Food Microbiology 26 (2009) 728733

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

Comparative study of culture media used for sourdough lactobacilli

Annabelle Vera a, b, *, Veronique Rigobello a, Yann Demarigny a
a
b

Departement Production Agroalimentaire et Qualite, Unite Ecosyste`mes microbiens complexes, ISARALyon, 23 rue Jean Baldassini, 69364 Lyon, France
Entreprise Philibert Savours, Le Jouvancy, 01290 Pont de Veyle, France

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 15 May 2009
Received in revised form
12 July 2009
Accepted 13 July 2009
Available online 18 July 2009

Eleven culture media were analysed to compare their exhaustiveness for the quantication of lactobacilli
type I sourdoughs. The media tested were MRS, maltose modied MRS, MRS5, SDB, SFM, MRS Vogel,
Rogosa as elective media, and FH, OH, LAMVAB, KCA as selective media. Six broth media were also tested
for colony subculture. Quantitatively, maltose MRS and MRS media showed the highest counts
respectively 9.47 and 9.43 mean log(cfu)/g. SFM and Rogosa media presented slighlty lower levels 9.1
and 9.24 log(cfu)/g respectively. Levels on the remaining media were signicantly lower lesser than
0.5 log(cfu)/g compared to maltose MRS and MRS media. From a qualitative standpoint, the bacterial
diversity, based on the comparison of the whole-cell protein patterns of isolates, was the largest on
maltose MRS medium. However, MRS5 medium allowed the isolation of a specic population only found
on this medium. For colony subculture, maltose MRS and MRS5 appeared the most appropriate broth
media. These results underline that there is no really efcient medium for the systemic study of sourdoughs. Therefore, the rst step before working on these ecosystems implies a deliberate choice of the
most appropriate media combination.
2009 Elsevier Ltd. All rights reserved.

Keywords:
Sourdough
Lactobacilli
Culture media

1. Introduction
Traditional type I sourdoughs are a mixture of our and water
fermented by lactic acid bacteria (LAB) and continuously propagated through backslopping at ambient temperature. Their pH is
generally close to 4. Sourdough microbiota is largely dominated by
LAB (>8 log(cfu)/g) and to a lesser extent by yeasts. The LAB:yeasts
ratio on average is close to 100:1. More than 50 LAB species have
been isolated from this ecosystem, mainly belonging to the genus
Lactobacillus (30 species) and more than 20 yeast species, Saccharomyces and Candida being the most frequent genera (De Vuyst and
Neysens, 2005; Hammes et al., 2005).
Sourdough is a complex food ecosystem in which lactobacilli are
highly adapted to the environmental conditions (temperature, pH,
acidity, maltose as the most abundant fermentable carbohydrate
and fructose as potentially metabolic electron acceptor, antimicrobial products, etc.). The process technology leads to select a ora
with specic nutrient requirements and growth conditions. As
a consequence, lactobacilli cultivation on laboratory media is
difcult to achieve and strain isolation strongly depends on both

* Corresponding author. Departement Production Agroalimentaire et Qualite,

Unite Ecosyste`mes microbiens complexes, ISARALyon, 23 rue Jean Baldassini,
69364 Lyon, France. Tel.: 33 427 85 85 71; fax: 33 427 85 86 38.
E-mail address: vera@isara.fr (A. Vera).
0740-0020/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2009.07.010

the medium composition and on the incubation conditions applied

(pH, temperature, aeration) (De Vuyst and Vancanneyt, 2007).
Several media have been proposed in order to study lactobacilli
population from a quantitative and qualitative point of view. These
media mainly differ on their composition. Culture conditions can
also change. De Man et al. (1960) proposed the MRS medium,
elective for the isolation and counting of lactobacilli from fermented food products. This rich medium contains glucose, main
source of carbohydrate. Later, Kline and Sugihara (1971) developed
the SourDough Bacteria (SDB) medium, specic for the detection of
Lactobacillus sanfranciscensis. It contains maltose as carbohydrate
source and freshly-prepared yeast extract (FYE) as growth stimulating factor. Afterwards, the Sanfrancisco medium (SFM) was
developed which allowed the isolation and description of Lactobacillus pontis and Lactobacillus mindensis (Vogel et al., 1994;
Ehrmann et al., 2003). This medium contains three carbohydrates
(maltose, fructose, glucose), FYE, cystein and rye or wheat bran.
Alternatively, Vogel et al. (1994) proposed a modied MRS medium
called later in this article MRS Vogel with higher pH value
(6.3). Recently, Meroth et al. (2003) developed the MRS5 medium,
composed of maltose, fructose, glucose, cystein and a mix of vitamins. Lactobacillus spicheri and Lactobacillus namurensis were
isolated from sourdough on this agar medium (Meroth et al., 2004;
Scheirlinck et al., 2007). The MRS medium was also widely modied on its carbohydrate and FYE compositions and concentrations.
Numerous other media for lactobacilli counting have been

A. Vera et al. / Food Microbiology 26 (2009) 728733

proposed and used for fermented products different from sourdoughs. Rogosa medium (Rogosa et al., 1951) mainly leads to the
enumeration and the isolation of lactobacilli population. Specic
media have been developed to select a part of the lactobacilli
population. FH (facultatively heterofermentative) and OH
(obligately heterofermentative) agar media are respectively used to
enumerate facultatively and obligately heterofermentative lactobacilli (Isolini et al., 1990). LAMVAB medium is commonly used to
quantify probiotic bacteria (Coeuret et al., 2004). KCA medium is
employed to count specic citrate degrading lactic acid bacteria
(Nickels and Leesment, 1964).
Among the different media available for lactobacilli cultivation,
none of them seems efcient to study sourdough ecosystem from
a quantitative as well as qualitative point of view. Consequently, the
aim of this study was to compare the efciency of 11 culture media
for sourdough lactobacilli enumeration, including counting levels
and colony biodiversity evaluation on the different media. This
work was achieved to select the most appropriate media prior to
a global dynamic study of type I sourdough fermented during
several consecutive days.
2. Materials and methods
2.1. Sourdough samples
Five samples of the same type I wheat sourdough were collected
before refreshment from a French industrial producer of sourdoughs. A 2-week period separated every sampling. The samples

were stored at 4 C less than 24 h before analysis.
2.2. Microbiological analysis

729

into 1 ml of sterile peptone water (1 g/l peptonecaseinmeat,

Biokar, Pantin, France). Each broth (5 ml) was then inoculated with

100 ml of the bacterial suspension and incubated at 30 C. Two trials
were performed. A total of 11 colonies from sourdough sample no. 1
isolated on MRS, maltose MRS, SDB, SFM, Rogosa, LAMVAB, FH and
OH plates were rstly subcultured on MRS, maltose MRS, SDB, and
Rogosa broths. The second trial tested the MRS, maltose MRS, MRS5
and MRS Vogel broths for the culture of 38 colonies from sourdough sample no. 4 and picked on MRS, maltose MRS, MRS5, MRS
Vogel, SDB, SFM, Rogosa, LAMVAB, FH and OH plates. The growth

of the isolates was checked before and after freezing at 80 C in
glycerol (10% nal concentration).
2.5. Whole-cell protein patterns
A total of 72 lactobacilli strains were isolated randomly and
characterised by SDS-PAGE analysis; 12 strains were picked from
MRS plates, 12 from maltose MRS, 14 from MRS5, 19 from MRS
Vogel, 2 from SDB, 5 from SFM, 5 from Rogosa, 2 from FH and 1
from OH plates. Isolates were rst cultivated in 5 ml of maltose MRS
broth (pH 5.4) for 4 days. The whole-cell proteins were prepared
according to Demarigny et al. (2006). Electrophoresis was performed in a denaturing gel (stacking gel: 9.67% acrylamide and
5.17% bisacrylamide; resolving gel: 18.15% acrylamide and 9.68%
bisacrylamide) on an electrophoresis apparatus (Protean II xi cell,
Biorad). Three runs were carried out: 90 V, 500 mA, 250 W, 30 min;
130 V, 500 mA, 250 W, 1 h; and 300 V, 500 mA, 250 W, 4 h. Gels
were stained with Coomassie blue and scanned. The band patterns
were normalised and processed by using the Gel-Compar 3.1
program (Applied Maths). Dendogramme was obtained using the
UPGMA method (Sokal and Michener, 1958).

Ten grams of sample were diluted in 90 ml of sterile peptone

water (1 g/l peptonecaseinmeat, Biokar, Pantin, France).
Depending on the needs, successive decimal dilutions were made
using the same diluent. For each dilution, 50 ml were spread in
duplicate on agar plates. Viable counts were performed after a oneweek incubation in order to improve the discrimination between
colonies based on their morphology. Following the medium, the


cultivation temperature was 30 C or 38 C. All cultivations were
achieved under anaerobic conditions (see below).

2.6. Genomic identication

2.3. Agar culture media and cultivation conditions

Statistical analyses (ANOVA) were performed using the STATITCF software (5th version, 1995, Institut Techniques des Cereales
et des Fourrages, Paris, France).

Eleven agar culture media available for lactobacilli were tested.

MRS, maltose MRS, SDB, SFM, MRS Vogel, MRS5 and Rogosa
media were elective for lactobacilli cultivation. KCA, FH, OH and
LAMVAB media were selective respectively for citrate positive,
facultatively heterofermentative, obligately heterofermentative
and probiotic lactobacilli. Characteristics of each medium are presented on Tables 1 and 2. MRS, maltose MRS, SDB, SFM, MRS
Vogel, MRS5, Rogosa and KCA media were incubated 7 days at


30 C. FH, OH and LAMVAB media were incubated 7 days at 38 C.
All cultivations were achieved under anaerobic conditions.
Anaerobiosis was chemically obtained with an oxygen trap system
linked with a carbon dioxide producer (GENbox anaer, Biomerieux,
Marcy-lEtoile, France).
2.4. Culture broth media
Six broth media were tested: MRS, maltose MRS, SDB, MRS
Vogel, MRS5 and Rogosa. MRS and Rogosa broths were sterilised


at 121 C for 15 min. The other broths were sterilised at 110 C,
10 min MRS5 broth was supplemented with the vitamin mix lter
sterilised (0.2 m) before use. Colonies were picked and suspended

A total of 20 strains isolated from this sourdough were subjected

to 16S rDNA sequence analysis. The complete 16S rDNA gene
sequence analysis was performed by COGENICS GENOME express
company (Grenoble, France) using two sets of two primers
(16S1FOR/16S3REV and 16S2FOR/16S2REV).
2.7. Statistical analysis

3. Results
3.1. Microbial enumeration
The results of the microbial analysis are presented on Fig. 1.
Maltose MRS and MRS media gave comparable results and showed
the highest counts respectively 9.47 and 9.43 log(cfu)/g. On the
contrary, SDB, MRS5 and LAMVAB media presented signicant
lower counts (p < 0.05) lesser than 0.5 log(cfu)/g compared to
maltose MRS and MRS media. SFM and Rogosa media showed
intermediate count levels and were not statistically different from
the other media. The MRS Vogel medium was excluded from the
variance analysis since bacterial levels were very low on this
medium: 8.37 mean log(cfu)/g. As expected, the effect of sourdough
sampling was signicant (p < 0.01) because of the continuous
evolution of this complex ecosystem. FH and OH being highly
selective, they target a specic part of the lactobacilli population
facultatively or strict heterofermentative lactobacilli. Thus, they
displayed low mean counts namely 8.02 log(cfu)/g on FH medium

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A. Vera et al. / Food Microbiology 26 (2009) 728733

Table 1
Characteristics and composition (g/l) of the elective media for lactobacilli ora.
MRSa

Maltose
MRSb

MRS5b

SFMb

Biokar,
France

Meroth et al. (2003)

Vogel et al. (1994)

Tryptone
Polypeptone
Trypticase
Meat extract
Yeast extract
Glucose
Maltose
Fructose
Tween 80
K2HPO4
KH2PO4
Sodium acetate
Sodium gluconate
Ammonium citrate
Ammonium
chloride
MgSO4
MnSO4
FeSO4
Cystein HCl
Supplement

10

10
5
20

1 ml
2

10

10
5

20

1 ml
2

10

5
5
5
10
5
1 ml
2.6
4
5

10

2
7
7
7
7
1 ml

2.5
5
2
5

10

5
5
7
7
7
1 ml
2.6

5
2
2

20

0.3 ml

10

5
20

1 ml

2
25

0.2
0.05

0.2
0.05

0.1
0.05

0.5
Vitamin mix,c
Cycloheximidec

0.2
0.05
0.01
0.5
Fresh bakers yeast, wheat bran

0.1
0.05

0.5

1.5% FYE

0.525
0.12
0.034

1.32 ml acetic acid

Final pH

5.4

5.4

5.8

5.4

6.3

5.6

5.4

Description

a
b
c

MRS Vogelb SDBb

Rogosab

Kline and Sugihara (1971) Rogosa et al. (1951)

Whole medium sterilised at 121 C, 15 min.


Sugars and cystein separately sterilised at 110 C, 10 min.
Component sterilised by ltration (0.2 m).

and 7.74 log(cfu)/g on OH medium. KCA medium revealed the

presence of citrate positive lactobacilli population but frequently at
levels lower than the detection limit (<6 log(cfu)/g).

irregular border and a diameter >4 mm F4 colonies showed a fried

egg like shape.
3.3. Biodiversity analysis

3.2. Colony differentiation

To evaluate the microbial diversity on each plate medium,
colonies were classied depending on their morphological form
(morphotype). For instance, SDB and SFM media revealed a unique
morphotype whereas at least four different morphotypes were
observed on maltose MRS, MRS, MRS5, MRS Vogel and Rogosa
media. As shown in Fig. 2, F1 colonies were characterised by
a round regular shape with a smooth surface and a 35 mm
diameter. The F2 morphotype was irregular with a thick and striped
border and a diameter 1 mm F3 colonies were rather translucent
with a smooth surface, an irregular border and a diameter 2 mm.
The F4 morphology was characterised by a rough surface, an

Biodiversity was investigated by the analysis of the whole-cell

protein patterns of colonies isolated from sourdough on the
different agar media, taking into account the colony morphologies
(mainly F1, F2). The F3 morphotypes were not included in this
analysis since most of the isolates were unable to grow in broth and
F4 colonies were not numerous enough to be representative.
Results are shown in Fig. 3. F1 isolates were always separated from
F2 isolates, irrespective of the medium from which they were
collected maltose MRS, MRS, MRS5 or MRS Vogel plates.
Maltose MRS medium revealed the highest diversity of strains
compared with the other media. Indeed, four different populations
were found on this medium two distinct subpopulations
presenting the same F1 morphology and two subpopulations

Table 2
Characteristics and composition (g/l) of the selective media for lactobacilli ora.
Description

LAMVABa

FHa

OHb

KCAd

Coeuret et al. (2004)

Isolini et al. (1990)

Isolini et al. (1990)

Nickels and Leesment (1964)

Composition
per liter

52.2 g MRS Broth (Biokar,

France), 0.25 g cystein HCl,
0.025 g bromocresol green,
0.02 g vancomycin
hydrochloridec
5.4

10 g polypeptone, 10 g meat extract, 1 g 10 g polypeptone, 10 g meat extract,

yeast extract, 20 g mannitol, 0.1 g MgSO4, 1 g yeast extract, 10 g rafnose, 10 g
0.1 g MnSO4, 200 ml acetate buffer, 1 ml melibiose, 0.1 g MgSO4, 0.1 g MnSO4,
tween 80, 0.05 g vancomycin
200 ml acetate buffer, 1 ml tween 80
hydrochloridec
Not adjusted
Not adjusted
Acetate buffer (per liter): 9.81 ml acetic acid, 96.94 g trisodium citrate; pH 5.4

Final pH
Remarks
a
b
c
d
e

Whole medium sterilised at 121 C, 15 min.


Sugars and cystein separately sterilised at 110 C, 10 min.
Component sterilised by ltration (0.2 m).

Whole medium sterilised at 120 C, 15 min.
TTC: 2,3,5-triphenyltetrazolium chloride.

20 g tryptone, 5 g yeast extract, 2.5 g

gelatine, 5 g glucose, 5 g lactose, 4 g NaCl, 2 g
sodium citrate, 8 g calcium lactate, 100 ml
whey-serum, 5 g calcium citrate, 0.1 g TTCe
6.6
TTCe lter sterilised (0.45 m)

A. Vera et al. / Food Microbiology 26 (2009) 728733

maltose MRS

9.47

MRS

9.43

Rogosa

9.24

SFM
MRS5
LAMVAB
8.00

AB

9.00

8.99

8.90
8.20

8.40

8.60

8.80

A
AB

9.10

SDB

9.00

B
9.20

9.40

9.60

9.80 10.00

count means log (cfu)/g

Fig. 1. ANOVA results of the microbiological analysis performed on sourdough samples
no. 1, 2, 3, and 5. A, AB, B: classication according to the Newman Keuls test.

presenting the same F2 morphology. Although MRS5 presented

a lesser biodiversity than maltose MRS (only two distinct populations), a specic population was isolated on this medium only.
Whole-cell protein patterns were compared to those of the reference strains Lactobacillus brevis CIP 102806T, Lactobacillus plantarum CIP 103151T, Lactobacillus reuteri CIP 101887T, Lactobacillus
rhamnosus CIP A157T and L. sanfranciscensis CIP 103252T. These
strains were chosen because of their frequent presence in sourdoughs. No strain isolated in this work was linked with one of these
reference strains.
A 16S rDNA sequence analysis assigned the MRS5 F1 isolates to
Lactobacillus panis. The three isolates Maltose F22, MRS F2 and
Vogel F2 could be linked with Lactobacillus amylolyticus.

3.4. Broth subculture

Over the 6 broth media tested, maltose MRS, MRS5 broths and to
a lesser extent MRS broth were the most tted for subculture.
Indeed, they allowed the development of around 60% of the
isolates. These results were obtained on young colonies, i.e. just
after the opening of the jars, and on older colonies (after a 7 day

storage delay). After storage at 80 C in glycerol solution, 100% of
the isolates grew in maltose MRS and MRS5 broths. In contrast, SDB
and Rogosa broths appeared unsuitable, respectively 90% and 80%

731

of the isolates being unable to grow. MRS Vogel was not suitable
either for 70% of the isolates. Isolates from SDB, SFM, LAMVAB, FH
and OH plates rarely grew in any broth media.
4. Discussion
This study was undertaken in order to compare the exhaustiveness of different culture media used to study the lactobacilli
population in type I sourdoughs. Such a study had only been made
once before and limited to three media MRS, SDB and SFM
(Picozzi et al., 2005). Our work is relevant in the sense that problems can arise when it is necessary to choose the appropriate
medium or compare the results obtained with previous data.
Eleven media were tested based on the study of the available
scientic literature, seven elective media mostly based on the
MRS composition and four selective media.
Considering the microbial load of the sourdoughs here studied,
the counts obtained with the elective media match those reported
by the literature for traditional sourdoughs, levels ranging from 7 to
9 log(cfu)/g (Reale et al., 2005; Valmorri et al., 2006; Scheirlinck
et al., 2008). On selective media namely FH, OH, KCA the results
were generally lower (<68.02 log(cfu)/g), which is not surprising
considering their selective goal. These media focus on populations
with specic metabolism, a part of the total population. Moreover,

FH and OH media include vancomycin and are cultivated at 38 C

instead of 30 C which increase the selectivity of these media.
Considering the elective media, the choice of the medium is
important since signicant differences were observed, both quantitatively and qualitatively. The largest lactobacilli numbers were
found on maltose MRS medium. This nding can be explained by
the presence of maltose as source of fermentable carbohydrate in
this medium. Indeed, sourdough lactobacilli are known to be highly
competitive in this environment where maltose is abundant. This
competitiveness is probably due to a specic maltose catabolism.
As an example, the L. sanfranciscensis carbohydrate metabolism was
largely studied. It seems to play a key role explaining the dominance of this population in the sourdough ecosystem (Vogel et al.,
1999; Ganzle et al., 2007). Despite low counts, the MRS5 medium
enabled the isolation of a specic population which was not found

Fig. 2. Colony morphologies observed on SFM, SDB, MRS5 and maltose MRS media. a: Direct plate observation; b: observation under binocular magnier; red arrow: form F1; blue
arrow: form F2; green arrow: form F3; yellow arrow: form F4. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this
article.)

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A. Vera et al. / Food Microbiology 26 (2009) 728733

200.00

60

70

80

90

100.00
90.00 80.00 70.00

60.00

50.00

40.00

30.00

100
CIP 102806 L. brevis
CIP 101887 L. reuteri
CIP 103151 L. plantarum
CIP 103252 L. sanfranciscensis
CIP A 157 L. rhamnosus
MRS F2
Maltose F2-2
Vogel F2
Maltose F2-1
MRS5 F2
Vogel F4
Maltos F1-2
Vogel F1
MRS F1
MRS5 F1
Maltose F1-1

Fig. 3. SDS-PAGE proles of the whole-cell proteins of isolates from maltose MRS, MRS, MRS5 and MRS Vogel media. The different clusters are framed or pointed by an arrow.

on the other media (called MRS5 F2 on Fig. 3). This population

probably needs high nutritional requirements provided by the
MRS5 medium as it is one of the richest. As noticed by Picozzi et al.
(2005), SFM medium presented higher levels than SDB medium.
This nding was expected since the composition of SFM medium is
more complex. However, contrary to Picozzi et al. (2005) ndings,
our results led us to think that MRS medium was relevant for
lactobacilli enumeration but not for biodiversity investigation. The
low cell levels found on MRS Vogel medium could be explained
by its higher pH (6.3) than the other media (pH 5.4). Lactobacilli are
probably more adapted to the acid conditions prevailing in this
sourdough (pH  3.5).
The 16S rDNA sequence analysis allowed us to classify a part of the
isolates into the species L. panis obligately heterofermentative
lactobacilli and L. amylolyticus obligately homofermentative lactobacilli. These species are generally found in type II sourdoughs and
not in type I sourdoughs. This nding can be explained by the
ambiguous industrial process applied to the sourdough. On one hand,
this sourdough can be classied as type I mother dough. It is
continuously propagated under non-controlled temperature conditions, a part is used as inoculum for other batches and it is refreshed
daily. On the other hand, this sourdough can be related to type II
sourdough. Effectively, the consistence of the sourdough is semi
uid, the propagation temperature exceeds 30 C and even some
times 35 C and the pH is around 3.5 (De Vuyst and Neysens, 2005).
Classical microbiological methods are known to lead to bacterial
counts possibly far from reality. Culture media only detect
microorganisms in cultivable state but not sub-lethally injured
cells. In such a complex ecosystem as sourdough, it is recognised
that microorganisms are submitted to many stresses (temperature
changes, pH, acidity) which can induce the cells to turn into viable
but non-cultivable state (Giraffa, 2004). Culture-independent
methods mostly molecular PCR-based techniques can be
specically adapted to by-pass this problem and to study sourdough biodiversity, quantitatively as well as qualitatively. They are
also fully used to monitor population dynamics (De Vuyst and
Vancanneyt, 2007). Recently, DGGE analysis performed on DNA and
RNA extracts revealed the presence of non-viable population
and lactobacilli species never detected on the agar media used
(Iacumin et al., 2009). However, these molecular methods are also
limited in their detection sensitivity for subdominant populations.
Polyphasic approaches based on culture-dependent and cultureindependent methods seem then the best strategy to have
a complete overview of sourdough ecology. Isolation of colonies on
plate media remains important since it allows strain phenotypical
characterizations.

Moreover, culture media are still routinely used in most food

plants to control daily the bacterial levels of the starters they use.
Sourdough biodiversity can be evaluated through cultivation on
plate medium as a rst step evaluation, even if some morphotypes
encompass several lactobacilli subpopulations. Thus, the deliberate
combination of several media can lead to the gross estimation of
the community biodiversity, as a sum of lactobacilli population and
to detect possible changes during the sourdough propagation.
5. Conclusion
To conclude, as stated before there is no universal medium
enabling to cover the complexity of sourdough biodiversity.
Considering type I sourdoughs studied here, the combination of
maltose MRS and MRS5 media would seem the most appropriate,
even if the information brought by these classical methods appear
limited to some extent. In this sense, the use of culture-independent methods in combination with agar media appears particularly
convenient. This work being limited to sourdough samples originating from the same plant it would be now interesting to enlarge
this study to other type I sourdoughs.
Acknowledgements
The authors would like to thank P. Philibert, O. Bourdon and
L. Nevoret for their technical assistance. They also thank
M. H. Ly-Chatain and M. Chareyron for proof-reading the paper and
the English corrections. This project was supported by Philibert
Savours company.
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