Accepted Manuscript

How the sourdough may affect the functional features of leavened baked goods

Marco Gobbetti, Carlo G. Rizzello, Raffaella Di Cagno, Maria De Angelis

PII: S0740-0020(13)00081-6
DOI: 10.1016/j.fm.2013.04.012
Reference: YFMIC 1964

To appear in: Food Microbiology

Received Date: 19 December 2012

Revised Date: 13 March 2013
Accepted Date: 9 April 2013

Please cite this article as: Gobbetti, M., Rizzello, C.G., Di Cagno, R., De Angelis, M., How the sourdough
may affect the functional features of leavened baked goods, Food Microbiology (2013), doi: 10.1016/
j.fm.2013.04.012.

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1 How the sourdough may affect the functional features of leavened baked

2 goods

3 Marco Gobbetti*, Carlo G. Rizzello, Raffaella Di Cagno, Maria De Angelis

4 Department of Soil, Plant and Food science, University of Bari, Italy

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7 Corresponding author. Tel.: + 39 0805442949; fax: +39 080 5442911.

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8 E-mail address: marco.gobbetti@uniba.it (M. Gobbetti).

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9 Abstract

10 Sourdough fermentation is one of the oldest food biotechnologies, which has been studied and

11 recently rediscovered for its effect on the sensory, structural, nutritional and shelf life properties

12 of leavened baked goods. Acidification, proteolysis and activation of a number of enzymes as

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13 well as the synthesis of microbial metabolites cause several changes during sourdough

14 fermentation, which affect the dough and baked good matrix, and influence the

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15 nutritional/functional quality. Currently, the literature is particularly rich of results, which show

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16 how the sourdough fermentation may affect the functional features of leavened baked goods. In

17 the form of pre-treating raw materials, fermentation through sourdough may stabilize or to

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18 increase the functional value of bran fractions and wheat germ. Sourdough fermentation may

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decrease the glycaemic response of baked goods, improve the properties and bioavailability of

dietary fibre complex and phytochemicals, and may increase the uptake of minerals. Microbial
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21 metabolism during sourdough fermentation may also produce new nutritionally active

22 compounds, such as peptides and amino acid derivatives (e.g., γ-amino butyric acid) with various
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23 functionalities, and potentially prebiotic exo-polysaccharides. The wheat flour digested via
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24 fungal proteases and selected sourdough lactobacilli has been demonstrated to be probably safe

25 for celiac patients.

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27 Keywords: sourdough, lactic acid bacteria, functional features, celiac disease

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28 1.0 Introduction

29 Under optimal processing conditions, functional microorganisms may contribute to food

30 functionality and lead to “food similar in appearance to conventional food that is intended to be

31 consumed as part of the normal diet, but has been modified to sub-serve physiological roles

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32 beyond the provision of simple nutrient requirements” (Roberfroid, 1999). In reality, the above

33 and well consolidated concept of functional foods originates since long time ago, from the Greek

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34 antiquity, and it was constantly reconsidered over time (Skiadas and Lascaratos, 2001). Just to

give a more detailed trace, the Latin “Tacuini sanitatis” (11th century) included foods and

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36 beverages among the six elements needed to keep human daily wellness (Codex 4182, Biblioteca

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37 Casanatense Rome). In particular, “white bread was considered to improve human wellness but it

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had to be completely fermented”.
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Cereal foods are, indeed, important elements of the daily diet, which mainly provide
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40 carbohydrates, proteins, dietary fibres and vitamins. Recently, the estimated annual intake of

41 bread in European countries is reported to range from 46 (Sweden, Great Britain, Finland and
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42 Austria) to 100 kg (Greece, Portugal, Spain and Italy) per person (Scazzina et al., 2009).
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43 Sourdough fermentation represents one of the oldest food biotechnologies to ferment cereal

44 matrices, which was mainly studied for its effect on the sensory, structural and shelf life
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45 properties of leavened baked goods. Sourdough could be considered as an indispensible tool to

46 exploit the potential of wheat, rye and wholegrain flours as well as that of bran, germ and gluten-
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47 free based products. Nowadays, the literature is also very rich of reports that show how the
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48 sourdough fermentation may affect the functional features of leavened baked goods. In the form

49 of pre-treating raw materials, fermentation through sourdough may stabilize or increase the

50 functional value of wheat germ and bran fractions. Sourdough fermentation may decrease the

51 glycaemic response of bread, improve the properties of the dietary fibre complex and increase

52 the uptake of minerals, vitamins and phytochemicals. Proteolysis via cereal endogenous or

53 exogenous proteases and peptidases from sourdough lactobacilli influences the allergy and
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54 intolerance responses of cereal sensitive individuals. Microbial metabolism during sourdough

55 fermentation may also produce new nutritionally active compounds, such as peptides and amino

56 acid derivatives (e.g., γ-amino butyric acid) with various functionalities, and potentially prebiotic

57 exo-polysaccharides.

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58 The continuous search for novel processes and products, providing ingredients with new

59 functionalities and cost-effective manufacturing, emphasizes the potential of food-grade

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60 fermentations and microbial bioconversion, being crucial for the production of functional

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61 metabolites (De Vos, 2005).

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63 2.0 Pre-treating raw materials

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Although sourdough fermentation is considered to be an important biotechnology option

for the manufacture of wholegrain products, especially rye bread, it may also be used to pre-treat
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66 and modify the techno-functionality of fibre-rich cereal ingredients such as bran and germ

67 (Katina and Poutanen, 2013). Most of the bran and some of the germ are removed during milling
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68 processes, therefore, the levels of nutrients of leavened baked goods is markedly lower compared
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69 to the potential of wholegrain (Piironen et al., 2002).

70 The outer layers of grain are rich of dietary fibre, phytochemicals, vitamins, minerals and
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71 endogenous enzymes (Poutanen et al., 2009). Overall, the conspicuous use of bran in the baked

72 good formulas is essentially limited due to the poor sensory and technology properties.
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73 Fermentation of wheat (Hassan et al., 2008; Salmenkallio-Marttila et al., 2001) and rye (Katina
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74 et al., 2007a and b) bran was effective to improve the sensory quality of bran containing bread,

75 and to degrade anti-nutritive factors such as the phytic acid (Hassan et al., 2008; Lioger et al.,

76 2007). The phytate breakdown was estimated to be up to 90%, which leads to an increased

77 magnesium and phosphorus solubility (Lopez et al., 2001). The addition of calcium carbonate, as

78 alkaline agent, during fermentation blunted the sourdough acidity and did not influence the

79 degradation of phytic acid (Lioger et al., 2007). Pre-fermentation of bran with yeasts and lactic
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80 acid bacteria increased the carbon dioxide retention of the dough and, consequently, the loaf

81 volume, and the crumb softness during storage of leavened baked goods (Katina et al., 2006;

82 Salmenkallio-Marttila et al., 2001). Most probably, fermented bran also showed changes on the

83 quality of dietary fibre and varied the bio-availability of phytochemicals (Broekaert et al., 2011).

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84 Wheat germ is particularly rich of vitamins, high quality proteins, lipids and contains a

85 significant amount of dietary fibre. The use of wheat germ in bread making is still moderate

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86 because of its poor shelf life stability. The high lipase and lipoxygenase activities cause

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87 sensitivity to oxidation, which leads to the release of free fatty acids and, consequently, to the

88 appearance of rancidity in baked goods. Sourdough fermentation stabilized, and enhanced some

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89 nutritional and chemical properties of the wheat germ (Figure 1). Due to lactic acidification, the

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lipase activity of the sourdough fermented wheat germ was markedly lower than that found in

the raw wheat germ. The fermentation of wheat germ was also noticed to enhance the volume of
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92 the bread and decrease the rate of firmness (Rizzello et al., 2010a and b).

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94 3.0 The glycaemic index/response

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95 The increased amount of rapidly digestible carbohydrates in the diet causes the rapid

96 rising of the blood glucose level (glycaemic index, GI) and the large demand of insulin (insulin
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97 index, II) during the postprandial period. Hyper-glycaemia is a well known risk factor, which is

98 mainly involved in the aetiology of diseases related to the metabolic syndrome (Barclay et al.,
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99 2008). Baked goods are among the major sources of digestible carbohydrates in the Western diet
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100 (Katina and Poutanen, 2013) and, unfortunately, the glycaemic responses to most of them is

101 high, also for those products made with wholemeal flour (Foster-Powell et al., 2002).

102 Various physiological factors affect the starch digestibility: binding of α-amylase to

103 substrates, gastric emptying, enzyme inhibitors, properties of digestive enzymes and viscosity at

104 the level of the digestive tract (Leloup et al., 2004; Zhang et al., 2008; Zhang et al., 2009). The

105 macro- and micro-structure of cereal foods also influences the digestibility of starch: the more
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106 gelatinised starch is, the more rapidly it is digested (e.g., white wheat bread) (Östman, 2003;

107 Singh et al., 2009). On the contrary, starch retro-gradation, which is the re-association of

108 amylose and amylopectin to form double helices and possible crystalline structures, promotes

109 slow digestibility. The high branch density of amylopectin also slows starch digestibility.

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110 Sourdough fermentation, especially in the presence of low values of pH (e.g., 3.5-4.0)

111 (De Angelis et al., 2007a; De Angelis et al., 2009; Lappi et al., 2010; Maioli et al., 2008; Najjar

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112 et al., 2009) and possibly combined with the addition of soluble fibre, is considered an effective

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113 tool to decrease GI (Fardet et al., 2006). The fermentation of wheat and rye flours with

114 sourdough lactic acid bacteria lowered the GI of wholemeal barley (Liljeberg et al., 1995;

Östman, 2003) and wheat breads ,(De Angelis et al., 2007a; De Angelis et al., 2009; Lappi et al.,

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2010; Maioli et al., 2008), and the II of rye breads with varying dietary fibre content (Juntunen et

al., 2003). The glucose response and GI of healthy volunteers was markedly higher (GI of
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118 72.0%) when feed with wheat flour bread started with baker’s yeast compared to sourdough

119 wheat flour/wholemeal flour bread (GI of 53.7%), enriched with oat fibre (5%). This latter bread
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120 was acid, had high specific volume, better cell crumb structure and more appreciated acidulous
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121 smell, taste and aroma (De Angelis et al., 2007a). Various hypotheses were proposed to explain

122 the effect of sourdough fermentation (Table 1). The synthesis of organic acids, especially lactic
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123 acid, was identified as one of the main causes. Although with variations depending on the type of

124 bread (Novotni et al., 2011), it seemed that lactic acid lowered the rate of starch digestion
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125 (Liljeberg et al., 1995), while acetic and propionic acids prolonged the gastric emptying rate
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126 (Liljeberg et al., 1998). Besides, the low pH increased the bread density, which promotes low GI

127 per se (Burton and Lightowler, 2006). Further, the chemical changes that occurred during

128 sourdough fermentation may diminish the degree of starch gelatinisation (Östman, 2003) and

129 promote the formation of resistant starch, which is less digestible. The same effects were not

130 found on the same types of bread, which were fermented with baker’s yeast alone (Scazzina et

131 al., 2009). Also the peptides and amino acids, and free phenolic compounds, which are liberated
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132 during sourdough fermentation, regulated the glucose metabolism (Katina et al., 2007a; Nilsson

133 et al., 2007), and lowered the GI and II (Novotni et al., 2011; Solomon and Blannin, 2007).

134 Although the abundant literature showed elsewhere, some reports (Hardman Fredensborg et al.,

135 2010) showed that not all the sourdoughs impact on GI/II but other factors, such as the addition

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136 of whole grains, are required. This would be not surprising since the various routes through

137 which sourdough may exert its effect, which probably are more or less pronounced depending on

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138 the conditions of using.

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139

140 4.0 Proteolysis and functional features

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141 The degradation of cereal proteins during sourdough fermentation markedly affects the

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overall quality of baked goods. Similarly to what described for other food proteins (e.g., caseins),

primary and secondary proteolysis, and the catabolism of free amino acids occur during
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144 sourdough fermentation (Figure 2). Acidification and the reduction of disulfide bonds of gluten

145 by hetero-fermentative lactobacilli promote the primary activity of cereal proteases, which lead
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146 to the liberation of various sized polypeptides. Intracellular peptidases of sourdough lactic acid
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147 bacteria complete proteolysis and liberated free amino acids, which, in turn, are subjected to

148 various catabolic reactions by the same microorganisms (Gaenzle et al., 2008). When optimized
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149 and tailored, the degradation of cereal proteins may also have important repercussions on several

150 functional features of leavened baked goods.

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151 4.1 Wheat and rye allergy

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152 Cereal proteins are one of the most frequent causes of food allergies. Wheat proteins

153 induce classical allergy affecting the skin, gut or respiratory tract and exercise induced

154 anaphylaxis, occupational rhinitis or asthma (Palosuo, 2003). Protein hydrolysis during

155 sourdough fermentation seemed to decrease the above allergenic properties. As shown by

156 immunoblotting with sera from allergic patients, wheat sourdough fermentation caused the

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157 disappearance of some IgE-binding proteins (albumins/globulins and gliadins mainly) compared

158 to the chemically acidified dough used as the control. The IgE-binding protein profile of wheat

159 and rye sourdough breads also differed from those of baker’s yeast breads. The signals of the

160 IgE-binding proteins contained in the sourdough breads disappeared after in vitro digestion with

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161 pepsin, trypsin and pancreatin. The same effect by digestive enzymes was not found for baker’s

162 yeast breads which showed persistent IgE-binding proteins (Rizzello et al., 2006). Similar results

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163 were found using the probiotic preparation VSL#3 as starter for bread making (De Angelis et al.,

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164 2007b). Overall, proteolysis by selected sourdough lactic acid bacteria may have an importance

165 during food processing to produce pre-digested wheat and rye dough, which contains IgE-

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166 binding proteins degradable by digestive enzymes (Rizzello et al., 2006).

167 4.2 Celiac disease AN

168 Celiac disease (gluten-sensitive enteropathy) is a chronic gastrointestinal tract disorder
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169 where the ingestion of gluten from wheat, rye and barley, and their cross-related varieties, leads

170 to damage of the small intestinal mucosa by an autoimmune mechanism in genetically

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171 susceptible individuals (Tye-Din and Anderson, 2008).

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172 Currently, baked goods are mainly manufactured by highly accelerated processes.

173 Especially at large scale industrial level, long-time fermentation by sourdough is largely replaced
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174 by the indiscriminate use of chemical and/or baker’s yeast leavening agents. Under these

175 technology conditions, cereal components (e.g.; proteins) are subjected to very mild or no
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176 degradation, which result in less easily digestible foods compared to traditional sourdough baked
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177 goods (Gobbetti et al., 2007). On the other side, although the wide spread of gluten containing

178 grains initiated ca. 10,000 years ago, wheat breeding was recently addressed to massive selection

179 of cultivars with an unusual and elevated content of gluten. The daily human exposure to such

180 elevated and not partially degraded levels of gluten suggested the possibility that these

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181 evolutionary challenges, together with other important medical factors, created conditions for

182 related human diseases, including celiac disease (Sapone et al., 2012).

183 Within the novel therapies for celiac disease, the enzyme strategy to detoxify gluten is

184 currently considered. It includes the oral administration of several bacterial endopeptidases,

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185 which showed a different degrees of tolerance to the gastrointestinal conditions, transamidation

186 of gliadin and the use of transglutaminase inhibitors (Caputo et al., 2010; Rauhavirta et al.,

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187 2011). Preliminary studies on intensive degradation of prolamins of wheat and rye (Di Cagno et

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188 al., 2002; Di Cagno et al., 2004; De Angelis et al., 2006a; 2006b; Gobbetti et al., 2007) opened

189 new possibilities to use these cereals in the gluten-free diet. Extensive prolamin hydrolysis also

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190 occurred in high-proteolytic-activity germinated-wheat sourdough, especially through cysteine

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proteinase activities of germinated wheat (Loponen et al., 2007). Peptidases from germinated

cereals were also shown to degrade both intact gluten proteins as well as gluten peptides,
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193 although relatively low specific activities is achieved (Schwalb et al., 2012). A biotechnology

194 protocol that used fungal proteases and a pool of selected sourdough lactobacilli degraded gluten
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195 to less than 100 ppm during long-time fermentation (Rizzello et al., 2007). Under these
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196 conditions, primary proteolysis mainly took place through the activity of fungal proteases. The

197 end products are various sized polypeptides, which, through a complex system of ABC and ATP
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198 transporters, namely Opp, DtpT and Dpp, are moved across the cytoplasmic membrane of

199 sourdough lactobacilli. Already after few minutes from the entry, the concentration of
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200 polypeptides markedly decreases, being about 100 times lower than that from the environment
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201 (De Angelis et al., 2010). Nine intracellular peptidases were partially purified from the pooled

202 cytoplasmic extract of the selected sourdough lactobacilli and used to simulate the hydrolysis

203 towards the 33-mer epitope. First, the combined activity of general aminopeptidase type N (EC

204 3.4.11.11; PepN), endopeptidase (EC 3.4.23; PepO) and prolyl endopeptidyl peptidase (EC

205 3.4.21.26; PEP) promoted the hydrolysis into five major fragments. Then, PepN and X-prolyl

206 dipeptidyl aminopeptidase (EC 3.4.14.5; PepX) mainly liberated dipeptides, which were mainly
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207 degraded into free amino acids via prolidase (EC 3.4.13.9; PepQ) and PepX. The only remaining

208 dipeptides were hydrolyzed through PepQ. In conclusion, five peptidases were responsible for

209 the complete degradation of the 33-mer or other synthetic immunogenic peptides, which

210 occurred within 14 h of incubation (De Angelis et al., 2010). Sweet baked goods were made

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211 using the complete hydrolyzed wheat flour and they were daily administered to celiac patients

212 for 60 days. Individuals ingested the equivalent of ca. 10 g of native gluten per day.

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213 Haematology, serology and intestinal permeability analyses showed the complete tolerance by all

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214 celiac patients during all time (Di Cagno et al., 2010). A second clinical challenge was carried

215 out on other celiac patients, who ingested the equivalent of ca. 8 g of native gluten per day.

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216 Serology but also morphometric and immunoistochemistry analyses were carried out during 60

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days. None of the CD patients had clinical complaints and none produced anti-TG2 antibodies or

had modification of the small intestinal mucosa. No increase of CD3 and gamma delta cells was
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219 found as well as the Marsh grade was unchanged after the challenge (Greco et al., 2011) (Figure

220 3). A third clinical challenge is under progress on a large number of volunteers, which is lasting
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221 six months.

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222 Obviously, such wheat preparations with extended fermentation and completely degraded

223 gluten need, as usually done for naturally gluten-free matrices, of baking improvers to be used
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224 into gluten-free recipes.

225 4.3 Naturally gluten-free products

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226 When compared to their wheat counterparts, naturally gluten-free (GF) baked goods show
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227 dry starch texture and more rapid staling, due to the absence of gluten network (Moore et al.,

228 2004), and poor mouth-feel and flavour (Gallagher et al., 2004). Compared to GF bread started

229 with baker’s yeast alone, the use of selected lactic acid bacteria give a number of advantages

230 (Clarke et al., 2002; Crowley et al., 2002; Ryan et al., 2006). Sourdough fermentation positively

231 influences texture, aroma, nutritional properties and shelf life of GF bread due to complex

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232 microbial metabolic activities. Lactic acid bacteria might be considered as “burgeoning” cell

233 factories to deliver functional biomolecules and food ingredients for the manufacture of high

234 quality GF products (Arendt et al. 2011; Zannini et al. 2012). L. sanfranciscensis LS40 and

235 LS41, and L. plantarum CF1 were selected and used as sourdough starters for the manufacture of

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236 GF bread following a two-step fermentation process. The nutritional, texture and flavour

237 characteristics of GF breads were improved (Di Cagno et al., 2008). A large industrial

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238 application of the sourdough biotechnology for making GF baked goods is emerging.

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239 4.4 Bioactive peptides and amino acid derivatives

240 Bioactive or biogenic peptides are defined as specific protein fragments that have a

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241 positive impact on the body function or condition, and may, ultimately, influence the human

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health (Kits, and Weiler, 2003). Usually, bioactive peptides correspond to cryptic sequences

243 from native proteins, which are mainly released through hydrolysis by digestive enzymes,
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244 microbial and plant proteolytic enzymes and, more in general, increase during food fermentation

245 (Korhonen, and Pihlanto, 2007). In vitro and some in vivo studies show a large spectrum of
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246 biological functions, which are attributed to bioactive peptides (Gobbetti et al., 2010).
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247 Recently, the interest for antioxidant peptides, deriving from food proteins, has increased,

248 and the evidences that bioactive peptides prevent oxidative stresses associated with numerous
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249 degenerative aging diseases (e.g., cancer and arteriosclerosis) are accumulating (Adebiyi et al.,

250 2009). Although synthetic antioxidants are more effective, natural antioxidants have a simpler
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251 structure, higher stability and non-hazardous immune-reaction (Sarmadi and Ismail, 2010). The
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252 capacity of selected lactic acid bacteria to release antioxidant peptides was shown during

253 sourdough fermentation of various cereal flours (Coda et al., 2012; Rizzello et al., 2008; Rizzello

254 et al., 2012b). The radical scavenging activity of water/salt-soluble extracts from sourdoughs was

255 markedly higher than that of chemically acidified doughs. The highest activity was found for

256 whole wheat, spelt, rye and kamut sourdoughs. Almost the same results were found for the

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257 inhibition of the linoleic acid autoxidation. Twenty-five peptides, consisting of 8 to 57 amino

258 acid residues, were identified in the active fractions from sourdough water-soluble extracts

259 (Table 2). Almost all sequences shared compositional features (e.g., abundant presence of

260 residues such as Tyr, Trp, Met, Lys, Pro, Cys, His, Val, Leu and Ala), which are typical of

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261 antioxidant peptides. The abundance of hydrophobic amino acids enhances the solubility of

262 peptides in lipids, thus facilitating the access to hydrophobic radical species and to hydrophobic

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263 polyunsaturated fatty acids (Sarmadi and Ismail, 2010). Fourteen of the above 25 sequences had

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264 hydrophobic ratios higher than 50%. All the purified fractions, which contained various peptides,

265 showed ex vivo antioxidant activity on mouse fibroblasts artificially subjected to oxidative stress.

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266 The potential of sourdough lactic acid bacteria to release lunasin during fermentation of cereal

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and nonconventional flours was exploited. Lunasin is a 43-amino acid peptide, which

corresponds to the small subunit peptide (Gm2S-1) of 2S soy albumin (Galvez and de Lumen,
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269 1999). Recently, lunasin was isolated also in some cereal and pseudo-cereal grains (e.g., wheat,

270 barley, amaranth). In vivo assays showed that lunasin has an inhibitory effect against the core
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271 histone acetylation of mammalian cells (Galvez et al., 2001; Jeong et al., 2002; Lam et al., 2003;
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272 de Lumen, 2005; Jeong et al., 2007a; Jeong et al., 2007b). This suggested its involvement in the

273 chromatin modification, the process implicated in cell-cycle control and suppression of
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274 carcinogenesis. The anti-inflammatory activity of lunasin was also demonstrated on RAW 264.7

275 macrophages (Dia et al., 2009). Compared to control douhgs, the concentration of lunasin
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276 increased up to 2-4 times after fermentation. Lactobacillus curvatus SAL33 and Lactobacillus
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277 brevis AM7 synthesized the highest concentrations of lunasin. Besides the presence of the entire

278 lunasin sequence, fragments containing the immune-reactive epitope RGDDDDDDDDD were

279 found.

280 γ-Amino butyric acid (GABA), a four-carbon non-protein amino acid, acts as the major

281 inhibitory neurotransmitter of the central nervous system (Krnjevic, 1974). GABA also has anti-

282 hypertensive, diuretic and tranquilizer effects, and prevents diabetes (Jakobs et al., 1993;
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283 Adeghate and Ponery, 2002; Cohen et al., 2002; Hagiwara et al., 2004; Komatsuzaki et al.,

284 2005). Glutamate decarboxylase is the enzyme, which catalyses the conversion of L-glutamate

285 (or its salts) onto GABA, through a single step α-decarboxylation (Ueno et al., 2000). L.

286 plantarum C48 and Lactococcus lactis subsp. lactis PU1 were used for sourdough fermentation

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287 of cereal, pseudo-cereal and leguminous flours. A blend of buckwheat, amaranth, chickpea and

288 quinoa flours was selected and subjected to sourdough fermentation under optimized conditions.

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289 The concentration of GABA (504 mg/kg) in the blend fermented with sourdough was markedly

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290 higher than that found for the blend of flours fermented with baker's yeast, and exceeded the

291 daily dose needed to show physiological effect (Inoue et al., 2003). An abundant concentration

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292 of GABA was also found during sourdough fermentation of micronized by-products from

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debranned durum wheat (Rizzello et al., 2012b) and wheat germ (Coda, et al., 2010; Rizzello et

al., 2008; Rizzello, et al., 2010)

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296 5.0 Fibres

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297 5.1 Dietary fibres

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298 Dietary fibre mainly consists of plant polysaccharides and lignin, which are resistant to

299 hydrolysis by human digestive enzymes. As well documented, high consumption of dietary fibre
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300 lowers the risk of cardiovascular disease, diabetes, hypertension, obesity and gastrointestinal

301 disorders (Anderson et al., 2009; Raninen et al., 2011). Cereal baked goods are an important
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302 source of dietary fibre, and as staple foods they may markedly increase the daily intake of dietary
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303 fibre. The physiological effect of dietary fibre depends on its chemical and physical

304 characteristics such as the degree of polymerisation of polysaccharides, the presence of side

305 chains and the degree of cross-linking, particle size and cell wall integrity (Raninen et al., 2011).

306 All these characteristics may be affected by acidification and activation of endogenous enzymes.

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307 Sourdough fermentation is considered to be one of the most suitable biotechnology for the

308 manufacture of wholemeal rye and wheat baked goods, which are very rich sources of dietary

309 fibre (Katina et al., 2005). Without sourdough wholemeal rye or wheat-rye flour mixes are very

310 difficult to process, and sourdough improves flavour, texture and shelf life of whole grain rye

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311 breads. Sourdough fermentation controls the endogenous microbiota of bran, the endogenous

312 xylanase acitivity and the subsequent solubilisation of arabinoxylans (Katina et al., 2012).

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313 Compared to conventional bread, partially baked frozen breads subjected to sourdough

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314 fermentation and addition of dietary fibre had significantly higher Chemical Score and Essential

315 Amino Acid Index (Kopec et al., 2011). Fractions from debranning of durum wheat (Triticum

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316 durum sp.) were subjected to micronization and air fractionation, obtaining coarse and fine

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fractions. Wheat flour (Triticum aestivum sp.) doughs, containing 5% of coarse or fine fractions,

were subjected to fermentation by baker's yeast alone or sourdough lactobacilli. Compared to

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319 wheat flour alone, the addition of micronized bran fractions increased the concentration of free

320 amino acids, total phenols and dietary fibre as well as the phytase and antioxidant activities of
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321 doughs (Rizzello et al., 2012b). Probably, due to the most accessible surface area of the
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322 micronized bran, which allows a larger contact with bacterial enzymes (Hemery et al., 2011),

323 sourdough fermentation further improved the nutritional features, and enhanced the texture and
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324 sensory properties of leavened baked goods containing debranning by-products, especially when

325 the coarse fraction was used (Rizzello et al., 2012b).

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326 5.2 Exo-polysaccharides

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327 Dietary non-digestible oligosaccharides modulate the composition and activity of

328 intestinal microbiota, and may exert human health benefits based on improved bowel functions,

329 prevention of overgrowth of pathogenic bacteria, through the stimulation of probiotic members

330 of the intestinal microbiota, and increased synthesis of short-chain fatty acids (Ketabi et al.,

331 2011).

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332 Sourdough lactic acid bacteria synthesize a large structural variety of exo-polysaccharides

333 (EPS) through the activity of glycosyltransferases. Since these bacteria are used as starters in

334 cereal fermentations, these polymers become available for food applications through the in situ

335 synthesis during processing (Bounaix et al., 2009; Tieking and Gaenzle, 2005). L.

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336 sanfranciscensis LTH2590 synthesized 0.5–1% levans (on flour basis) during 24 h fermentation

337 of wheat and rye flours (Korakli and Gaenzle, 2002). Levans from L. sanfranciscensis are

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338 preferentially degraded by bifidobacteria at the level of the human intestinal tract (Korakli and

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339 Gaenzle, 2002). The synthesis of EPS (fructans and glucans) with prebiotic potential was also

340 shown during sourdough fermentation of wheat or sorghum flours by Lactobacillus frumenti,

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341 Lactobacillus pontis, Lactobacillus acidophilus, Lactobacillus reuteri and Weissella cibaria

342

343
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(Schwab et al., 2008; Tieking et al., 2003a and b). The addition of 12% sucrose (flour weight) to

the dough was always considered. Since the autochthonous microbiota of traditional sourdoughs
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344 typically consists of two to five strains, it is likely that one EPS-forming strain is always present.

345 L. reuteri LB 121 synthesized two types of EPS from sucrose, a 4,6-disubstituted a-glucan
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346 (reuteran) and levan. The enzymes responsible for the synthesis of EPS (glucansucrase and
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347 fructosyltransferase) were purified and characterized (Kralj et al., 2002; van Geel-Schutten et al.,

348 1998; van Hijumet al., 2001). A silent gene was identified in L. reuteri strain LB 121, which was
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349 found to code for inulosucrase by heterologuous expression in Escherichia coli (van Hiju et al.,

350 2002).
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351 More recently, EPS-forming sourdough lactic acid bacteria were used for making GF
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352 products (Galle et al., 2011; 2012). The in situ formation of EPS was responsible for the

353 significant decrease of dough strength and elasticity, and dextran showed the best shelf life

354 improvement. Microbial EPS, which are synthesized during sourdough fermentation, may be

355 considered as promising alternatives to replace hydrocolloids for the manufacture of GF

356 products.

357
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358 6.0 Vitamins

359 Wholegrain cereal foods are an important source of vitamins such as thiamine, vitamin E

360 and folates. Overall, fermentation by yeasts increased the folate content of wheat flour, bran

361 (Kariluoto et al., 2004; Katina et al., 2007a) and rye (Kariluoto et al., 2004; Katina et al., 2007a

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362 and b; Liukkonen et al., 2003). Contrarily to lactic acid bacteria, some strains of sourdough

363 yeasts showed a marked capability to increase the concentration of folate in rye sourdough

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364 (Hjortmo et al., 2005 and 2008; Kariluoto et al., 2006). Nevertheless, some reports (Gujska et al.,

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365 2009) showed a decrease of the folate content during yeast and lactic acid bacteria fermentation.

366 The concentration of thiamine increased during extensive yeast fermentation (Batifoulier et al.,

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367 2005; Ternes and Freund, 1998) or decreased under other baking processes (Martinez-

368

369
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Villaluenga et al., 2009). Contrarily to short processes, prolonged sourdough fermentation

maintained the original content of vitamin B1 in whole wheat baked goods. Whole wheat bread
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370 making with yeast under long-time fermentation resulted in the fortification of riboflavin. The

371 use of both yeast and sourdough did not have a synergistic effect on B vitamin levels (Batifoulier
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372 et al., 2005). The synthesis of vitamin B2 to enrich pasta and bread was strain dependent
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373 (Capozzi et al., 2011). Decreased levels of vitamin E were found during sourdough preparation

374 and dough making (Wennermark and Jägerstad, 1992), and the levels of tocopherol and
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375 tocotrienol decreased during rye sourdough baking (Liukkonen et al., 2003). Probably, due to the

376 numerous microbial activities and technology parameters, which may affect the content of
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377 vitamins in sourdough baked goods, the potential of sourdough, regarding this aspect, should be
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378 more clearly investigated.

379

380 7.0 Phytase/phytic acid activities

381 Whole grains and cereal baked goods are sources of minerals, mainly calcium, potassium,

382 magnesium, iron, zinc and phosphorus. Grains also contain 3-22 mg of phytic acid (myo-inositol

383 hexaphosphate) per gram, which is concentrated in the aleurone layers and it is indispensible for
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384 seedling growth (Garcia-Estepa et al., 1999). Phytic acid has strong chelating capacity and forms

385 insoluble complexes with dietary cations, which impairs mineral absorption. Phytase

386 dephosphorylates phytic acid and forms free inorganic phosphate and inositol phosphate esters

387 (Figure 4). These compounds have less chelating capacity, decreasing the influence on mineral

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388 bioavailability.

389 The activity of grain endogenous phytase is stimulated under the acidic conditions, which

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390 are promoted through sourdough fermentation. The optimum value of pH for wheat phytase is ca.

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391 5.0 (Türk et al., 1996). During sourdough fermentation, a moderate acidification to pH 5.5

392 decreased the phytate content of whole wheat flour by 70% because of the enhanced activity of

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393 flour endogenous phytase (Leenhart et al., 2005). Compared to baking yeast bread, the levels of

394

395
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hemoglobin, hematocrite, ferritin and iron were significantly higher in mice fed with traditional

sourdough. A significant decrease of the excreted iron levels was also found (Chaoui et al.,
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396 2006). Absorption of zinc, magnesium and iron was higher in rats when bread was made using

397 sourdough (Lopez et al., 2003). Although some of the results are somewhat ontradictory, it
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398 seemed that most of the lactic acid bacteria and yeasts also possess phytase activity but certainly
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399 their contribute to hydrolysis of phytic acid is markedly lower than that of the flour endogenous

400 enzymes (Chaoui et al., 2003; Reale et al., 2004). Phytase activity was found in commercial
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401 baker´s yeasts (Türk et al., 2000). Regarding lactic acid bacteria, the phytase activity could be

402 considered strain specific and largely variable depending on environmental and assay conditions.
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403 The screening of a large number of sourdough lactic acid bacteria revealed no intense phytase
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404 activity (Reale et al., 2007). Nevertheless, other authors (Lopez et al., 2000; Shirai et al., 1994)

405 found that other sourdough strains used phytic acid as the only carbon source and the phytase

406 activity of L. sanfranciscensis was proven in vitro and in situ (De Angelis et al., 2003).

407

408 8.0 Phytochemicals

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409 Phytochemicals are biologically active compounds, which are mainly located in the outer

410 layers (e.g., bran) of cereal grains (Slavin, 2003). Various chemical classes are included in

411 phytochemicals, even though they mainly consist of phenolic acids, alkylresorcinols, lignans,

412 phytosterols and tocols.

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413 The antioxidant properties of baked goods are affected by the variable content and

414 bioavailability of phytochemicals (Liukkonen et al., 2003; Mattila et al., 2005), which is mainly

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415 determined during milling and food processing (Bondia-Pons et al., 2009; Slavin et al., 2000).

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416 The antioxidant potential of wheat bran-flour mixture and rye flour was higher than that found in

417 conventional products when subjected to sourdough fermentation (Liukkonen et al., 2003;

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418 Rizzello et al., 2012b). Overall, the antioxidant potential of sourdough rye breads is higher than

419

420
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that of white wheat breads (Martinez-Villaluenga et al., 2009; Michalska et al., 2007).

Fermentation increases the levels of easily extractable phenolic compounds (Liukkonen et al.,
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421 2003). Yeast, lactic acid or mixed fermentation had variable effects on native and germinated

422 wholemeal rye flour. Tailored fermentation offers the possibility to increase the phytochemical
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423 potential of wholemeal rye flour. The levels of folates, free phenolic acids, total phenolic
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424 compounds, lignans and alkylresorcinols increased during mixed fermentation of germinated rye,

425 which also caused a lower pH compared to native rye flour. After germination and fermentation,
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426 the level of folate and free phenolic acids increased up to seven- and ten-fold, respectively

427 (Katina et al., 2007b). On the other side, the levels of phytate (Frolich et al., 1986; Larsson and
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428 Sandberg, 1991), alkylresorcinols (Verdeal and Lorenz, 1977) and tocopherols (Piironen et al.,
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429 1987) decreased during sourdough baking process, whereas the levels of lignans did not change

430 (Nilsson et al., 1997). Fermentation of rye or wheat bran with yeast and, especially, with added

431 cell wall degrading enzymes increased the level of free ferulic acid (Katina et al., 2007a and

432 2012; Mateo Anson et al., 2009). Ferulic acid is a structural component of the grain cell walls,

433 which is cross-linked to arabinoxylan. Overall, the supplementation of wheat bread with

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434 processed bran increased the in vitro and in vivo bioavailability of phenolic compounds (Mateo

435 Anson et al., 2009 and 2011).

436

437 9.0 Future trends

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438 Although not yet exhaustive, a very abundant literature clearly shows the functional

439 potential of the sourdough fermentation under several perspectives. The interim prospect would

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440 probably be to consider the sourdough like a cell factory to modify cereals and other materials

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441 for functional and nutritional tailored food or feed. The formation or modification of bioactive

442 compounds during sourdough fermentation should expand the toolset to develop sourdough

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443 baked goods with specific nutritional functionality.

444

445 References
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859 Zannin, E., Pontonio, E., Waters, D. M., & Arendt, E.K. (2012). Applications of microbial

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861 and Biotechnology, 93, 473-485.

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864 fine structure. Journal of Agricultural and Food Chemistry, 56, 4686–4694.

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867 Legends to Figures

868 Figure 1. Sourdough fermentation of wheat germ: direct effect on the raw material and effects of

869 sourdough fermented wheat germ when used as an ingredient in bread making (adapted from

870 Rizzello et al., 2010a and b).

PT
871 Figure 2. Schematic representation of the proteolysis during sourdough fermentation. (A) Primary

872 proteolysis triggered by the acidification and the reduction of disulfide bonds of gluten by hetero-

RI
873 fermentative lactobacilli, which, in turn promote the primary activity of cereal proteases, which lead

SC
874 to the liberation of various sized polypeptides. (B) Secondary proteolysis by intracellular peptidases

875 of sourdough lactic acid bacteria, which complete the proteolysis and liberated free amino acids. (C)

U
876 Catabolism of free amino acids by sourdough lactic acid bacteria: example of catabolic reaction

877

878
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involving phenylalanine (adapted from Gaenzle et al., 2007; 2008).

Figure 3. Density of gamma delta intraepithelial lymphocytes cells in jejunal biopsy from celiac
M
879 patient at the beginning (panel A) and after 60 days of challenge (panel B) with fully hydrolyzed

880 baked goods (8 ppm residual gluten). Arrows refer to gamma delta intraepithelial lymphocytes
D

881 (adapted from Greco et al., 2011).

TE

882 Figure 4. Schematic representation of the phytase activity during sourdough fermentation. Phytase

883 dephosphorylates phytic acid and forms free inorganic phosphate and inositol phosphate esters.
EP

884 These compounds have less chelating capacity, which increases the mineral, proteins, peptides and

885 amino acids bioavailability. During sourdough fermentation, acidification promoted the activity of
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886 grain endogenous phytase.

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844 Table 1. Effect of sourdough fermentation on glycaemic index/response of leavened baked goods.

Biochemical effect In vivo effect References

PT
Synthesis of lactic acid Lowering the rate of starch digestion Liljeberg et al., 1995; De Angelis et al., 2007; De Angelis et

al., 2009

RI
Synthesis of acetic and propionic acids Lowering the gastric emptying rate Liljeberg et al., 1998; De Angelis et al., 2007

SC
Increasing the interaction between starch and cereal Reducing the starch bioavailability Östman, 2003

proteins

U
Synthesis/release of peptides and amino acids Regulating glucose metabolism Novotni et al., 2011; Solomon and Blannin, 2007

AN
Synthesis of free phenolic compounds Increasing glucose tolerance and insulin Katina et al., 2007; Solomon and Blannin, 2007

M
sensitivity

Acidification, increase of arabinoxylan Increasing of insulin resistance by Lappi et al., 2009

D
solubilisation and proteolysis lowering postprandial responses
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845
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Table 2. Sequences of antioxidant peptides purified from the water/salt-soluble extracts (WSE) of whole wheat, spelt, rye and kamut sourdoughs

fermented with selected lactic acid bacteria (adapted from Coda et al., 2012).

PT
Sequencea

RI
Sourdough Peptide Molecular Source Protein

number mass NCBI accession number

SC
Whole 1 MAPAAVAAAEAGSK GH32_ORYSJ, Probable indole-3-acetic acid-amidosynthetase;
1243.0196
wheat P0C0M2

U
2 DNIPIVIR AKH2_MAIZE, Bifunctionalaspartokinase/homoserine dehydrogenase

AN
937.5898
2; P49080

M
Spelt 3 AIAGAGVLSGYDQLQILFFGK 2166.0752 ADT1_MAIZE, ADP,ATP carrierprotein 1, mitochondrial; P04709

4 GNQEKVLELVQR 1412.4122 FH16_ORYSJ, Formin-like protein 16

D
5 PAGSAAGAAP 769.4197 C3H31_ORYSJ, Zinc finger CCH domain-containing protein 31;
TE Q7XPK1

6 EALEAMFL 923.5322 IAA31_ORYSJ, Auxin-responsive protein IAA31; P0C133

EP

7 AAGAAAAARSAGQCGR SAP17_ORYSJ, Zinc finger AN1 domain-containing stress-associated

1388.3942
protein 17; Q6H595
C
AC

8 ITFAAYRR 997.6323 NIP41_ORYSJ, Aquaporin NIP4-1; Q9ASI1

9 HPVPPKKK 912.5011 CCF11_ORYSJ, Putative cyclin-F1-1; Q6K1Z1

Rye 10 VFVDEGLEVLGWRPVPFNVSVVGRNAK GLTB_MAIZE, Ferredoxin-dependent glutamate synthase,

2983.0406
chloroplastic; P23225
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11 RLSLPAGAPVTVAVSP CSLD4_ORYSJ cellulose synthase-like protein d4 Oryza sativa subsp

1534.9327
japonica; Q2QNS6

12 NANGELCPNNMCCSQWGYCGLGSEFCGNGCQSGACCPEK 4032.5905 AGI_ORYSJ, Lectin; Q0JF21

PT
13 LCPVHRAADL 1094.6324 CSLD4_ORYSJ cellulose synthase-like protein d4; Q2QNS6

RI
14 PAEMVAAALDR 1483.8343 SLY1_OTYSJ SEC1 family transport protein SLY1; Q851W1

15 KVALMSAGSMH 1131.3802 MLOH1_HORVU, MLO protein homolog 1; O49873

SC
16 DLADIPQQQRLMAGLALVVATVIFLK 2822.9826 CP51_SORBI, Obtusifoliol 14-alpha demethylase; P93846

U
17 KNGSIFNSPSATAATIIHGHNYSGLAYLDFVTSK 3581.6329 KSL6_ORYSJ, Ent-isokaur-15-ene synthase; A4KAG8

AN
18 GTIFFSQEGDGPTSVTGSVSGLKPGLHGFHVHALGDTTNG SODC2_ORYSJ, Superoxidedismutase [Cu-Zn]; P28757
5338.2306
CMSTGPHFNPTGK

M
Kamut 19 YEWEPTVPNFDVAKDVTDM 2254.3380 KRP3_ORYSJ, Cyclin-dependent kinase inhibitor 3; Q2R185

20 GVSNAAVVAGGH 1038.6974 SUT5_ORYSI, Sucrose transport protein SUT5; A2X6E6

D
21 DAQEFKR 891.7372 HFN40_MAIZE, Suppressorprotein HFN40; P82865

22 PPGPGPGPPPPPGAAGRGGGG
TE 1703.9566 FH18_ORYSJ Formin-like protein 18; Q6MWG9

23 HKEMQAIFDVYIMFIN 1999.0342 ZRP4_MAIZE O-methyltransferase ZRP4; P47917

EP

24 TGGGSTSSSSSSSSSLGGGASRGSVVEAAPPATQGAAAAN SLR1_ORYSJ, DELLA protein SLR1; Q7G7J6

5124.3480
C

APAVPVVVVDTQEAGIR
AC

25 DTAAGYVAPPDPAVSTGDYGLAGAEAPHPHESAVMSGAA DHR25_ORYSJ, Dehydrin Rab25; P30287

4920.3312
AAAVAPGGEAYTR

a
The single-letter amino acid code is used.
 ACCEPTED MANUSCRIPT
854 Figure 1.
855
Direct effects
Anti-nutritional factors:
- Decrease of raffinose concentration
- Increase of phytase activity
(Rizzello et al., 2010a)
Stabilisation:
- Decrease of lipase activity
- Low concentration of hexanal and
other markers of lipid oxidation
during long-term storage
(Rizzello et al., 2010a)
Nutritional/functional properties:
- Increase of free amino-acids concentration
- GABA synthesis (Glu conversion)
- Increase of antioxidant activity
(Rizzello et al., 2010a)
D
856
TE
C EP
AC
Effects on baked goods
PT
Nutritional/functional properties:
- Increase of free amino-acids concentration
- Increase of antioxidant activity
RI
- Increase of in vitro-digestibility
(Rizzello et al., 2010b)
SC
Sourdough
fermentation of Sensory and structural properties:
wheat germ - More appreciate acidic taste and
flavour; salty taste, elasticity
U
- Increase of loaf volume and crumb
softness
(Rizzello et al., 2010b)
AN
Shelf life extension:
- Firmness delay (Rizzello et al., 2010b)
M
- Synthesis of antifungal compounds
(organic acids and peptides)
(Rizzello et al., 2012)
40
 ACCEPTED MANUSCRIPT

857 Figure 2.

(A) Primary proteolysis

gliadin
LMW glutenin
y-HMW glutenin

x-HMW glutenin

PT
degradation products
peptide

Ψredox - pH

RI
Sourdough fermentation

SC
Endogenous enzymes
(cereal proteases)

U
AN
(B) Secondary proteolysis
M
-
acid
+
PepQ alkaline
D

PepX PepO
DtpT
TE

PepN
Amino acids
PepP
Opp
H+
EP

(C) Catabolism of free amino acids

C
AC

Transaminase Dehydrogenase

Phenylalanine Phenylpyruvate Phenyllactate

Decarboxylase

Dehydrogenase Dehydrogenase
Phenylacetate Phenylacetaldehyde Phenylethanol
858
41
 ACCEPTED MANUSCRIPT

859 Figure 3.

PT
RI
SC
860

U
AN
M
D
TE
C EP
AC

42
 ACCEPTED MANUSCRIPT

1 Figure 4.
2
Sourdough fermentation

5.6 pH

5.0

PT
Activation 4.2
Grain endogenous Microbial phytases
phytase (lactic acid bacteria and yeasts)

RI
Inorganic phosphate;

SC
Phytase Myo-inositol phosphate esters;
EC 3.1.3.8 Free minerals (e.g., Fe3+, Zn2+, Ca2+);
EC 3.1.3.26

U
Proteins, peptides, amino acids.

Phytic acid (myo-inositol hexaphosphate)

AN
3
M
D
TE
C EP
AC