Abstract

Sourdough fermentations are characterized by the combined activity of lactic acid bacteria and yeasts. An investigation of the microbial composition of 21 artisan sourdoughs from 11 different Belgian bakeries yielded 127 yeast isolates. Also, 12 spontaneous 10-day laboratory sourdough fermentations with daily backslopping were performed with rye, wheat, and spelt flour, resulting in the isolation of 217 yeast colonies. The isolates were grouped according to PCR-fingerprints obtained with the primer M13. Representative isolates of each M13 fingerprint group were identified using the D1/D2 region of the large subunit rRNA gene, internal transcribed spacer sequences, and partial actin gene sequences, leading to the detection of six species. The dominant species in the bakery sourdoughs were Saccharomyces cerevisiae and Wickerhamomyces anomalus (formerly Pichia anomala), while the dominant species in the laboratory sourdough fermentations were W. anomalus and Candida glabrata. The presence of S. cerevisiae in the bakery sourdoughs might be due to contamination of the bakery environment with commercial bakers yeast, while the yeasts in the laboratory sourdoughs, which were carried out under aseptic conditions with flour as the only nonsterile component, could only have come from the flour used.

Introduction

Sourdough fermentation has enjoyed an increasing popularity due to its beneficial effects on the flavour, texture, shelf-life, and nutritional and health-promoting properties of the resulting breads (Hansen, 2004; De Vuyst & Neysens, 2005). Sourdough develops by spontaneous fermentation of yeasts and lactic acid bacteria in mixtures of cereal flour(s) and water, with the lactic acid bacteria being responsible for the acidification of the dough and the yeasts for the leavening action via CO2 production. Usually, a stable microbial community arises during periodic refreshments of the flour/water mixture, closely depending on external factors such as temperature and pH, propagation cycles, and the type of cereal (De Vuyst & Vancanneyt, 2007; Vogelmann, 2009).

Extensive research efforts have been directed towards the study of the species diversity and identification of lactic acid bacteria involved in sourdough fermentation processes (Corsetti & Settanni, 2007; De Vuyst & Vancanneyt, 2007; Vogel & Ehrmann, 2008). In contrast, fewer studies on sourdough yeast species diversity and identification are available (Vogel & Ehrmann, 2008). In no small measure, this apparently lower interest in sourdough yeasts is due to the fact that wild-type yeasts have almost been eliminated from bakeries by the use of commercial bakers yeast, Saccharomyces cerevisiae, as a starter culture for bread production. However, a variety of yeasts can dominate a spontaneous sourdough fermentation process, depending on the flour type, process conditions, environment, and location, all of which substantially add to the diversity and originality of sourdough products (Rossi, 1996; Mantynen, 1999; Meroth, 2003; Succi, 2003; Pulvirenti, 2004; Hammes, 2005).

The most frequently reported yeasts in both wheat and rye sourdoughs are the species S. cerevisiae and Candida humilis (synonym Candida milleri); other frequently reported species are Pichia kudriavzevii (formerly Issatchenkia orientalis, anamorph Candida krusei) and Kazachstania exigua [formerly Saccharomyces exiguus, anamorph Candida (Torulopsis) holmii] (for reviews, see Rossi, 1996; De Vuyst & Neysens, 2005, and further Gullo, 2003; Vernocchi, 2004a, b; Garofalo, 2008; Vogelmann, 2009). Less frequently reported species include, among others, Wickerhamomyces anomalus (formerly Pichia anomala; Rocha & Malcata, 1999), Wickerhamomyces (formerly Pichia) subpelliculosa (Barber, 1983), Candida glabrata (Meroth, 2003; Succi, 2003), Kazachstania unispora (formerly Saccharomyces unisporus; Salovaara & Savolainen, 1984), and Torulaspora delbrueckii (Almeida & Pais, 1996). Using culture-dependent and -independent approaches, Meroth (2003) could isolate S. cerevisiae, P. kudriavzevii, C. humilis, and C. glabrata from rye sourdough with added bakers yeast, while they also detected Dekkera bruxellensis, Emericella nigrum, and unculturable ascomycetes by culture-independent PCR-denaturing gradient gel electrophoresis (PCR-DGGE). In contrast, Gatto & Torriani (2004) found no diversity in the yeast population and revealed the dominance of S. cerevisiae in a traditional wheat sourdough preparation.

A first reference point for accurate yeast identification is the constantly growing D1/D2 large subunit (LSU) rRNA sequence database, encompassing virtually all known yeast species (Kurtzman & Robnett, 1998; Fell, 2000). However, some distinct species such as W. anomalus and Pichia myanmarensis (Nagatsuka, 2005) as well as Kazachstania bulderi and Kazachstania barnettii (Middelhoven, 2000) show a low sequence divergence of two and three substitutions in this region, a variation that can also be observed among strains of the same species. Therefore, the use of D1/D2 LSU sequences was complemented by PCR amplification of repetitive DNA elements (e.g. Vassart, 1987; Lieckfeldt, 1993; Groenewald, 2008) and by determination of additional gene sequences such as the internal transcribed spacer (ITS) region of the rRNA gene cluster (Scorzetti, 2002) or protein-coding genes such as the actin gene (ACT1; Daniel & Meyer, 2003), and the mitochondrial cytochrome oxidase 2 (COX2) and cobalamin-independent methionine synthase (MET6) genes (Gonzáles, 2006). The evaluation of conventional classification criteria (Van der Walt & Yarrow, 1984; Kreger-van Rij, 1987; Robert, 1997; Barnett, 2000) allows the comparison of molecular with conventional identification results and conclusions on the physiological adaptation of the yeast community to the investigated environment. In recognition of the artificial nature of several yeast genera, a series of taxonomic changes will gradually transform the current phenotype-based classification towards a phylogeny-based classification. These changes concern, among others, the species P. anomala, which has been shown by multigene sequence analyses to be more appropriately classified in a newly described genus as W. anomalus (Kurtzman, 2008).

This study aimed at characterizing the diversity of yeast species in Belgian artisan sourdoughs, as well as gaining an understanding of the contribution of naturally occurring yeast species towards a stable microbiota during spontaneous sourdough fermentations. Therefore, both traditional sourdoughs with daily propagation cycles as well as spontaneous laboratory sourdoughs, daily backslopped for 10 days, were sampled. The results are discussed in relationship with associated studies on the bacterial diversity of the same samples (Scheirlinck, 2007; Van der Meulen, 2007).

Materials and methods

Yeast isolates

A total of 127 yeast isolates were obtained from 21 artisan sourdoughs collected from 11 Belgian bakeries, located in seven of the 10 provinces of Belgium. The sampled sourdoughs were produced without the addition of bakers yeast and were based on wheat, spelt, and rye flours, as well as mixtures thereof (Table 1). The sampling and isolation methods were described by Scheirlinck (2007). A total of 217 yeast isolates were obtained from 12 spontaneous laboratory sourdough fermentations. The sampling and isolation methods of these were described by Van der Meulen (2007). The laboratory sourdough fermentations were carried out in closed, presterilized fermentors with rye, spelt, or wheat flour, derived from two different mills in Belgium, as the only nonsterile component. Temperature, dough yield, and time between backsloppings mimicked bakery sourdough conditions as closely as possible. In each case, up to 10 yeast colonies were isolated from yeast extract–glucose agar (20 g L−1 glucose, 5 g L−1 yeast extract, 20 g L−1 agar) supplemented with 0.1 g L−1 of chloramphenicol. Selected strains, representing each species from each individual sourdough, were deposited in the Mycothèque de l'Université catholique de Louvain (MUCL), Belgium, under the numbers MUCL 51207–MUCL 51263.

Table 1

Yeast species distribution and main characteristics of 21 Belgian artisan sourdoughs

       Log CFU g−1 Yeast species identified (number of isolates) 
       
 

 
Sample designation Province Flour Age (years) Temp. (°C) Time (h) pH Yeasts LAB S. cerevisiae W. anomalus Others 
D01WW01T01 East Flanders Wheat 30 10 3.83 7.68 8.9  
D02WR01T01 West Flanders Wheat/rye 25 AT <24 4.01 4.2 8.86   
D02WR01T02  Wheat/rye 25 AT 22 3.8 3.89 9.02  T. delbrueckii (1) 
D02WW01T01  Wheat AT 22 3.83 5.72 9.15   K. barnettii (5) 
D03WW01T01 West Flanders Wheat 30 4.12 7.57 8.97 10   
D04WW01T01 Brabant Wallon Wheat AT 3.12 8.29   
D05WW01T01 East Flanders Wheat 28 24 3.63 7.14 8.6   
D06SS01T01 Liège Spelt 1.5 21–23 5.04 4.55 7.67   
D06WW01T01  Wheat 12 21–23 4.75 4.77 7.5  T. delbrueckii (1) 
D07WR01T01 Hainaut Wheat/rye 1 0.8 26 13 3.83 7.46 8.67   
D07WR01T02  Wheat/rye 2 0.8 26 13 3.86 7.46 8.19  
D07WR02T01  Wheat/rye 3 0.01 AT 12 3.78 6.98 8.9  
D08WW01T01 Namur First wheat, then spelt 15 AT 3.8 6.84 7.99   
D09ME01T01 Namur Wheat/rye/spelt AT <24 3.78 7.54 8.68   
D10SS01T01 Luxemburg spelt 28 4.28 5.63 8.03   
D10WW01T01  Wheat 28 4.09 5.94 8.3  
D10WR01T01  Wheat/rye 28 3.96 5.92 8.14  
D11RR01T01 Namur Rye AT 12 3.79 7.67 9.25  
D11WW01T01  Wheat 1 AT 12 3.72 7.26 9.08  K. unispora (1) 
D11WW02T01  Wheat 2 AT 12 3.85 7.62 9.17  
D11SS01T01  Spelt AT 12 3.83 7.29 9.02   
       Log CFU g−1 Yeast species identified (number of isolates) 
       
 

 
Sample designation Province Flour Age (years) Temp. (°C) Time (h) pH Yeasts LAB S. cerevisiae W. anomalus Others 
D01WW01T01 East Flanders Wheat 30 10 3.83 7.68 8.9  
D02WR01T01 West Flanders Wheat/rye 25 AT <24 4.01 4.2 8.86   
D02WR01T02  Wheat/rye 25 AT 22 3.8 3.89 9.02  T. delbrueckii (1) 
D02WW01T01  Wheat AT 22 3.83 5.72 9.15   K. barnettii (5) 
D03WW01T01 West Flanders Wheat 30 4.12 7.57 8.97 10   
D04WW01T01 Brabant Wallon Wheat AT 3.12 8.29   
D05WW01T01 East Flanders Wheat 28 24 3.63 7.14 8.6   
D06SS01T01 Liège Spelt 1.5 21–23 5.04 4.55 7.67   
D06WW01T01  Wheat 12 21–23 4.75 4.77 7.5  T. delbrueckii (1) 
D07WR01T01 Hainaut Wheat/rye 1 0.8 26 13 3.83 7.46 8.67   
D07WR01T02  Wheat/rye 2 0.8 26 13 3.86 7.46 8.19  
D07WR02T01  Wheat/rye 3 0.01 AT 12 3.78 6.98 8.9  
D08WW01T01 Namur First wheat, then spelt 15 AT 3.8 6.84 7.99   
D09ME01T01 Namur Wheat/rye/spelt AT <24 3.78 7.54 8.68   
D10SS01T01 Luxemburg spelt 28 4.28 5.63 8.03   
D10WW01T01  Wheat 28 4.09 5.94 8.3  
D10WR01T01  Wheat/rye 28 3.96 5.92 8.14  
D11RR01T01 Namur Rye AT 12 3.79 7.67 9.25  
D11WW01T01  Wheat 1 AT 12 3.72 7.26 9.08  K. unispora (1) 
D11WW02T01  Wheat 2 AT 12 3.85 7.62 9.17  
D11SS01T01  Spelt AT 12 3.83 7.29 9.02   

Main characteristics are the region of origin (Belgian province), the flour, the time during which the sourdough starter culture has been maintained (age), the sourdough fermentation temperature (temp.), the fermentation time since the inoculation of a fresh lot of flour with the maintained starter culture, and pH of the sample. SDs of the CFU of yeasts and bacteria were reported in Scheirlinck (2007).

*

Sample designations used in a complementary study on lactic acid bacteria (Scheirlinck, 2008).

AT, ambient temperature; LAB, lactic acid bacteria.

M13 PCR-fingerprinting

To determine groups of isolates based on hypervariable minisatellites, PCR-fingerprinting with a single primer derived from the core sequence within the protein III gene of the bacteriophage M13 was used. DNA extraction and PCR reactions were performed as described before (Groenewald, 2008). For cluster analysis, the similarity among digitized profiles was calculated using the Pearson correlation coefficient, and an unweighted pair group with arithmetic averages dendrogram was derived from the profiles using bionumerics version 5.1 (Applied Maths N.V., Sint-Martens-Latem, Belgium).

DNA sequencing

Ribosomal DNA regions and ACT1 gene fragments were amplified and sequenced as described previously (Daniel, 2009). Selected isolates of S. cerevisiae were further characterized by COX2 gene sequences, which were amplified using the primers COII-3 and COII-5 (Belloch, 2000), and by MET6 gene sequences, which were amplified using the primers MET6-5 and MET6-3 (Gonzáles, 2006). An approximately 600-bp fragment of both genes was generated and sequenced using a CEQ 2000 XL capillary automated sequencer (Beckman Coulter, Fullerton, CA). After initial blast searches for the most similar sequences, alignments were performed using bioedit 7.0.5.3 (Hall, 1999) for comparisons with the corresponding sequences of type strains and determination of substitutions and potential insertions or deletions (indels). To find gene sequences of type strains, the CBS database (http://www.cbs.knaw.nl/yeast/BioloMICS.aspx) was used as an additional resource. The gene sequences obtained were deposited in the EMBL databank (http://www.ebi.ac.uk/EMBL; Hinxton, UK) with accession numbers FN393977FN393993, FN435838 (D1/D2 LSU), FN393994FN394008, FN435839 (ITS), FN394009FN394023, FN435840 (ACT1), FN394066FN394073 (MET6), and FN394074FN394080 (COX2).

Physiological and morphological properties as conventional classification criteria

Physiological and morphological profiles of representative isolates were determined using the automated microplate method Allev/Biolomics (BioAware SA, Hannut, Belgium) of Robert (1997), a yeast identification system based on standard taxonomic criteria (Van der Walt & Yarrow, 1984; Kreger-van Rij, 1987).

Sourdough fermentation-related physiological characterization

To investigate the physiological adaptation of the yeast community to the sourdough fermentation environment, the following physiological characteristics were determined for a subset of strains: capacity to assimilate maltose, glucose, fructose, and sucrose; low pH tolerance; and the ability to grow in the presence of acetic acid. Tests were carried out in duplicate on a 10-mL scale and were incubated aerobically with periodic agitation at 30 °C for 48 h, except for the K. barnettii isolate D02WW01T02-1, which was incubated at 23 °C. The test medium was composed of 6.2 g L−1 of yeast–nitrogen base (YNB; Difco, Basingstoke, UK) and 5 g L−1 of carbohydrate (glucose, fructose, sucrose, or maltose); YNB without an added carbon source was used as a negative control. To test low pH tolerance, YNB medium with 5 g L−1 of glucose was adjusted to pH 2.5, 3.5, and 5.0 with 5 M HCl. To test the ability to grow in the presence of acetic acid, YNB medium with 5 g L−1 of glucose was supplemented with 1% w/v of acetic acid, after which the pH was corrected to 5.0. All tubes were inoculated with 1% v/v of a yeast culture, which was grown at 30 °C (23 °C for D02WW01T02-1) for 24 h. Growth was determined by measurements of OD600 nm.

Results

Clustering of yeast isolates

In total, 344 yeast isolates were typed by M13 PCR-fingerprinting (Figs 1 and 2). Cluster analysis of the M13 PCR-fingerprints resulted in six clusters and two single (ungrouped) isolates, referred to as A (n=177), B1 (n=91), B2 (n=6), C (n=5), D (n=1), E1 (n=61), E2 (n=1), and F (n=2). Artisan sourdough isolates were grouped into five clusters and one single isolate (Fig. 1), while spontaneous laboratory sourdough isolates were grouped into three clusters and one single isolate (Fig. 2).

Fig. 1

Dendrogram generated by the unweighted pair-group method with arithmetic averages based on M13 PCR-fingerprints of yeasts isolated from 21 artisan sourdoughs, originating from 11 Belgian bakeries. Isolate numbers are given in the order of appearance of the profiles in the tree. Isolates that were chosen for sourdough fermentation-related physiological characterization and sequence analysis are marked with a cross and an asterisk, respectively.

Fig. 1

Dendrogram generated by the unweighted pair-group method with arithmetic averages based on M13 PCR-fingerprints of yeasts isolated from 21 artisan sourdoughs, originating from 11 Belgian bakeries. Isolate numbers are given in the order of appearance of the profiles in the tree. Isolates that were chosen for sourdough fermentation-related physiological characterization and sequence analysis are marked with a cross and an asterisk, respectively.

Fig. 2

Dendrogram generated by the unweighted pair-group method with arithmetic averages based on M13 PCR-fingerprints of yeasts isolated from 12 spontaneous laboratory sourdough fermentations. Isolate numbers are given in the order of appearance of the profiles in the tree. Isolates that were chosen for sourdough fermentation-related physiological characterization and sequence analysis are marked with a cross and an asterisk, respectively.

Fig. 2

Dendrogram generated by the unweighted pair-group method with arithmetic averages based on M13 PCR-fingerprints of yeasts isolated from 12 spontaneous laboratory sourdough fermentations. Isolate numbers are given in the order of appearance of the profiles in the tree. Isolates that were chosen for sourdough fermentation-related physiological characterization and sequence analysis are marked with a cross and an asterisk, respectively.

Molecular identification of yeast isolates

Species identification using multiple gene sequences was performed for 18 isolates, which were selected to represent the six clusters and two single isolates found by PCR-fingerprinting. This selection included isolates from all subclusters and those with atypical profiles observed in the combined similarity tree of bakery and laboratory sourdough isolates (not shown). For the selected isolates, 18 D1/D2 LSU sequences, 16 ITS sequences, and 16 partial ACT1 gene sequences were generated (Supporting Information, Table S1). Sequence variations detected within species, as indicated by D1/D2 LSU, ITS, and ACT1 sequences, were zero to one nucleotide differences in D1/D2 LSU sequences, zero to nine nucleotide differences in ITS sequences, and zero to four nucleotide differences in the ACT1 gene. The fingerprint clusters of S. cerevisiae (B1, B2) and the cluster and the single isolate of C. glabrata (E1, E2) were considered as intraspecies variants, based on the low variation observed in the ACT1 gene, which is evolving faster than ribosomal sequences. The morphological and physiological characterization of representative isolates was in agreement with the sequence-based species identification, with the exception of the isolates representing cluster C, identified as K. barnettii, which resembled S. cerevisiae and K. exigua physiologically (Tables S1, S2).

To further characterize selected S. cerevisiae and K. barnettii isolates, eight partial MET6 and seven partial COX2 gene sequences were determined as additional protein-coding gene markers. The identification of the selected K. barnettii isolate was confirmed by its COX2 sequence, which was identical to that of the K. barnettii type strain, and by its MET6 sequence, which was largely different from S. cerevisiae. The MET6 sequences of S. cerevisiae isolates of both clusters B1 and B2 were identical to S. cerevisiae, while their COX2 sequences revealed the presence of two major sequence types differing by 20–25 nucleotide substitutions. The first sequence type was highly similar (two substitutions) to the type strain of S. cerevisiae and was only represented by isolate D01WW01T01-2. The five isolates belonging to the second sequence type showed nucleotide substitutions in six positions among themselves. In comparison with the type strains of the most closely related species, the five isolates showed 11–14 substitutions to Saccharomyces cariocanus, 17–21 substitutions to Saccharomyces mikatae, and 18–23 substitutions to S. cerevisiae (Table S1). This considerable variation between the second COX2 sequence type determined here and the type strains of related and recognized species was contrasted by only one to five substitutions in comparison with the COX2 sequence of the Saccharomyces italicus type strain. Saccharomyces italicus is considered a synonym of S. cerevisiae based on high DNA reassociation values (96%; Vaughan Martini & Kurtzman, 1985).

Physiological characterization

The physiological properties of representative yeast strains as determined in microplates are reported in Table S2.

All yeast isolates tested for their sourdough-relevant properties in tubes, marked in Figs 1 and 2, were capable of assimilating glucose, fructose, and sucrose, and were capable of growth at pH 3.5 and 5.0. Most isolates could assimilate maltose, except for the seven C. glabrata isolates and the single K. barnettii and K. unispora isolates tested. The capacity to grow at pH 2.5 was variable, with one W. anomalus isolate out of 23 tested (R11-96h3), nine out of 17 S. cerevisiae tested (R9-24h2, R9-72h2, R9-96h5, D01WW01T01-6, D03WW01T01-3, D06SS01T01-2, D11WW01T01-5, D11WW02T01-1, and D11SS01T01-4), and the single tested T. delbrueckii isolate (D06WW01T01-2) incapable of growing under these conditions. Most isolates grew in the presence of 1% acetic acid, except for three isolates of W. anomalus (R7-96h2, R9-24h3, and R15-192h) and one isolate of S. cerevisiae (D10SS01T01-7).

Species distribution

The 127 artisan sourdough isolates were identified as S. cerevisiae (68%), W. anomalus (26%), and K. barnettii (4%). Kazachstania unispora and T. delbrueckii were represented by one and two isolates, respectively (Fig. 1). In contrast, the 217 spontaneous laboratory sourdough isolates were identified as W. anomalus (66%) and C. glabrata (29%); S. cerevisiae (5%) was rarely detected (Fig. 2).

The 21 sourdoughs of 11 artisan bakeries in seven provinces of Belgium were dominated by the presence of S. cerevisiae (in 18 sourdoughs), followed by W. anomalus (in nine sourdoughs) (Table 1). While 11 of the doughs led to the isolation of only one yeast species, the other 10 were comprised of two species. The species diversity was not correlated with the flour type (Table 1). Some bakeries maintained pure S. cerevisiae doughs and doughs with two different yeast species in parallel; in some cases, the non-S. cerevisiae yeast even dominated a specific dough (D07WR01T02, D07WR02T01, and D11RR01T01). However, pure W. anomalus doughs were only found in two bakeries (D08 and D09), from which no S. cerevisiae doughs were known and a pure K. barnettii dough was found in a bakery (D02) that also maintained two S. cerevisiae-dominated doughs (Table 1). The yeast diversity did not indicate a correlation with temperature, pH, time after refreshment, or age (Table 1).

The 12 spontaneous laboratory sourdough fermentations with flour from two different Belgian mills were dominated by W. anomalus (found in 11 sourdoughs), followed by C. glabrata (in nine sourdoughs), and the occasional detection of S. cerevisiae (in three sourdoughs). Although some fermentations led to the isolation of only one yeast species, the majority contained a combination of species, typically of W. anomalus and C. glabrata. The yeast diversity did not indicate a correlation with the mill or the flour (Table 2).

Table 2

Detection of yeast species during 12 spontaneous laboratory sourdough fermentations

    Colony number obtained from sample collected at time (h): 
    
 
Fermentation Mill Flour Yeast species 24 48 72 96 120 144 168 192 216 240 
R6 D12 Spelt W. anomalus  
Spelt A   C. glabrata        
  S. cerevisiae           
R13 D12 Spelt W. anomalus         
  C. glabrata            
  S. cerevisiae            
R8 D12 Wheat W. anomalus          
Wheat A   C. glabrata           
  S. cerevisiae            
R12 D12 Wheat W. anomalus    
  C. glabrata            
  S. cerevisiae            
R7 D12 Rye W. anomalus      
  C. glabrata        
  S. cerevisiae            
R14 D12 Rye W. anomalus        
  C. glabrata   
  S. cerevisiae          
R11 D13 Spelt W. anomalus      
Spelt B   C. glabrata            
  S. cerevisiae            
R16 D13 Spelt W. anomalus      
  C. glabrata          
  S. cerevisiae            
R10 D13 Wheat W. anomalus       
Wheat B   C. glabrata       
  S. cerevisiae            
R15 D13 Wheat W. anomalus      
  C. glabrata      
  S. cerevisiae            
R9 D13 Rye W. anomalus          
  C. glabrata        
  S. cerevisiae       
R17 D13 Rye W. anomalus            
  C. glabrata     
  S. cerevisiae            
    Colony number obtained from sample collected at time (h): 
    
 
Fermentation Mill Flour Yeast species 24 48 72 96 120 144 168 192 216 240 
R6 D12 Spelt W. anomalus  
Spelt A   C. glabrata        
  S. cerevisiae           
R13 D12 Spelt W. anomalus         
  C. glabrata            
  S. cerevisiae            
R8 D12 Wheat W. anomalus          
Wheat A   C. glabrata           
  S. cerevisiae            
R12 D12 Wheat W. anomalus    
  C. glabrata            
  S. cerevisiae            
R7 D12 Rye W. anomalus      
  C. glabrata        
  S. cerevisiae            
R14 D12 Rye W. anomalus        
  C. glabrata   
  S. cerevisiae          
R11 D13 Spelt W. anomalus      
Spelt B   C. glabrata            
  S. cerevisiae            
R16 D13 Spelt W. anomalus      
  C. glabrata          
  S. cerevisiae            
R10 D13 Wheat W. anomalus       
Wheat B   C. glabrata       
  S. cerevisiae            
R15 D13 Wheat W. anomalus      
  C. glabrata      
  S. cerevisiae            
R9 D13 Rye W. anomalus          
  C. glabrata        
  S. cerevisiae       
R17 D13 Rye W. anomalus            
  C. glabrata     
  S. cerevisiae            

Flour of different cereals processed at two different Belgian mills was used.

*

Sample designations used in a complementary study on lactic acid bacteria (Van der Meulen, 2007).

Discussion

Spontaneous sourdoughs usually develop a stable microbiological community (De Vuyst & Neysens, 2005; De Vuyst & Vancanneyt, 2007), which was supported by the low species diversity of yeasts identified during this study. To our knowledge, this is the first study investigating the spontaneously developing yeast microbiota in controlled laboratory fermentations without the addition of a starter culture. Meroth (2003) have shown the dominance of S. cerevisiae and C. humilis during controlled fermentations, but these yeasts originated from the bakers yeast and commercial sourdough starters used, respectively. Moreover, this is the first study of Belgian artisan sourdoughs.

In the present study, the yeast species identification was achieved by M13 PCR-fingerprinting, followed by multiple gene sequencing and physiological characterization of representative isolates of each fingerprinting group. This revealed the presence of primarily S. cerevisiae and W. anomalus in artisan bakery sourdoughs and W. anomalus and C. glabrata in spontaneous laboratory sourdoughs. The clusters and the single isolate labelled with a common letter and distinguished by subscript numbers (B1/B2, E1/E2) were determined by DNA sequences to represent the same species (S. cerevisiae and C. glabrata, respectively). The separation of clusters B1 from B2 and of E1 from E2, as well as the fingerprint variation within clusters, was attributed to intraspecies variation, indicating the presence of genetically different lineages among the sourdough yeast isolates. Among these postulated genetic lineages, only the six S. cerevisiae isolates in cluster B2 representing a single sourdough from bakery D05 indicated the selection of a particular strain in a bakery.

Most detected species, with the exception of K. barnettii and K. unispora, are well known to occur in sourdough, although some typically sourdough-associated species such as K. exigua (anamorph C. holmii), C. humilis (synonym C. milleri), and P. kudriavzevii (formerly I. orientalis, anamorph C. krusei) were not found (Pulvirenti, 2001; Meroth, 2003; Succi, 2003; Vernocchi, 2004b; Hammes, 2005). Apparently, many existing studies have been based on Italian sourdoughs (Corsetti, 2001; Pulvirenti, 2001; Succi, 2003; Foschino, 2004; Vernocchi, 2004b) and significant differences in the range of the detected yeast species may be due to process-related differences.

During the current study, the species composition differed between bakery and laboratory sourdoughs. In Belgian bakery sourdoughs, the frequently reported species S. cerevisiae (Corsetti, 2001; Pulvirenti, 2001; Meroth, 2003; Succi, 2003; Vernocchi, 2004b) might originate from the bakery's environment in most cases, and was rarely introduced into the process by the flour (Meroth, 2003). As an additional difference, C. glabrata was detected regularly and exclusively in the laboratory fermentations. Although documented before as an occasional component (Succi, 2003; Vogelmann, 2009), the present paper is, to our knowledge, the first report of a sourdough fermentation dominated by C. glabrata. Its growth relies mainly on glucose as an energy source. Kazachstania barnettii, a maltose-negative yeast species identified for the first time from sourdough, was the only yeast isolated from the bakery sourdough D02W01T01. This species might have been misidentified as S. cerevisiae or K. exigua in studies using phenotypical identification methods. This same sourdough was the only bakery sourdough dominated by a maltose-negative yeast species, which conforms to a mutualistic association with maltose-utilizing Lactobacillus sanfranciscensis (Ottogalli, 1996). The best-known association of yeasts and lactic acid bacteria in sourdough is that of the maltose-negative yeasts K. exigua or C. humilis with the maltose-positive L. sanfranciscensis in San Francisco sourdough and Panettone, respectively (Gobbetti, 1994; Ottogalli, 1996). This stable association has been attributed to the fact that, unlike L. sanfranciscensis, K. exigua and C. humilis are unable to ferment maltose, thereby avoiding nutritional competition and catabolite repression by glucose (Gobbetti, 1994). This interaction is further enhanced by the reported excretion of glucose upon maltose consumption through maltose phosphorylase activity by L. sanfranciscensis, which can then serve as an energy source for the maltose-negative yeasts (Gobbetti, 1994; Stolz, 1996). The species K. unispora and T. delbrueckii can be considered as minor components of the sourdough microbiota, as they were represented by single isolates in doughs dominated by S. cerevisiae.

Sequencing of two additional protein-coding genes (MET6, COX2) allowed a better understanding of the genetic variation in S. cerevisiae isolates. The observed large intraspecies variation of the mitochondrial COX2 gene sequences in S. cerevisiae has been noted before and might be due to a high evolutionary rate of the COX2 gene in this species (Kurtzman & Robnett, 2003), leading to conspecific populations that developed distinctive COX2 genes. The occurrence of the same COX2 gene sequences in isolates from three different bakeries (D03WW01T01-1, D05WW01T01-1, and D07WR02T01-2) indicated that a common origin of the strains present in different bakeries is possible.

The ability of all W. anomalus and S. cerevisiae isolates to strongly assimilate maltose and sucrose, the major carbohydrates of flour, might be considered as an advantage of these species, leading to their dominance in laboratory sourdough fermentations and their frequent occurrence in bakery sourdough fermentations. The known osmotolerance of W. anomalus, but generally not of S. cerevisiae, may add to the first species' advantage, as invertase activity provided by the yeast may lead to an increase of glucose from sucrose in sourdough (Lues, 1993; Gobbetti, 1994). Saccharomyces cerevisiae showed the least tolerance to the lowest pH. This may be another reason why this yeast species did not dominate spontaneous laboratory sourdough fermentations, as it could not outcompete more acid-tolerant strains of W. anomalus. From only one laboratory fermentation with rye flour, S. cerevisiae was isolated up to the end of the experiment, while this species was temporarily present in two other fermentations. The isolate obtained at the end of the experiment was able to grow at a pH 2.5 in contrast to those obtained at earlier time points of the same fermentation, indicating an adaptation or selection towards acid tolerance.

No clear correlation could be established between the yeast and lactic acid bacteria composition in artisan bakery sourdoughs, although it is noteworthy that, among others, the sourdoughs of two bakeries (D08 and D09), from which W. anomalus was isolated as the sole yeast species, lack the maltose-positive L. sanfranciscensis (Scheirlinck, 2008). Previously, a difference in the lactic acid bacteria composition of sourdoughs based on different cereals has been reported, namely that Lactobacillus fermentum is absent from spelt sourdoughs, while present in rye and wheat sourdoughs (Van der Meulen, 2007). Although the same sourdoughs were included in the present study, no such cereal-related difference could be observed for the composition of the yeast population.

In laboratory fermentations, typically, the final species composition was established after the first week, although not all species could be cultured at all sampling times. The bacterial populations and metabolite profiles of the four fermentations analysed by Van der Meulen (2007) had stabilized after the same fermentation time.

To conclude, the 127 yeast isolates from artisan bakery sourdoughs were dominated by S. cerevisiae (68%), followed by W. anomalus (26%), whereas the 217 isolates from spontaneous laboratory fermentations were dominated by W. anomalus (66%), followed by C. glabrata (29%). This study indicated a specific diversity of yeasts in artisan Belgian sourdoughs that can partly be assigned to the local flours as potential inoculum and the influence of the environment and bakery practice on the growth of yeasts during traditional sourdough fermentation, as evidenced by the dominance of the yeast populations during spontaneous laboratory sourdough fermentations.

Acknowledgements

The authors would like to acknowledge their financial support from the Research Council of the Vrije Universiteit Brussel (OZR, GOA, and IOF projects), the Fund for Scientific Research – Flanders, the Institute for the Promotion of Innovation through Science and Technology in Flanders, in particular, the SBO project ‘New Strategy for the Development of Functional and Performant Starter Cultures for Foods in Function of “Food Qualitomics”’, the Federal Research Policy, in particular, the project of the Action for the Promotion of and Cooperation with the Belgian Coordinated Collections of Microorganisms (contracts BCCM C3/10/003 and C4/00/001), and the European Commission's Marie Curie Mobility Actions contract MIRG-CT-2005-016539. H.M.D. thanks M.-C. Moons and P. Evrard for morphological and physiological analyses as well as S. Huret and C. Bivort for sequence analyses.

References

Almeida
MJ
Pais
CS
(
1996
)
Characterization of the yeast population from traditional corn and rye bread doughs
.
Lett Appl Microbiol
 
23
:
154
158
.
Barber
S
Baguena
R
Martinez-Anaya
MA
Torner
MJ
(
1983
)
Microflora de masa madre panaria. Identificacion y propiedades funcionales de microorganismos de masas madre industriales elaboradas con harina de trigo
.
Rev Agroquim Tecnol
 
23
:
552
562
.
Barnett
JA
Payne
RW
Yarrow
D
(
2000
)
Yeasts: Characteristics and Identification
 ,
3rd edn
.
Cambridge University Press
,
Cambridge
.
Belloch
C
Querol
A
García
MD
Barrio
E
(
2000
)
Phylogeny of the genus Kluyveromyces inferred from the mitochondrial cytochrome-c oxidase II gene
.
Int J Syst Evol Micr
 
50
:
405
416
.
Corsetti
A
Settanni
L
(
2007
)
Lactobacilli in sourdough fermentation
.
Food Res Int
 
40
:
539
558
.
Corsetti
A
Lavermicocca
P
Morea
M
Baruzzi
F
Tosti
N
Gobbetti
M
(
2001
)
Phenotypic and molecular identification and clustering of lactic acid bacteria and yeasts from wheat (species Triticum durum and Triticum aestivum) sourdoughs of Southern Italy
.
Int J Food Microbiol
 
64
:
95
104
.
Daniel
HM
Meyer
W
(
2003
)
Evaluation of ribosomal RNA and actin gene sequences for the identification of ascomycetous yeasts
.
Int J Food Microbiol
 
86
:
61
78
.
Daniel
HM
Vrancken
G
Takrama
JF
Camu
N
De Vos
P
De Vuyst
L
(
2009
)
Yeast diversity of Ghanaian cocoa bean heap fermentations
.
FEMS Yeast Res
 
9
:
774
783
.
De Vuyst
L
Neysens
P
(
2005
)
The sourdough microflora and metabolic interactions
.
Trends Food Sci Tech
 
16
:
43
56
.
De Vuyst
L
Vancanneyt
M
(
2007
)
Biodiversity and identification of sourdough lactic acid bacteria
.
Food Microbiol
 
24
:
120
127
.
Fell
JW
Boekhout
T
Fonseca
A
Scorzetti
G
Statzell-Tallman
A
(
2000
)
Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis
.
Int J Syst Evol Micr
 
50
:
1351
1371
.
Foschino
R
Gallina
S
Andrighetto
C
Rossetti
L
Galli
A
(
2004
)
Comparison of cultural methods for the identification and molecular investigation of yeasts from sourdoughs for Italian sweet baked products
.
FEMS Yeast Res
 
4
:
609
618
.
Garofalo
C
Silvestri
G
Aquilanti
L
Clementi
F
(
2008
)
PCR-DGGE analysis of lactic acid bacteria and yeast dynamics during the production processes of three varieties of Panettone
.
J Appl Microbiol
 
105
:
243
254
.
Gatto
V
Torriani
S
(
2004
)
Microbial population changes during sourdough fermentation monitored by DGGE analysis of 16S and 26S rRNA gene fragments
.
Ann Microbiol
 
54
:
31
42
.
Gobbetti
M
Corsetti
A
Rossi
J
(
1994
)
The sourdough microflora – interactions between lactic acid bacteria and yeasts – metabolism of carbohydrates
.
Appl Microbiol Biot
 
41
:
456
460
.
Gonzáles
SS
Barrio
E
Gafner
J
Querol
A
(
2006
)
Natural hybrids from Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces kudriavzevii in wine fermentations
.
FEMS Yeast Res
 
6
:
1221
1234
.
Groenewald
M
Daniel
HM
Robert
V
Poot
GA
Smith
MTh
(
2008
)
Polyphasic re-examination of Debaryomyces hansenii strains and reinstatement of D. hansenii, D. fabryi and D. subglobosus
.
Persoonia
 
21
:
17
27
.
Gullo
M
Romano
AD
Pulvirenti
A
Giudici
P
(
2003
)
Candida humilis– dominant species in sourdoughs for the production of durum wheat bran flour bread
.
Int J Food Microbiol
 
80
:
55
59
.
Hall
TA
(
1999
)
BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT
.
Nucl Acids Symp Ser
 
41
:
95
98
.
Hammes
WP
Brandt
MJ
Francis
KL
Rosenheim
J
Seitter
MFH
Vogelmann
SA
(
2005
)
Microbial ecology of cereal fermentations
.
Trends Food Sci Tech
 
16
:
4
11
.
Hansen
AS
(
2004
)
Sourdough bread
.
Handbook of Food and Beverage Fermentation Technology
  (
Hui
YH
Meunier-Goddik
L
Hansen
AS
Josephsen
J
Nip
WK
Stanfield
PS
Toldrá
F
, eds) pp.
729
755
.
Marcel Dekker
,
New York
.
Kreger-van Rij
NJW
(
1987
)
Classification of yeasts
.
The Yeasts
 , Vol.
1
,
2nd edn
(
Rose
AH
Harrison
JS
, eds) pp.
5
61
.
Academic Press
,
London
.
Kurtzman
CP
Robnett
CJ
(
1998
)
Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences
.
Antonie van Leeuwenhoek
 
73
:
331
371
.
Kurtzman
CP
Robnett
CJ
(
2003
)
Phylogenetic relationships among yeasts of the ‘Saccharomyces complex’ determined from multigene sequence analyses
.
FEMS Yeast Res
 
3
:
417
432
.
Kurtzman
CP
Robnett
CJ
Basehoar-Powers
E
(
2008
)
Phylogenetic relationships among species of Pichia, Issatchenkia and Williopsis determined from multigene sequence analysis, and the proposal of Barnettozyma gen. nov., Lindnera gen. nov. and Wickerhamomyces gen. nov
.
FEMS Yeast Res
 
8
:
939
954
.
Lieckfeldt
E
Meyer
W
Börner
T
(
1993
)
Rapid identification and differentiation of yeasts by DNA and PCR fingerprinting
.
J Basic Microb
 
33
:
413
426
.
Lues
JFR
Viljoen
BC
Miller
M
Prior
BA
(
1993
)
Interaction of nonculture microbial flora on dough fermentation
.
Food Microbiol
 
10
:
205
213
.
Mantynen
VH
Korhola
M
Gudmundsson
H
Turakainen
H
Alfredsson
GA
Salovaara
H
Lindström
K
(
1999
)
A polyphasic study on the taxonomic position of industrial sour dough yeasts
.
Syst Appl Microbiol
 
22
:
87
96
.
Meroth
CB
Hammes
WP
Hertel
C
(
2003
)
Identification and population dynamics of yeasts in sourdough fermentation processes by PCR-denaturing gradient gel electrophoresis
.
Appl Environ Microb
 
69
:
7453
7461
.
Middelhoven
WJ
Kurtzman
CP
Vaughan-Martini
A
(
2000
)
Saccharomyces bulderi sp. nov., a yeast that ferments gluconolactone
.
Antonie van Leeuwenhoek
 
77
:
223
228
.
Nagatsuka
Y
Kawasaki
H
Seki
T
(
2005
)
Pichia myanmarensis sp. nov., a novel cation-tolerant yeast isolated from palm sugar in Myanmar
.
Int J Syst Evol Micr
 
55
:
1379
1382
.
Ottogalli
G
Galli
A
Foschino
R
(
1996
)
Italian bakery products obtained with sour dough: characterization of the typical microflora
.
Adv Food Sci
 
18
:
131
144
.
Pulvirenti
A
Caggia
C
Restuccia
C
Gullo
M
Giudici
P
(
2001
)
DNA fingerprinting methods used for identification of yeasts isolated from Sicilian sourdoughs
.
Ann Microbiol
 
51
:
107
120
.
Pulvirenti
A
Solieri
L
Gullo
M
De Vero
L
Giudici
P
(
2004
)
Occurrence and dominance of yeast species in sourdough
.
Lett Appl Microbiol
 
38
:
113
117
.
Robert
V
Evrard
P
Hennebert
GL
(
1997
)
BCCM/ALLEV 2.00 an automated system for the identification of yeasts
.
Mycotaxon
 
64
:
455
463
.
Rocha
JM
Malcata
FX
(
1999
)
On the microbiological profile of traditional Portuguese sourdough
.
J Food Protect
 
62
:
1416
1429
.
Rossi
J
(
1996
)
The yeasts in sourdough
.
Adv Food Sci
 
18
:
201
211
.
Salovaara
H
Savolainen
J
(
1984
)
Yeast type isolated from Finnish sour rye dough starter
.
Acta Aliment Pol
 
10
:
241
246
.
Scheirlinck
I
Van der Meulen
R
Van Schoor
A
Vancanneyt
M
De Vuyst
L
Vandamme
P
Huys
G
(
2007
)
Influence of geographical origin and flour type on diversity of lactic acid bacteria in traditional Belgian sourdoughs
.
Appl Environ Microb
 
73
:
6262
6269
.
Scheirlinck
I
Van der Meulen
R
Van Schoor
A
Vancanneyt
M
De Vuyst
L
Vandamme
P
Huys
G
(
2008
)
Taxonomic structure and stability of the bacterial community in Belgian sourdough ecosystems as assessed by culture and population fingerprinting
.
Appl Environ Microb
 
74
:
2414
2423
.
Scorzetti
G
Fell
JW
Fonseca
A
Statzell-Tallman
A
(
2002
)
Systematics of basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions
.
FEMS Yeast Res
 
2
:
495
517
.
Stolz
P
Hammes
WP
Vogel
RF
(
1996
)
Maltose-phosphorylase and hexokinase activity in lactobacilli from traditionally prepared sourdoughs
.
Adv Food Sci
 
18
:
1
6
.
Succi
M
Reale
A
Andrighetto
C
Lombardi
A
Sorrentino
E
Coppola
R
(
2003
)
Presence of yeasts in southern Italian sourdoughs from Triticum aestivum flour
.
FEMS Microbiol Lett
 
225
:
143
148
.
Van der Meulen
R
Scheirlinck
I
Van Schoor
A
Huys
G
Vancanneyt
M
Vandamme
P
De Vuyst
L
(
2007
)
Population dynamics and metabolite target analysis of lactic acid bacteria during laboratory fermentations of wheat and spelt sourdoughs
.
Appl Environ Microb
 
73
:
4741
4750
.
Van der Walt
JP
Yarrow
D
(
1984
)
Methods for the isolation, maintenance, classification and identification of yeasts
.
The Yeasts, a Taxonomic Study
  (
Kreger-van Rij
NJW
, eds) pp.
45
104
.
Elsevier
,
London
.
Vassart
G
Georges
M
Monsieur
R
Brocas
H
Lequarre
AS
Christophe
D
(
1987
)
A sequence in M13-phage detects hypervariable minisatellites in human and animal DNA
.
Science
 
235
:
683
684
.
Vaughan Martini
A
Kurtzman
C
(
1985
)
Deoxyribonucleic acid relatedness among species of the genus Saccharomyces Sensu Stricto
.
Int J Syst Bacteriol
 
35
:
508
511
.
Vernocchi
P
Valmorri
S
Dalai
I
Torriani
S
Gianotti
A
Suzzi
G
Guerzoni
ME
Mastrocola
D
Gardini
F
(
2004a
)
Characterization of the yeast population involved in the production of a typical italian bread
.
J Food Sci
 
69
:
182
186
.
Vernocchi
P
Valmorri
S
Gatto
V
Torriani
S
Gianotti
A
Suzzi
G
Guerzoni
ME
Gardini
F
(
2004b
)
A survey on yeast microbiota associated with an Italian traditional sweet-leavened baked good fermentation
.
Food Res Int
 
37
:
469
476
.
Vogel
RF
Ehrmann
MA
(
2008
)
Sourdough fermentations
.
Molecular Techniques in the Microbial Ecology of Fermented Foods
  (
Cocolin
L
Ercolini
D
, eds) pp.
119
144
.
Springer
,
New York
.
Vogelmann
SA
Seitter
M
Singer
U
Brandt
MJ
Hertel
C
(
2009
)
Adaptability of lactic acid bacteria and yeasts to sourdough prepared from cereals, pseudocereals and cassava and use of competitive strains as starters
.
Int J Food Microbiol
 
130
:
205
212
.

Supporting Information

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

Author notes

Editor: Cletus Kurtzman