of a quinoa sourdough, obtained with selected authoctonous lactic acid bacteria, as

647 ingredient for breadmaking, may enhance the nutritional, structural and sensory features of the

648 baked goods.

649

650 Acknowledgements

651 The authors Davide Minervini (Molini Tandoi, Corato, BA-IT) for rheology analysis.

652
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653

654 References

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819 growth curve. Appl. Environ. Microbiol. 56, 1875-81.

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Legends to figures

Figure 1. Dendrogram obtained by combined random amplification of polymorphic DNA patterns

for the isolates from raw quinoa flour (T0); quinoa flour dough incubated at 30°C for 16 h (T1);
quinoa type I sourdough (T6) using primers M13, P1 and P4. The codes used to identify isolates, A,
B, and C, refer to the media used for isolation (MRS, mMRS, or SDB, respectively). Cluster
analysis was based on the simple matching coefficient and unweighted pair group with arithmetic
average.

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Figure 2. Decrease of pH value (∆pH, panel A) and final concentration of total free amino acids (B)
in quinoa sourdoughs (DY 160) fermented at 30°C for 16 h with lactic acid bacteria isolated from
raw quinoa flour, quinoa flour dough incubated at 30°C for 16 h, and quinoa type I sourdough. Data

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are the means of three independent analyses. a-fValues with different superscript letters within the
same amino acid, differ significantly (P<0.05). Bars of standard deviations are also represented.

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Figure 3. Concentration of free amino acids and their derivatives (mg/kg) of the experimental
breads (dough yield 160). QSB, quinoa sourdough bread, made with quinoa sourdough at the
percentage of 20% (w/w); QB, baker’s yeast bread containing the same percentage of quinoa flour
of QSB (12.5% w/w); WB baker’s yeast wheat bread (WB) produced without the addition of quinoa

U
flour. Data are the means of three independent analyses. Three-letters amino acid code (IUPAC) is
used. a-cValues with different superscript letters within the same amino acid, differ significantly
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(P<0.05). Bars of standard deviations are also represented.

Figure 4. Rate of starch hydrolysis of the experimental breads following chewing, incubation with
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pepsin, and further incubation with pancreatic α-amilase in dialysis tubing. QSB, quinoa sourdough
bread, made with quinoa sourdough at the percentage of 20% (w/w); QB, baker’s yeast bread
containing the same percentage of quinoa flour of QSB (12.5% w/w); WB baker’s yeast wheat
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bread (WB) produced without the addition of quinoa flour (reference). a-cValues obtained at the
same time with different superscript letters differ significantly (P<0.05).
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Figure 5. Spider web chart of the sensory analysis data for experimental breads. QSB, quinoa
sourdough bread, made with quinoa sourdough at the percentage of 20% (w/w); QB, baker’s yeast
bread containing the same percentage of quinoa flour of QSB (12.5% w/w); WB baker’s yeast
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wheat bread (WB) produced without the addition of quinoa flour.

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Table 1. Proximal composition of quinoa (QF) and sourdough fermented quinoa (QS) flours.

QF QS*

Moisture (%) 11.4 ± 0.6 10.6 ± 0.2

Proteins (% of d.m.) 14.1 ± 0.6 13.6 ± 0.7

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Lipids (% of d.m.) 6.0 ± 0.3 6.3 ± 0.3

Carbohydrate (% of d.m.) 67.9 ± 3.3 68.2 ± 3.5

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Soluble fibers (% of d.m.) 1.4 ± 0.3b 4.2 ± 0.8a

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Insoluble fibers (% of d.m.) 8.9 ± 1.8a 5.8 ± 0.0b

Ash (%) 2.2 ± 0.2 2.3 ± 0.2

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* The sourdough fermented quinoa flour was obtained by milling a freeze-dried the quinoa sourdough.
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The data are the means of three independent experiments ± standard deviations (n = 3).
a-b
Values in the same row with different superscript letters differ significantly (P<0.05).
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Table 2. Chemical characteristics of the quinoa sourdough (QS), started with Lactobacillus
plantarum T6B10 and Lactobacillus rossiae T0A16 (initial cell density of lactic acid bacteria, 7.0
log cfu/g) and fermented at 30°C for 16 h. A dough, made with quinoa flour and produced without
the inoculum of the starters was incubated in the same conditions, and used as control (QD). The
dough yield was 160.

QD QS
a
3.83 ± 0.20b

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pH 5.82 ± 0.20
TTA (ml of 0,1 N NaOH/10g) 7.4 ± 1b 30 ± 2a
Lactic acid (mmol/kg) 3 ± 1b 171 ± 12a

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Acetic acid (mmol/kg) n.d. 12 ± 2
FQ n.d. 13.7 ± 3

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b
TFAA (mg/kg) 5403 ± 75 22650 ± 239a
Total phenols (mmol/kg) 8.17 ± 0.23b 16.19 ± 1.12a

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Antioxidant acticity1 41.7 ± 2.3b 71.8 ± 2.8a
Phytase activity (U)2 1.73 ± 0.72b 4.76 + 0.55a
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Condensed tannins (mg/kg) 457 ± 11a 85 ± 23b
In vitro protein digestibility (%) 53.36 ± 2.23b 78.17 ± 3.01a
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1
The antioxidant activity was determined based on the scavenging activity towards DPPH radical after 10
min of reaction.
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2
One unit (U) of phytase activity was defined as the amount of enzyme required to release 1 µmol of
phosphate per min under the assay conditions.
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The data are the means of three independent experiments ± standard deviations (n = 3).
a-b
Values in the same row with different superscript letters differ significantly (P<0.05).
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Table 3. Characteristics of doughs and breads (dough yield 160): WB made with wheat flour alone;
QB, containing 12.5% (w/w) of raw quinoa flour, and SQB, containing 20% (w/w) of quinoa
sourdough (QS, corresponding to 12.5% fermented quinoa flour).

WB QB SQB
Doughs
Chemical characteristics
5.76 ± 0.15a 5.68 ± 0.20a 4.48 ± 0.18a

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pH
Total titratable acidity (TTA) 2.0 ± 0.1c 3.2 ± 0.2b 7.2 ± 0.2a
Total free amino acids (mg/kg) 522 ± 24c 1767 ± 43b 4556 ± 61a
Lactic acid (mmol/kg) 0.2 ± 0.1b 0.8 ± 0.2b 37.5 ± 0.2a

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Acetic acid (mmol/kg) 0.6 ± 0.2b 0.8 ± 0.2b 3.5 ± 0.4a
Phytase Activity (U)1 2.03 ± 0.20c 3.65 ± 0.31b 6.15 ± 0.35a
Total phenols (mmol/kg) 5.05 ± 0.18c 6.09 ± 0.21b 8.78 ± 0.22a

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Antioxidant activity (%)2 35 ± 2c 53 ± 3b 65 ± 6a
Breads
Chemical characteristics
Water activity 0.98 ± 0.01 0.97 ± 0.01 0.97 ± 0.01

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Moisture (%) 28 ± 2 27 ± 2 27 ± 2
Proteins (% ) 10.2 ± 0.2b 11.1 ± 0.4a 11.3 ± 0.5a
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Lipids (%) 0.9 ± 0.0b 1.8 ± 0.1a 1.9 ± 0.1a
Saturated fats (%) 0.20 ± 0.02b 0.26 ± 0.02a 0.26 ± 0.02a
Carbohydrates (%) 58 ± 3a 55 ± 3b 54 ± 3b
Sugars (% ) 1.0 ± 0.2a 1.2 ± 0.2a 0.7 ± 0.1b
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Ash (%) 0.5 ± 0.1b 0.7 ± 0.2a 0.8 ± 0.2a

Total dietary fibers (%) 3.8 ± 0.7b 6.0 ± 1.2a 6.0 ± 1.1a
Soluble fibers (%) 0.3 ± 0.1c 1.3 ± 0.3b 2.4 ± 0.5a
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Insoluble fibers (%) 3.5 ± 0.7b 4.7 ± 0.9a 3.6 ± 0.5b

Structural characteristics
Specific volume (cm3/g) 3.06 ± 0.13a 2.73 ± 0.24b 3.09 ± 0.28a
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Hardness (g) 9625 ± 120b 10111 ± 210a 7700 ± 145c

Resilience 0.77 ± 0.12b 0.85 ± 0.15a 0.65 ± 0.15c
Fracturability (g) 8743 ± 188a 6624 ± 201c 7520 ± 224b
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Image analysis
Black pixel area (%) 45.9 ± 0.5a 36.6 ± 0.7b 46.3 ± 0.5a
Color analysis (crust)
L 58.7 ± 1.5a 58.8 ± 1.1a 55.8 ± 1.8b
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a -0.8 ± 0.2b -0.3 ± 0.1a -0.3 ± 0.3a

b 12.0 ± 0.2b 14.1 ± 0.3a 13.4 ± 0.1a
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dE 35.5 ± 1.3a 36.6 ± 1.1a 34.9 ± 1.7b

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

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Table 4. Nutritional indexes of the experimental breads (dough yield 160): WB, made with wheat flour alone; QB containing 12.5% (w/w) of raw
quinoa flour; SQB, containing 20% (w/w) of quinoa sourdough (QS, corresponding to12.5% fermented quinoa flour).

Breads WB QB SQB

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Energy value (kJ/100g) 1200 ± 70 1211 ± 88 1260 ± 62
In vitro protein digestibility (%) 88.2 ± 1.2a 71.0 ± 1.0c 84 ± 2.2b

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Chemical score (%)
Histidine 48.9 ± 0.8c 52.5 ± 1.0b 54.7 ± 0.5a

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Isoleucine 43.9 ± 1.1c 42.4 ± 1.1b 48.3 ± 0.5a
Leucine 61.3 ± 0.5a 47.7 ± 1.0b 60.9 ± 0.4a

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Lysine 13.0 ± 0.6c 17.8 ± 0.9b 22.7 ± 0.8a

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Cystine 110.1 ± 0.5b 111.8 ± 0.7a 119.8 ± 0.8a
Methionine 30.0 ± 1.2b 31.1 ± 0.7b 36.5 ± 0.9a
Phenylalanine + tyrosine 62.0 ± 1.0a 53.1 ± 0.8b 59.7 ± 1.0a

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Threonine 37.9 ± 0.8b 37.3 ± 0.7b 43.5 ± 1.1a
Valine 53.4 ± 0.3b 52.8 ± 0.5b 56.6 ± 1.2a

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Tryptophan 32.2 ± 0.5c 40.1 ± 1.0b 59.8 ± 0.5a

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Sequence of limiting essential amino Lysine Lysine Lysine
acids (EAA) Methionine Methionine Methionine
Tryptophan Tryptophan Threonine
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Protein score (%) 13.0 ± 0.4c 17.8 ± 0.8b 22.7 ± 0.5a
Essential Amino Acid Index (EAAI) 43.3 ± 0.5b 46.1 ± 0.8a 48.6 ± 0.3a
C

Biological Value (BV) 35.5 ± 0.6b 38.5 ± 0.2a 41.3 ± 0.6a

AC

Protein Efficiency Ratio (PER) 21.9 ± 0.6a 16.5 ± 0.3b 21.7 ± 0.7a
Nutritional Index (NI) 2.82 ± 0.14b 2.65 ± 0.22b 5.01 ± 0.13a

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

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Table 5. Friedman ANOVA rank values of the sensory analysis scores. χ2 and p-values obtained for each attribute are also reported.

Sensory attributes WB QB SB χ2 p

PT
Elasticity 2.60 1.75 1.65 5.89189 0.0525
Crust color 1.00 2.10 2.90 18.2000 0.0001

RI
Crumb color 1.25 2.35 2.40 9.6571 0.0080
Acid flavor 1.60 2.05 2.35 3.8000 0.1496

SC
Acid taste 1.50 1.80 2.70 8.6667 0.0131

U
Sweetness 2.40 2.45 1.15 11.7297 0.0028

AN
Dryness 1.95 1.75 2.30 1.7714 0,4124
Salty 1.45 2.10 2.45 6.2424 0.0441

M
Taste 1.60 2.10 2.30 2.8889 0.2359

D
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Highlights

- Lactic acid bacteria were isolated from quinoa flour and spontaneously fermented doughs

- strains were selected based on acidification and proteolytic activities

- a quinoa sourdough made with the selected strains was characterized

- sourdough fermentation caused the improvement of the nutritional profile

PT
- a wheat bread including quinoa sourdough was made and characterized

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