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et al., 1999; Meroth et al., 2003; Succi et al., 2003; Pulvirenti recognition of the artificial nature of several yeast genera, a
et al., 2004; Hammes et al., 2005). series of taxonomic changes will gradually transform the
The most frequently reported yeasts in both wheat and current phenotype-based classification towards a phylo-
rye sourdoughs are the species S. cerevisiae and Candida geny-based classification. These changes concern, among
humilis (synonym Candida milleri); other frequently re- others, the species P. anomala, which has been shown by
ported species are Pichia kudriavzevii (formerly Issatchenkia multigene sequence analyses to be more appropriately
orientalis, anamorph Candida krusei) and Kazachstania classified in a newly described genus as W. anomalus (Kurtz-
exigua [formerly Saccharomyces exiguus, anamorph Candida man et al., 2008).
(Torulopsis) holmii] (for reviews, see Rossi, 1996; De Vuyst & This study aimed at characterizing the diversity of yeast
Neysens, 2005, and further Gullo et al., 2003; Vernocchi species in Belgian artisan sourdoughs, as well as gaining an
et al., 2004a, b; Garofalo et al., 2008; Vogelmann et al., 2009). understanding of the contribution of naturally occurring
Less frequently reported species include, among others, yeast species towards a stable microbiota during sponta-
c 2010 Federation of European Microbiological Societies FEMS Yeast Res 10 (2010) 471–481
Published by Blackwell Publishing Ltd. All rights reserved
Yeast species composition in sourdough 473
Table 1. Yeast species distribution and main characteristics of 21 Belgian artisan sourdoughs
1
Log CFU g Yeast species identified (number of isolates)
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 et al. (2007).
Sample designations used in a complementary study on lactic acid bacteria (Scheirlinck et al., 2008).
AT, ambient temperature; LAB, lactic acid bacteria.
the bacteriophage M13 was used. DNA extraction and PCR alignments were performed using BIOEDIT 7.0.5.3 (Hall,
reactions were performed as described before (Groenewald 1999) for comparisons with the corresponding sequences of
et al., 2008). For cluster analysis, the similarity among type strains and determination of substitutions and poten-
digitized profiles was calculated using the Pearson correla- tial insertions or deletions (indels). To find gene sequences
tion coefficient, and an unweighted pair group with arith- of type strains, the CBS database (http://www.cbs.knaw.nl/
metic averages dendrogram was derived from the profiles yeast/BioloMICS.aspx) was used as an additional resource.
using BIONUMERICS version 5.1 (Applied Maths N.V., Sint- The gene sequences obtained were deposited in the EMBL
Martens-Latem, Belgium). databank (http://www.ebi.ac.uk/EMBL; Hinxton, UK)
with accession numbers FN393977–FN393993, FN435838
(D1/D2 LSU), FN393994–FN394008, FN435839 (ITS),
DNA sequencing
FN394009–FN394023, FN435840 (ACT1), FN394066–
Ribosomal DNA regions and ACT1 gene fragments were FN394073 (MET6), and FN394074–FN394080 (COX2).
amplified and sequenced as described previously (Daniel
et al., 2009). Selected isolates of S. cerevisiae were further
Physiological and morphological properties as
characterized by COX2 gene sequences, which were ampli-
conventional classification criteria
fied using the primers COII-3 and COII-5 (Belloch et al.,
2000), and by MET6 gene sequences, which were amplified Physiological and morphological profiles of representative
using the primers MET6-5 and MET6-3 (Gonzáles et al., isolates were determined using the automated microplate
2006). An approximately 600-bp fragment of both genes was method Allev/Biolomics (BioAware SA, Hannut, Belgium)
generated and sequenced using a CEQ 2000 XL capillary of Robert et al. (1997), a yeast identification system based on
automated sequencer (Beckman Coulter, Fullerton, CA). standard taxonomic criteria (Van der Walt & Yarrow, 1984;
After initial BLAST searches for the most similar sequences, Kreger-van Rij, 1987).
Sourdough fermentation-related physiological zero to nine nucleotide differences in ITS sequences, and
characterization zero to four nucleotide differences in the ACT1 gene. The
fingerprint clusters of S. cerevisiae (B1, B2) and the cluster
To investigate the physiological adaptation of the yeast
and the single isolate of C. glabrata (E1, E2) were considered
community to the sourdough fermentation environment,
as intraspecies variants, based on the low variation observed
the following physiological characteristics were determined
in the ACT1 gene, which is evolving faster than ribosomal
for a subset of strains: capacity to assimilate maltose,
sequences. The morphological and physiological character-
glucose, fructose, and sucrose; low pH tolerance; and the
ization of representative isolates was in agreement with the
ability to grow in the presence of acetic acid. Tests were
sequence-based species identification, with the exception of
carried out in duplicate on a 10-mL scale and were in-
the isolates representing cluster C, identified as K. barnettii,
cubated aerobically with periodic agitation at 30 1C for 48 h,
which resembled S. cerevisiae and K. exigua physiologically
except for the K. barnettii isolate D02WW01T02-1, which
(Tables S1, S2).
was incubated at 23 1C. The test medium was composed of
c 2010 Federation of European Microbiological Societies FEMS Yeast Res 10 (2010) 471–481
Published by Blackwell Publishing Ltd. All rights reserved
Yeast species composition in sourdough 475
100
10
20
30
40
50
60
70
80
90
+
*
*
*
+
*
Saccharomyces cerevisiae
D01WW01T01-6 , D01WW01T01-9, D01WW01T01-5,
D03WW01T01-1*, D07WR01T01-6, D07WR02T01-2*,
D07WR01T02-2*, D06SS01T01-2 , D07WR01T02-6,
D03WW01T01-2, D03WW01T01-4, D01WW01T01-2*,
D01WW01T01-3, D07WR01T01-1, D07WR01T01-3,
+ D07WR01T01-4, D03WW01T01-6, D03WW01T01-8,
D03WW01T01-5, D03WW01T01-10, D06SS01T01-3,
D06SS01T01-4, D06SS01T01-6, D06SS01T01-5,
+
+
+
Kazachstania barnettii
D02WW01T02-2, D02WW01T02-5, D02WW01T02-6,
+* C D02WW01T02-1 *, D02WW01T02-3
Saccharomyces cerevisiae
B2 D05WW01T01-2, D05WW01T01-4, D05WW01T01-3,
D05WW01T01-5, D05WW01T01-6, D05WW01T01-1*
*+*
D Kazachstania unispora
+ D11WW01T01-4 *
Wickerhamomyces anomalus
D07WR02T01-3, D08WW01T01-1, D09ME01T01-6 ,
D01WW01T01-7, D01WW01T01-10, D01WW01T01-1,
D01WW01T01-4, D01WW01T01-8, D07WR01T02-5,
+ D07WR02T01-1, D09ME01T01-3, D09ME01T01-4,
D09ME01T01-1, D09ME01T01-2, D07WR01T02-1 ,
A
+ D09ME01T01-5, D07WR01T02-3, D07WR01T02-4,
D11RR01T01-5 , D11RR01T01-6, D11RR01T01-4,
D11RR01T01-1, D11RR01T01-2, D10WW01T01-6a ,
+ D10WR01T01-1a, D08WW01T01-3, D08WW01T01-4,
D08WW01T01-5 , D08WW01T01-6, D08WW01T01-2,
+ D11WW02T01-6, D07WR02T01-5, D07WR02T01-6
Torulaspora delbrueckii
+* F D02WR01T02-3*, D06WW01T01-2
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.
100
20
30
40
50
60
70
80
90
10 Candida glabrata
Saccharomyces cerevisiae
R9-24h2 *, R14-24h2, R9-48h2, R9-96h2, R9-72h2 ,
B1
R9-72h3, R14-72h3, R9-240h1 , R9-48h3, R6-PR48-72h, R9-96h5
Wickerhamomyces anomalus
R14-72-1, R14-120-1, R14-144h, R15-96-3, R15-96-4,
R10-240h1, R12-72h1, R6-PR36-48h, R7-24h2*,
R18R19-24h3, R11-240h2 , R6-PR124-192h, R11-240h1, R11-0h1 ,
R11-96h3 , R15-48-3, R16-48h, R11-72h3,
R16-24h, R6-PR48-72h, R11-48h5, R11-72h2,
R6-PR78-120h, R8R27-24h4, R15-144h1, R6-PR138-216h, R6-
PR140-216h, R12-95h1, R13-48-1, R13-72-1,
R6-PR47-72h, R6-PR19-24h, R14-24-1, R16-144-3,
R6-PR73-120h, R6-PR123-192h, R6-PR107-168h,
R6-PR108-168h, R6-PR35-48h, R12-240h1, R16-72h,
R15-48h1, R15-96-1, R15-24-1, R15-72h , R16-120h,
R12-144h1, R12-168h1, R12-120h1, R12-120h2,
R12-192h1, R12-216h1b, R12-216h1a, R15-96-5*,
R7-48h4, R10-48h5, R10-24h2, R10-24h3b, R10-24h3a, R10-48h1,
R10-48h2, R10-24h1, R7-72h3, R10-72h5,
R10-96h4, R10-240h3, R11-96h4, R10-96h3, R10-240h4, R10-72h3,
A R13-24h1, R13-24h2, R7-72h2, R7-48h2,
R7-72h1, R7-48h, R7-48h3, R7-144h4, R7-144h5, R7-48h5, R7-
72h4, R7-240h4, R7-144h3, R6-PR80-120h,
R6-PR125-192h, R6-PR61-96h, R6-PR122-192h,
R6-PR92-144h, R7-24h1*, R13-24h4, R13-48-2, R13-72-2, R13-
24h3, R10-72h4, R10-96h2, R6-PR141-216h,
R7-240h5, R7-240h1 R6-PR34-48h, R6-PR60-96h,
R8R28-24h3, R8R19-24h1, R8R19-24h2, R11-24h2 ,
R11-72h5, R11-96h2, R10-72h1, R9-24h3 , R9-24h4 ,
R9-24h5, R6-PR93-144h, R11-72h1, R9-24h1 , R11-96h5, R16-96h,
R6-PR18-120h, R9-240h2, R11-48h3,
R6-PR94-144h, R6-PR49-72h , R6-PR139-216h,
R11-48h4, R8R27-24h13 , R8R28-24h1 , R8R28-24h2,
R6-PR143-216h, R6-PR155-240h, R6-PR158-240h,
R6-PR160-240h, R6-PR156-240h , R6-PR157-240h, R8R27-24h2,
R8R27-24h1 , R11-24h3, R7-96h2 ,
R11-72h4 *, R11-240h5, R11-48h2, R11-96h1, R11-48h1, R6-
PR62-96h , R11-240h3, R11-240h4 , R15-192h *
Candida glabrata
E2
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.
c 2010 Federation of European Microbiological Societies FEMS Yeast Res 10 (2010) 471–481
Published by Blackwell Publishing Ltd. All rights reserved
Yeast species composition in sourdough 477
of 17 S. cerevisiae tested (R9-24h2, R9-72h2, R9-96h5, laboratory fermentations without the addition of a starter
D01WW01T01-6, D03WW01T01-3, D06SS01T01-2, culture. Meroth et al. (2003) have shown the dominance of
D11WW01T01-5, D11WW02T01-1, and D11SS01T01-4), S. cerevisiae and C. humilis during controlled fermentations,
and the single tested T. delbrueckii isolate (D06WW01T01- but these yeasts originated from the bakers yeast and
2) incapable of growing under these conditions. Most commercial sourdough starters used, respectively. Moreover,
isolates grew in the presence of 1% acetic acid, except for this is the first study of Belgian artisan sourdoughs.
three isolates of W. anomalus (R7-96h2, R9-24h3, and R15- In the present study, the yeast species identification was
192h) and one isolate of S. cerevisiae (D10SS01T01-7). achieved by M13 PCR-fingerprinting, followed by multiple
gene sequencing and physiological characterization of re-
Species distribution presentative isolates of each fingerprinting group. This
revealed the presence of primarily S. cerevisiae and
The 127 artisan sourdough isolates were identified as
W. anomalus in artisan bakery sourdoughs and W. anomalus
S. cerevisiae (68%), W. anomalus (26%), and K. barnettii
Fermentation Mill Flour Yeast species 0 24 48 72 96 120 144 168 192 216 240
R6 D12 Spelt W. anomalus 2 3 2 3 3 3 3 4 5 5
Spelt A C. glabrata 2 1 1 1
S. cerevisiae 1
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 et al., 2007).
identified for the first time from sourdough, was the only best-known association of yeasts and lactic acid bacteria in
yeast isolated from the bakery sourdough D02W01T01. This sourdough is that of the maltose-negative yeasts K. exigua or
species might have been misidentified as S. cerevisiae or C. humilis with the maltose-positive L. sanfranciscensis in
K. exigua in studies using phenotypical identification meth- San Francisco sourdough and Panettone, respectively
ods. This same sourdough was the only bakery sourdough (Gobbetti et al., 1994; Ottogalli et al., 1996). This stable
dominated by a maltose-negative yeast species, which con- association has been attributed to the fact that, unlike
forms to a mutualistic association with maltose-utilizing L. sanfranciscensis, K. exigua and C. humilis are unable to
Lactobacillus sanfranciscensis (Ottogalli et al., 1996). The ferment maltose, thereby avoiding nutritional competition
c 2010 Federation of European Microbiological Societies FEMS Yeast Res 10 (2010) 471–481
Published by Blackwell Publishing Ltd. All rights reserved
Yeast species composition in sourdough 479
and catabolite repression by glucose (Gobbetti et al., 1994). from spelt sourdoughs, while present in rye and wheat
This interaction is further enhanced by the reported excre- sourdoughs (Van der Meulen et al., 2007). Although the
tion of glucose upon maltose consumption through maltose same sourdoughs were included in the present study, no
phosphorylase activity by L. sanfranciscensis, which can then such cereal-related difference could be observed for the
serve as an energy source for the maltose-negative yeasts composition of the yeast population.
(Gobbetti et al., 1994; Stolz et al., 1996). The species In laboratory fermentations, typically, the final species
K. unispora and T. delbrueckii can be considered as minor composition was established after the first week, although
components of the sourdough microbiota, as they were not all species could be cultured at all sampling times. The
represented by single isolates in doughs dominated by bacterial populations and metabolite profiles of the four
S. cerevisiae. fermentations analysed by Van der Meulen et al. (2007) had
Sequencing of two additional protein-coding genes stabilized after the same fermentation time.
(MET6, COX2) allowed a better understanding of the To conclude, the 127 yeast isolates from artisan bakery
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