ARTICLE IN PRESS

FOOD
MICROBIOLOGY
Food Microbiology 24 (2007) 592–600
www.elsevier.com/locate/fm

Identification of subdominant sourdough lactic acid bacteria and their

evolution during laboratory-scale fermentations
Aldo Corsetti, Luca Settanni, Sara Valmorri, Mario Mastrangelo, Giovanna Suzzi
Dipartimento di Scienze degli Alimenti, Sezione di Microbiologia Agro-Alimentare ed Ambientale, Università degli Studi di Teramo,
V.C.R. Lerici 1, 64023 Mosciano Sant’Angelo, Teramo, Italy
Received 22 September 2006; received in revised form 12 December 2006; accepted 2 January 2007
Available online 12 January 2007

Abstract

Presumptive lactic acid bacterial cocci were found in six sourdoughs (out of 20) from the Abruzzo region (central Italy) and subjected
to phenotypic and genotypic characterization. A total of 21 isolates, recognized as seven strains by randomly amplified polymorphic
DNA (RAPD)–polymerase chain reaction (PCR) typing, were identified by a polyphasic approach, consisting of 16S rRNA gene
sequencing, multiplex PCR assays and physiological features, as Enterococcus faecium and Pediococcus pentosaceus. Four strains
belonging to those species and previously isolated from wheat kernels were inoculated in sterile flour to verify their capacity to grow in
sourdough environment. Doughs with several dual bacterial combinations, including Lactobacillus sanfranciscensis, were propagated for
11 days and pH measurements and bacterial counts were carried out.
r 2007 Elsevier Ltd. All rights reserved.

Keywords: Co-fermentations; Enterococcus faecium; Lactobacillus sanfranciscensis; Pediococcus pentosaceus; Sourdough; Lactic acid bacteria

1. Introduction et al., 2002; Meroth et al., 2003). Italian wheat sourdoughs

The modern biotechnology of baked goods largely uses
sourdough in fermentation because of the many advan-
tages it offers over baker’s yeast. In a mature sourdough, a
mixed population of different lactic acid bacteria (LAB)
and yeasts represent the characteristic fermenting micro-
flora (Corsetti et al., 1996). LAB are fundamental for the
properties of sourdough: lactic fermentation, proteolysis,
synthesis of volatile compounds, anti-mould and anti-
ropiness are the most important activities during dough
leavening (Gobbetti, 1998; Hammes and Gänzle, 1998).
Typical sourdough LAB mainly belong to the genus
Lactobacillus and include obligately and facultatively
heterofermentative and obligately homofermentative spe-
cies (Hammes and Vogel, 1995).
Many works have been forwarded to the characteriza-
tion of LAB population of sourdoughs produced in
different countries (Galli et al., 1988; Gabriel et al., 1999;
Rocha and Malcata, 1999; Corsetti et al., 2001; De Vuyst
Corresponding author. Tel.: +39 861 266896; fax: +39 85 8071509.
E-mail address: acorsetti@unite.it (A. Corsetti).
have been extensively studied (Gobbetti et al., 1994;
Corsetti et al., 1998, 2001, 2003; Foschino et al., 1999;
Settanni et al., 2005a; Catzeddu et al., 2006; Valmorri et al.,
2006) and the dominant Lactobacillus species are reported
to be Lactobacillus sanfranciscensis, Lactobacillus brevis,
Lactobacillus fermentum, Lactobacillus fructivorans and
Lactobacillus rossiae belonging to the obligately hetero-
fermentative group; Lactobacillus plantarum and Lactoba-
cillus alimentarius, belonging to the facultatively
heterofermentative group; and Lactobacillus acidophilus,
Lactobacillus delbrueckii subsp. delbrueckii and Lactoba-
cillus farciminis, belonging to the obligately homofermen-
tative lactobacilli.
Besides the well characterized group of lactobacilli, some
works reported on the presence of non-Lactobacillus
species belonging to the group of LAB: Enterococcus
casseliflavus, Enterococcus durans, Enterococcus faecium,
Lactococcus lactis, Leuconostoc gelidum, Leuconostoc
mesenteroides subsp. mesenteroides, Leuconostoc mesenter-
oides subsp. dextranicum, Pediococcus spp., Pediococcus
acidilactici, Pediococcus cerevisiae, Pediococcus pentosa-
ceus, Streptococcus constellatus, Streptococcus equinus,

0740-0020/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2007.01.002
 ARTICLE IN PRESS
A. Corsetti et al. / Food Microbiology 24 (2007) 592–600
Weissella viridescens, Weissella confusa and Weissella
cibaria (Azar et al., 1977; Spicher, 1984; Spicher, 1987;
Infantes and Tourneur, 1991; Faid et al., 1994; Coppola
et al., 1996; Hamad et al., 1997; Galli et al., 1988; Rocha
and Malcata, 1999; Corsetti et al., 2001; De Vuyst et al.,
2002; Neysen, 2004; Gül et al., 2005; Valmorri et al., 2006).
The present study is part of a project aimed at
characterizing the microflora of 20 regional Abruzzo
sourdoughs (see also Valmorri et al., 2006). The objectives
of this study were to count, isolate and identify LAB cocci
isolated from the Abruzzo region sourdoughs; and to
follow the fate of some E. faecium and P. pentosaceus
strains, previously isolated from wheat grains, mixed at the
same cell concentration of Lactobacillus sanfranciscensis in
doughs made with sterile wheat flour.
2. Materials and methods
2.1. Strains and growth conditions
E. faecium DSM 20477T, E. durans DSM 20633T,
Lactococcus lactis subsp. lactis DSM 20481T, Leuconostoc
mesenteroides DSM 20343T, P. acidilactici LMG 11384T
and P. pentosaceus DSM 20336T were the type strains used
in this study to evaluate strain polymorphism of isolates
from the Abruzzo region sourdoughs by randomly
amplified polymorphic DNA (RAPD)–polymerase chain
reaction (PCR) analysis. The strains were propagated for
24 h as indicated by the respective culture collection. In
order to perform sourdough co-fermentation experiments,
some strains belonging to culture collection of the
Department of Food Science (Section of Agri-Food and
Environmental Microbiology) of University of Teramo
(Italy) were used: E. faecium WGJ4.1 and WGK64,
P. pentosaceus WGX15.1 and WGK32.2, isolated from
wheat grains (Corsetti et al., under submission), were
cultured in MRS (Oxoid, Milan, Italy) broth at 30 1C for
24 h; Lactobacillus sanfranciscensis CH8f, identified from
sourdough (Valmorri et al., 2006), was propagated in
sourdough bacteria (SDB) broth (Kline and Sugihara,
1971) at 30 1C for 24 h.
2.2. Sourdough characteristics, isolation and preliminary
characterization of LAB cocci
The characteristics of 20 sourdoughs collected through-
out the Abruzzo region used for microbiological analyses
and the isolation of LAB have been reported in a paper
recently published by Valmorri et al. (2006). Gram-positive
(by Gregersen’s KOH methods (Gregersen, 1978)), cata-
lase-negative (determined by transferring fresh colonies
from agar medium to a glass slide and adding 5% H2O2),
non-motile, cocci (n ¼ 21) were maintained at 80 1C in
glycerol stocks.
In this study, the cultures were purified by successive
subculturing in M17 (Oxoid) agar. In order to perform a
593
first clustering, the isolates were subjected to physiological
tests as described by Corsetti et al. (2001).
2.3. DNA extraction
For the preparation of genomic DNA for PCR assays of
sourdough isolates, cells from 2 ml of overnight cultures
grown in M17 at 30 1C were harvested and DNA was
extracted according to the method of De Los Reyes-
Gavilán et al. (1992). The final concentration of lysozyme
used for cell lysis was 2 mg ml1. The concentration and
purity of DNA were assessed by a biophotometer
(Eppendorf AG, Hamburg, Germany).
DNA of colonies collected from plate counts of
sourdough co-fermentation experiments was extracted as
follows: colonies were suspended in 20 ml of microLYSISTM
buffer (Labogen, Terrazzano di Rho, Italy) and subjected
to a heating–cooling treatment, performed by means of a
thermal cycler (MyCycler; Bio-Rad, Hercules, CA, USA)
as indicated by the buffer supplier.
2.4. RAPD–PCR analysis and genotypic identification of
LAB cocci
In order to perform a strain differentiation, all isolates
from sourdough were analysed by RAPD–PCR using
primers P4 (Corsetti et al., 2003) and M13 (Stendid et al.,
1994), with arbitrarily chosen sequences. Reaction mixtures
contained 200 mmol l1 of each 20 -deoxynucleoside 50 -tripho-
sphate, 1 mmol l1 primer, 1 mmol l1 MgCl2, 1.25 U of Taq
DNA polymerase (Life Technologies, Milan, Italy), 2.5 ml of
10  PCR buffer, ca. 25 ng of DNA, and sterile bi-distilled
water to bring the final volume to 25 ml. The PCR programme
reported by Corsetti et al. (2003) was used for primers P4,
whereas that reported by Zapparoli et al. (1998) was used for
primer M13. The same RAPD–PCR method was applied to
examine colonies isolated from doughs produced for co-
fermentation studies with the following modification: 1 ml of
cell lysate was used as template DNA. RAPD profiles were
acquired using the Gel Doc EQ System (Bio-Rad), compared
by Fingerprinting II InformatixTM Software (Bio-Rad) and
subjected to cluster analysis performed with Pearson
similarity coefficient and unweighted pair group method
using arithmetic averages (UPGMA) dendrogram type.
Eleven representative strains were subjected to 16S
rRNA gene sequence analysis using the LacbF/LacbR
primer pair following the method of Corsetti et al. (2004).
A sodA gene-based multiplex PCR system (Jackson et al.,
2004) was also necessary to verify E. faecium identity, while
a multiplex PCR targeting the 16S rRNA and ldhD genes
(Mora et al., 1997) was applied to distinguish between
P. pentosaceus and P. acidilactici.
2.5. Dough production, propagation and analysis
In order to verify their ability to grow in sourdough,
except Lactobacillus sanfranciscensis CH8F which is known
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594 A. Corsetti et al. / Food Microbiology 24 (2007) 592–600

for the fermentation of only glucose and maltose, the other
strains used as sourdough starters were first inoculated in
MRS containing one of the main four fermentable sugars
of flour (glucose, fructose, sucrose and maltose) or starch
at a concentration of 20 g l1 and checked for their growth
at 30 1C for 3 days. After that, starter cultures were
cultivated in glucose MRS at 30 1C for 24 h. Cells were
harvested by centrifugation at 6500g for 10 min, washed
twice with sterile distilled water, and then re-suspended in
sterile Ringer’s solution (Sigma Aldrich Chemical Co,
Milwaukee, WI, USA); the cell suspension, diluted 1:10,
gave an OD620 of 1.25, which roughly corresponds to a
concentration of 109 cfu ml1. Before mixing, the cell
suspensions were further diluted until 105 cfu ml1. Wheat
flour, containing 12.5% moisture, 11% of dry matter (dm)
proteins (N  5.70), 1.82% of dm fat and 0.60% of dm ash,
was sterilized by g-ray (25 kGy) treatment (Gammatom,
Guanzate, Italy). Wheat flour (50 g), cellular suspension
(7.6 ml for each strain) and tap water [enough to bring the
volume of the cellular suspension(s) to 30 ml], were
manually mixed to produce doughs with a dough yield
(weight of the dough/weight of the flour  100) of 160 and
with a cellular concentration of about 104 cfu g1. A
reference dough (R) without starter was also produced.
Doughs (80 g) were individually placed in sterile plastic
beakers and incubated at 30 1C for 6 h. At the end of this
fermentation the sourdoughs were stored at 4 1C for about
18 h after which each sourdough (16 g) was mixed with
wheat flour (40 g) and tap water (24 ml), incubated at 30 1C
for 6 h and stored again at 4 1C for about 18 h. Following
this procedure the sourdoughs were propagated daily for a
period of 11 days at 30 1C.
Sourdough samples were taken and analysed immedi-
ately after mixing and after every 6 h of fermentation. Ten
grams of each sample were suspended in 90 ml of sterile
physiological solution, homogenized in a Stomacher (LAB
Blender 400, Seward Medical, London, UK) for 2 min at
260 rev min1 and serially diluted. Depending on the LAB
added as starter, plate counts were performed using SDB
and MRS containing lactose instead of glucose (lMRS) at
30 1C for 48 h. The presence of microorganisms added as
starter was also confirmed by microscopic analysis and,
after colony isolation, by RAPD–PCR analysis. The pH
was determined by a MP 220 pH meter (Mettler-Toledo,
Schwerzenbach, Switzerland).
3. Results
3.1. Grouping and identification of LAB cocci
Twenty-one bacterial cocci, collected from six of a total
of 20 sourdoughs previously analysed by Valmorri et al.
(2006), were, following Gram (positive) and catalase
(negative) reactions, considered as presumptive LAB.
Doughs and isolate distribution are reported in Table 1.
LAB cocci were found at concentrations approximately
ranging between 104 and 106 cfu g1. The isolates were
physiologically divided into two main groups on the basis
of different phenotypic characteristics, including morphol-
ogy, growth temperature, CO2 production from glucose,
NH3 production from arginine, esculin hydrolysis (Corsetti
et al., 2001) and growth on kanamycin aesculin azide
(KAA) medium (Oxoid). In Table 1, only morphology and
growth on KAA are reported. Genotypic characterization,
by RAPD–PCR analysis, was fully in agreement with
physiological grouping, since also RAPD–PCR profiles
recognized two clusters: A (n ¼ 7) and B (n ¼ 14) (results
not shown). Eleven strains (four from dough AV, two from
doughs CT and OF and one from doughs CO, SU and TC)
were further analysed by 16S rRNA gene sequencing and
multiplex PCRs and they were identified as E. faecium
(n ¼ 8) and P. pentosaceus (n ¼ 3).
3.2. Evaluation of the interaction between subdominant
sourdough LAB and Lactobacillus sanfranciscensis
On the basis of the LAB cocci species (E. faecium and
P. pentosaceus) isolated from the Abruzzo region sour-
doughs and since the same species were often isolated from
wheat grains (Corsetti et al., under submission), in the
present study different doughs (Table 2, Figs. 1–3) were
prepared and propagated in order to follow the evolution of
the representative subdominant sourdough LAB in combi-
nation with the key sourdough LAB Lactobacillus san-
franciscensis (Gobbetti and Corsetti, 1997). In particular,
four strains (two E. faecium and two P. pentosaceus) of
wheat seed origin were coupled with the sourdough
Lactobacillus sanfranciscensis CH8F (four combinations)
and inoculated in sterile flour to mimic a real situation when
microorganisms from flour are mixed to those of sour-
dough. Prior mixing, flour was subjected to g-ray treatment

Table 1
Characteristics of LAB cocci isolated from sourdoughs

Sourdougha Concentration (cfu g1) No. of isolates LAB morphology Growth on KAA Species found No. of strains

AV 106 4 Ovoid, short chains Positive E. faecium 2

CO 104 3 Ovoid, short chains Positive E. faecium 1
CT 104 4 Spherical, tetrads Negative P. pentosaceus 1
OF 106 4 Ovoid, short chains Positive E. faecium 1
SU 106 3 Ovoid, pairs Positive E. faecium 1
TC 105 3 Spherical, tetrads Negative P. pentosaceus 1
a
See Valmorri et al. (2006) for sourdough origin.
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A. Corsetti et al. / Food Microbiology 24 (2007) 592–600 595

Table 2
Kinetics of growth and acidification of selected LAB during co-fermentation experiments in sourdoughs propagated for 11 days

Sourdoughsa Startersb pH T0 log cfu g–1 T0c pH 6 h log cfu g–1 6 h

A WGJ4.1; CH8f 5.8870.04 3.6270.23; 3.7070.12 4.9070.05 6.6570.07; 3.9870.08

A1 5.6970.04 NR 4.4170.03 7.7570.14; 4.6370.06
A2 5.4470.05 NR 4.2970.11 8.1770.08; 6.3670.16
A3 5.3670.01 NR 4.3270.07 8.4370.05; 7.3770.12
A4 5.2570.04 NR 4.1370.08 7.4670.22; 8.3870.14
A5 5.1070.01 NR 4.0470.07 7.0370.04; 8.2870.11
A6 5.1670.05 NR 4.0670.04 6.9070.07; 8.4170.07
A7 5.1970.05 NR 3.9270.04 6.6070.09; 8.3170.06
A8 5.0970.03 NR 3.9570.04 6.5670.10; 8.3570.07
A9 5.1070.03 NR 3.9070.02 6.4570.07; 8.4070.08
A10 5.1070.01 NR 3.9170.04 6.5170.07; 8.3370.06
B WGK64; CH8f 5.8570.04 3.4570.24; 3.7770.03 4.9970.02 6.5170.05; 3.8770.04
B1 5.7470.04 NR 4.4270.04 7.5770.11; 4.4870.07
B2 5.4870.03 NR 4.3670.07 8.0870.05; 6.2970.09
B3 5.4170.08 NR 4.3870.06 8.3570.10; 7.5470.13
B4 5.3170.04 NR 4.1070.04 7.5470.10; 8.3370.06
B5 5.0670.04 NR 3.9870.02 7.1570.12; 8.4270.07
B6 5.1470.08 NR 4.0270.04 6.9370.11; 8.3970.07
B7 5.1170.04 NR 3.8970.06 6.5470.11; 8.2370.10
B8 5.1270.04 NR 3.9370.03 6.5070.09; 8.3770.07
B9 5.0970.01 NR 3.9370.03 6.4870.04; 8.3270.11
B10 5.1070.03 NR 3.9370.04 6.5470.12; 8.2970.08
C WGX15.1; CH8f 5.8870.02 3.5170.07; 3.7370.08 4.9370.04 6.7870.07; 4.0170.10
C1 5.7670.01 NR 4.0070.03 7.8070.04; 4.7370.05
C2 5.0170.01 NR 3.9570.04 8.5270.02; 6.6270.14
C3 4.9270.04 NR 3.9170.02 8.1270.12; 7.6870.10
C4 4.9370.04 NR 4.0170.04 7.7970.05; 8.4470.11
C5 4.9270.08 NR 3.9570.05 7.4470.07; 8.3270.09
C6 5.1370.08 NR 3.8870.02 6.9970.03; 8.4270.05
C7 5.0970.04 NR 3.8870.06 6.8670.10; 8.3970.11
C8 5.0770.04 NR 3.9170.04 6.9070.08; 8.3570.09
C9 5.0970.04 NR 3.8770.03 6.3670.07; 8.3670.06
C10 5.1070.02 NR 3.9070.03 6.4170.10; 8.2870.08
D WGK32.2; CH8f 5.8470.03 3.7270.08; 3.6070.26 4.9170.01 6.8070.07; 3.9670.05
D1 5.7170.06 NR 3.9570.04 7.8670.12; 4.5870.03
D2 5.0070.01 NR 3.9370.01 8.4270.14; 6.5270.17
D3 4.9070.06 NR 3.8970.00 8.2470.09; 7.4170.13
D4 4.9070.07 NR 3,9870.09 7.8770.02; 8.2070.11
D5 4.8970.12 NR 3.9570.07 7.3470.08; 8.3670.09
D6 5.0370.07 NR 4.0370.07 7.0170.01; 8.4070.10
D7 5.1470.07 NR 3.9370.04 6.8670.10; 8.3970.11
D8 5.0770.05 NR 3.9370.03 7.0370.08; 8.3870.09
D9 5.0570.05 NR 3.8870.06 6.4470.06; 8.2670.06
D10 5.0770.03 NR 3.9170.01 6.3970.10; 8.3070.05
R Without starter 5.95 0 5.95 0
R1 5.94 ND 5.95 ND
R2 5.92 ND 5.92 ND

NR, not reported; ND, not determined.

a
Letters not followed by any numbers indicate the sourdough started with cellular suspension. After 6 h of fermentation each sourdough was used as a
starter for the next dough. The number indicate the step of propagation.
b
Microorganisms used as starter cultures are termed by the collection reference abbreviation: WGJ4.1 and WGK64, E. faecium; WGX15.1 and
WGK32.2, P. pentosaceus; CH8F, Lactobacillus sanfranciscensis.
c
Results, except those of R for which experiments were carried out in single repetitions, indicate mean7SD of two independent experiments.

to kill viable microbial cells while avoiding modifications of
its chemical composition due to the thermal sterilization;
after that the raw material was chemically identical to that
employed in the industrial processes.
To approach the microflora dynamics during a realistic
situation of competition, all LAB strains included in the
present work were added to the doughs at a starting
concentration of about 104 cfu g1. This is about 103-fold
lower as compared with the inoculation levels of typical
LAB added or present in a fermented sourdough used as
starter.
The results of sourdough fermentation obtained by
individual and mixed starters are reported in Table 2 and a
representative dual combinations (A, Table 2) is schema-
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596 A. Corsetti et al. / Food Microbiology 24 (2007) 592–600

6.5

5.5

5
pH

4.5

3.5

3
0T0 06h 1T0 16h 2T0 26h 3T0 36h 4T0 46h 5T0 56h 6T0 66h 7T0 76h 8T0 86h 9T0 96h 10T0106h
Step of propagation

Fig. 1. Kinetics of acidification of selected LAB during individual fermentation of sourdoughs propagated for 11 days. Symbols: E, E. faecium WGJ4.1;
J, E. faecium WGK64; ’, P. pentosaceus WGX15.1; n, P. pentosaceus WGK32.2; &, Lactobacillus sanfranciscensis CH8f.

9.00

8.00

7.00
log cfu g-1

6.00

5.00

4.00

3.00
0 1 2 3 4 5 6 7 8 9 10
Step of propagation

Fig. 2. Kinetics of growth of selected LAB during individual fermentation of sourdoughs propagated for 11 days. Results refer to cell concentration at the
end of each propagation step. Symbols: E, E. faecium WGJ4.1; J, E. faecium WGK64; ’, P. pentosaceus WGX15.1; n, P. pentosaceus WGK32.2; &,
Lactobacillus sanfranciscensis CH8f.

tically presented in Fig. 3. Doughs with single starter were
produced to follow the individual evolution of the strains
and, especially, to evaluate the capacity of E. faecium and
P. pentosaceus strains to grow in sourdough (Figs. 1 and 2).
Propagation of doughs were carried out for 11 days. The
value of bacterial concentration and pH did not change
significantly from the third step onward for E. faecium and
P. pentosaceus and from the fifth refreshment onward in
case of Lactobacillus sanfranciscensis CH8f. Starting from a
pH of 5.94–5.95 at time zero (T0), doughs prepared with
individual starters reached the lowest pH at different times.
In particular, E. faecium strains decreased the pH of
doughs till 4.31–4.39 at the end of 6 h fermentation of the
first propagation step while P. pentosaceus were stronger
dough acidifier reaching, after the first refreshment, a pH
of 3.84–3.89 (Fig. 1). In both cases, the pH values reached
at the end of the third refreshments were maintained till the
end of the study and cell counts were higher than
8.00 log cfu g1 from the third propagation step onward
(Fig. 2), thus demonstrating the ability of those strains to
use sugars of flour and grow in sourdough ecosystems. In
broth media, the four bacterial cocci were able to ferment
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A. Corsetti et al. / Food Microbiology 24 (2007) 592–600 597

9.00

8.00

7.00
log cfu g-1

6.00

5.00

4.00

3.00
0 1 2 3 4 5 6 7 8 9 10
Step of propagation

Fig. 3. Evolution of E. faecium WGJ4.1 (’) and Lactobacillus sanfranciscensis CH8f (J) during sourdough co-fermentation. Results refer to cell
concentration at the end of each propagation step. The panel is representative of dough A of Table 2.

glucose, maltose, fructose and sucrose, but not starch. On
the contrary, Lactobacillus sanfranciscensis CH8f was
found to be a slower acidifier. In fact, the dough started
with this strain (initial pH ¼ 5.96) reached the lowest pH
(3.89) (Fig. 1) concomitantly with the highest cell
concentration (8.50 log cfu g1) (Fig. 2), after the fifth
propagation step. Comparable results were observed till the
eleventh refreshment.
A selective growth of E. faecium WGJ4.1 and WGK64,
P. pentosaceus WGX15.1 and WGK32.2 and Lactobacillus
sanfranciscensis CH8f was possible using lMRS and SDB.
The former medium was useful to easily distinguish lactose
fermenting enterococci and pediococci from the lactose-
negative Lactobacillus sanfranciscensis after growth in
association. All five strains were able to develop in SDB.
The presence and identity of inoculated strains (E. faecium
WGJ4.1 and WGK64, P. pentosaceus WGX15.1 and
WGK32.2 and Lactobacillus sanfranciscensis CH8f) was
monitored following isolation from dough, by microscopic
inspection and comparing their RAPD profiles to those
obtained from the original cultures (data not shown). The
doughs started with the associations E. faecium WGJ4.1/
Lactobacillus sanfranciscensis CH8f and E. faecium
WGK64/Lactobacillus sanfranciscensis CH8f showed, at
the end of the first refreshment, a decrease of pH till the
value obtained in the individual fermentations of enter-
ococci. The highest cell counts for LAB cocci
(8.08–8.35 log cfu g1), similarly to individual propagation,
were observed at the end of the second and third
propagation step (Table 2, Fig. 3). The same behaviour
was found in case of the associations P. pentosaceus
WGX15.1/Lactobacillus sanfranciscensis CH8f and
P. pentosaceus WGK32.2/Lactobacillus sanfranciscensis
CH8f. In all four combinations, a decrement of LAB cocci
counts were obtained from the fourth refreshment
(Table 2) concomitantly with the increase till maximum
concentration (8.20–8.44 log cfu g1) of Lactobacillus san-
franciscensis. The decrement of LAB cocci was registered
until the seventh propagation step for enterococci and the
ninth for pediococci, after that they remained constant
during the rest of the experiment (Table 2). Lactobacillus
sanfranciscensis CH8f was counted at about 109 cfu g1
from the fourth refreshment onward (Table 2, Fig. 3). No
acidifying activity was showed by the reference dough (R)
produced without starters and processed as the doughs
added with starter cultures; pH values and plate counts
(Table 2) confirmed the absence of spontaneous flour
microflora after g-ray treatment.
4. Discussion
Knowledge of fermenting microorganisms plays a
defining role in the process of fermentation standardization
and it is also of paramount importance to have an
exhaustive view of microbial interactions. This work was,
in fact, aimed towards the characterization of the
subdominant sourdough LAB and then, on the basis of
those results, to the study of the influence on the
interactions between LAB during laboratory-scale sour-
dough fermentations.
Concentrations of LAB cocci were in the range of
10–104 cfu g1 lower than rod LAB previously counted in
the 20 sourdoughs of the Abruzzo region (Valmorri et al.,
2006). The species detected were E. fecium and
P. pentosaceus that are reported to be isolated from
sourdough matrices of different geographical origin. In
particular, E. fecium was detected in traditional maize
Portuguese (Rocha and Malcata, 1999) and wheat German
(Kitahara et al., 2005) sourdough while P. pentosaceus was
identified in rye Swedish (Lönner et al., 1986), wheat
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598 A. Corsetti et al. / Food Microbiology 24 (2007) 592–600
Moroccan (Boraam et al., 1993), French (Infantes and
Tourneur, 1991), Belgian (Neysen, 2004) and German
(Kitahara et al., 2005) sourdoughs. Commonly, all
sourdough LAB (rods and cocci) are counted together
and plate counts refer to the concentrations of lactobacilli
that are present at the highest dilutions, thus in general,
LAB cocci numbers, being lower than rod LAB, are not
reported. However, in one case (Kitahara et al., 2005), it
was reported that the cell counts of E. fecium and
P. pentosaceus which were 6.30 and 7.30 log cfu g1,
respectively, concentrations about 2–3 log cycles lower
than that of Lactobacillus sanfranciscensis (9.60 log cfu g1)
detected in that work. Our data and those so far published
highlight the limited importance that enterococci and
pediococci may have in mature sourdoughs where lacto-
bacilli dominate. Enterococci are natural inhabitants of the
intestine of warm-blooded animals (Devriese et al., 1992)
and, as members of the group of LAB, they generally
appear and participate in food and feed fermentations
(Franz et al., 1999). In particular, E. faecium appears to be
frequent in different types of processed foods (Stiles et al.,
1978), such as cheeses (Saavedra et al., 2003), sausages
(Herranz et al., 2001) and olives (Lavermicocca et al.,
1998). P. pentosaceus is usually found in fermented
vegetables (Fleming and McFeeters, 1981), such as olives
(Oliveira et al., 2004) and suerkrauts (Pederson and
Albury, 1969).
Cereal grains are naturally contaminated by eucaryotic
(moulds and yeasts) and procaryotic (bacteria) organisms.
Those microorganims might follow the different phases of
flour preparation and, thus, be found in products made
with flour, such as sourdoughs. From our recent char-
acterization of LAB associated with wheat kernels (Cor-
setti et al., under submission), it emerged that the species
found, mainly enterococci, pediococci and atypical lacto-
bacilli, are different from those characteristics of mature
sourdoughs; thus, in the present study, we performed
several co-fermentations in order to better understand the
evolution of sourdough LAB during manufacturing. With
the aim of studying sourdough LAB interactions, in
previous papers (Corsetti et al., 2004; Settanni et al.,
2005b), we demonstrated that in situ active bacteriocin-
producing LAB influence the microbial consortium of
sourdough LAB and that Lactococcus lactis ssp. lactis
M30, a strain isolated from barley grains (Hartnett et al.,
2002), may support the dominance of insensitive strains,
such as those belonging to Lactobacillus sanfranciscensis
species, useful to confer good characteristics to the dough.
Although enterococci occur as commensals of the
gastrointestinal tract of warm-blooded animals, they may
also display subtle virulence traits (Jett et al., 1994; Mundy
et al., 2000) and certain strains of enterococci have emerged
as leading causes of nosocomial infection, including
urinary tract infections, wound infections, and bacteraemia
(Franz et al., 1999). The use of certain Enterococcus strains
as starters in food fermentation must consider the absence
of virulence and the inability to transfer antibiotic
resistance to other microorganisms and to acquire resis-
tance to vancomycin themselves. Enterococcal starter
strains should be chosen among those proposed as
probiotics and they should also be able to produce
bacteriocins showing a broad inhibitory spectrum, includ-
ing pathogenic, toxigenic and food-spoilage bacteria.
Regarding sourdough fermentation, there are no reports
on the use of E. faecium as starter culture. On the contrary,
some works have dealt with the application of
P. pentosaceus strains to start doughs: Katina et al.
(2002) employed a P. pentosaceus strain (VTT E-90390)
able to inhibit the growth of rope-forming Bacillus strains
in order to evaluate their capacities in situ; Paramithiotis
et al. (2005) evaluated the effects on the total metabolite
production of some LAB, including P. pentosaceus,
representing the complementary microflora of sourdough,
when combined with the major microflora consisting of
Saccharomyces cerevisiae and Lactobacillus sanfranciscensis
during a traditional Greek three-stage procedure. Further-
more, P. pentosaceus was reported to be the typical
microorganism during the production of Sudanese Kisra,
a traditional sourghum flour fermentation (Mohammed et
al., 1991): it was dominant at the end of the first 24-h
fermentation, after flour and water mixing, and in three
consecutive 9 h refreshments. To our knowledge there is no
work reporting on the prevalence of E. faecium strains in
sourdough environments at the end of fermentation.
Our results on the co-fermentations of E. faecium/
Lactobacillus sanfranciscensis and P. pentosaceus/Lactoba-
cillus sanfranciscensis, carried out in the conditions of type
I sourdough (continuous daily refreshments at 30 1C),
showed that subdominant LAB such as E. faecium and
P. pentosaceus are stronger acidifiers than Lactobacillus
sanfranciscensis at the beginning of sourdough production.
Those species that, inhibiting indigenous microorganisms
other than LAB by lowering the pH, might prepare the
environment for the establishment of the typical species
(e.g. Lactobacillus sanfranciscensis) of mature sourdoughs.
Nevertheless, once Lactobacillus sanfranciscensis colonized
doughs (ca. 109 cfu g1), it determined a rapid decrement of
LAB cocci which remained at subdominant concentrations
(ca. 107 cfu g1). Lactobacillus sanfranciscensis, due to
utilization of specific amino acids and peptides, presence
of maltose phosphorylase and pronounced proteolytic
activity, is physiologically well adapted to the sourdough
environment (Gobbetti and Corsetti, 1997).
The present work showed that E. faecium and
P. pentosaceus are the main LAB cocci found in the
regional Abruzzo sourdoughs and that strains belonging to
those species, isolated from wheat grains, determine a
quick acidification of doughs. Thus, they are confirmed to
be species important in sourdough fermentation and their
most crucial role is played at the beginning of sourdough
production. Further investigations are needed in order to
evaluate subdominant LAB evolution and persistence
during sourdough propagation with non-sterile flour in
presence of contaminant microorganims.
 ARTICLE IN PRESS
A. Corsetti et al. / Food Microbiology 24 (2007) 592–600
Acknowledgements
The authors wish to thank the company Gammatom
(Guanzate, Italy) for kindly sterilizing flour samples by
gamma-ray treatment.
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