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7/25/2019 Characterization of Sourdough Lactic Acid Bacteria
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Characterization of sourdough lactic acid bacteria based ongenotypic and cell-wall protein analyses
A. Corsetti1, M. De Angelis1,2, F. Dellaglio3, A. Paparella4, P.F. Fox5, L. Settanni1
and M. Gobbetti61Dipartimento di Scienze degli Alimenti, Sezione di Tecnologie e Biotecnologie degli Alimenti, Universitadegli Studi di Perugia, Perugia,
Italy, 2Institute of Sciences of Food Production, CNR, Bari, Italy, 3Dipartimento Scientifico e Tecnologico, Universita`degli Studi di
Verona, Verona, Italy, 4Dipartimento di Strutture, Funzioni, Patologie Animali e Biotecnologie, Universitadegli Studi di Teramo, Teramo,
Italy, 5Food Chemistry, Food Science and Technology Department, University College Cork, Ireland, and 6Dipartimento di Protezione
delle Piante e Microbiologia Applicata, Universitadegli Studi di Bari, Bari, Italy
2002/406: received 23 October 2002, revised and accepted 9 December 2002
ABSTRACT
A . C O R S E T T I , M . D E A N G E L I S , F . D E L L A G L I O , A . P A P A R E L L A , P . F . F O X , L . S E T T A N N I
AND M. GOBBETTI. 2003.
Aims: To evaluate the effectiveness of two independent methods in differentiating a large population of lactic acid
bacteria (LAB) isolated from wheat flours and sourdoughs and to correlate eventual differences/similarities among
strains with their geographical origin and/or process parameters.
Methods and Results: One hundred fifty strains belonging to Lactobacillusspp. andWeissellaspp., plus eight type
strains, one for each species, and two unidentified isolates, were characterized by randomly amplified polymorphic
DNA (RAPD) and SDS-PAGE of cell-wall proteins. The RAPD analysis separated the eight type strains but did
not always assign all the strains of a species to the same group, while SDS-PAGE cell-wall protein profiles were
species-specific. Frequently, strains isolated from sourdoughs of the same geographical origin or produced by
similar raw material/process parameters showed similar RAPD and/or cell-wall profiles.
Conclusions: The combined use of the RAPD and cell-wall protein analysis represents a useful tool to classify
large adventitious microbial populations and to discriminate the diversity of the strains.
Significance and Impact of the Study: This study represents a typing of a large collection of flour/sourdough
LAB and provides evidence of the advantage of using two independent methods in the classification and traceability
of microorganisms.
Keywords: Cell-wall proteins, lactic acid bacteria, Lactobacillus, PCR-RAPD, SDS-PAGE, sourdough, typing,
Weissella.
INTRODUCTION
By one definition (Anon 1994), sourdough is described as a
dough, the microflora (especially lactic acid bacteria (LAB)
and yeasts) of which originate from sourdough or a
sourdough starter and is metabolically active or can be
reactivated. Upon addition of flour and water, the micro-
organisms continue to produce acids.
The modern biotechnology of baked goods largely usessourdough as a natural leavening agent because of the many
advantages it offers over bakers yeast. LAB are fundamental
for the properties of sourdough: lactic fermentation, proteo-
lysis, synthesis of volatile compounds, anti-mould and anti-
ropiness are the most important activities during dough
leavening (Gobbetti 1998; Hammes and Ganzle 1998).
Endogenous factors in cereal products (carbohydrates,
nitrogen sources, minerals, lipids and free fatty acids, and
enzyme activities) and process parameters (temperature,
dough yield, water activity, oxygen, fermentation time and
Correspondence to: Aldo Corsetti, Dipartimento di Scienze degli Alimenti, Sezione di
Tecnologie e Biotecnologie degli Alimenti, Universita degli Studi di Perugia, Via S.
Costanzo, 06126 Perugia, Italy (e-mail: [email protected]).
2003 The Society for Applied Microbiology
Journal of Applied Microbiology2003, 94, 641654
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number of sourdough propagation steps) markedly influence
the microflora of the sourdough and the features of leavened
baked goods (Hammes and Ganzle 1998). Numerous species
of LAB, mainly belonging to the Lactobacillus genus, have
been isolated from sourdoughs and identified, although
selection occurs during propagation leading to the establish-ment of, usually, one or two species at numbers three or four
orders of magnitude above those of the adventitious micro-
bial flora (Hammes et al., 1996; Hammes and Ganzle 1998).
Studies on the diversity of sourdough LAB, especially in
countries such Italy where more than 200 different types of
bread are produced and where different bread-making
processes are used (Gobbetti et al. 1994; Corsetti et al.
2001), may be helpful for differentiating baked goods,
establishing the effects of technological parameters on
specific differences in the microbial flora and gaining
information about the diversity of a large adventitious
population. Different methods are available in order toevaluate the microbial biodiversity. Randomly amplified
polymorphic DNA (RAPD) analysis is a less time consu-
ming, PCR-based method, which provides good levels of
discrimination and is applicable to a large number of strains
(Vincentet al. 1998). This method has been used to estimate
the diversity ofLactobacillusspecies and strains (Tailliezet al.
1996; Nigatu et al. 2001), to type strains of Lactobacillus
plantarum (Johansson et al. 1995) and to study the popu-
lation of non-starter lactic acid bacteria (NSLAB) in mature
commercial cheese (Fitzsimons et al. 1999). Regarding the
microflora of sourdough, the RAPD analysis has been used
to differentiate Lactobacillus sanfranciscensis strains (Zappar-
oli et al . 1998) and to distinguish several species oflactobacilli using a 21-mer primer (Hamad et al. 1997).
Analysis of cell-wall protein profiles has already been used
to study andcompare several strains of lactobacilli (Yasui etal.
1995; Bootet al. 1996) and to differentiate the thermophilic
lactobacilli present in natural or selected starters used to
produce several Italian cheeses (Gatti et al. 1997). This
method has been found to be reliable and rapid for
characterizing large numbers of strains and relating differ-
ences in cell-wall protein profiles of strains to adaptation to
different ecological niches and technological processes.
We previously characterized, by RAPD and cell-wall
protein analyses, NSLAB isolated from Italian ewe cheeses(De Angelis et al. 2001). We found differences between the
two methods for resolving the classification of NSLAB and
useful information on the microbial diversity in cheeses and
on the influence of geographical and technological factors in
determining NSLAB heterogeneity. We concluded that
both methods should be used to obtain complete and
integrated information.
In this paper, we describe genotypic (RAPD analysis) and
cell-wall protein characterization of LAB isolated from
Italian sourdoughs and flours.
MATERIALS AND METHODS
Origin of LAB and sourdough characteristics
LAB had been isolated previously from 45 sourdoughs from
the Centre (Umbria region) and South (Puglia region) of
Italy (Gobbetti et al. 1994; Corsetti et al. 2001) and fromfour Triticum aestivum organic flours (sample nos. 4649 in
Table 1) of the Centre of Italy (Marche region) (Corsettiet al.
1998). The sourdoughs from the Centre and South of Italy
were produced from T. aestivum, T. durum or a mixture of
the two varieties. The time of fermentation varied from 3 to
24 h (Table 1) depending on the bread-making protocol
while the dough yield [(weight of the dough/weight of the
flour) 100] was in the range 140160. Overall, the
sourdoughs contained two or more species of LAB that
belonged mainly to the genus Lactobacillus (Gobbetti et al.
1994; Corsetti et al. 2001).
A total of 150 strains of LAB, Lb. sanfranciscensis (57strains),Lb. fermentum(three strains),Lb. brevis(28 strains),
Lb. alimentarius (24 strains), Lb. farciminis (nine strains),
Lb. plantarum (17 strains), Lb. fructivorans (six strains),
Weissella confusa (four strains), plus two unidentified
Lactobacillus spp. and eight type strains, one for each
identified species, were used for genotypic and cell-wall
protein characterization.
Genotypic characterization
LAB were genotypically characterized by RAPD-PCR
analysis. Genomic DNAs from all the strains were extracted
as reported by De Los Reyes-Gavilanet al. (1992) from 2-mlsamples of overnight cultures grown in SDB broth at 30 or
37C. The final concentration of lysozyme used for cell
lysis was 2 mg ml)1. The concentration and purity of
DNA were assessed by determining the optical densities at
260 and 280 nm, as described by Sambrook et al. (1989);
the concentration of each DNA sample was adjusted to
25 ng ll)1 Ten primers (Life Technologies, Milan, Italy),
with arbitrarily chosen sequences, were tested at a final con-
centration of 1 lmol l)1. The sequences were the following:
P1 5 ACGCGCCCT 3; P 2 5 ATGTAACGCC 3;
P3 5 CTGCGGCAT 3; P4 5 CCGCAGCGTT 3; P5 5
TGCTCTGCCC 3; P 6 5 GTCCACACGG 3; P 7 5AGCAGCGTGG 3; P8 5 CGTACAGGCT 3; P9
5 TCACCGTCGC 3; and P10 5 ACTGGCTCCG 3
(De Angelis et al. 2001). Each reaction mixture contained
200lmol l)1 of each 2-deoxynucleoside 5-triphosphate,
1 lmol l)1 primer, 15 mmol l)1 MgCl2, 125 U of Taq
DNA polymerase (Life Technologies), 25 ll of PCR buffer,
25 ng of DNA, and enough sterile bi-distilled water to bring
the volume to 25 ll. The PCR program comprised 45 cycles
of denaturation for 1 min at 94C, annealing for 1 min at
35C, and extension for 2 min at 72C; the cycles were
642 A . C O R S E T T I ET AL.
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Table 1 Geographical origin and technological characteristics of Italian sourdough and organic wheat flours
Sourdough* and
wheat flour number
City of
production Strain Type of wheat flour
Fermentation
time (h)
1 Lecce Lb. brevis 1D, 1Q, 1F Triticum durum 24
2 Lecce Lb. sanfranciscensis 2A; Lb. alimentarius 2S T. durum 63 Lecce Lb. alimentarius DA70, 3D T. durum 24
4 Lecce N.I. 4R T. durum 24
5 Lecce Lb. sanfranciscensis 5D; Lb. alimentarius 5Q, 5S,
5A, 5a; Lb. brevis 5Z
T. durum 24
6 Lecce Lb. brevis 6L T. durumand T. aestivum 10
7 Bari Lb. sanfranciscensis 7A, 7H, 7M, 7N T. aestivum 5
8 Bari Lb. alimentarius 8D; W. confusa 8L, 8V T. aestivum 3
9 Bari Lb. sanfranciscensis 9F, 9N; Lb. brevis 9V Whole T. durum 4
10 Bari Lb. brevis 10A, 10D, 10I, 10R, 10a T. durum 3
11 Bari N.I. 11N T. aestivum 3
12 Bari Lb. sanfranciscensis 91 Whole T. aestivum 3
13 Bari Lb. sanfranciscensis 13R T. durum 3
14 Bari W. confusa 14R, 14S T. durum 3
15 Brindisi Lb. alimentarius 15A, 15F, 15M, 15b; Lb. brevis 15R T. durumand T. aestivum 9
16 Brindisi Lb. alimentarius 16A, 16B, 16I, 16M, 16R, 16a, 16c T. durum and whole T. aestivum 12
17 Foggia Lb. alimentarius 17D T. aestivum 12
18 Foggia Lb. brevis18C, 18F T. aestivum 24
19 Foggia Lb. plantarum 19A T. aestivum 24
20 Foggia Lb. plantarum 20B; Lb. brevis 20E, 20T T. aestivum 3
21 Foggia Lb. plantarum 21A, 21B; Lb. brevis 21S T. durum and whole T. aestivum 18
22 Foggia Lb. brevis 24A, 24V T. durum 24
23 Foggia Lb. brevis 25K T. durum 24
24 Perugia Lb. sanfranciscensis 57, 57cur T. aestivum 8
25 Perugia Lb. sanfranciscensis I1; Lb. fermentum I2;
Lb. alimentarius I4; Lb. brevis I5
T. aestivum 6
26 Perugia Lb. sanfranciscensis E3, E5, E6, E7, E9, E10, E12, E13,
E14, E15, E16, E17, E18, E19, E20, E21, E22, 73
T. aestivum 3
27 Perugia Lb. sanfranciscensis A2Z; Lb plantarum P2 T. aestivum 3
28 Perugia Lb. sanfranciscensis A1, A4, A6, A7, A15, A17, A22 T. aestivum 3
29 Perugia Lb. sanfranciscensis 79, 174, 274, 77St T. aestivum 3
30 Perugia Lb. fermentum6E T. aestivum 6
31 Perugia Lb. plantarum CF1, 7C5 T. aestivum 6
32 Perugia Lb. brevis DE9 T. aestivum 6
33 Perugia Lb. plantarum 13, 18, 20, 30, DB200, DC400 T. aestivum 8
34 Perugia Lb. alimentarius O9; Lb. fructivorans P4, P9 T. aestivum 8
35 Perugia Lb. sanfranciscensis D17 T. aestivum 12
36 Amelia Lb. fructivorans DA110, DD7, DD10 T. aestivum 9
37 Terni Lb. sanfranciscensis 12, BB12 T. aestivum 3
38 Terni Lb. sanfranciscensis 62 T. aestivum 4
39 Terni Lb. sanfranciscensis CB1; Lb. fructivorans DD8 T. aestivum 4
40 Terni Lb. sanfranciscensis 72, 125 T. aestivum 12
41 Marsciano Lb. alimentarius F13; Lb. sanfranciscensis H1, H3, H4,
H5, H6, H7, H10
T. aestivum 3
42 Foligno Lb. brevis DA64 T. aestivum 12
43 Foligno Lb. brevis AM7, AM8; Lb. alimentarius AN2 T. aestivum 3
44 Foligno Lb. fermentumCD5 T. aestivum 24
45 Foligno Lb. plantarum AD4, 2A1 T. aestivum 12
46 Pesaro Lb. brevis 1xF5; Lb. farciminis 5xF12, 5xF14 T. aestivum
47 Pesaro Lb. plantarum 2F3; Lb. farciminis 2xA3, 2xA6, 5C1 T. aestivum
M O L E C U L A R T Y P I N G O F S O U R D O U G H L A B 643
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preceded by denaturation at 94C for 4 min and followed by
extension at 72C for 5 min (Rossi et al. 1998).
PCR products (15 ll) were separated by electrophoresis at
120 V for 4 h on 15% (wt/vol) agarose gel (Gibco BRL,
France) and the DNA was detected by UV transillumination
after staining with ethidium bromide (05 lg ml
)1
). Themolecular sizes of the amplified DNA fragments were
estimated by comparison with a 123-bp ladder DNA (Gibco
BRL, France).
Photographs of RAPD-PCR gels were obtained with a
high-performance charge-coupled device camera (Cohu,
Inc., San Diego, CA, USA) and were scanned by using an
HP Scanject II cx scanner (Hewlett Packard Co., Palo Alto,
CA, USA). Electrophoretic profiles were compared using
GelCompar 40 software (Applied Maths, Kortrijk, Bel-
gium). Three series of RAPD-PCR profiles were combined
to obtain a unique dendrogram. The combined RAPD
patterns were analysed using the Pearson product moment
correlation coefficient and the unweighted pair-groupmethod using arithmetic average (UPGMA), from which a
dendrogram showing the relationships between LAB was
obtained. The reproducibility of RAPD fingerprints was
determined from triplicate loading of independent, triplicate
RAPD reaction mixtures prepared from eight strains on
three gels; cluster analysis was performed as described
above.
Cell-wall protein characterization
Cell-wall protein was extracted by using a slightly modified
version of the method of Gatti et al. (1997). Twenty-four-hour cells (stationary phase) of sourdough lactobacilli
cultivated in modified MRS broth were harvested, washed
twice in 005 mol l)1 TrisHCl, pH 75, containing
01 mol l)1 CaCl2, and resuspended in 1 ml of the same
buffer at an A600 of 10 (measured on a 1 : 10 diluted cell
suspension). After centrifugation at 8000 gfor 5 min, cell-
wall proteins were extracted from the pellets with 1 ml of
extraction buffer, pH 80, containing 001 mol l)1 EDTA,
001 mol l)1 NaCl and 2% (wt/vol) SDS. Suspensions
were stored at room temperature for 60 min, heated at
100C for 5 min and centrifuged at 11 600 gfor 10 min at
4C. The supernatants were analysed by SDS-PAGE using
a Phast system (Pharmacia Uppsala, Sweden) and stained
with Comassie blue (Heukeshoven and Dernik 1988). The
mobility of individual proteins was calculated and the
protein profile of the strains compared. The 70-kit molecularweight protein standard (molecular weight range, 14 300
66 000; 54 lg of total protein) in addition to a2-macroglob-
ulin (molecular weight, 170 000; 6 lg of protein) and b-
galactosidase (molecular weight 116 400; 8 lg of protein)
was used (Sigma Chemical Company, St Louis, MO, USA).
The reproducibility of the SDS-PAGE was estimated by
loading two independent, triplicate cell-wall protein extracts
from eight strains on two gels. The relative error ( E) for
each band in each gel was calculated as follows:
E [(Rf) Rfm)/Rfm] 100, where Rf is the distance of
a protein band from the top of the separating gel and Rfmthe
meanRffor the band obtained in different gels. Comparison
between pairs of banding patterns was evaluated calculatingan index of similarity by the simple matching coefficient
(Sokal and Michener 1958). Electrophoretic profiles were
analysed using the NTSYS.PC package, version 1 8 (Rohlf
1993). Cluster analysis was carried out with the UPGMA
clustering method.
RESULTS
Genotypic characterization
Primers P2, P3, P5, P6, P8, P9 and P10 only gave one or a
few bands, despite extended annealing times at a lowtemperature and increasing the concentration of MgCl2.
Similar results were obtained when the same primers were
used to study the diversity of cheese-related NSLAB (De
Angelis et al. 2001). Primers P1, P4 and P7 generated the
most diverse pattern and were selected for genotypic
characterization. The reproducibility of RAPD fingerprints
was assessed by comparing the PCR products obtained with
primers P1, P4 and P7 and DNA prepared from three
separate cultures of the same strain. Eight strains were
studied, and the patterns for the same strain were 9295%
Table 1 (Contd.)
Sourdough* and
wheat flour number
City of
production Strain Type of wheat flour
Fermentation
time (h)
48 Ancona Lb. brevis8C6; Lb. farciminis F3, 3xA4;
Lb. plantarum 3xA6
T. aestivum
49 Ancona Lb. farciminis 9xC8, 10xF6; Lb. brevis DE5 T. aestivum
*Sourdough number also indicates the manufacturer number.
Wheat flour.
Lecce, Bari, Brindisi and Foggia are in the Puglia region; Perugia, Amelia, Terni, Marsciano and Foligno are in the Umbria region; Pesaro and
Ancona are in the Marche region2 .
644 A . C O R S E T T I ET AL.
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similar, indicating that the reproducibility of the technique
under the conditions used was high (data not shown).
The three primers P1, P4 and P7 showed distinct band
patterns following agarose gel electrophoresis (data not
shown). In particular, primers P1 and P4 produced different
bands ranging from 500 to ca. 4000 bp, while primer P7gave bands from 246 to ca. 4000 bp. Nevertheless, none of
the three primers selected was useful for obtaining species-
specific bands.
The combined RAPD profiles generated with primers P1,
P4 and P7 for 150 isolates and eight type strains produced
the UPGMA dendrogram shown in Fig. 1. In general, the
eight different Lactobacillus andWeissella species, including
the respective ATCC type strains, were discriminated from
each other and grouped into nine clusters separated at a
similarity level of about 30%. Details of each cluster are
given in Table 2. The strains identified as Lb. alimentarius
grouped into two separate clusters (nos. 5 and 7) while, atthe above similarity level, some strains (Lb. plantarum 19A,
Lb. plantarum P2 and W. confusa 8V), which produced a
unique RAPD pattern, did not belong to any cluster. Not all
strains of a species formed homogeneous groups. Cluster no.
2 comprised all the Lb. farciminis isolates besides the
type strain ATCC 29644 and the strains Lb. fermentumCD5
and Lb. brevis DE5. Cluster no. 4 contained all the
Lb. fructivorans isolates and one Lb. brevis (strain DE9),
which did not group within the majorLb. breviscluster no. 1
(Fig. 1 and Table 2).
At a similarity level of 59%, each cluster included
different sub-clusters (Fig. 1). Overall, considering the 24
sub-clusters formed, and excluding sub-clusters 4b and 5b,which contained only one isolate besides the type strain, 17
sub-clusters included strains isolated from the same region
(sub-clusters 1a, 2b, 5d, 6b, 6d, 6f, 6h, 7a, 8a and 9a) or from
the same city (sub-clusters 1d, 3a and 5a) or from the same
manufacturer (sub-clusters 1c, 4a, 5c and 6c) (Fig. 1 and
Table 1), while the remaining five sub-clusters (1b, 2a, 6a,
6e and 6g) contained mainly strains from the same region
(from four strains in sub-cluster 2a to 10 strains in sub-
cluster 6a) besides one or two strains of different origin.
In particular, sub-clusters 1a and 1c contained all the
Lb. brevisstrains isolated from the Puglia region, sub-cluster
1b included the other five Lb. brevis isolates from Pugliasourdoughs and a strain (I5) from a manufacturer located in
the Umbria region, while sub-cluster 1d comprised all the
other strains from the Umbria region. At a similarity level of
59%, the strains isolated from organic flours (Lb. brevis1xF5
and 8C6) and strain 25K from the Puglia region did not
belong to any sub-cluster.
Considering the major cluster, no. 6, which included all
the Lb. sanfranciscensis isolates and the two unidentified
strains, 4R and 11N (overall 40% of the microorganisms
studied), it was observed that some sub-clusters (e.g. 6b, 6c,
6d and 6f) included strains that were isolated from
sourdough of the same manufacturer or produced from a
common type of flour and time of fermentation (sourdough
nos. 24, 26, 27, 28 and 41) (Fig. 1 and Table 1).
The five strains of Lb. sanfranciscensis (7N, 5D, 7A, 7H
and 91) isolated from sourdough from various manufactur-ers in the Puglia region were included, besides isolates from
the Umbria region, in the sub-clusters 6a, 6e and 6g. In
these cases, the three manufacturers from the Puglia region
(nos. 5, 7 and 9) used a different type of flour and time of
fermentation (Table 1).
The speciesLb. plantarum showed the highest number of
strains with unique RAPD profiles. While 10 strains
grouped together with the type strain ATCC 14917 in
sub-cluster 8a, the remaining seven isolates did not belong to
any sub-cluster (Fig. 1).
Cell-wall protein characterization
The reproducibility of the SDS-PAGE method was esti-
mated by loading two independent, triplicate cell-wall
protein extracts from eight strains on two gels. The relative
error for each band in each gel was less than 1% (Gomez-
Zavaglia et al. 1999). Based on preliminary assays, the
resolving power of SDS-PAGE was higher when 12%
acrylamide was used (data not shown). Representative SDS-
PAGE cell-wall protein profiles for the eight species of LAB
(sevenLactobacillus spp. and one Weissella spp.) are shown
in Fig. 2. Following the SDS-PAGE analysis, two main
groups including Lb. alimentarius strains were formed. For
this reason, two strains, representative ofLb. alimentariusgroups I and II, were analysed for that species.
A protein band ofca.50 kDa was found, at different levels,
for all the isolates;Lb. sanfranciscensis,Lb. brevis,Lb. alimen-
tarius group II, Lb. plantarum and W. confusa showed the
highest level of expression of that protein. Moreover, all
the strains, with the exception of those belonging to the
Lb. alimentarius group II, showed another common protein
ofca. 95 kDa. In general, each species showed some protein
bands common to all the strains of that species and other
bands present in only some strains. All Lb. sanfranciscensis
strains showed three well-defined proteins ofca. 95, 50 and
135 kDa. Lb. fermentum strains were characterized by twocommon protein bands of 123 and 44 kDa, while Lb. brevis
had a very intense band at 50 kDa and a less intense band at
ca.37 kDa. Four bands at molecular masses of 95, 55, 50 and
14 kDa characterized the species Lb. farciminis. Lb. alimen-
tariusgroup I comprised all those strains producing at least
seven well-defined bands ranging from 95 to 14 kDa, while
the Lb. alimentarius strains of the group II expressed three
main proteins of 48, 40 and 31 kDa. All the Lb. plantarum
strains were characterized by six well-marked proteins in the
range 6535 kDa. Both Lb. fructivorans and W. confusa
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100908070605040302010
Lb. brevis10D
Lb. brevis20T
Lb. brevis21SLb. brevis20E
Lb. brevis24V
Lb. brevis24A
Lb. brevis6LLb. brevis10R
Lb. brevis10I
Lb. brevis18C
Lb. brevis9VLb. brevis10A
Lb. brevisATCC 14869T
Lb. brevis18F
Lb. brevisI5Lb. brevis15R
Lb. brevis102
Lb. brevis5Z
Lb. brevis 1QLb. brevis1xF5
Lb. brevis25K
Lb. brevis1D
Lb. brevis1FLb. brevis8C6
Lb. brevisDA64
Lb. brevisAM7
Lb. brevisAM8Lb. farciminis5xF12
Lb. farciminis2xA6
Lb. farciminisF3
Lb. fermentumCD5Lb. brevis DE5Lb. farcimins10xF6
Lb. farciminis5xF14
Lb. farciminis2xA3Lb. farciminisATCC 29644T
Lb.farciminis9xC8
Lb.farciminis5C1
Lb.farciminis3xA4Lb. fermentum6E
Lb. fermentumI2
Lb. fermentumATCC 14931T
Lb. fructivoransDA110Lb. fructivoransDD7
Lb. fructivoransDD10
Lb. brevisDE9
Lb. fructivorans ATCC 8288T
Lb. fructivoransP9
Lb. fructivoransDD8
Lb. fructivoransP4
Lb. alimentarius3DLb. alimentariusDA70
Lb. alimentarius5A
Lb. alimentarius2SLb. alimentariusF13
Lb. alimentariusATCC 29643T
Lb. alimentarius5S
Lb. alimentarius52Lb. alimentariusAN2
Lb. alimentariusO9
Lb. alimentariusI4
N.I. 11NN.I. 4R
Lb. sanfranciscensis13R
Lb. sanfranciscensisE20
Lb. sanfranciscensisA15Lb. sanfranciscensisE5
Lb. sanfranciscensisA4
Lb. sanfranciscensisE13
Lb. sanfranciscensis62Lb. sanfranciscensisBB12
Lb. sanfranciscensis12
Lb. sanfranciscensisCB1
Lb. sanfranciscensisH4Lb. sanfranciscensis7N
Lb. sanfranciscensis5D
Lb. sanfranciscensis7M
Lb. sanfranciscensisA1Lb. sanfranciscensis125
Lb. sanfranciscensis72
Lb. sanfranciscensis57
Lb. sanfranciscensis57curLb. sanfranciscensisI1
Lb. sanfranciscensisE18
Lb. sanfranciscensisE21
Lb. sanfranciscensisE19
Lb. sanfranciscensis73Lb. sanfranciscensisH6
Lb. sanfranciscensisH5
Lb. sanfranciscensisH1Lb. sanfranciscensisH3
Lb. sanfranciscensis274
Lb. sanfranciscensis77st
Lb. sanfranciscensis7ALb. sanfranciscensis174
Lb. sanfranciscensis79
Lb. sanfranciscensisE12
Lb. sanfranciscensisDI7Lb. sanfranciscensisE17
Lb. sanfranciscensisE16
Lb. sanfr. ATCC 27651T
Lb. sanfranciscensisE15Lb. sanfranciscensisE10
Lb. sanfranciscensisH7
Lb. sanfranciscensisE22
Lb. sanfranciscensisE9Lb. sanfranciscensisE7
Lb. sanfranciscensisE6
Lb. sanfranciscensisE3
Lb. sanfranciscensisH10Lb. sanfranciscensisA2Z
Lb. sanfranciscensisA17
Lb. sanfranciscensisA22
Lb. sanfranciscensisE14Lb. sanfranciscensisA7
Lb. sanfranciscensis7H
Lb. sanfranciscensis91
Lb. sanfranciscensisA6Lb. sanfranciscensis9N
Lb. sanfranciscensis2A
Lb. sanfranciscensis9F
Lb. alimentarius16BLb. alimentarius15
Lb. alimentarius16I
Lb. alimentarius16M
Lb. alimentarius8DLb. alimentarius16R
Lb. alimentarius15A
Lb. alimentarius15M
Lb. alimentarius17DLb. alimentarius15F
Lb. alimentarius162
Lb. alimetarius16ALb. alimentarius16
Lb. alimentarius5Q
Lb. plantarumDC400
Lb. plantarumDB200Lb. plantarumATCC 14917T
Lb. plantarumCF1
Lb. plantarum7C5
Lb. plantarum2A1Lb. plantarumAD4
Lb. plantarum13
Lb. plantarum20
Lb. plantarum18Lb. plantarum30
Lb. plantarum2F3
Lb. plantarum3xA6
Lb. plantarum21BLb. plantarum21A
Lb. plantarum20B
Lb. plantarum19A
Lb. plantarumP2W. confusa8VW. confusa DSM 20196T
W. confusa8L
W. confusa14SW. confusa14R
Similarity (%)
1
3
4
5
6
7
8
9
a
b
c
d
a
b
a
a
b
a
bcd
a
bc
d
e
f
g
h
a
a
a
Cluster no.
2
Sub-cluster
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showed seven protein bands ranging from 95 to 45 kDa, and
from 95 to 31 kDa, respectively.
The variability in cell-wall protein production among the
strains is summarized in the UPGMA dendrogram in
Fig. 3. Details concerning each cluster are given in Table 2.
At a similarity level of 82%, the type strains and the isolates
from sourdough and organic flours grouped into nine
clusters that separated the entire LAB belonging to the
different species (Fig. 3). At the above similarity level, only
the two unidentified strains, 4R and 11N, did not group
into any cluster. Owing to a different protein pattern,
Lb. alimentarius strains formed two clusters (nos. 4 and 6)joined at a similarity level ofca. 74%.
At a similarity level of ca. 93%, LAB fell into 24 sub-
clusters, 17 of which included strains from the same origin
(Fig. 3 and Table 1). In particular, sub-clusters 1e, 2a, 3a, 4a,
4b, 5a, 5b, 6a and 8a grouped isolates from the same region;
sub-clusters 1d, 7a and 9a comprised isolates from the same
city and sub-clusters 1g, 3b, 3c, 6b and 7c contained strains
isolated from dough produced by the same manufacturer
(Fig. 3 and Table 1). The other sub-clusters (1a, 1b, 1f, 3d,
4c and 7b) comprised, besides strains of the same origin, a
number of strains, ranging from one (sub-cluster 1a) to five
(sub-cluster 1b), from other geographic area.
Unlike RAPD analysis, the SDS-PAGE-based cluster-ing showed that some strains (e.g. Lb. sanfranciscensis BB12
and 12, Lb. sanfranciscensis E10, E15, E16 and E17,
Lb. plantarum 13 and 18, as well as other strains) (Fig. 3)
had an identical profile, being characterized by a similarity
level of 100%.
DISCUSSION
LAB isolated from four organic flours and 45 sourdoughs
produced in the Centre and South of Italy were mainly
Lactobacillus spp. and Weissella spp. A combination of two
techniques, RAPD-PCR and cell-wall protein analysis, wasused to differentiate the 152 isolates and eight type strains,
one for each species identified. RAPD-PCR was recently
used to differentiate 56 Lb. sanfranciscensis strains isolated
from Italian sourdoughs (Zapparoli et al. 1998) and to
discriminate a total of 36 species ofLactobacillus and three
species ofWeissella (Nigatu et al. 2001).
In this study, we applied the RAPD analysis to a large
number of strains belonging to eight different species. The
combination of three primers, P1, P4 and P7, was useful for
differentiating eight type strains and, with some exceptions,
to separate the species at a similarity level of ca. 30%.
Nigatuet al. (2001) stated that the use of a large number of
strains of each species is very important for evaluating the
effectiveness of the RAPD analysis. Moreover, the same
authors observed that the inclusion of many isolates can
muddle the discrimination between species due to thevariation in band patterns and random similarities occurring
within and between field and type strains. Vogel et al.
(1996) and Kurzak et al. (1998) used the PCR-RAPD
technique to characterize sourdough and gut-associated
LAB, respectively, and reported a non-perfect separation
among different species, even tough, under well-defined
experimental conditions, the majority of the strains could be
correctly attributed to their proper species (Vogel et al.
1996).
On the other hand, Zapparoli et al. (1998), who applied
RAPD typing to a large number of strains of Lb. sanfranci-
scensis, found that this technique was useful for differenti-ating the strains within species.
As previously observed in one of our studies on the
characterization of cheese-relatedLactobacillusspp. by using
the same three primers, P1, P4 and P7 (De Angelis et al.
2001), we did not find a species-specific DNA band, even
though each primer produced a specific combination of
bands for individual clusters. The combined use of primers
P1, P4 and P7 separated the 25 Lb. alimentarius strains,
previously identified by phenotypic assays and, in some
cases, by partial 16S rDNA sequencing (Gobbetti et al.
1994; Corsetti et al . 2001), in two clusters below the
similarity level (30%) useful for distinguishing different
species. In a study on taxonomic characterization of LABisolated from sourdough, Cai et al. (1999) found some
strains, which, on the basis of phenotypic characteristics
and of 16S rRNA sequencing analysis, were similar to
Lb. alimentarius. Nevertheless, DNADNA hybridization
studies indicated that those strains did not belong to
Lb. alimentarius and for that reason the authors proposed a
new name for them, Lb. paralimentarius sp. nov.
The discriminatory power of RAPD analysis seemed to
be useful for resolving intraspecific differences among
strains, in most cases according to the geographical origin
and/or the technology used to produce the sourdoughs.
For example, even though some strains (I5, CD5, 7N, 5D,7A, 7H and 91) were sub-clustered besides strains isolated
from sourdough from a different region, it could be
observed that, some of them (7N, 7A, 7H and 91), had
been isolated from sourdoughs produced by similar flours
(T. aestivum) and with a short time of fermentation (35 h)
(Tables 1 and 2). A possible effect of the technological
processes and geographic area on the selection of genetic-
ally diverse groups of lactobacilli has been presumed by
some authors (Zapparoli et al. 1998; De Angelis et al.
2001).
Fig. 1 Dendrogram obtained from combined RAPD patterns with
three primers ofLactobacillus and Weissella isolates from flours and
sourdoughs and type strains. A cluster analysis was conducted with
similarity estimates by using UPGMA
b
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RAPD cluster* CWP cluster City of production Strain
6 1 Bari Lactobacillus sanfranciscensis 9F
6 1 Terni Lb. sanfranciscensis BB12
6 1 Terni Lb. sanfranciscensis 12
6 1 Perugia Lb. sanfranciscensis E56 1 Terni Lb. sanfranciscensis CB1
6 1 Perugia Lb. sanfranciscensis 77St
6 1 Marsciano Lb. sanfranciscensis H3
6 1 Perugia Lb. sanfranciscensis E7
6 1 Bari Lb. sanfranciscensis 7M
6 1 Lecce Lb. sanfranciscensis 2A
6 1 Bari Lb. sanfranciscensis 7N
6 1 Perugia Lb. sanfranciscensis E6
6 1 Lecce Lb. sanfranciscensis 5D
6 1 Perugia Lb. sanfranciscensis E3
6 1 Perugia Lb. sanfranciscensis I1
6 1 Perugia Lb. sanfranciscensis 57
6 1 Bari Lb. sanfranciscensis 13R
6 1 Perugia Lb. sanfranciscensis E17
6 1 Perugia Lb. sanfranciscensis E16
6 1 Perugia Lb. sanfranciscensis E15
6 1 Perugia Lb. sanfranciscensis E10
6 1 Perugia Lb. sanfranciscensis 274
6 1 Perugia Lb. sanfranciscensis 174
6 1 Perugia Lb. sanfranciscensis 79
6 1 Perugia Lb. sanfranciscensis 73
6 1 Perugia Lb. sanfranciscensis A6
6 1 Bari Lb. sanfranciscensis 7H
6 1 Bari Lb. sanfranciscensis 91
6 1 Perugia Lb. sanfranciscensis E9
6 1 Marsciano Lb. sanfranciscensis H10
6 1 Perugia Lb. sanfranciscensis E226 1 Perugia Lb. sanfranciscensis A7
6 1 Perugia Lb. sanfranciscensis A22
6 1 Perugia Lb. sanfranciscensis E14
6 1 Perugia Lb. sanfranciscensis A17
6 1 Lb. sanfranciscensis ATCC 27651T
6 1 Perugia Lb. sanfranciscensis E13
6 1 Perugia Lb. sanfranciscensis E20
6 1 Marsciano Lb. sanfranciscensis H1
6 1 Marsciano Lb. sanfranciscensis H7
6 1 Marsciano Lb. sanfranciscensis H4
6 1 Perugia Lb. sanfranciscensis A15
6 1 Terni Lb. sanfranciscensis 62
6 1 Terni Lb. sanfranciscensis 125
6 1 Perugia Lb. sanfranciscensis DI7
6 1 Terni Lb. sanfranciscensis 72
6 1 Perugia Lb. sanfranciscensis E12
6 1 Bari Lb. sanfranciscensis 7A
6 1 Perugia Lb. sanfranciscensis E18
6 1 Marsciano Lb. sanfranciscensis H6
6 1 Marsciano Lb. sanfranciscensis H5
6 1 Perugia Lb. sanfranciscensis 57cur
6 1 Perugia Lb. sanfranciscensis A2Z
6 1 Bari Lb. sanfranciscensis 9N
Table 2 Characteristics of the lactic acid
bacteria type strains and isolates from Italian
sourdoughs and flours
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Table 2 (Contd.)RAPD cluster* CWP cluster City of production Strain
6 1 Perugia Lb. sanfranciscensis E19
6 1 Perugia Lb. sanfranciscensis E21
6 1 Perugia Lb. sanfranciscensis A1
6 1 Perugia Lb. sanfranciscensis A43 2 Perugia Lb. fermentum 6E
3 2 Perugia Lb. fermentum I2
3 2 Lb. fermentum ATCC 14931T
2 2 Foligno Lb. fermentum CD5
1 3 Lecce Lb. brevis1Q
1 3 Lecce Lb. brevis6L
1 3 Foggia Lb. brevis24A
1 3 Bari Lb. brevis10D
1 3 Foggia Lb. brevis20T
1 3 Foggia Lb. brevis24V
1 3 Foggia Lb. brevis25K
1 3 Foggia Lb. brevis21S
1 3 Foggia Lb. brevis20E
1 3 Lecce Lb. brevis1D
1 3 Lecce Lb. brevis1F
1 3 Foligno Lb. brevisAM7
1 3 Foligno Lb. brevisAM8
4 3 Perugia Lb. brevisDE9
2 3 Ancona Lb. brevisDE5
1 3 Pesaro Lb. brevis 1xF5
1 3 Bari Lb. brevis 10R
1 3 Lb. brevis ATCC 14869T
1 3 Bari Lb. brevis9V
1 3 Foggia Lb. brevis18C
1 3 Foggia Lb. brevis18F
1 3 Perugia Lb. brevisI5
1 3 Foligno Lb. brevisDA641 3 Bari Lb. brevis10A
1 3 Bari Lb. brevis10I
1 3 Lecce Lb. brevis5Z
1 3 Bari Lb. brevis10a
1 3 Brindisi Lb. brevis15R
1 3 Ancona Lb. brevis8C6
7 4 Brindisi Lb. alimentarius 17D
5 4 Foligno Lb. alimentarius AN2
5 4 Marsciano Lb. alimentarius F13
5 4 Perugia Lb. alimentarius O9
5 4 Lecce Lb. alimentarius 5a
5 4 Lecce Lb. alimentarius 3D
5 4 Lecce Lb. alimentarius DA70
5 4 Lecce Lb. alimentarius 5S
5 4 Perugia Lb. alimentarius I4
5 4 Lecce Lb. alimentarius 2S
5 4 Lecce Lb. alimentarius 5A
5 4 Lb. alimentarius ATCC 29643T
2 5 Ancona Lb. farciminis 9xC8
2 5 Ancona Lb. farciminis 3xA4
2 5 Pesaro Lb. farciminis 5C1
2 5 Pesaro Lb. farciminis 2xA3
2 5 Ancona Lb. farciminis 10xF6
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RAPD cluster* CWP cluster City of production Strain
2 5 Pesaro Lb. farciminis 5xF14
2 5 Ancona Lb. farciminis F3
2 5 Pesaro Lb. farciminis 2xA6
2 5 Pesaro Lb. farciminis 5xF122 5 Lb. farciminis ATCC 29644T
7 6 Brindisi Lb. alimentarius 15b
7 6 Brindisi Lb. alimentarius 16c
7 6 Brindisi Lb. alimentarius 16M
7 6 Bari Lb. alimentarius 8D
7 6 Brindisi Lb. alimentarius 16A
7 6 Brindisi Lb. alimentarius 16a
7 6 Brindisi Lb. alimentarius 16R
7 6 Brindisi Lb. alimentarius 15A
7 6 Lecce Lb. alimentarius 5Q
7 6 Brindisi Lb. alimentarius 16I
7 6 Brindisi Lb. alimentarius 16B
7 6 Brindisi Lb. alimentarius 15M
7 6 Brindisi Lb. alimentarius 15F
8 7 Perugia Lb. plantarum 7C5
8 7 Foligno Lb. plantarum 2A1
8 7 Ancona Lb. plantarum 3xA6
8 7 Pesaro Lb. plantarum 2F3
8 7 Foggia Lb. plantarum 21A
8 7 Perugia Lb. plantarum DB200
8 7 Perugia Lb. plantarum DC400
8 7 Lb. plantarum ATCC 14917T
8 7 Perugia Lb. plantarum 30
8 7 Foggia Lb. plantarum 20B
8 7 Perugia Lb. plantarum 20
8 7 Foligno Lb. plantarum AD4
8 7 Foggia Lb. plantarum 21B8 7 Perugia Lb. plantarum CF1
8 7 Perugia Lb. plantarum 13
8 7 Perugia Lb. plantarum 18
SC 7 Foggia Lb. plantarum 19A
SC 7 Perugia Lb. plantarum P2
4 8 Amelia Lb. fructivorans DD10
4 8 Perugia Lb. fructivorans P4
4 8 Amelia Lb. fructivorans DD7
4 8 Lb. fructivorans ATCC 8288T
4 8 Amelia Lb. fructivorans DA110
4 8 Perugia Lb. fructivorans P9
4 8 Perugia Lb. fructivorans DD8
9 9 Weissella confusa DSM 20196T
9 9 Bari W. confusa 14S
9 9 Bari W. confusa 14R
9 9 Bari W. confusa 8L
SC 9 Bari W. confusa 8V
6 SC Lecce N.I. 4R
6 SC Bari N.I. 11N
CWP, cell-wall proteins.
*Cluster numbers that refer to the dendrogram of Fig. 1.
Cluster numbers that refer to the dendrogram of Fig. 3.
SC, single cluster in the related dendrogram.
Table 2 (Contd.)
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Other authors (Costaset al. 1990; Pot et al. 1993; Samelis
et al. 1995) used cell protein analysis to differentiate bacterial
strains, but there have been no studies on the application of
this technique to LAB isolated from sourdough.Confirming the results of our previous study (De Angelis
et al. 2001), we obtained, by SDS-PAGE, cell-wall pro-
tein profiles that could resolve, better than the RAPD
analysis, the LAB at species level (Figs 1 and 3). Using the
same technique for the characterization of thermophilic
cheese starters, Gatti et al . (1997) identified different
combinations of protein bands that were useful to classify
the speciesLb. helveticus, Lb. delbrueckii, Lb. acidophilus and
Lb. fermentum.
As shown in the dendrogram of Fig. 3, the species of LAB
showed different degrees of overall similarity. Lb. farciminis
andLb. sanfranciscensis, with an overall similarity level ofca.83 and 84%, respectively, appeared as the most heteroge-
neous species, while Lb. fructivorans, with a similarity
level of 96%, seemed the least variable. As for the RAPD
analysis, typing of cell-wall proteins resolved the strains of
Lb. alimentariusspecies into two major clusters (nos. 4 and 6)
(Fig. 3); moreover, with the exception of strain 17D, both
clusters included the same strains both with RAPD and cell-
wall protein analysis (Figs 1 and 3), supporting the hypo-
thesis of the presence, into the Lb. alimentarius spp., of two
well-separated lines.
At the similarity level (82%) that resolved the eight
species considered in this study, the two unidentified strains,
4R and 11N, did not belong to any cluster but showed the
highest similarities with the clusters Lb. sanfranciscensisLb. fermentum and W. confusa, respectively (Fig. 3). This
result partially reflects that obtained with the RAPD
analysis, which included both strains in the Lb. sanfranci-
scensiscluster 6 (Fig. 1). Over the similarity level of 93%, it
was possible, as with RAPD analysis, to justify the presence
of some strains in the same sub-cluster on the basis of a
common geographic origin and/or similar technological
parameters. Gatti et al. (1997), in a study on cell-wall
protein profiles of dairy thermophilic lactobacilli reported
that, for most of the microorganisms studied, it seemed to be
possible to discern a relationship between the source of the
strains and their cell-wall pattern, suggesting that differ-ences in cell-wall protein profiles might be related to strain
adaptation to different ecological niches and cheese tech-
nology.
On the basis of the cell-wall protein analysis, some
strains showed the same protein pattern. When isolated
from the same sourdough, as for Lb. sanfranciscensis
strains E10, E15, E16 and E17, they could represent
different isolates of the same strain or, at least, very
similar isolated ones as also confirmed by the high
similarity level among those strains within the RAPD
1kDa
220160
120100
908070
60
50
40
30
25
20
15
10
2 3 4 5 6 7 8 9 10
Fig. 2 SDS-PAGE patterns of cell-wall proteins from Lactobacillus and Weissella isolates. Lane 1, standard proteins (see Materials and Methods);
lane 2, Lb. sanfranciscensis 9F; lane 3, Lb. fermentum I2; lane 4, Lb. brevis 10A; lane 5, Lb. farciminis F3; lane 6, Lb. alimentarius 5A; lane 7, Lb.
alimentarius 8D; lane 8, Lb. plantarum DC400; lane 9, Lb. fructivorans DA110; lane 10, W. confusa 14R
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Lb. sanfranciscensis9F
Lb. sanfranciscensisBB12Lb. sanfranciscensis12Lb. sanfranciscensisE5Lb. sanfranciscensisCB1Lb. sanfranciscensisH3Lb. sanfranciscensis77StLb. sanfranciscensisE7Lb. sanfranciscensis7MLb. sanfranciscensis2A
Lb. sanfranciscensis7N
Lb. sanfranciscensisE6
Lb. sanfranciscensis5DLb. sanfranciscensisE3Lb. sanfranciscensisI1Lb. sanfranciscensis57Lb. sanfranciscensis13RLb. sanfranciscensisE17Lb. sanfranciscensisE16Lb. sanfranciscensisE15
Lb. sanfranciscensisE10Lb. sanfranciscensis274
Lb. sanfranciscensis174
Lb. sanfranciscensis79Lb. sanfranciscensis73Lb. sanfranciscensisA6Lb. sanfranciscensis7HLb. sanfranciscensis91Lb. sanfranciscensisE9Lb. sanfranciscensisH10Lb. sanfranciscensisE22Lb. sanfranciscensisA7
Lb. sanfranciscensisA22Lb. sanfranciscensisE14
Lb..sanfranciscensisA17
Lb sanfranciscensisATCC 27651T
Lb. sanfranciscensisE13
Lb. sanfranciscensisE20Lb. sanfranciscensisH1
Lb. sanfranciscensisH7Lb. sanfranciscensisH4Lb. sanfranciscensisA15Lb. sanfranciscensisD17Lb. sanfranciscensis125Lb. sanfranciscensis62Lb. sanfranciscensis72
Lb. sanfranciscensisE12Lb. sanfranciscensis7A
Lb. sanfranciscensisE18
Lb. sanfranciscensisH6Lb. sanfranciscensisH5Lb. sanfranciscensis57curLb. sanfranciscensisA2ZLb. sanfranciscensis9NLb. sanfranciscensisE19Lb. sanfranciscensisE21Lb. sanfranciscensisA1Lb. sanfranciscensisA4
Lb. fermentum6ELb. fermentum I2
Lb. fermentumCD5Lb. fermentumATCC 14931T
N.I. 4RLb. brevis1QLb. brevis6LLb. brevis24ALb. brevis10DLb. brevis20TLb. brevis24V
Lb. brevis25K
Lb. brevis21SLb. brevis20E
Lb. brevis1DLb. brevis1FLb. brevisAM7Lb. brevisAM8Lb. brevis1xF5Lb. brevis10RLb. brevis8C6Lb. brevis9VLb. brevis18CLb. brevis18F
Lb. brevis I5Lb. brevisDE9
Lb. brevisDA64Lb. brevis10A
Lb. brevis10ILb. brevisDE5
Lb. brevis5Z
Lb. brevis102Lb. brevis15RLb. brevisATCC 14869T
Lb. alimentarius O9Lb. alimentarius AN2Lb. alimentarius F13Lb. alimentarius 17DLb. alimentarius 52Lb. alimentarius 3D
Lb. alimentarius DA70Lb. alimentarius 5S
Lb. alimentarius I4
Lb. alimentarius 2SLb. alimentarius 5ALb.alimentariusATCC29643TLb. farciminis 9xC8Lb. farciminis 3xA4Lb. farciminis 5C1Lb. farciminis 2xA3Lb. farciminis 10xF6Lb. farciminis 5xF14Lb. farciminis F3Lb. farciminis 2xA6
Lb. farciminis 5xF12Lb. farciminisATCC 29644T
Lb. alimentarius15Lb. alimentarius16Lb. alimentarius16M
Lb. alimentarius8DLb. alimentarius16A
Lb. alimentarius162Lb. alimentarius16RLb. alimentarius15ALb. alimentarius5QLb. alimentarius16ILb. alimentarius16BLb. alimentarius15MLb. alimentarius15F
Lb. plantarum 20B
Lb. plantarum 21ALb. plantarum 21B
Lb. plantarum 19ALb. plantarum ATCC 14917T
Lb. plantarum 2A1
Lb. plantarum DB200Lb. plantarum DC400Lb. plantarum 2F3Lb. plantarum 30Lb. plantarum 7C5Lb. plantarum 20Lb. plantarum AD4Lb. plantarum 21BLb. plantarum CF1Lb. plantarum P2Lb. plantarum 13Lb. plantarum 18
Lb. fructivorans DA110Lb. fructivorans DD7
Lb. fructivorans DD10Lb. fructivorans P4
Lb. fructivorans DD8Lb. fructivorans P9Lb. fructivoransATCC 8288TW. confusa 20196T
W. confusa 14SW. confusa 14RW. confusa 8VW. confusa 8LN.I. 11N
Cluster no.
2
4
6
8
a
b
c
d
e
f
1
2
4
6
8
1
g
a
a
bc
d
a
b
c
a
b
a
b
a
b
c
a
a
50 75 10025 Similarity (%)
1
2
3
4
5
6
7
8
9
Sub-cluster
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clusters. In other cases, the differences among strainsshowing an identical cell-wall protein pattern (e.g. Lb.
plantarum AD4 and 21B) (Fig. 3) but having a different
origin (Table 1) could be resolved only by the RAPD
analysis, which joined the above strains at a similarity
level of about 32%, near to the level used to differentiate
the species (Fig. 1).
Based on the RAPD and cell-wall protein typing of a large
number of LAB strains isolated from Italian flours and
sourdoughs, the following conclusions could be drawn: (i) by
using the RAPD analysis, some strains cannot be assigned to
the correct species; (ii) cell-wall protein analysis represents a
more useful tool for the correct grouping of a large numberof isolates of LAB belonging to many species; (iii) both
methods of analysis can resolve some intraspecific differ-
ences (e.g. classification of the Lb. alimentarius strains into
two groups); (iv) in some cases, RAPD analysis improves the
ability of cell-wall protein analysis to differentiate the
strains; (v) geographic origin and technological parameters
may be responsible for the selection of similar lines of
microorganisms.
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