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.

2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 641654


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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 .




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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. sanfranciscensis62Lb. sanfranciscensisBB12

Lb. sanfranciscensis12

Lb. sanfranciscensisCB1

Lb. sanfranciscensisH4Lb. sanfranciscensis7N

Lb. sanfranciscensis5D

Lb. sanfranciscensis7M

Lb. sanfranciscensisA1Lb. sanfranciscensis125



Lb. sanfranciscensis57curLb. sanfranciscensisI1




Lb. sanfranciscensis73Lb. sanfranciscensisH6

Lb. sanfranciscensisH5

Lb. sanfranciscensisH1Lb. sanfranciscensisH3


Lb. sanfranciscensis77st

Lb. sanfranciscensis7ALb. sanfranciscensis174



Lb. sanfranciscensisDI7Lb. sanfranciscensisE17


Lb. sanfr. ATCC 27651T

Lb. sanfranciscensisE15Lb. sanfranciscensisE10

Lb. sanfranciscensisH7


Lb. sanfranciscensisE9Lb. sanfranciscensisE7



Lb. sanfranciscensisH10Lb. sanfranciscensisA2Z



Lb. sanfranciscensisE14Lb. sanfranciscensisA7

Lb. sanfranciscensis7H


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




7/14

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




8/14

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 Lecce Lb. sanfranciscensis 5D


6 1 Perugia Lb. sanfranciscensis I1

6 1 Perugia Lb. sanfranciscensis 57

6 1 Bari Lb. sanfranciscensis 13R









6 1 Perugia Lb. sanfranciscensis A6

6 1 Bari Lb. sanfranciscensis 7H

6 1 Bari Lb. sanfranciscensis 91



6 1 Perugia Lb. sanfranciscensis E226 1 Perugia Lb. sanfranciscensis A7




6 1 Lb. sanfranciscensis ATCC 27651T









6 1 Perugia Lb. sanfranciscensis DI7



6 1 Bari Lb. sanfranciscensis 7A




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




9/14

Table 2 (Contd.)RAPD cluster* CWP cluster City of production Strain




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




10/14

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 Foligno Lb. plantarum AD4

8 7 Foggia Lb. plantarum 21B8 7 Perugia Lb. plantarum CF1



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.)




11/14

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




12/14

Lb. sanfranciscensis9F

Lb. sanfranciscensisBB12Lb. sanfranciscensis12Lb. sanfranciscensisE5Lb. sanfranciscensisCB1Lb. sanfranciscensisH3Lb. sanfranciscensis77StLb. sanfranciscensisE7Lb. sanfranciscensis7MLb. sanfranciscensis2A

Lb. sanfranciscensis7N


Lb. sanfranciscensis5DLb. sanfranciscensisE3Lb. sanfranciscensisI1Lb. sanfranciscensis57Lb. sanfranciscensis13RLb. sanfranciscensisE17Lb. sanfranciscensisE16Lb. sanfranciscensisE15

Lb. sanfranciscensisE10Lb. sanfranciscensis274


Lb. sanfranciscensis79Lb. sanfranciscensis73Lb. sanfranciscensisA6Lb. sanfranciscensis7HLb. sanfranciscensis91Lb. sanfranciscensisE9Lb. sanfranciscensisH10Lb. sanfranciscensisE22Lb. sanfranciscensisA7

Lb. sanfranciscensisA22Lb. sanfranciscensisE14

Lb..sanfranciscensisA17

Lb sanfranciscensisATCC 27651T


Lb. sanfranciscensisE20Lb. sanfranciscensisH1

Lb. sanfranciscensisH7Lb. sanfranciscensisH4Lb. sanfranciscensisA15Lb. sanfranciscensisD17Lb. sanfranciscensis125Lb. sanfranciscensis62Lb. sanfranciscensis72

Lb. sanfranciscensisE12Lb. sanfranciscensis7A


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




13/14

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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Characterization of Sourdough Lactic Acid Bacteria

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