Accepted Manuscript

Sourdough lactic acid bacteria: exploration of non-wheat cereal-based fermentation

Rossana Coda, Raffaella di Cagno, Marco Gobbetti, Carlo Giuseppe Rizzello

PII: S0740-0020(13)00132-9
DOI: 10.1016/j.fm.2013.06.018
Reference: YFMIC 1999

To appear in: Food Microbiology

Received Date: 18 December 2012

Revised Date: 15 May 2013
Accepted Date: 27 June 2013

Please cite this article as: Coda, R., Cagno, R.d., Gobbetti, M., Rizzello, C.G., Sourdough lactic acid
bacteria: exploration of non-wheat cereal-based fermentation, Food Microbiology (2013), doi: 10.1016/
j.fm.2013.06.018.

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 ACCEPTED MANUSCRIPT

2 Sourdough lactic acid bacteria: exploration of non-wheat

3 cereal-based fermentation

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5 Rossana Coda, Raffaella di Cagno, Marco Gobbetti,Carlo Giuseppe Rizzello*

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

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13 *Corresponding author: Department of Soil, Plant and Food science, Via G. Amendola 165/a, 70126

14 Bari, Italy. Phone: 39 080 5442948. E-mail: carlogiuseppe.rizzello@uniba.it

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1 Abstract

2 Cereal-based foods represent a very important source of biological as well as of cultural

3 diversity, as testified by the wide range of derived fermented products. A trend that is

4 increasingly attracting bakery industries as well as consumers is the use of non-conventional

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5 flours for the production of novel products, characterised by peculiar flavour and better

6 nutritional value. Lactic acid bacteria microbiota of several non-wheat cereals and pseudo-

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7 cereals has been recently deeply investigated with the aim of studying the biodiversity and

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8 finding starter cultures for sourdough fermentation. Currently, the use of ancient or ethnic

9 grains is mainly limited to traditional typical foods and the bread making process is not well

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10 standardized with consequent negative effects on the final properties. The challenge in

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fermenting such grains is represented by the necessity to combine good technology and

sensory properties with nutritional/health benefits. The choice of the starter cultures has a
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13 critical impact on the final quality of cereal-based products, and strains that dominate and

14 outcompete contaminants should be applied for specific sourdough fermentations. In this

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15 sense, screening and characterization of the lactic acid bacteria microbiota is very useful in
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16 the improvement of a peculiar flour, from both a nutritional and technological point of view.

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1 1. Introduction

2 Cultivation of cereals dates back to 7000 B.C. for wheat and barley, 4500 B.C. for rice and

3 maize, 4000 B.C. for millet and sorghum, 400 B.C. for rye, and 100 B.C. for oat. Triticale is

4 the only cereal (since 1930) of our time (McGee, 1984). Several cereals are cultivated, but on

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5 a worldwide basis, wheat and rice are the most important crops, accounting for over 50% of

6 the world’s cereal production. Moreover, FAO has predicted that the 2012 world wheat

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7 production will be the second highest with a record of 690 million tonnes (FAO, 2012).

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8 Despite the huge amount produced, wheat is not a typical cereal in developing or emerging

9 countries where other crops like sorghum (Sorghum bicolor L. Moench) (Rooney and Awika

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10 2005), millet (Pennisetum glaucum) (Joshi et al. 2008; Balasubramanian and Viswanathan,

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2010), acha or white fonio (Digitaria exilis), iburu or black fonio (Digitaria iburua), teff

(Eragrostis tef), maize (Zea mais), and rice (Oryza sativa), constitute the staple diet for
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13 human consumption and play an essential role in providing healthy food for the poorest

14 populations and regions (Jideani and Jideani, 2011; McKevith, 2004). For this reason and for
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15 their numerous useful properties, research and development on ethnic and ancient grains gets
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16 worldwide renewed interest. It raises a great deal of recent interest that, besides wheat, other

17 crops such as buckwheat, oat, barley, spelt, rye, quinoa and amaranth constitute highly
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18 nutritional grain ingredients for healthy food production and special dietary uses (Coda et al.,

19 2010a; Moroni et al., 2010a; Vogelmann 2009). Several of these are considered ancient crops
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20 and/or minor cereals (e.g. kamut, barley, spelt, rye, einkorn, millet, oat, sorghum) and are
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21 often underutilised or only consumed locally; others are pseudo-cereals (e.g. quinoa,

22 amaranth, buckwheat), crops evolutionarily distant from cereals, which produce grains (Sterr

23 et al., 2009; Correia et al., 2010; Coda et al., 2010b; Guyot 2012). The use of minor cereals

24 and pseudo-cereals is of nutritional interest because of their better healthy composition,

25 especially regarding minor components present in grains (dietary fibre, resistant starch,

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1 minerals, vitamins, phenolic compounds). Besides, many of them, such as spelt, emmer, teff,

2 fonio and other indigenous cereal grains, are low-input plants, suitable for growing without

3 the use of pesticides in harsh ecological conditions and marginal areas of cultivation (Moroni

4 et al 2010b, Coda et al 2010b, Jideani and Jideani 2011). They are well suited to local

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5 conditions, being reasonably resistant to drought, and help to maintain the environment by

6 providing a covering of vegetation on ground which is ecologically fragile, and considered of

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7 little value (Jideani and Jideani, 2011). In addition, they are often consumed as whole grains

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8 (Bonafaccia et al. 2000; Buerli, 2006; Jideani and Jideani, 2011). In these last years these

9 grains are attracting bakery industries and consumers of western countries, mainly as niche

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10 products with proposed healthier and more natural features compared to modern wheat.

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Besides the cereals outlined above, there are several other crops which, although not

important on a global level, may have a key role in certain parts of the world. For example,
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13 pseudo-cereals such as buckwheat, amaranth and quinoa show nutritional and texture

14 features, which make them suitable for replacing, at least in part, traditional cereal-based
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15 products (Coda et al., 2010a; Sterr, 2009; Aluko and Monu, 2003; Escudero et al., 2004;
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16 Fessas et al., 2008). Buckwheat (Fagopyrum esculentum) is eaten as a cooked grain, porridge

17 or baked into pancakes in many countries (Moroni et al., 2012). It has a high concentration of
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18 dietary fibres and essential micro-nutrients (Schoenlechner et al., 2008) and has been

19 considered as alternative crop for the production of functional foods (Coda et al., 2010a;
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20 Moroni et al., 2012). Amaranth (Amaranthus hypochondriacus) has a high protein content
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21 and a very balanced amino acid composition (Olusegun, 1983). The grain quinoa

22 (Chenopodium album) native from South America, is commonly used in Chile and Peru to

23 make bread (Bender and Bender, 1999). Its nutritional properties are mainly due to high

24 content of minerals, vitamins, fatty acids and antioxidants (Vega-Gàlvez et al., 2010).

25 Exploitation of these crops for the development of healthy or speciality foods like bread,

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1 biscuit, cookies, traditional drinks, or flakes, is gaining interest. Although the production is

2 mainly increasing, sometimes crop yields remain low, especially in not developing countries

3 (Jideani and Jideani, 2011; Vega-Gàlvez et al., 2010). This may be attributed to poor

4 agricultural practices and, as a consequence, farmers will not spend money to improve

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5 productivity unless there is a profitable market at an economical price. However, a constant

6 supply of reliably quality grain available at the economical price is of high importance at

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7 industrial or semi-industrial level. The opportunity to supplement or completely replace

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8 common cereal grains with a cereal or pseudo-cereal of higher nutritional value is a strategy

9 that extends to the food chain, from farm to processed food, and that can be inherently

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10 beneficial for the public interest.

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Compared to their large diversity, only a few studies have dealt with grains different from

wheat. This review describes the problematic related to the fermentation of cereals and
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13 pseudo-cereals, mainly focusing on the lactic acid bacteria microbiota and the selection of

14 starters for baked goods manufacturing.

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16 2. Non-wheat cereal and pseudo-cereal fermentation

17 Non-wheat grains and flours are commonly used for making fermented products. In
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18 particular, they are prepared as fermented beverages, gruels, porridges, soups, etc. and

19 designated with specific names (Beuchat, 1997). They often are semi-solid cooked doughs
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20 and porridges, and liquid beverages, prepared mainly in Africa and Asia, but also in Latin
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21 America and the Pacific Islands. These fermented products play an important role in the daily

22 diet not only for the attractiveness but also for the improvement of shelf-life and nutritional

23 properties that derive from fermentation. In western countries, food fermentations are often

24 integrated in marketing strategies to construct nutritional claims, in response to the increased

25 attention paid by consumers to a healthy way of life, and to address specific organoleptic

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1 characteristics (Humblot and Guyot, 2008). Recently, new consumer demands for food

2 products with improved nutritional value or health benefit have involved also the baking

3 industry, posing new challenges. Even though the market for new novel bakery products

4 produced with alternative cereals or pseudo-cereals is increasing, the use of such flours is

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5 restricted due to their low baking quality, as well as the final sensory quality of the baked

6 products (Gallagher et al., 2004). It has been shown that lactic acid bacteria fermentation of

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7 these alternative flours can improve both the sensory and baking qualities and plays an

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8 important role in developing countries, providing wholesome food with attractive flavour and

9 texture. Moreover, from a nutritional point of view, it offers the opportunity of increasing

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10 nutrient and energy density and of decreasing anti-nutritional factors, like tannins and phytic

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acid (Coda et al., 2010a; Coda et al., 2011a; Moroni et al., 2012). Phytic acid, in particular,

can be present at high concentration in some non-wheat cereals or pseudo-cereals, especially

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13 when milled as whole grains. As well known, sourdough fermentation increases phytase

14 activity indirectly, determining more suitable conditions of pH for the activity of endogenous
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15 phytases, or directly, through the enzymatic activity of lactic acid bacteria (Rizzello et al.,
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16 2010). The use of non-wheat cereal and pseudo-cereal flours often corresponds to a lower

17 hydrolysis index (HI) compared to wheat products, as the consequence of the higher
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18 concentration in dietary fibres and resistant starch (Coda et al., 2010a). Moreover, when

19 sourdough fermentation is applied, a further decrease of the rate of starch hydrolysis and HI
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20 due to the biological acidification is observed in several cereal, leguminous and pseudo-
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21 cereal flours (Coda et al., 2010a; Coda et al., 2011c). On the basis of the above

22 considerations, the improvement of fermentation technology for the flours alternative to

23 wheat can be considered as an important opportunity also for western countries, satisfying the

24 demand for more natural and healthy food.

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1 2.1 Lactic acid bacteria microbiota of non-wheat cereal and pseudo-cereal flours and

2 sourdoughs

3 Cereal fermentation processes are affected by specific variables that should be controlled in

4 the most proper way in order to obtain products characterized by a well-defined quality

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5 (Hammes and Gänzle, 1998). Among these variables, the type of cereal is one of the most

6 important, since it affects the amount and quality of carbohydrates as primary fermentation

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7 substrates, nitrogen sources and growth factors (Hammes et al., 2005). The native microbiota

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8 of the grains and consequently of the flour, is affected by a number of factors. Primarily, it is

9 influenced by the climate in the particular country (temperature and humidity), the cultivation

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10 and the storage conditions (De Vuyst and Neysens, 2005). However endogenous factors in

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flours and technological parameters employed for sourdough processing have a key role in

influencing the microbial communities (Hammes and Gänzle, 1998).

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13 Traditional non-wheat cereal and pseudo-cereal flours and their derived foods, are very

14 original ecological niches that represent a true opportunity to find new microbial biodiversity
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15 for food processing. This diversity should be considered not only at species level but also at
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16 strain level, since important peculiar characteristics may be harboured (Guyot, 2012). Studies

17 on the lactic acid bacteria microbiota of non-wheat cereals and pseudo-cereals such as those
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18 of African and American origin are increasing in these last years, revealing in many cases a

19 large biodiversity and opening new perspectives for their exploitation for a larger
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20 consumption not only in native regions (Moroni et al., 2010a; Coda et al., 2011a).
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21 According to previous studies, L. plantarum is commonly found in sourdoughs ecosystems

22 (De Vuyst and Neysens, 2005), and its prevalence in cereal fermentations has been mainly

23 attributed to the versatile metabolism of carbohydrates (Kleerebezem et al., 2003; Minervini

24 et al., 2010). Its dominance has been confirmed in African traditional cereal foods produced

25 with natural fermentation of millet, maize and sorghum (Oguntoyinbo et al., 2011), amaranth

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1 spontaneous fermentation (Scheirlinck et al., 2007); spelt and emmer flours (Van der Meulen

2 et al., 2007; Coda et al., 2010b). L. plantarum has often been reported in association with L.

3 fermentum in spontaneous fermentation of cereal products (Lei and Jacobsen, 2004; Kalui et

4 al., 2010). L. fermentum was as the dominant species in African fermented foods such as

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5 sorghum sourdough bread (Hamad et al., 1992), maize dough (Halm et al., 1993), and millet,

6 maize and sorghum beverages such as ogi and kunu-zaki (Oguntoyinbo et al., 2011).

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7 Although the population of lactic acid bacteria in raw cereals is usually dominated by species

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8 belonging to the Lactobacillus genus (De Vuyst and Neysens, 2005), Pediococcus is also

9 commonly encountered. Pediococcus pentosaceus is endemic in Ethiopian traditional

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10 fermented foods and was also identified in Swedish rye (Lönner et al., 1986), Moroccan

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(Boraam et al., 1993), French (Infantes and Tourneur, 1991), Belgian (Neysen, 2004) and

German (Kitahara et al., 2005) wheat sourdoughs as well as in the spontaneous fermentation
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13 of the sorghum flour (Mohammed et al., 1991) and other African grains (Nout, 2009; Coda et

14 al., 2010c) showing a large capacity of environmental adaptation. It has shown to be a

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15 competitive specie in amaranth and buckwheat sourdoughs (Sterr et al., 2009; Moroni et al.,
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16 2010b; Moroni et al., 2012). However, a wide variety of lactic acid bacteria has been

17 observed during the investigation of the microbiota of several grains, showing the presence of
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18 species such as Leuconostoc holzapfelii, Leuconostoc gallinarum, Leuconostoc graminis

19 Leuconostoc vaginalis and Weissella cibaria, which are not endemic in traditional
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20 sourdoughs but more often isolated from tropical fermented products (Moroni et al., 2011).
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21 Among these L. vaginalis and L. gallinarum have been characterized as dominant species in

22 sorghum, rice, and rye sourdoughs (Hamad et al., 1997; Meroth et al., 2004).

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24 3. Use of starter cultures in non-wheat cereal-based fermentation

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1 In recent years, the increasing interest toward bakery products made with flours different

2 from wheat has leaded to a diversification in the market and to the necessity of developing

3 more appropriate starter cultures for specific products. With the words “starter culture” one

4 refers to a microbial preparation of cells that is added to a raw material to produce a

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5 fermented food by accelerating and driving its fermentation process (Leroy and De Vuyst,

6 2004). The need to identify proper starter cultures with functional features for the

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7 fermentation of non-wheat cereals and pseudocereals appears important when considering the

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8 limited knowledge about processing of these alternative grains. There are very few reports

9 debating the technological performances of autochthonous or commercial starters for non-

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10 wheat grain substrates and more knowledge would improve their utilisation with important

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consequences on industry and consumers acceptability (Mouquet-Rivier et al., 2008; Vieira-

Dalode et al., 2008). This necessitates of a comprehensive characterisation of microbial

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13 composition and fermentation and their relative contribution to product development. The use

14 of lactic acid bacteria as starter cultures for industrial bread making, resembling the
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15 traditional sourdough fermentation has been extensively explored during these last years.
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16 Fermentation of flours for wheat sourdough production is commonly carried out through

17 back-slopping (type I sourdough). This protocol, characterised by daily propagation, has the
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18 largest application in traditional products and results in the dominance of the best adapted

19 strain. Back-slopping represents a more or less conscious method for selecting the more
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20 adaptable starter strains in order to shorten the fermentation process and to reduce the risk of
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21 fermentation failure (Leroy and De Vuyst, 2004). Failure of fermentation processes can result

22 in spoilage and/or the survival of pathogens, thereby creating unexpected health risks in food

23 products. For these reasons, the use of specific starter cultures appears important when well-

24 defined characteristics are desired in food. Spontaneous fermentations typically result from

25 the competitive activities of a variety of autochthonous and contaminating microorganisms.

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1 Those best adapted to the conditions during the fermentation process will eventually

2 dominate. Under these technological circumstances, several species of the genus

3 Lactobacillus are predominant and vary depending on the type of sourdoughs and

4 environmental factors (De Vuyst et al., 2002; Gobbetti et al., 2005). Generally, facultatively

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5 and obligately heterofermentative lactobacilli such as L. plantarum, L. alimentarius, L.

6 sanfranciscensis, L. pontis, L. brevis and L. reuteri are associated in sourdoughs (Gobbetti,

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7 1998; Gobbetti et al., 2005). For industrial bread making, back-slopping of sourdough type I

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8 should occur less frequently (weekly or even monthly), but in this case starter cultures have

9 to be used (Minervini et al., 2010).

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10 Notwithstanding the value of spontaneous sourdough fermentation, especially in the case of

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traditional baked goods production, the use of selected starters is also industrially important,

especially when nutritional, technological and sensory specific standards are desired.
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13 Industries have shown an increasing interest in type II sourdoughs, but they require faster

14 acidification without losing the baking and sensorial properties (Messens and De Vuyst,
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15 2002). Currently, traditional and industrial processes have specific needs such as constant
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16 rheology and flavour properties of the bread, so, daily propagation of sourdoughs must be

17 reduced to lower time-consuming processes and risks and sourdough must be adapted for fast
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18 fermentation processes without losing the major properties of lactic acid bacteria (Gaggiano

19 et al., 2007).
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21 3.1 Selection of starters for sourdough fermentation

22 So far, design of starters for bread making has been based on functional selection of strains,

23 considering mainly properties such as acidification, proteolysis and synthesis of volatile

24 compounds during sourdough fermentation (Collar, 1996; Stoltz and Böcker, 1996; Corsetti

25 et al., 1998; Hammes and Gänzle, 1998). However, functional starters need to adapt well to

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1 sourdough to assure a stable persistence in the environment (De Vuyst, 2000; De Vuysts and

2 Neysens, 2005). It has been recently shown that during propagation of sourdough type I, only

3 three out of nine strains of L. sanfranciscensis were dominant throughout ten days of

4 propagation in wheat flour sourdough (Siragusa et al., 2009). A similar result was obtained in

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5 the case of L. plantarum, where two out of seven selected strains markedly decreased from

6 sourdoughs during propagation (Siragusa et al., 2009; Minervini et al., 2010). This indicates

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7 that functional selection is only the first step to get efficient starter cultures. Indeed,

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8 robustness of sourdough starters is indispensable, especially because autochthonous strains

9 may frequently outcompete starters. Nowadays, bread and other bakery products are expected

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10 to offer not only food for nourishment but also for hedonistic satisfaction (Geyzen et al.,

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2012). Modern consumers pay a lot of attention to the relation between nutrition, health and

sensory properties of food and all these aspects are valuable, even though with different
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13 degree of importance, for western and for developing countries as well. In the bakery

14 industry, the use of autochthonous and commercial starters has been extensively documented
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15 (Vogelmann et al., 2009; Moroni et al; 2010b; Minervini et al., 2010; Moroni et al., 2012). A
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16 distinction between autochthonous and allochthonous starters has also been made.

17 Autochthonous starters are isolated from, and used for the same raw matrix, while
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18 allochthonous starters are isolated from certain raw matrices but used to ferment various

19 products. Commonly, commercial starters, which are not necessarily selected to ferment a
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20 specific matrix, are used to ferment a variety of foods, and they mostly coincide with the
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21 above definition of allochthonous strains (Di Cagno et al., 2013). According to some authors

22 the use of autochthonous starters is more suitable when functional performance is required,

23 since they have less competition during fermentation (Minervini et al., 2010; Leroy and De

24 Vuyst 2004).

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1 The main criteria used to select starters can be divided in to three categories: (i)

2 technological; (ii) sensory; and (iii) nutritional. Technological factors of interest for

3 sourdough fermentation are growth and acidification rate (Sterr et al., 2009; Coda et al.,

4 2010b; Coda et al., 2011a,c); salt tolerance (Gobbetti, 1998; Gaggiano et al., 2007); growth at

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5 low temperature (Gaggiano et al., 2007); synthesis of antimicrobial compounds (Coda et al.,

6 2011b; Messens and De Vuyst, 2002). Regarding the sensory characteristics of cereal-based

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7 products, usually hetero-fermentative metabolism, synthesis of aroma compounds or their

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8 precursors, and release of free amino acids through proteolysis are considered important

9 (Gobbetti, 1998; Gobbetti et al., 2005). Among nutritional properties, synthesis of biogenic

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10 compounds (e.g. bioactive peptides), degradation of antinutritional factors (e.g. phytic acid),

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increase of the antioxidant activity (ex. total phenols), synthesis of exo-polysaccharides (e. g.

glucan and fructan) are desirable (Coda et al., 2012; Rizzello et al., 2010; Rizzello et al.,
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13 2012; Di Cagno et al., 2006). Nevertheless, according to the particular raw matrix the strains

14 are intended for, other different characteristics should be taken into account during selection,
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15 since lactic acid bacteria have to be adapted to the intrinsic characteristics of the raw material
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16 to get desirable properties. In flours, in fact, besides concentration of fermentable

17 carbohydrates, buffering capacity and pH, also the presence of inhibitory compounds can
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18 affect the growth and acidification of lactic acid bacteria (Banu et al., 2011). Tolerance to

19 high concentrations of specific substances contained in some flours, such as tannins in

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20 sorghum and buckwheat (Steadman et al., 2001; Correia et al., 2010) and saponins in quinoa
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21 (Vega-Gàlvez et al., 2010) should be also considered.

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23 3.2 Autochthonous starters

24 The use of alternative flours is often limited due to their low baking and sensory quality

25 (Gallagher et al., 2004) and the challenge in fermenting these cereals is mainly based on the

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1 capacity to combine good technology and sensory properties with demonstrated nutritional

2 and health benefits (Guyot, 2012). Selection of proper strains within the lactic acid bacteria

3 microbiota of cereals and flours is indispensable to guarantee optimal performance during

4 fermentation. Some recent studies have shown that the use of selected autochthonous lactic

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5 acid bacteria to ferment sourdough is a suitable biotechnology to exploit the potential of non-

6 wheat cereals and pseudo-cereals in bread making (Sterr et al., 2009; Coda et al 2010a,b;

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7 Moroni et al., 2010a,b). This is particularly evident if considering that these flours often

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8 possess characteristics different from wheat, from nutritional, technological, and sensory

9 points of view. Defined multi-species sourdough starter have already been proposed to satisfy

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10 industrial demands and quality of baked goods which would be difficult to reproduce

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otherwise (Gaggiano et al., 2007).
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Some recent studies underlined how sourdough fermentation with selected autochthonous
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13 mixed starters is indispensable to improve the nutritional and sensory potential of ancient

14 grains different from wheat (Coda et al., 2010b). For amaranth sourdough production, one
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15 strain of L. plantarum and one of P. pentosaceus were selected since they showed positive
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16 behavior to acidification and growth rate in the first 24 h, dominated toward the other

17 autochthonous microbiota and showed positive behavior to incubation temperature (Sterr et

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18 al., 2009). The use of mixed starters was considered functional to completely exploit the

19 nutritional and sensory potential of spelt and emmer flours. Mixed obligate and facultative
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20 heterofermentative lactic acid bacteria starters such as Lactobacillus brevis, W. confusa and
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21 L. plantarum strains for spelt and two strains of L. plantarum and one strain of W. confusa for

22 emmer were selected on the basis of rapid growth and acidification and the capacity to

23 liberate free amino acids (Coda et al., 2010b). Mixtures of strains with different carbohydrate

24 metabolism are frequently used because it may guarantee optimal acidification and sensory

25 properties (Gobbetti, 1998). With the same approach, strains of P. pentosaceus, L. curvatus,

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1 and L. plantarum that improved nutritional and sensory characteristics of acha and iburu flour

2 were selected on the basis of acidifying properties, liberation of free amino acids and

3 synthesis of acetic acid and acetoin (Coda et al., 2011a). Sourdough fermentation also

4 improved the technological properties of acha and iburu flours, allowing the obtainment of

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5 higher specific volume and cell-total area of bread crumb compared to the use of baker’s

6 yeast alone, as well as better textural and structural features (e.g. hardness, fracturability,

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7 resilience) (Coda et al., 2011a). Moreover, compared to breads started with baker’s yeast

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8 alone, sourdough fermentation with selected autochthonous lactic acid bacteria increased the

9 value of in vitro protein digestibility, thanks to the proteolysis and to the inactivation of some

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10 anti-nutritional factors such as trypsin inhibitors (Coda et al., 2011a).

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12 3.3 Allochthonous and commercial starters

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13 Sometimes, industrial starter cultures lack the necessary properties for product diversification

14 and the biodiversity of commercial starters might be limited if considering all the peculiar
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15 features of different flours, with the risk of causing a loss of the most important
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16 characteristics. For the industrial production of wheat and rye sourdough bread commercial

17 starters are widely used, and the appropriate selection of the starter culture has a critical
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18 impact on the final quality of bread (De Vuyst et al., 2009). Commercial/allochthonous starter

19 cultures may present several limitations: (i) the selection does not consider other features than
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20 rapid acidification; (ii) the adaptation to the main sensory and functional properties of the
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21 matrix is poor; (iii) the metabolic flexibility is low; and (iv) the diversity does not reflect the

22 ecosystem where they have to be used (Oberman and Libudzisz, 1998; Leroy and De Vuyst,

23 2004). Consequently, highly performing commercial/allochthonous starter cultures are not

24 very common. Investigation on the adaptability of several lactic acid bacteria strains to

25 sourdoughs made from different cereals, pseudo-cereals and cassava, showed that only few

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1 strains of L. fermentum, L. helveticus, L. paralimentarius, L. plantarum, L. pontis, and L.

2 spicheri were competitive on the autochtonous microbiota (Vogelmann et al., 2009). A

3 competitive strain belonging to the L. paralimentarius species was characterized for the high

4 tolerance to the presence of substances common in pseudo-cereal flours, such as tannins,

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5 rutin and saponins (Steadman et al., 2001; Correia et al., 2010; Vega-Gàlvez et al., 2010).

6 Interactions among microorganisms and the substrate quality can affect the competitiveness

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7 of lactic acid bacteria in specific sourdough environments (Vogelmann et al., 2009). Under

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8 specific conditions applied, only some of the strains can persist over the propagation process,

9 while autochthonous lactic acid bacteria become part of the stable biota of the mature

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10 sourdoughs. It was the case of L. plantarum strain, showing high competitiveness during

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fermentation of buckwheat flour, whereas L. paralimentarius dominated buckwheat and teff

fermentations (Moroni et al., 2010b). Based on these results, it can be concluded that the
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13 capability to adapt to a specific substrate depends strongly not only on the composition of the

14 substrate itself but also on the specific strain, since some lactic acid bacteria are able to adapt
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15 to many different substrates and occur more often, and in higher cell counts, than others. The
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16 reasons for this are not yet well known, but an understanding of these complex interactions

17 would offer new possibilities to control cereal fermentations.

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18

19 4. Interactions between starters and raw matrix

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20 Beyond the potential of sourdough fermentation, the selection of cereals and pseudo-cereals
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21 based on their nutritional and healthy potential is also of key importance to get optimal

22 technology, sensory and healthy properties. The nature of the substrate has an influence also

23 on the persistence of microorganisms, as shown in the case of some lactic acid bacteria

24 starters in continuously propagated buckwheat and teff sourdoughs (Moroni et al., 2010b). In

25 this sense the use of a proper selection of lactic acid bacteria and flours with high nutritional

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1 potential may lead to the manufacture of sourdough fermented baked goods with peculiar

2 nutritional or technological features. In fact, when specific requirements or objectives are

3 needed, allochthonous highly performing starters can be still considered. The use of a strain

4 of Lactobacillus buchneri isolated from bread drink (a German fermented cereal beverage) as

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5 starter culture for sorghum sourdoughs significantly influenced the rheology, showing the

6 possibility of successful application in gluten-free baking (Galle et al., 2012). A good

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7 interaction between the raw matrix and selected starters starters can lead to the enhancement

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8 of functional properties of leavened baked goods, such as the enrichment in bioactive

9 compounds. Strains belonging to L. plantarum and Lactococcus lactis species previously

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10 selected for the capacity to synthesize the bioactive non-proteic amino acid γ-aminobutyric

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12 flours, showing that

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acid (GABA), were used for sourdough fermentation of cereal, pseudo-cereal and leguminous

amaranth, quinoa, buckwheat, and chickpea were the flours most

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13 suitable to be enriched in GABA (Coda et al., 2010a). In particular, the use of a blend of

14 buckwheat, amaranth, chickpea and quinoa flours allowed the manufacture of GABA
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15 enriched bread (ca. 500 mg/kg of GABA), demonstrating also the successful replacement of
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16 wheat flour with pseudo-cereals or leguminous flours for manufacturing of baked goods (Tosi

17 et al., 2002; Schoenlechner et al., 2008; Coda et al., 2010a). Lactic acid bacteria may possess
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18 specific proteinase and peptidase activities, retained to be the prerequisites to release

19 bioactive peptides from native oligopeptide sequences. It has been shown that a pool of
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20 selected lactic acid bacteria had the capacity to release antioxidant peptides from spelt and
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21 kamut under appropriate sourdough fermentation conditions (Fig.2) (Coda et al., 2012).

22 Moreover, the ex-vivo antioxidant activity of the purified peptide fractions derived from spelt

23 and kamut sourdoughs, tested on mouse fibroblasts artificially subjected to oxidative stress,

24 was comparable to α-tocopherol (Coda et al., 2012).

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1 A recent study showed that fermentation of cereal, pseudo-cereal, and leguminous flours with

2 sourdough lactic acid bacteria selected for their proteolytic activity successfully increased the

3 concentration of the anti-cancer peptide lunasin, suggesting new possibilities for the

4 formulation of innovative functional foods (Rizzello et al., 2012).

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5

6 5. Conclusions and future perspectives

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7 World-wide, the diversity of cereal and pseudo-cereal fermentation represents an important

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8 opportunity to develop foods more able to satisfy different needs. In developing countries,

9 traditional cereal-based fermented foods are staple foods and often used as weaning foods for

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10 infant and young children but they are still produced at household level or on small industrial

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scale (Kalui et al., 2010). In developing countries, the increasing interest for local cereals and

pseudo-cereals, from consumers and small enterprises as well, demonstrates the possibility
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13 for the development of good quality products. These grains have received some attention

14 from consumers of western countries and show a huge potential for wider use (Jideani and
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15 Jideani, 2011). Mainly, the characteristics that are making these crops attractive are
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16 nutritional quality and healthier properties, and the low environmental impact. However some

17 practical problems still remain to be solved to improve the cultivation and export of cereals
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18 and pseudo-cereals. Different solutions should be pursued to upgrade quality and

19 competitiveness, such as the use of adapted varieties, appropriate production and farming
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20 systems, and innovation in processing and in marketing systems for local and export markets
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21 (Jideani and Jideani, 2011; Guyot, 2012). In some cases, it has been possible to improve

22 household fermentation technologies to an industrial scale in order to provide value added

23 products that meet urban population demands for traditional products (Gadaga et al., 1999;

24 Belton and Taylor, 2004). One of the prerequisites for the establishment of small-scale

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1 industrial production of fermented foods in developing countries is actually the design of

2 starter cultures (Sanni, 1993).

3 Sourdough is an established technology in improving and diversifying the sensory quality of

4 bread, and is finding good use also in the case of fermentation of cereals different from wheat

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5 and pseudo-cereals. Studies on the sourdough microbiota of these crops revealed a large

6 biodiversity of the microflora and contributed to increase the interest for starter cultures,

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7 especially for those showing the properties of enhancing nutritional and sensory quality

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8 (Corsetti and Settanni, 2007). In the future, it can be foreseen that sourdough processing

9 could be used to improve the traditional technology as well as to design food with particular

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10 characteristics. This aspect is also more important if considering that the introduction of some

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industrial foods risks to displace the traditional fermented foods. In this context, for example,

different protocols for manufacturing functional emmer non-alcoholic beverages by the use
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13 of autochtonous lactic acid bacteria, based on traditional processes, were recently proposed

14 (Coda et al., 2011c). Because of the very important role indigenous crops and fermented
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15 foods play for the growing food needs of developing countries, it is necessary that the
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16 knowledge on their production is not lost and, at the same time, is necessary to improve the

17 efficiency of processing in order to obtain value added food meeting the demand for healthier
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18 products.

19
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13 Yousif, N. M. K., Huch, M., Schuster, T., Cho, G. S., Dirar, H. A., Holzapfel, W. H., &

14 Franz, C. M. (2010). Diversity of lactic acid bacteria from Hussuwa, a traditional African
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15 fermented sorghum food. Food Microbiology, 27, 757–768.

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1 Legends to Figures

2 Figure 1. Lactic acid bacteria microbiota in emmer and spelt flours: dendrogram obtained by

3 combined random amplification of polymorphic DNA patterns for the strains isolated from

4 flours using three different primers with arbitrarily chosen sequences. The codes identify the

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5 source: “E”, emmer or “S”, spelt. (Adapted by Coda et al., 2010b).

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6 Figure 2. Antioxidant activity of different flours fermented with sourdough lactic acid

7 bacteria. The activity was measured as lipid peroxidation inhibition under a linoleic acid

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8 oxidation system, for 8 days. Butylatedhydroxytoluene (BHT) and α-tocopherol were used as

9 the positive controls; a negative control, without antioxidants, was also considered (ct).

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10 (Adapted by Coda et al., 2012).

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