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Food Microbiology 26 (2009) 666–675

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Food Microbiology

journal homepage: www.elsevier .com/locate/ fm

Biodiversity, ecological determinants, and metabolic exploitationof sourdough microbiota

L. De Vuyst*, G. Vrancken, F. Ravyts, T. Rimaux, S. WeckxResearch Group of Industrial Microbiology and Food Biotechnology, Faculty of Sciences and Bio-engineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium

a r t i c l e i n f o

Article history:Received 20 May 2009Received in revised form13 July 2009Accepted 13 July 2009Available online 18 July 2009

Keywords:Sourdough fermentationLactic acid bacteriaYeastsBiodiversity

* Corresponding author. Tel.: þ32 2 6293245; fax:E-mail address: ldvuyst@vub.ac.be (L. De Vuyst).

0740-0020/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.fm.2009.07.012

a b s t r a c t

Sourdough is a microbial ecosystem of lactic acid bacteria (LAB) and yeasts in a matrix of mainly cereal flourand water. Culture-dependent and culture-independent microbiological analysis together with metabolitetarget analyses of different sourdoughs enabled to understand this complex fermentation process. It isdifficult to link the species diversity of the sourdough microbiota with the (geographical) type of sour-dough and the flour used, although the type and quality of the latter is the main source of autochthonousLAB in spontaneous sourdough fermentations and plays a key role in establishing stable microbial con-sortia within a short time. Carbohydrate fermentation targeted towards maltose catabolism, the use ofexternal alternative electron acceptors, amino acid transamination reactions, and/or the arginine deimi-nase pathway are metabolic activities that favour energy production, cofactor (re)cycling, and/or tolerancetowards acid stress, and hence contribute to the competitiveness and dominance of certain species of LABfound in sourdoughs. Also, microbial interactions play an important role. The availability of genomesequences for several LAB species that are of importance in sourdough as well as technological advances inthe fields of functional genomics, transcriptomics, and proteomics enable new approaches to studysourdough fermentations beyond the single species level and will allow an integral analysis of themetabolic activities and interactions taking place in sourdough. Finally, the implementation of selectedstarter cultures in sourdough technology is of pivotal importance for the industrial production of sour-doughs to be used as flavour carrier, texture-improving, or health-promoting dough ingredient.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Sourdough is a mixture of mainly cereal flour and water, which ismade metabolically active by a heterogeneous population of lacticacid bacteria (LAB) and yeasts, either by spontaneous fermentationor by fermentation initiated through the addition of a sourdoughstarter culture, whether or not involving backslopping (De Vuyst andNeysens, 2005; De Vuyst and Vancanneyt, 2007; Corsetti and Set-tanni, 2007). The major metabolic activities are acidification (LAB),flavour formation (LAB and yeasts), and leavening (yeasts and LAB),resulting in fermented doughs with low pH (around 4.0) and(sourdough) breads with desirable texture and flavour. Sourdoughsare used in the production of common, yeast-leavened bread (asflavour carrier or bread quality enhancer, both active and inactivesourdough preparations), artisan specialty breads (use of sourdoughas a natural leavening agent for its high sensory quality), andtraditional rye breads (to achieve the baking ability of rye doughs).Also, cakes and crackers, pizza, and various sweet baked goods are

þ32 2 6292720.

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manufactured with sourdough. The stability of a ripe sourdoughdepends on the environmental microbiota (the flour and otheringredients used, the house microbiota, etc.), its metabolic activity(amylase activity of flour and/or microorganisms, cofactor regener-ation capability and energy production ability of the microorgan-isms involved, etc.), and specific technological process parameters(chemical and enzymatic composition of the flour, leavening andstorage temperature, pH and redox potential, dough hydration andyield, number of sourdough refreshment steps, fermentation timebetween refreshments, the use of starters and/or bakers yeast, etc.)(Hammes et al., 1996; Gobbetti et al., 2005; Arendt et al., 2007;Corsetti and Settanni, 2007; Ganzle et al., 2007). As a consequence ofthe heterogeneity of these ecological determinants, mature sour-doughs differ in species diversity and metabolic activity (De Vuystet al., 2002; De Vuyst and Neysens, 2005). This review deals witha state-of-the-art on the species diversity, ecological determinantsand metabolic potential of the sourdough microbiota, in particularLAB, as a follow-up of the reviews published in the proceedings ofthe international conferences on sourdough held in Verona (Vogel,1996), Brussels (De Vuyst and Ganzle, 2005) and Bari (Gobbetti andGanzle, 2007), and hence only in particular cases will be referred topapers already cited herein.

L. De Vuyst et al. / Food Microbiology 26 (2009) 666–675 667

2. Population dynamics of the sourdough microbiota

2.1. Methodologies

In-depth studies of the species diversity of sourdoughs and of thepopulation dynamics during sourdough fermentation are funda-mental to understand this complex process and to manufacturestandardized, high-quality end-products (Van der Meulen et al.,2007b; Vogel and Ehrmann, 2008). Initially, mainly culture-depen-dent methods have been used to study the microbiology of sour-dough ecosystems, whether or not involving molecularidentification techniques as part of a polyphasic approach for speciesdesignation of the cultivable isolates (Ehrmann and Vogel, 2005; DeVuyst and Vancanneyt, 2007). Currently, the combination of culture-dependent and culture-independent methodologies are used for themicrobiological analysis of sourdough ecosystems, focusing on boththe cultivable and (viable or non-viable) non-cultivable (if any) LABspecies present, thereby making use of mostly denaturing gradientgel electrophoresis (DGGE) of polymerase chain reaction (PCR)amplicons of specific fragments of the 16S rRNA gene (Meroth et al.,2003b, 2004; Gatto and Torriani, 2004; Randazzo et al., 2005;Settanni et al., 2006; Valmorri et al., 2006; Van der Meulen et al.,2007b; Garofalo et al., 2008; Scheirlinck et al., 2007c, 2008, 2009a;Vogelmann et al., 2009), the rpoB gene (Siragusa et al., 2009), or the28S rRNA gene (yeasts; Meroth et al., 2003a; Garofalo et al., 2008;Vogelmann et al., 2009), as well as making use of temporaltemperature gradient gel electrophoresis (TTGE) (Ferchichi et al.,2007). Also, PCR-based classification and identification techniques,such as random amplification of polymorphic DNA PCR (RAPD-PCR)and repetitive element sequence (rep)-PCR fingerprinting, as well asmonoplex or multiplex PCR and multi-locus sequence analysistechniques, have been optimized for the rapid and accurate detec-tion and/or monitoring of sourdough LAB (Muller et al., 2001; Realeet al., 2005; Settanni et al., 2005, 2006; Corsetti et al., 2007b;Valcheva et al., 2007; Ferchichi et al., 2007, 2008a,b; Scheirlincket al., 2007c, 2008; Iacumin et al., 2009). Whereas DGGE analysisallows differentiation and semi-quantitative comparison of thepredominant species in sourdough populations, culture-indepen-dent real-time PCR-based assays are considered more sensitive andprovide quantitative data on the relative occurrence of specific LABin sourdough ecosystems (Scheirlinck et al., 2009a).

2.2. Source, evolution, and stability of the sourdough microbiota

2.2.1. The sourdough LAB microbiotaCereal flours and other sourdough ingredients are not micro-

biologically sterile neither are they heat treated before use. Whenthe flour is mixed with water, the contaminating microorganismsstart to grow and competitive yeasts and LAB reach numbers abovethose of the adventitious microbiota (De Vuyst and Neysens, 2005).While certain ingredients, such as (added) sugars and salt or otheradjuncts, may favour one or another group of yeasts and/or LAB,a strict (geographical) correlation can hardly be made between thetype of species of yeasts and/or LAB dominating the fermentingflour/water mixture and the type of sourdough produced (De Vuystand Neysens, 2005; De Vuyst and Vancanneyt, 2007). Whether thetype of flour used mainly directs the growth of sourdough LABseems evident but remains controversial (Rosenquist and Hansen,2000; Corsetti et al., 2001, 2007b; Kitahara et al., 2005; Scheirlincket al., 2007c; Van der Meulen et al., 2007b; Siragusa et al., 2009;Vogelmann et al., 2009). However, spontaneous sourdoughfermentations carried out in the laboratory indicate that the typeand quality of the cereal flour used is indeed the source ofautochthonous LAB and plays a key role in establishing stablemicrobial consortia within a short time (Van der Meulen et al.,

2007b). In addition, it has been shown that previous introduction offlour into the bakery environment helps to build up a housemicrobiota that may serve as an important inoculum for subse-quent sourdough fermentations, as sourdough and bakery envi-ronment isolates are genetically indistinguishable and the adaptedLAB strains are repetitively introduced in consecutive sourdoughbatches during backslopping (Scheirlinck et al., 2009a). Conse-quently, not only the flour but also the environment (apparatus, air,etc.) cause contamination of sourdough batches (Scheirlinck et al.,2009a). Also, the occurrence of lactobacilli of mainly intestinalorigin may be the result of cross-contamination (Du Toit et al.,2003; Ehrmann and Vogel, 2005; De Angelis et al., 2006). Ingeneral, species of Lactobacillus, in particular Lactobacillus brevis,Lactobacillus plantarum, Lactobacillus paralimentarius, Lactobacillusrossiae, and Lactobacillus sanfranciscensis dominate sourdoughfermentation processes that are characterized by low incubationtemperatures and continuous backslopping (traditional, typeI sourdoughs); industrialized, type II sourdoughs, which are char-acterized by higher temperatures, longer fermentation times, andhigher water contents compared to type I doughs, harbour heat-and acid-tolerant lactobacilli such as Lactobacillus amylovorus,Lactobacillus fermentum, Lactobacillus pontis, and Lactobacillus reu-teri (Table 1). As a result of a wide biosprospection of type I and typeII sourdoughs and an evolving taxonomy of LAB, several newspecies have been described in the last decade (Table 1). It turns outthat obligate heterofermentative species of Lactobacillus are typicalfor most sourdough ecosystems. Species belonging to the generaEnterococcus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus,and Weissella are much less frequently encountered, althoughseveral of these species are present in the cereal kernels and flour(De Vuyst and Neysens, 2005; Corsetti et al., 2007a) or during theearly fermentation process (Corsetti et al., 2007b; Van der Meulenet al., 2007b), and may even dominate some sourdoughs (Rochaand Malcata, 1999; Corsetti et al., 2007b; Zotta et al., 2008).Although in most cases the Lactobacillus species mentioned aboveare not solely specific for the sourdough ecosystem, as they may befound in many other (food) ecosystems as well, some of themevolved to optimize their fitness, for instance through specializa-tion or optimization of their metabolism (Hammes et al., 1996;Gobbetti and Corsetti, 1997). For instance, it appears that Lb. san-franciscensis can mostly be found in type I wheat sourdoughs,wherein it occurs as a stable association with certain yeasts thanksto its highly specialized, energetically favourable, hetero-fermentative carbohydrate metabolism (Hammes et al., 1996;Gobbetti et al., 2005). This sourdough species is selected for only bythe type of technology applied, whereby backslopping practices,temperature of incubation, and pH of the dough seem to be themost important factors of influence on its prevalence (Ganzle et al.,1998; Kitahara et al., 2005; Picozzi et al., 2006; Ferchichi et al.,2007; Siragusa et al., 2009). The dominance of Lb. reuteri in type IIsourdough fermentation processes may be ascribed to its compet-itiveness, not only due to its carbohydrate metabolism but also toits antimicrobial potential (Ganzle and Vogel, 2003). In contrast, thewidespread occurrence of certain sourdough LAB species mayreflect not only their adaptability to the prevailing fermentationconditions but also their intraspecific heterogeneity, as revealed byintraspecific genotyping of Lb. rossiae sourdough isolates (Settanniet al., 2005; Di Cagno et al., 2007a; Scheirlinck et al., 2009b).

2.2.2. Sourdough LAB associationsCereal flours have a highly heterogeneous physicochemical

composition, which offers the possibility for the simultaneousoccupation of multiple niches by adapted microbial species and/orstrains, whereby coexisting species and/or strains often interactthrough nutritional or trophic relationships (Gobbetti, 1998;

Table 1Common and new species associated with sourdoughs.

Lactic acid bacteria Reported sources

Common species (Corsetti et al., 2001; Randazzo et al., 2005; Catzeddu et al., 2006;Valmorri et al., 2006; Ferchichi et al., 2007; Iacumin et al., 2009; Scheirlinck et al., 2007c;Garofalo et al., 2008; Zotta et al., 2008; Siragusa et al., 2009; Vogelmann et al., 2009)

- Obligately heterofermentativeLactobacillus brevis Sourdough, sauerkraut, kimchi, pickles, olives, kefir, silage, .

Lactobacillus fermentum Sourdough, ogi, kisra, boza, whisky, cocoa, gari, .

Lactobacillus reuteri Sourdough, dairy, human and animal intestines, mother’s milk, .

Lactobacillus sanfranciscensis Sourdough

- Facultative heterofermentativeLactobacillus plantarum Sourdough, sausage, cocoa, cheese, sauerkraut, kimchi, pickles,

olives, human saliva, animal intestines, silage, .

Lactobacillus (par)alimentarius Sourdough

- Obligately homofermentativeLactobacillus amylovorus Sourdough, corn steep liquor, pig intestines, .

New species since 2000, originally isolated from sourdough (first reference between brackets)

- Obligately heterofermentativeLactobacillus acidifarinae (Vancanneyt et al., 2005) SourdoughLactobacillus crustorum (Scheirlinck et al., 2007a) SourdoughLactobacillus frumenti (Muller et al., 2000) SourdoughLactobacillus hammesii (Valcheva et al., 2005) SourdoughLactobacillus mindensis (Ehrmann et al., 2003) SourdoughLactobacillus namurensis (Scheirlinck et al., 2007b) SourdoughLactobacillus nantensis (Valcheva et al., 2006) SourdoughLactobacillus nodensis (Kashiwagi et al., 2009) SourdoughLactobacillus panis (Wiese et al., 1996) Sourdough, distillers’ grain, intestines, chicken faecesLactobacillus pontis (Vogel et al., 1994) Sourdough, distillers’ grain, intestines, faecesLactobacillus rossiae (formerly Lb. rossii, Corsetti et al., 2005) Sourdoughs, pig faecesLactobacillus secaliphilus (Ehrmann et al., 2007) SourdoughLactobacillus siliginis (Aslam et al., 2006) SourdoughLactobacillus spicheri (Meroth et al., 2004) SourdoughLactobacillus zymae (Vancanneyt et al., 2005) Sourdough

L. De Vuyst et al. / Food Microbiology 26 (2009) 666–675668

De Vuyst and Neysens, 2005; Corsetti and Settanni, 2007; Vogel-mann et al., 2009). Carbohydrate fermentation targeted towardsmaltose catabolism (encompassing maltose phosphorylaseactivity), the use of external alternative electron acceptors (such asfructose), and/or the arginine deiminase (ADI) pathway are meta-bolic activities that favour energy production, cofactor (re)cyclingand/or tolerance towards acid stress (Fig. 1), and hence contributeto the competitiveness and dominance of certain species of LABfound in sourdoughs, such as Lb. sanfranciscensis, Lb. reuteri, Lb.brevis, Lb. pontis, and Lb. fermentum (Hammes et al., 1996; Gobbettiet al., 2005; Ganzle et al., 2007). Also, differences in the use of allfour flour carbohydrates (maltose, sucrose, fructose, and glucose)may result in a non-competitive association of different species ofLAB, such as in the stable association between Lb. sanfranciscensisand Lb. plantarum, the former species preferentially utilizingmaltose and generally unable to ferment fructose, while the latterspecies preferentially ferments glucose and fructose with maltosemetabolism being subject to carbon catabolite repression(Gobbetti, 1998; Corsetti et al., 2001). Further, the rate and intensityof acidification may determine the stability of the final populationof LAB encountered in mature sourdoughs. For instance, entero-cocci, pediococci, lactococci, leuconostocs, and weissellas mayoccur in growth association with lactobacilli during early sour-dough fermentation, often paving the way for subsequent growthof more sourdough-specific aciduric lactobacilli (Van der Meulenet al., 2007b; Corsetti et al., 2007b), but can be encountered in thefinal dough as well, for instance depending on the recipe butcertainly in the case of appropriate refreshment steps, fermentationtimes, and/or pH values (Kitahara et al., 2005; Ferchichi et al., 2007;Garofalo et al., 2008). In addition, it has been shown that cell–cell interactions may play an important role in the stabilizationof associations of sourdough LAB (Di Cagno et al., 2007b).

However, whether the stable association of Lb. sanfranciscensis andLb. plantarum as mentioned above or that of Lb. sanfranciscensis andLb. paralimentarius, which occurs frequently too (Corsetti et al.,2001), solely depends on the type and quality of the wheat flourused remains unsolved. In particular, although Lb. sanfranciscensisemerges as one of the predominant species in traditional wheatsourdoughs, the source of the dominant strains (autochthonous orstarters) and the factors (cereals and/or technology), influencingthe stable association, remain controversial (Scheirlinck et al.,2007c; Siragusa et al., 2009; Vogelmann et al., 2009).

2.2.3. The sourdough yeast microbiota and its interactions with LABYeasts are present in most sourdoughs (Rossi, 1996; Gullo et al.,

2002; Succi et al., 2003; De Vuyst and Neysens, 2005; Hammes et al.,2005). Saccharomyces cerevisiae, Kazachstania exigua [formerlySaccharomyces exiguus, anamorph Candida (Torulopsis) holmii] andCandida humilis (synonym Candida milleri) are most frequentlyencountered, followed by Pichia kudriavzevii (formerly Issatchenkiaorientalis, anamorph Candida krusei) (Garofalo et al., 2008; Iacuminet al., 2009; Vogelmann et al., 2009). Whereas the stability of somesourdoughs depends on the specific cooperation between certainspecies of yeast and LAB (Gobbetti, 1998), the presence of S. cerevisiae(bakers yeast) may be ascribed to its presence in the bakery envi-ronment (use of bakers yeast, bakery air contamination, etc.; Corsettiet al., 2001; Meroth et al., 2003a; Succi et al., 2003; Pulvirenti et al.,2004; Vernocchi et al., 2004a,b; Garofalo et al., 2008), althoughautochthonous strains of S. cerevisiae may occur (G. Vrancken, L. DeVuyst, R. Van der Meulen, G. Huys, P. Vandamme and H.-M. Daniel,unpublished results). Stable, non-competitive associations existbetween the maltose-negative, acid-tolerant K. exigua or C. humilisand the maltose-positive, wheat sourdough-specific Lb. sanfranci-scensis in, for instance, San Francisco sourdough and Panettone,

ArginineOrnithine

ArginineOrnithine

CitrullineCarbamoyl-phosphate

NH3 + CO2

NH3

ATP

ADP

Proteins

Amino acids

Peptides

α-ketoglutarate

Glutamic acid NAD+

NADH + H+

Keto acid

Aldehyde

Alcohol Carboxylic acid

Ester

Hydroxyacid

Acyl-CoA

Acyl-P

NAD+

NADH + H+

NAD+

NADH + H+

CO2

NADH + H+

NAD+

ATP

ADP

Alcohol

CoASH

Pi

NAD+

NADH + H+CoA

CO2

Maltose H+

Maltose

Glucose Glucose-1-P

H+

Glucose-6-P

Xylulose-5-P

2 NADH + H+

2 NAD+

Glyceraldehyde-3-P

Pyruvate

LactateNAD+

NADH + H+

NAD+

NADH + H+

Acetate

NADH + H+

Acetyl-P

ADP

ATP

CO2

Pi

NAD+

Ethanol

Pi

CoA2 ADP

2 ATP

Fructose

Fructose

Mannitol

Fructose-6-P

ATP

ADP

SUGAR AND CITRATE METABOLISM AMINO ACID CATABOLISM ADI PATHWAY

I

II

IIIIV

V

VI

VIII

VII IXX

XI

ATP

ADP

Oxaloacetate

Citrate

Citrate

Acetate

CO2

Amino acids

Citrulline

BCAA

Acetyl-CoA

Acetaldehyde

NADH + H+

NAD+

ArginineOrnithine

ArginineOrnithine

CitrullineCarbamoyl-phosphate

NH3 + CO2

NH3

ATP

ADP

Proteins

Amino acids

Peptides

α-ketoglutarate

Glutamic acid NAD+

NADH + H+

Keto acid

Aldehyde

Alcohol Carboxylic acid

Ester

Hydroxyacid

Acyl-CoA

Acyl-P

NAD+

NADH + H+

NAD+

NADH + H+

CO2

NADH + H+

NAD+

ATP

ADP

Alcohol

CoASH

Pi

NAD+

NADH + H+CoA

CO2

Maltose H+

Maltose

Glucose Glucose-1-P

H+

Glucose-6-P

Xylulose-5-P

2 NADH + H+

2 NAD+

Glyceraldehyde-3-P

Pyruvate

LactateNAD+

NADH + H+

NAD+

NADH + H+

Acetate

NADH + H+

Acetyl-P

ADP

ATP

CO2

Pi

NAD+

Ethanol

Pi

CoA2 ADP

2 ATP

Fructose

Fructose

Mannitol

Fructose-6-P

ATP

ADP

I

II

IIIIV

V

VI

VIII

VII IXX

XI

ATP

ADP

Oxaloacetate

Citrate

Citrate

Acetate

CO2

Amino acids

Citrulline

BCAA

Acetyl-CoA

Acetaldehyde

NADH + H+

NAD+

Fig. 1. Pathways active in typical heterofermentative sourdough lactic acid bacteria. Left panel. Maltose, fructose, and citrate metabolism. Middle panel (based on Liu et al., 2008).Generic conversion of branched-chain amino acids (valine, leucine, and isoleucine), aromatic amino acids (tyrosine, tryptophan, and phenylalanine), and sulphur-containing aminoacids (methionine), as initiated by transamination in LAB. The dotted box represents branched-chain amino acid conversion through oxidative decarboxylation. Right panel.Arginine deiminase (ADI) pathway. Key enzymes involved in these pathways and directly or indirectly related to enhanced competitiveness, acid stress response, and/or flavourformation are designated with Roman numbers: I, maltose phosphorylase; II, phosphoketolase; III, lactate dehydrogenase; IV, acetate kinase; V, mannitol dehydrogenase; VI,aminotransferase; VII, glutamate dehydrogenase; VIII, a-keto acid decarboxylase; IX, arginine deiminase; X, ornithine transcarbamoylase; XI, carbamate kinase.

L. De Vuyst et al. / Food Microbiology 26 (2009) 666–675 669

respectively. Lb. sanfranciscensis hydrolyses maltose, taken up bymaltose/Hþ symport, by an intracellular maltose phosphorylase toproduce unphosphorylated glucose plus glucose-1-phosphate(without the use of ATP) that is metabolized further intracellularly;the unphosphorylated glucose can be excreted outside the cell toavoid intracellular accumulation and hence can be utilized bymaltose-negative yeasts or induces glucose repression in maltose-positive yeasts (Hammes et al., 1996; Stolz et al., 1996; Gobbetti,1998). Furthermore, stable associations exist between S. cerevisiaeand Lb. plantarum, both utilizing all four flour carbohydrates butbeing susceptible towards carbon catabolite repression, and betweenCandida spp. (maltose-negative) and Lb. brevis (maltose-positive)(Corsetti and Settanni, 2007; Iacumin et al., 2009).

2.2.4. Stability of ripe sourdoughsA combination of culture-dependent and culture-independent

microbiological analysis of spontaneous sourdough laboratoryfermentations has revealed that a stable population of LAB occursthrough a three-phase evolution within a few days, which does notdiffer significantly between wheat, spelt, or rye flours (Van derMeulen et al., 2007b). The same population dynamics have beenobserved when Lb. sanfranciscensis starters are used (Siragusa et al.,2009). These population dynamics may reflect the ability to utilizecarbohydrates and peptides/amino acids efficiently, efficient regu-lation of the redox balance, as well as tolerance towards acidificationand other stress factors (Van der Meulen et al., 2007b). In the studyof Meroth et al. (2003b), temperature was a determining factor forthe few Lactobacillus strains becoming dominant early into thefermentation process, when using commercially available sourdoughstarter mixtures. Besides the type and quality of the substrate(s) andthe applicable processing conditions, interactions among microor-ganisms - not only nutritional and trophic relationships as

mentioned above but also antagonistic interactions – may beresponsible for the competitiveness and subsequent dominance ofLAB in sourdoughs (Messens and De Vuyst, 2002; De Vuyst andNeysens, 2005; Corsetti and Settanni, 2007; Corsetti et al., 2008;Vogelmann et al., 2009). For instance, it has been shown that thestable persistence of Lb. reuteri in a German rye sourdough (SER),prepared for producing a commercially available baking aid, is due tothe production of the antibiotic reutericyclin (Ganzle and Vogel,2003). Also, it has been shown that the bacteriocin producer Lb.amylovorus DCE 471 is a competitive starter culture for type IIsourdough fermentations (Leroy et al., 2007). Finally, it has beenshown that artisan bakery sourdoughs, characterized by eitherrestricted or wide species diversity, are remarkably stable, whichmay be the result of a combination of factors, including a stablehouse microbiota (Scheirlinck et al., 2007c, 2009a).

3. Genomics, transcriptomics and proteomics analysisof sourdough lactic acid bacteria

The availability of genome sequences for several LAB speciesthat are of importance in sourdough as well as technologicaladvances in the fields of functional genomics, transcriptomics, andproteomics enable new approaches to study sourdough fermenta-tions beyond the single species level and will allow an integralanalysis of the metabolic activities and interactions in mixedcultures of sourdough. As most studies on the ‘omics’ of sourdoughare still in progress, few papers are available on this topic.

3.1. Genomics

During the last decade, the genomes of Lb. brevis ATCC 367(Makarova et al., 2006), Lb.plantarumWCFS1 (Kleerebezemet al., 2003),

L. De Vuyst et al. / Food Microbiology 26 (2009) 666–675670

and Lb. reuteri JCM 1112T and Lb. fermentum IFO 3956 (Morita et al.,2008), species that are frequently found in sourdoughs, have beenpublished. However, none of the sequenced strains were isolatedfrom sourdough. In addition, only two strains, namely Lb. brevisATCC 367 and Lb. fermentum IFO 3956, are food-related, the othershaving been isolated from human intestine (Lb. reuteri JCM 1112T)and human saliva (Lb. plantarum WCFS1). The fact that genomesequences of sourdough isolates of Lb. sanfranciscensis and Lb. pontiswill be publicly available soon is promising (http://www.foodscience.ws). Based on a more general comparative genomicstudy of LAB, it appears that lactobacilli with a rather small genome,such as Lb. brevis, witness a metabolic simplification during LABevolution (Makarova et al., 2006). This evolutionary process hasresulted in strains that are well adapted to their microbial niche,with a large set of genes coding for transporters and enzymesinvolved in sugar and peptide/amino acid metabolism and onlya limited set of biosynthetic pathways, indicating the importance tocope with competitiveness and survival. Larger LAB genomes,encoding a large set of enzymes involved in different metabolicpathways, reflect a larger flexibility to grow under different condi-tions, as is the case for Lb. plantarum (Boekhorst et al., 2004).Genome annotation analysis shows that Lb. reuteri JCM 1112T and Lb.fermentum IFO 3956 both possess the mapA gene coding for maltosephosphorylase, an enzyme that gives a competitive advantage tostrains when growing in a sourdough environment. Lactobacillusplantarum WCFS1 has four genes coding for maltose phosphorylase,i.e., map1, map2, map3, and map4, indicating the potential involve-ment of different enzymes in different physiological processes(Vogel et al., 2002). Lactobacillus brevis ATCC 367 has no maltosephosphorylase gene. Of the four sequenced strains, only Lb. fer-mentum IFO 3856 harbours all four genes of the ADI pathway (Fig.1)and has these genes positioned adjacent to each other, in the orderornithine carbamoyltransferase, carbamate kinase, arginine deimi-nase, and the arginine–ornithine antiporter. Sequencing of the ADIoperon in the genome of Lb. fermentum IMDO 130101 revealed thesame genes in the same order (G. Vrancken, T. Rimaux, S. Weckx, L.De Vuyst and F. Leroy, unpublished results). Also, Lb. reuteri JCM1112T possesses all four genes of the ADI pathway, but not all fourpositioned in the same genomic location. In Lb. brevis ATCC 367, thegene coding for the arginine–ornithine antiporter is missing; in Lb.plantarum WCFS1, the genes coding for ornithine carbamoyl-transferase and the arginine–ornithine antiporter are missing. Fromthis analysis, it turns out that the food-related Lb. fermentum IFO3956 (and Lb. fermentum IMDO 130101) has the two key traits to beable to survive in a sourdough environment, namely the ability toconsume maltose and to cope with acid stress through the ADIpathway. However, the genome analysis presented here might notbe representative for other strains of the same species, in particularthose isolated from sourdough, as strain-dependent differences willoccur. In addition, improved genome annotations will certainlycontribute to further insights into gene functionalities (Liu et al.,2008). Until now, no metagenomic analysis studies have been per-formed on sourdough fermentation samples. However, sequencingof the metagenomes of the sourdough microbiota will undoubtedlyboost research on the species diversity, population dynamics,genetic and metabolic potential of particular strains, and onmicrobial interactions within the complex sourdough ecosystem,and thus support the development of appropriate starter cultures.

3.2. Transcriptomics

Recently, transcriptome analysis with strains of Lb. reuteri grownin a type II rye bran sourdough has been carried out. Applying invivo expression technology (IVET), 29 conditionally expressedgenes of Lb. reuteri LTH 5531, an isolate from a type II sourdough,

have been identified (Dal Bello et al., 2005). These genes areinvolved in cellular processes, such as the metabolism of aminoacids and nucleotides and stress responses. However, detectinggenes with elevated levels of expression using IVET is a ratherelaborate method that involves the use of gene constructs. There-fore, microarray analysis with whole-genome microarrays orfunctional gene microarrays (species-independent by focusing ondedicated functional genes) is promising (Zhou, 2003). Usinga whole-genome microarray, transcriptome analysis of Lb. reuteriATCC 55730, a strain isolated from human mother’s milk, hasrevealed significant changes of mRNA levels for 101 genes, whenthe strain is grown in a bran sourdough compared with growth ina chemically defined medium (Hufner et al., 2008). Of these genes,58 show an elevated expression and 43 are repressed. All genes areinvolved in diverse cellular processes, such as carbohydrate andenergy metabolism, cell envelope biosynthesis, exopolysaccharideproduction, stress responses, signal transduction, and cobalaminbiosynthesis. Furthermore, gene expression data concerning theutilization of starch and non-starch carbohydrates, the rearrange-ment of the cell wall and the regulatory function of two-componentsystems for cell wall biogenesis and metabolism have indicatedtheir importance for growth in and adaptation of Lb. reuteri tosourdough. Metatranscriptome analysis of wheat and spelt sour-dough samples with a species-independent functional gene LABmicroarray, which is composed of 406 genes involved in importanttraits of sourdough LAB and represented by 2269 oligos (S. Weckx,J. Allemeersch, R. Van der Meulen, G. Vrancken, G. Huys, P. Van-damme, P. Van Hummelen and L. De Vuyst, unpublished results),revealed the activation of different key metabolic pathways, theability to use the different energy sources present in sourdough,versatile cofactor recycling, and acid and oxidative stress responses(S. Weckx, J. Allemeersch, G. Vrancken, R. Van der Meulen,I. Scheirlinck, G. Huys, P. Vandamme, P. Van Hummelen and L. DeVuyst, unpublished results). Furthermore, this metatranscriptomeanalysis confirmed the three-stage population dynamics of thespontaneous sourdough fermentation process mentioned above.

3.3. Proteomics

Proteomic analysis related to sourdough fermentation is limitedto stress response and cell–cell communication studies. In addition,experimental data on the proteome (and transcriptome) level ofLb. sanfranciscensis, affected by sublethal hydrostatic pressure, areavailable (Drews et al., 2002; Vogel et al., 2005). Compared withcold, heat, salt, acid, and starvation stress at the proteome level,Lb. sanfranciscensis appears to use overlapping subsets of stress-inducible proteins rather than stereotype responses (Hormannet al., 2006). Acid stress response of non-adapted, acid-adapted,and acid-tolerant mutants of Lb. sanfranciscensis CB1, studied atprotein level, shows changes in the level of expression of 63proteins (De Angelis et al., 2001). Of these proteins, some arecommon between the acid-adapted and acid-tolerant mutants.Also, the acid-tolerant mutants show a faster and higher acidifi-cation rate during sourdough fermentation, compared to theparental strain, indicating a competitive advantage. In a morerecent proteomic study, focusing on cell–cell communication(Di Cagno et al., 2007b), co-cultures of Lb. sanfranciscensis CB1 withLb. plantarum DC400, Lb. brevis CR13, or Lb. rossiae A7, have beeninvestigated. Whereas the Lb. sanfranciscensis strain has the sameproteins induced in the three co-cultures at the mid-exponentialgrowth phase, much more proteins are induced at the late-stationary phase, with a maximal number of proteins induced inthe co-culture with the Lb. plantarum strain, underlying a stressresponse of Lb. sanfranciscensis. Nineteen of these stress responseproteins have been identified, most of them being regulated by the

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LuxS-mediated signalling pathway. Since this pathway is known tobe important in quorum sensing, the molecular mechanisms ofquorum sensing in Lb. plantarum DC400, when co-cultured withLb. sanfranciscensis DPPMA174 and Lb. rossiae A7, have been studied(Di Cagno et al., 2009). Although the growth and survival of Lb.plantarum DC400 are not effected in the co-cultures, this studyshows that proteins related to quorum sensing and stress responsemechanisms are overexpressed in a co-culture compared to growthin monoculture.

4. Metabolomics of carbohydrate and peptide/amino acidutilization during sourdough fermentation

Metabolite target analyses of sourdough fermentations havebeen studied from both a microbiological and organoleptic qualitypoint of view (Hammes et al., 1996; Gobbetti et al., 2005; Ganzleet al., 2007; Guerzoni et al., 2007). These studies have revealeda link between the metabolites produced during fermentation and/or present in the end-products (sourdoughs and/or bakery prod-ucts) and the microbial diversity of the sourdough ecosystemsstudied (De Vuyst et al., 2002; De Vuyst and Neysens, 2005; Corsettiand Settanni, 2007; De Vuyst and Vancanneyt, 2007; Paramithiotiset al., 2007; Scheirlinck et al., 2007c; Van der Meulen et al., 2007b;Siragusa et al., 2009). The available genomic information of LAB,often involving non-sourdough LAB strains but considering LABspecies relevant for sourdough fermentation, allows insight intopathways for carbohydrate and amino acid utilization, includingflavour formation. However, the analysis presented here might notbe representative for all strains of all sourdough LAB species. Future(multi)genome-scale metabolic models and associated constraint-based modelling techniques will undoubtedly boost research onthe growth, physiology, metabolite production and interactions ofsourdough yeasts and LAB.

4.1. Carbohydrate utilization and organic acid production

Sourdough yeasts may be divided into maltose-negative andmaltose-positive yeasts, which is responsible for their degree ofcompetition with a maltose-specific or non-specific sourdough LABmicrobiota, respectively. Maltose-negative yeasts primarily useglucose, which remains unattached by the maltose-positive sour-dough LAB, for conversion into ethanol via the Emden–Meyerhof–Parnas pathway. Maltose-positive yeasts normally use all fourcommon sourdough carbohydrates, their consumption beingsusceptible to carbon catabolite repression. Sourdough LAB oftenutilize all four flour soluble carbohydrates too, namely starch-derived maltose, sucrose, fructose, and glucose, as well asmonosaccharides from plant-derived polysaccharides such as ara-binoxylan and arabinogalactan. This may reduce direct metaboliccompetition between, for instance, common LAB and Lb. san-franciscensis present in the sourdough. The use of maltose as thepreferred energy source through a dedicated catabolic pathway,including maltose/Hþ symport, maltose phosphorylase activity,possible glucose excretion (avoiding glucose repression), and thephosphoketolase pathway (elaborating lactic acid, ethanol/aceticacid, and CO2) is a key characteristic of species of LAB adapted to thesourdough environment (Fig. 1), such as Lb. sanfranciscensis, Lb.reuteri, Lb. brevis, Lb. pontis, and Lb. fermentum (Hammes et al.,1996; Gobbetti et al., 2005; Ganzle et al., 2007). The use of carbo-hydrates other than glucose by sourdough strains of LAB, lackingmaltose phosphorylase activity, is subjected to carbon cataboliterepression (Leroy et al., 2006; Vrancken et al., 2008). Maltoseutilization, made possible through an energy-efficient pathway,results in a favourable fermentation quotient (i.e., the ratio betweenlactic acid and acetic acid) of 2.5, although this process parameter

may vary considerably (Van der Meulen et al., 2007b). Whereasacetic acid and lactic acid both contribute to the acidity and texture-forming components of sourdough, acetic acid is an importantflavour volatile and displays anti-ropiness and antifungal activitiestoo (Gobbetti et al., 2005; Arendt et al., 2007).

The use of fructose as alternative external electron acceptor byLb. sanfranciscensis, Lb. reuteri, Lb. pontis, and Lb. fermentum favourstheir competitive advantage in sourdough (Hammes et al., 1996;Gobbetti et al., 2005; Ganzle et al., 2007; Vrancken et al., 2008). Thedirect conversion of fructose into mannitol by mannitol dehydro-genase (Fig. 1) enables the production of increased amounts ofacetic acid from acetyl-phosphate, as ethanol production is nofurther necessary for NADþ regeneration, favouring flavourformation. This is accompanied with the synthesis of an extra ATP,which favours competitiveness. The same competitive advantageresults from the use of oxygen, citrate, short-chain aldehydes, andoxidised glutathione (Ganzle et al., 2007; Jansch et al., 2007). Theuse of citrate by Lb. sanfranciscensis results in increased both acetateand lactate levels. Indeed, strains of this species use the pyruvatebranch to metabolise citrate, whereby the cofactor regeneration inthe lactate dehydrogenase reaction enables the additional forma-tion of acetate via the acetate kinase reaction out of maltose (Fig. 1).

Sourdough LAB can use sucrose through intracellular sucrosephosphorylase (e.g. Lb. reuteri) or extracellular glycosyltransferases(e.g. Lb. reuteri and Lb. sanfranciscensis) (Schwab et al., 2007;Waldherr et al., 2008). The use of sucrose through glycansucrasescontributes to the formation of extracellular homopolysaccharides,which is of importance for the texture of (rye) bread (Decock andCappelle, 2005; Tieking and Ganzle, 2005; Tieking et al., 2005; DiCagno et al., 2006; Arendt et al., 2007; Ganzle et al., 2007; Lacazeet al., 2007; Kaditzky et al., 2008b; Schwab et al., 2008). Also, it hasbeen shown that such homopolysaccharides exert a protectiveeffect on the producing strain against low pH (Kaditzky et al.,2008a). The potential to produce heteropolysaccharides seems tobe absent among sourdough LAB (Van der Meulen et al., 2007a).

Pentoses, such as arabinose and xylose, are usually utilized byheterofermentative LAB (not Lb. sanfranciscensis), by the facultativeheterofermentative Lb. plantarum, and rarely by obligately homo-fermentative LAB species. Moreover, simultaneous use of pentosesand maltose during sourdough fermentation has been reported forboth heterofermentative and homofermentative LAB, but itsmetabolic regulation needs further investigation (Gobbetti et al.,1999, 2000). However, expression of both the homo- and hetero-fermentative pathway in sourdough LAB is more common thanassumed previously, as revealed by metatranscriptome analysis(S. Weckx, J. Allemeersch, G. Vrancken, R. Van der Meulen,I. Scheirlinck, G. Huys, P. Vandamme, P. Van Hummelen and L. DeVuyst, unpublished results).

4.2. Peptide/amino acid utilization and flavour formation

Proteolysis and further amino acid conversions by sourdoughLAB contribute to their competitiveness (ATP production, cofactorregeneration, and/or acid stress response) and flavour activity(Ganzle et al., 2007; Van der Meulen et al., 2007b). Whereasproteolytic activity by the LAB themselves is rather limited insourdoughs, acidification through their sugar breakdown activatesendogenous cereal proteases that liberate peptides and amino acidsthat can be taken up by the microorganisms present; also, aminoacids can be accumulated by strain-specific peptidases (Ganzleet al., 2007; Wieser et al., 2008). In general, LAB use amino acids forprotein synthesis, as an energy source (e.g. the ADI pathway), toregulate the intracellular pH in an acidic environment [e.g. throughdecarboxylation of amino acids, leading to the production ofbiogenic amines (e.g. out of tyrosine) and g-aminobutyric acid

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(out of glutamate)], to regenerate cofactors, and for bioconversions.Amino acid conversions encompass the production of flavourprecursors and flavour-active compounds (such as aldehydes andthe corresponding alcohols) through the transamination/elimina-tion reactions (LAB) or Ehrlich pathway (yeasts) during thesourdough fermentation process (Fig. 1), while extra flavours(aldehydes and the corresponding carboxylic acids) and breadflavour-intensive compounds (e.g. 2-acetyl-1-pyrroline) aregenerated through Maillard reactions and Strecker degradationduring the baking process (Czerny and Schieberle, 2002; Demyt-tenaere et al., 2002; Thiele et al., 2002; Hansen and Schieberle,2005; Smit et al., 2009). In general and as shown for dairy LABduring cheese ripening, aminotransferases are involved in theconversion of amino acids (branched-chain amino acids, aromaticamino acids, and sulphur-containing amino acids) into the corre-sponding a-keto acids, followed by decarboxylation into the cor-responding aldehydes (Ardo, 2006; Liu et al., 2008; Smit et al.,2009; Fig. 1). a-Ketoglutarate serves as the preferred aminoacceptor in the transamination reaction and is converted intoglutamate; it has to be recycled by NADþ-dependent glutamatedehydrogenase, a strain-specific enzyme among LAB. The trans-amination reaction is an important bottleneck in the conversion ofamino acids by LAB. Interestingly, the cofactor-dependency ofglutamate dehydrogenase in Lb. sanfranciscensis links glutamaterecycling and the metabolic flux through the transaminase reactionto NADHþHþ regeneration in central carbohydrate metabolism(Ganzle et al., 2007). Whether fructose and/or citrate conversion asmentioned above is linked to amino acid conversion, in particularglutamate conversion, needs further investigation. Also, in general,the a-keto acid decarboxylase gene is not widespread among LAB,for which the full genome sequence is available, in particular instarter culture strains used in the food fermentation industry today.However, in the case of branched-chain amino acids, an alternativeroute through oxidative decarboxylation of the a-keto acids can befollowed (catalysed by the a-keto acid dehydrogenase complex),making use of the cofactors NADþ and CoA and generating extraATP (Fig. 1), hence contributing to competitiveness as well. Hydroxyacids (flavourless) may be formed from the keto acids as long asNADþ is needed to run glycolysis (Fig. 1). In general, most of theflavour-forming conversions enable the cell to oxidise NADHþHþ orreduce NADþ, depending on the needs of the cells. Alcohols may beproduced from the aldehydes as long as NADþ is needed, whileproduction of carboxylic acids generates NADHþHþ. However,subsequent regeneration of NADHþHþ needs oxygen or oxidisedcompounds, which sets limits for these reactions in an anaerobicenvironment. In the case of methionine, both a transaminationroute (initiated by an aminotransferase) and elimination route(catalysed by carbon–sulphur lyases) occur generally. Finally, estersand/or thioesters are formed by condensation of carboxylic acidsand alcohols and/or methanethiol, originating from methionine,respectively, which are important flavour compounds too.Comparative genomics of enzymes involved in flavour-formingpathways from amino acids reveals their presence in lactococci andcertain lactobacilli (e.g. glutamate dehydrogenase in the genome ofLb. plantarum), but their absence in a number of lactobacilli, such asthe type strains of Lb. brevis and Lb. reuteri (Liu et al., 2008).Alternatively, experimental evidence on leucine, glutamine, andphenylalanine conversions in sourdough LAB and its relationshipwith bread flavour exists (Thiele et al., 2002; Ganzle et al., 2007).Glutamate decarboxylase activity has been evidenced by theproduction of g-aminobutyric acid (Rizzello et al., 2008). Also,activity of cystathionine g-lyase has been shown in strains of Lb.fermentum and Lb. reuteri (De Angelis et al., 2002; Lo et al., 2009).However, the presence of some of these (flavour-forming) enzymescan vary between strains from the same species, which explains an

extra competitive advantage for sourdough strains possessing thiscapability, in particular when external electron acceptors aredepleted while energy sources are not (Van der Meulen et al.,2007b). Furthermore, it is fundamental for the diversity of (artisan)fermented foods, including sourdoughs.

Specific dedicated pathways may be involved in flavour forma-tion too. For instance, Lb. sanfranciscensis and Lb. reuteri have beenfound to display glutaminase activity, which converts glutamineinto glutamate. This deamidation improves the acid tolerance ofthese lactobacilli and influences wheat bread flavour (Vermeulenet al., 2007). The conversion of arginine into ornithine via the ADIpathway is of special importance during sourdough fermentation inthe presence of Lb. reuteri, Lb. pontis, Lb. fermentum, Lb. brevis, Lb.frumenti, and Lb. sakei (Gobbetti et al., 2005; Ganzle et al., 2007;G. Vrancken, T. Rimaux, F. Leroy and L. De Vuyst, unpublishedresults). The ADI pathway enhances the competitiveness of thesesourdough species (by allowing production of an extra ATP),improves their tolerance towards an acidic environment (byconsumption of two protons and liberating ammonia) and elabo-rates ornithine (as the final end-product of the pathway) (Fig. 1).Ornithine is a well-known precursor of 2-acetyl-1-pyrroline, whichis formed during the baking process and represents the charac-teristic flavour of baked wheat bread crust (Demyttenaere et al.,2002; Hansen and Schieberle, 2005; Ganzle et al., 2007).

Finally, amino acid conversions do not produce flavour volatilessolely. For instance, besides flavours, phenylalanine conversionproduces the antifungal compound phenyllactic acid, which is anantimicrobial agent and therefore of importance in determining thecomposition of a stable sourdough microbiota (Lavermicocca et al.,2003; Gobbetti et al., 2005; Schnurer and Magnusson, 2005; Ganzleet al., 2007). The physiological relevance of their production can befound in their contribution to redox balancing too (Ganzle et al.,2007; Van der Meulen et al., 2007b).

4.3. Metabolite exploitation of the sourdough microbiota

Besides carbohydrate and peptide utilization, organic acidproduction, and flavour formation, sourdough LAB and yeastspossess numerous metabolic activities that can be of interestduring sourdough fermentation (Corsetti and Settanni, 2007). Forinstance, controlled proteolysis of gluten results in bread suited forceliac patients (Katina et al., 2005; Gobbetti et al., 2005, 2007;Arendt et al., 2007; Corsetti and Settanni, 2007). Biodegradation ofphytate, an antinutritional factor that complexes certain mineralsand hence does not make them bioavailable, has been reported tobe activated by endogenous cereal phytase activity through sour-dough fermentation (Lopez et al., 2000; Katina et al., 2005; Corsettiand Settanni, 2007; Reale et al., 2007). Bacteriocin production aidsmicroorganisms to dominate the sourdough ecosystem (Leroyet al., 2007; Settanni and Corsetti, 2008); also, inhibition of rope-forming bacilli by LAB bacteriocins has been shown (Settanni andCorsetti, 2008). Homopolysaccharides produced by sourdough LABinfluence the viscoelastic properties of the dough and beneficiallyaffect the texture and shelf-life (in particular starch retrogradation)of bread (Arendt et al., 2007; Lacaze et al., 2007).

5. Starter culture development

From the above it is clear that, besides the ingredients used, theimplementation of selected starter cultures in sourdough tech-nology is of pivotal importance for the industrial production ofsourdoughs to be used as flavour carrier or texture-improving,antifungal, or health-promoting dough ingredient. In this context,strain robustness and fitness towards microbial competitors andenvironmental conditions have to be considered as one of the main

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criteria for selecting useful starters to be used in industrial sour-dough fermentation processes (Meignen et al., 2001; Vogel et al.,2002; Meroth et al., 2003b; Katina et al., 2004; Pepe et al., 2004;Hansen and Schieberle, 2005; Paramithiotis et al., 2005; Robertet al., 2006; Leroy et al., 2007; Siragusa et al., 2009; Vogelmannet al., 2009). However, starter cultures in use often have beenselected for a certain property only (e.g. acidification or flavourformation) and are not competitive enough or do not use thesourdough substrates (maltose, peptides, arginine, electronacceptors) efficiently to be optimally functionally active, as straindifferences occur frequently (De Angelis et al., 2007; Di Cagno et al.,2007a; Zotta et al., 2007). Moreover, studies on industrial startercultures are scarce (Brandt, 2007; Carnevali et al., 2007). It is ofcourse well-known that fermentation temperature and timeinfluence the flavour intensity of sourdough, homolactatefermenters being used at high temperature/short time (e.g. 37 �Cfor 36 h) and heterolactate fermenters at low temperature/longtime (e.g. 25 �C for 48 h), which results in flavoursome sourdoughscharacterized by the presence of mainly lactic acid or acetic acid(without excessive pungent flavour), respectively (Decock andCappelle, 2005; Katina et al., 2006). However, the impact offermentation technology on functional properties other than acidflavour has been neglected. Therefore, intellectual high-throughputscreening strategies, taking into account characteristics to survivethe sourdough ecosystem, besides desirable functional propertiesas those described above and being expressed under the prevailingtechnological conditions, are of high significance to find and/orimplement new sourdough starter strains.

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