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doi:10.1016/j.sy

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Microbiologia

Michele a/A (T

Systematic and Applied Microbiology 30 (2007) 561–571

www.elsevier.de/syapm

A taxonomic survey of lactic acid bacteria isolated from wheat

(Triticum durum) kernels and non-conventional flours

Aldo Corsettia,�, Luca Settannia,1, Clemencia Chaves Lopeza, Giovanna E. Felisb,Mario Mastrangeloa, Giovanna Suzzia

aDipartimento di Scienze degli Alimenti, Sezione di Microbiologia Agro-Alimentare ed Ambientale,

Universita degli Studi di Teramo, V.C.R. Lerici 1, 64023 Mosciano Sant’Angelo (TE), ItalybDipartimento Scientifico e Tecnologico, Universita degli Studi di Verona, Strada Le Grazie 15, I-37134 Verona, Italy

Received 26 June 2007

Abstract

In order to explore the correspondence between raw material- and mature sourdough-lactic acid bacterial (LAB)communities, 59 Italian wheat (Triticum durum) grain samples, one bran and six non-conventional flour samples wereanalyzed through a culture-dependent approach. The highest cell count by an agar medium specific for LAB was2.16 logCFU/g. From about 2300 presumptive LAB (Gram-positive and catalase-negative) colonies collected, a totalof 356 isolates were subjected to identification by a genetic polyphasic strategy consisting of RAPD-PCR analysis,partial 16S rRNA gene sequencing, species-specific and multiplex PCRs. The isolates were recognized as 137 strainsbelonging to Aerococcus, Enterococcus, Lactobacillus, Lactococcus and Pediococcus genera and a phylogram based onpartial 16S rRNA genes was constructed. The species most frequently found were Enterococcus faecium, Enterococcus

mundtii and Lactobacillus graminis, which are not generally reported to be typical in mature sourdoughs.r 2007 Elsevier GmbH. All rights reserved.

Keywords: Culture-dependent methods; Genetic polyphasic approach; Lactic acid bacteria; Non-conventional flours; Sourdough;

Triticum durum

Introduction

The discovery of fossil kernels revealed that theutilization of cereals by mankind commenced in theNeolithic era. Millennia BC wheat was already one ofthe most important cultivated cereals. Thanks to its

e front matter r 2007 Elsevier GmbH. All rights reserved.

apm.2007.07.001

ing author. Tel.: +39 (0)861 266896;

071509.

ess: acorsetti@unite.it (A. Corsetti).

ess: Istituto Agrario di San Michele all’Adige (IASMA),

ntale – Dipartimento Qualita Agro-Alimentare, Unita

e Tecnologie Alimentari, V. E. Mach 1, 38010 San

N), Italy.

great adaptability (with its numerous varieties) atdifferent environmental conditions (extremely at highor low temperature and humidity). Nowadays, wheat iscultivated worldwide for human and animal consump-tion [54]. Generally, Triticum durum and Triticum

aestivum are the wheat species used in pasta- andbread-making, respectively. However, in some southernItalian regions, T. durum flour, alone or in associationwith T. aestivum flour, is employed for bread produc-tion, such as Altamura bread produced in the Pugliaregion with durum wheat flour [14].

Cereal grains are naturally contaminated by eucar-yotic (moulds and yeasts) and procaryotic (bacteria)

ARTICLE IN PRESSA. Corsetti et al. / Systematic and Applied Microbiology 30 (2007) 561–571562

organisms. The total microbial population and therelative species proportion on wheat grains can beaffected by many factors, mainly climatic conditionssuch as temperature and rainfall, physical damage dueto insects or mould attacks and application of insecti-cides and fungicides. Micro-organisms of grains mightfollow the different phases of flour preparation and canbe found in products made with flour, such as cereal-based fermented foods and sourdough. Among rawmaterial-associated bacteria, lactic acid bacteria (LAB)play a crucial role in the preservation and the microbialsafety in fermented foods [8], thus promoting themicrobial stability of the final products of fermentation[42]. The protection of foods is due to the production oforganic acids, carbon dioxide, ethanol, hydrogen per-oxide and diacetyl [3,21], antifungal compounds such asfatty acids [12] or phenyllactic acid [37], bacteriocins [21]and antibiotics such as reutericyclin [33].

LAB from mature sourdoughs [15] and wheat flours[13] have been isolated, identified and characterized fortheir many features, but to our knowledge, no papersdealing with LAB associated with wheat kernels andnon-conventional flours, the latter used to producebread for celiac sprue-affected people and in someregional traditional recipes, are available. Thus, in orderto give a more complete overview of raw material-associated indigenous LAB involved in sourdoughproduction, the aims of this study were isolation,identification, molecular characterization and phyloge-netic analysis of LAB naturally occurring on wheatkernels and non-conventional flours.

Materials and methods

Raw materials and isolation of LAB

Fifty-nine wheat samples (Triticum durum), reportedin Table 1, were collected from several Italian regionsand areas. In addition, six non-conventional floursamples and one bran sample were purchased in a retailmarket.

LAB from raw cereals and non-conventional flourswere isolated according to Hartnett et al. [32]. There-after, 1ml of cell suspension was serially diluted.Dilutions were plated onto four agar media, some ofwhich are generally used to isolate LAB associated tosourdough environments: Sour Dough Bacteria (SDB)[35], San Francisco Medium (SFM) [56], MRS (Oxoid,Milan, Italy) and M17 (Oxoid). In order to avoid yeastgrowth, cycloeximide (10 mg/ml) was added to themedia. Petri dishes were anaerobically incubated for 2days at 30 1C; after that, colonies were counted andthose of various shapes were randomly picked from agarplates and transferred to the corresponding broth media.Microbiological counts were performed in duplicate. In

the absence of growth, isolation of LAB was carried outafter the enrichment procedure. All samples (10 g) wereinoculated in 50ml of modified-MRS (mMRS) broth(maltose, lactose and fresh yeast extract were added tothe final concentrations of 1, 1 and 10%, respectively,and the final pH was adjusted to 5.6) and, after 4 days ofincubation at 30 1C, pH was measured (Table 1). For theisolation of LAB, 1ml of cell suspension was plated asdescribed above.

Gram-positive (by Gregersen’s KOH methods [29]),catalase-negative (determined by transferring freshcolonies from an agar medium to a glass slide andadding 5% H2O2), non-motile, cocci and rods werepurified by successive sub-culturing. The purity waschecked microscopically and the cultures were main-tained at �80 1C in glycerol stocks.

Strains and growth conditions

Enterococcus casseliflavus DSM 20680T, Enterococcus

durans DSM 20633T, Enteroccus faecalis DSM 20468T,Enterococcus faecium DSM 20477T, Enterococcus mund-

tii DSM 4838T, Lactobacillus coryniformis subsp.coryniformis DSM 20001T, Lactobacillus curvatus subsp.curvatus DSM 20019T, Lactobacillus graminis DSM20719T, Lactococcus garvieae DSM 20684T and Pedio-

coccus pentosaceus DSM 20336T were the type of strainsused in this study to evaluate strain polymorphism byRAPD-PCR analysis. All strains were cultivated asindicated by the Deutsche Sammlung von Mikroorga-nismen und Zellkulturen (DSMZ).

DNA isolation and LAB differentiation

For the preparation of genomic DNA for PCRassays, cells from 2ml of overnight cultures wereharvested and DNA was extracted according to themethod of De Los Reyes-Gavilan et al. [19]. The finalconcentration of lysozyme used for cell lysis was 2mg/ml. The concentration and purity of DNA was assessedby a biophotometer (Eppendorf, Hamburg, Germany).

In order to perform a first-strain differentiation, theisolates belonging to the same sample were analyzed byRAPD-PCR (at least twice) using primers P4 [11] andM13 [52]. PCR products were separated by electrophor-esis through 1.5% (w/v) agarose gel (Gibco BRL, CergyPontoise, France) containing ethidium bromide (0.5 mg/ml), and the DNA was detected by UV transillumina-tion. RAPD-PCR profiles were acquired using the GelDoc EQ System (Bio-Rad, Munich, Germany) andvisually inspected for comparison.

RAPD patterns from the isolates recognized asdifferent strains were then compared using Fingerprint-ing II InformatixTM Software (Bio-Rad), and weresubjected to a cluster analysis performed with the

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Table 1. Wheat grain and non-conventional flour characteristics

Sample Matrix Cultivar Geographical

origin

LAB counts (log CFU/g)a pH of

enrichment

broths

No of

isolates

LAB species (number of

strains)

MRS M17 SDB SFM

1 Durum wheat grain NR Abruzzo region 1.83�1.91 1.47�1.61 1.30�1.50 1.33�1.41 3.84 7 E. faecium (1), P.

pentosaceus (1)

2 Durum wheat grain NR Abruzzo region 1.24�1.66 1.58�1.90 1.09�1.31 1.10�1.52 4.02 7 E. faecium (1)

3 Durum wheat grain NR Abruzzo region o1.00 o1.00 o1.00 o1.00 4.32 4 E. faecium (1)

4 Durum wheat grain NR Abruzzo region 1.87�2.07 1.94�2.06 1.45�1.65 1.33�1.51 4.01 7 E. faecium (1), Lb.

graminis (2)

5 Durum wheat grain NR Abruzzo region 1.66�1.94 1.8172.07 o1.00�1.16 1.13�1.31 3.86 7 E. faecium (1), P.

pentosaceus (1)

6 Durum wheat grain NR Abruzzo region 2.03�2.13 1.54�1.96 1.34�1.66 1.18�1.32 3.79 7 E. faecium (1), L.

garvieae (1), P.

pentosaceus (1)

7 Durum wheat grain NR Abruzzo region 1.58�1.72 1.86�2.10 1.27�1.43 1.25�1.49 3.88 5 E. faecium (1)

8 Durum wheat grain NR Abruzzo region 1.42�1.84 1.57�1.83 1.56�1.74 1.41�1.53 4.07 8 E. faecium (1), Lb.

coryniformis subsp.

coryniformis (2)

9 Durum wheat grain NR Abruzzo region 1.75�1.99 1.66�1.88 1.16�1.38 o 1.00 4.06 7 E. faecium (1), L.

garvieae (2)

10b Durum wheat grain NR Abruzzo region 1.81�2.03 1.97�2.11 1.79�2.01 1.18�1.56 4.12 6 E. mundtii (1), Lb.

graminis (1)

11c Durum wheat grain NR Puglia region 1.76�1.86 1.32�1.62 1.21�1.35 1.08�1.17 4.37 7 E. faecium (1), E.

mundtii (2)

12c Durum wheat grain NR Puglia region 1.58�1.74 1.24�1.42 1.21�1.49 1.29�1.69 4.15 7 E. faecium (1), Lb.

graminis (2)

13c Durum wheat grain NR Puglia region – – – – 5.86 – –

14c Durum wheat grain NR Puglia region o1.00 o1.00 o1.00 o1.00 4.38 5 E. faecium (1)

15c Durum wheat grain NR Puglia region 1.9570.07 1.43�1.75 1.17�1.43 1.27�1.63 4.21 7 E. faecium (1), P.

pentosaceus (1)

16c Durum wheat grain NR Puglia region o1.00 o1.00 o1.00 – 4.80 6 E. mundtii (2)

17c Durum wheat grain NR Puglia region 1.7870.00 1.62�2.04 1.23�1.47 o1.00�1.16 4.47 5 E. faecium (1)

18c Durum wheat grain NR Puglia region – – – – 5.74 – –

19c Durum wheat grain NR Puglia region 1.44�1.68 o1.00 1.30�1.50 1.33�1.41 4.33 5 Lb. graminis (2)

20c Durum wheat grain NR Puglia region 1.26�1.56 1.34�1.66 o1.00 o1.00 4.63 5 E. mundtii (3)

21c Durum wheat grain NR Puglia region o1.00 o1.00 o1.00 o1.00 4.45 4 E. faecium (1)

22c Durum wheat grain NR Puglia region 1.55�1.69 1.34�1.54 1.22�1.46 1.00�1.30 4.18 8 E. casseliflavus (3), E.

faecium (1)

23d Durum wheat grain NR Abruzzo region o1.00 – o1.00 – 3.85 5 Lb. graminis (1)

24d Durum wheat grain NR Abruzzo region o1.00 – o1.00 – 3.89 4 Lb. graminis (1)

25d Durum wheat grain NR Abruzzo region 2.02�2.16 1.59�1.87 1.57�1.83 1.18�1.32 4.19 7 E. faecium (1), E.

mundtii (2), Lb. graminis

(1)

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Table 1. (continued )

Sample Matrix Cultivar Geographical

origin

LAB counts (log CFU/g)a pH of

enrichment

broths

No of

isolates

LAB species (number of

strains)

MRS M17 SDB SFM

26d Durum wheat grain NR Abruzzo region 1.87�2.11 1.53�1.83 1.13�1.41 1.14�1.44 4.15 8 E. durans (3), E. faecium

(1), Lb. graminis (1), L.

garvieae (1)

27d Durum wheat grain NR Abruzzo region o1.00 o1.00 o1.00 o1.00 3.83 6 Lb. graminis (2)

28d Durum wheat grain NR Abruzzo region 1.70�1.70 1.52�1.74 1.03�1.11 1.06�1.24 4.36 5 E. faecium (1)

29d Durum wheat grain NR Abruzzo region 1.43�1.65 1.62�1.98 o1.00 o1.00 4.56 5 E. mundtii (4)

30d Durum wheat grain NR Abruzzo region o1.00 o1.00 o1.00 o1.00 4.87 6 E. casseliflavus (3)

31d Durum wheat grain NR Abruzzo region 1.89�1.97 1.71�1.95 1.25�1.57 1.16�1.44 4.42 6 E. faecium (1), E.

mundtii (2)

32d Durum wheat grain NR Abruzzo region o1.00 o1.00 o1.00 – 4.27 5 P. pentosaceus (2)

33d Durum wheat grain NR Abruzzo region 1.15�1.45 1.34�1.56 1.37�1.63 o1.00 4.43 5 E. faecium (1)

34d Durum wheat grain NR Abruzzo region 1.27�1.43 1.59�1.87 o1.00�1.09 – 4.42 7 L. garvieae (1), P.

pentosaceus (1)

35 Bran NR Unknown o1.00 o1.00 o1.00 o1.00 3.86 5 E. faecium (1)

36 Amaranth flour NR Unknown o1.00�1.08 o1.00�1.10 1.34�1.64 o1.00 3.76 7 E. faecium (1), P.

pentosaceus (2)

37 Chickpea flour NR Unknown 1.32�1.68 1.72�1.96 1.0970.21 1.15�1.43 4.13 7 E. faecium (1), Lb.

graminis (2)

38 Corn flour NR Unknown 1.48�1.48 o1.00 1.01�1.19 o1.00 3.99 4 Lb. curvatus subsp.

curvatus (2)

39 Rice flour NR Unknown 1.33�1.57 1.26�1.42 1.21�1.43 o1.00 4.23 5 E. faecium (1)

40 Quinoa flour NR Unknown 1.42�1.58 1.78�2.06 1.11�1.32 1.20�1.38 4.10 8 E. faecium (1), E.

mundtii (4) , L. garvieae

(1)

41 Potato flour NR Unknown – – – – 5.92 – –

50 Durum wheat grain Avispa Padanian area o1.00 o1.00 o1.00 o1.00 4.65 5 E. mundtii (1)

51 Durum wheat grain Claudio Padanian area o1.00 o1.00 o1.00 o1.00 4.67 6 E. mundtii (2)

52 Durum wheat grain Creso Padanian area 1.34�1.56 1.71�1.85 o1.00 o1.00 4.57 6 E. mundtii (3)

53 Durum wheat grain Duilio Padanian area 1.81�2.03 1.56�1.90 1.48�1.60 o1.00 4.69 6 E. mundtii (3)

54 Durum wheat grain Iride Padanian area 1.17�1.43 1.30. 1.56 o1.00 o1.00 4.41 6 E. mundtii (1), L.

garvieae (1)

60 Durum wheat grain Avispa Adriatic area o1.00 – o1.00 – 4.21 4 Lb. curvatus (2)

61 Durum wheat grain Claudio Adriatic area 1.60�1.72 1.44�1.62 1.03�1.16 o1.00�1.12 4.45 6 E. faecium (3)

62 Durum wheat grain Creso Adriatic area 1.83�1.91 1.81�1.97 1.47�1.79 1.42�1.64 4.33 6 E. faecium (3), Lb.

graminis (1)

63 Durum wheat grain Duilio Adriatic area o1.00 o1.00 o1.00 o1.00 4.39 6 E. faecium (1)

64 Durum wheat grain Iride Adriatic area 1.34�1.54 1.22�1.46 1.10�1.40 o1.00 4.52 6 E. faecium (2)

70 Durum wheat grain Avispa Appenninic

area

o1.00 – o1.00 o1.00 4.44 5 Lb. graminis (1)

71 Durum wheat grain Claudio Appenninic

area

1.45�1.63 1.68�1.88 1.44�1.52 1.17�1.39 4.28 6 Lb. graminis (1), L.

garvieae (1)

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S72 Durum wheat grain Creso Appenninic

area

o1.00 o1.00 o1.00 o1.00 4.78 4 E. mundtii (1)

73 Durum wheat grain Duilio Appenninic

area

o1.00 o1.00 – – 5.23 4 A. viridans (1)

74 Durum wheat grain Iride Appenninic

area

1.00�1.02 o1.00 1.11�1.28 o1.00 4.43 5 E. faecium (2)

80 Durum wheat grain Avispa Ionian area 1.10�1.34 1.32�1.65 o1.00 o1.00 4.71 5 E. casseliflavus (2)

81 Durum wheat grain Claudio Ionian area o1.00 – o1.00 o1.00 3.98 5 Lb. graminis (2)

82 Durum wheat grain Creso Ionian area 1.28�1.32 1.22�1.77 1.20�1.46 1.06�1.14 4.28 5 E. faecium (1), Lb.

graminis (1)

83 Durum wheat grain Duilio Ionian area 1.09�1.21 1.15�1.45 o1.00 o1.00 4.93 5 E. mundtii (2)

84 Durum wheat grain Iride Ionian area 1.75�1.99 1.50�1.68 1.27�1.62 o1.00�1.17 4.23 8 E. faecium (2), Lb.

graminis (1), L. garvieae

(1)

90 Durum wheat grain Avispa Sicily region 1.39�1.66 o1.00�1.25 o1.00�1.22 o1.00 4.54 5 E. faecalis (2)

91 Durum wheat grain Claudio Sicily region 1.52�1.74 1.17�1.47 o1.00 o1.00 4.58 5 E. faecalis (1)

92 Durum wheat grain Creso Sicily region – – – – 5.84 – –

93 Durum wheat grain Duilio Sicily region – – – – 5.68 – –

94 Durum wheat grain Iride Sicily region 1.72�2.10 1.51�1.89 o1.00 1.24�1.66 4.26 8 E. casseliflavus (2), E.

faecium (1), Lb. graminis

(1), L. garvieae (1)

NR, not reported.

– No LAB were found and collected.aCell counts of two independent experiments.bOriginating from organic agriculture field.c,dOriginating from parcels of the same field subjected to different fungicide treatments (period and active principle).

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Pearson similarity coefficient, and UPGMA dendro-gram-type. LAB cocci and LAB rods were analyzedseparately.

Genotypic identification of LAB

The different strains were first subjected to a 16SrRNA gene sequence analysis using the LacbF/LacbRprimer pair following the method of Corsetti et al. [17].PCR products were separated by electrophoresis asreported above, and purified by the GFXTM PCR DNAand Gel Band Purification Kit (Amersham Biosciences,Piscataway, NJ, USA). DNA sequencing reactionswere performed by MWG Biotech AG (Ebersberg,Germany). The identities of the sequences, obtainedafter analysis of amplified PCR products, were verifiedby a BlastN [1] search against the NCBI non-redundantsequence database located at http://www.ncbi.nlm.nih.gov. In the case that identity percentage resulted inbeing lower than 100% but at least 97% [51], the speciesidentity was further verified by means of species-specificPCRs or multiplex PCRs. Species belonging to the Lb.

graminis/Lactobacillus sakei/Lb. curvatus 16S rRNAgene group were processed with the primers 16, Lc, Lgand Ls as described by Berthier and Ehrlich [6], whilethe isolates that were allotted to the L. garvieae/Lactococcus lactis 16S rRNA gene group were analyzedwith the pLG-1/pLG-2 primer pair, specific for L.

garvieae, as reported by Zlotkin et al. [57]. MultiplexPCR assays were necessary to better identify enterococciand pediococci: the sodA gene-based strategy developedby Jackson et al. [34] with primers DU1, DU2, FL1,FL2, FM1, FM2, MA1 and MA2 specific for E. durans,E. faecalis, E. faecium and Enterococcus malodoratus,

CA1, CA2, GA1, GA2, SO1 and SO2 specific for E.

casseliflavus, Enterococcus gallinarum and Enterococcus

solitarius, FV1, FV2, MU1, MU2, SU1 and SU2 specificfor Enterococcus flavescens, E. mundtii and Enterococcus

sulfureus and the multiplex PCR targeting the 16SrRNA and ldhD genes with primers Pac, Ppe, Pu, ldhDFand ldhDR specific for Pediococcus acidilactici and P.

pentosaceus as reported by Mora et al. [43].

Phylogenetic analysis

Strains showing the most diverse RAPD-PCR profileswere subjected to phylogenetic analysis. Sequence align-ment was performed with CLUSTALX [53]. Sequenceand alignment manipulations and calculation of similar-ity values and nucleotide compositions of sequenceswere performed with GENEDOC and BIOEDIT.Positions available for analysis were 590bp. Phyloge-netic and molecular evolutionary analyses were con-ducted using MEGA version 3.1 [36] with differentmodels for distance calculations (Kimura, Tajima-Nei,

Tamura three parameters) and the two possible treereconstruction methods (neighbor-joining and minimumevolution).

Results

Enumeration, isolation and grouping of LAB

Plate counts (Table 1) from wheat grain, bran andnon-conventional flour samples, when positive (at least1.00 logCFU/g) ranged between 1.00 and 2.16 logCFU/g. From those samples, presumptive LAB (Gram-positive and catalase-negative) were isolated beforeenrichment, whereas the samples whose LAB countswere lower than 1.00 logCFU/g (not isolable from 10�1

plate count) were isolated instead after enrichment inmMRS broth. Samples 13, 18, 41, 92 and 93 (Table 1),even after enrichment, did not show the presence ofLAB; these results were also confirmed by the high pHvalue of the enrichment broths ranging between 5.68and 5.92. On the other hand, the pH of brothsinoculated with samples harboring LAB ranged between3.76 and 4.93; the highest pHs were determined bystrains of E. mundtii while the lowest pHs wereregistered for E. faecium alone or in combination.

At first, about 10 colonies from each medium (ca. 40per sample) were selected and subjected to microscopicinspection. From a total of ca. 2300 colonies, 356isolates (285 cocci and 71 rods) were further analyzed byRAPD-PCR. RAPD profiles were used for straindifferentiation: 137 different LAB strains were recog-nized. Identical patterns from the same sample wereconsidered to belong to the same strain. On the basis ofRAPD-PCR cluster analysis, represented in Fig. 1 bythe expanded branch of each group, LAB strains werethen divided into 11 groups, eight for cocci (n ¼ 108)(Fig. 1A) and three for rods (n ¼ 29) (Fig. 1B).

Identification of LAB and phylogenetic relationships

A total of 137 strains were identified by partial 16SrRNA gene sequencing and those showing sequencehomology between 97% and o100% were furtherprocessed by species-specific PCRs or multiplex PCRs.LAB species found for each sample are reported inTable 1, while the number of strains for each species arereported in Fig. 2. In particular, the most frequent LABisolated from wheat grains, bran and non-conventionalflours (49 of 66 samples) were represented by enter-ococci (E. casseliflavus, E. durans, E. faecalis, E. faecium

and E. mundtii). Lactobacilli (Lb. coryniformis subsp.coryniformis, Lb. curvatus subsp. curvatus and Lb. graminis)were isolated from 19 samples while L. garvieae andP. pentosaceus were identified from nine and seven

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10080604020

E. durans (n = 3)

E. mundtii (n = 31)

E. faecalis (n = 3)

E. casseliflavus (n = 10)

E. faecium (n = 41)

A. viridans (n = 1)P. pentosaceus (n = 9)

L. garvieae (n = 10)

100706050 9080

Lb. graminis (n = 23)

Lb. curvatus subsp. curvatus (n = 4)Lb. coryniformis subsp. coryniformis (n = 2)

Fig. 1. Dendrograms obtained from combined RAPD-PCR patterns of LAB isolates from wheat kernels and non-conventional

flours. (A) LAB cocci; (B) LAB rods. Upper lines indicate the percentage of similarity.

A. Corsetti et al. / Systematic and Applied Microbiology 30 (2007) 561–571 567

samples, respectively. Aerococcus viridans was alsoisolated from one sample (73, Table 1) and itrepresented the sole LAB species found in that sample.

In order to show the relationships of the LABassociated with wheat grains, bran and non-conven-tional flours, a phylogram (Fig. 2) was constructed usingthe partial 16S rRNA gene sequence of strains (n ¼ 36)representative of each RAPD group. Some strainscollected from samples of different geographical areas(e.g. E. mundtii WGWT10.1a and WGK53) presentedlittle differences in the RAPD profile and had closelyrelated sequences, while isolates of the same geographi-cal area (e.g. E. faecium WGW11.1 and WGJ17.2)showing a different RAPD profile were phylogeneticallyless close, thus demonstrating a good correlationbetween RAPD and phylogenetic analysis.

Discussion

In general, the plant-associated habitat is a dynamicenvironment in which many factors may affect thestructure and species composition of bacterial commu-nities. Microbial interactions represent one of the mostimportant factors for determining changes in speciescomposition [2]. Fresh vegetables are known to harborlow numbers of LAB [45].

This study was mainly aimed at characterizing LABpopulations isolated from a large number of T. durum

wheat samples (n ¼ 59) collected throughout Italy and itrepresents the first study to explore the composition ofwheat-associated LAB in this country. In addition, sixsamples of non-conventional flours and a sample ofbran were examined, in order to investigate their LABcontribution to traditional preparations. The 66 sampleswere analyzed by a culture-dependent approach andwere shown to harbor between less than 1.00 and2.16 logCFU/g of LAB. Similar LAB values have beenreported for other plant materials, such as 102–103 CFUof Lactobacillus, Pediococcus and Leuconostoc permilliliter of freshly extracted grape juice [24,27], ando102 CFU of Enterococcus, Lactobacillus, Lactococcus

and Weissella per gram of wine grapes [4].In the present study, enterococci (E. casseliflavus, E.

durans, E. faecalis, E. faecium and E. mundtii) were themost frequently isolated LAB since they were identifiedin 49 samples; in particular, they were mainly repre-sented by E. faecium and E. mundtii. Enterococci arenatural inhabitants of the intestine in warm-bloodedanimals [20] and, being members of the group of LAB,they generally appear and participate in food and feedfermentations [26]. With particular regard to E. faecium,E. casseliflavus, E. mundtii, E. sulfureus and Enterococ-

cus hirae [5,7,9,41,55], enterococci are also known to

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Fig. 2. Phylogenetic relationships of representative strains of LAB isolated from wheat grains and non-conventional flours based on

partial 16S rRNA gene sequences. The tree was calculated with the Tamura 3 parameters model as a distance matrix formula and

minimum evolution as a tree reconstruction method. The bar indicates the number of nucleotide substitutions per site.

A. Corsetti et al. / Systematic and Applied Microbiology 30 (2007) 561–571568

normally occur on the above-ground parts of vegetables,cereals and forage plants [44].

Most of the other species identified in the presentstudy are normally found associated with plant material:

Lb. graminis is commonly isolated from grass silage;P. pentosaceus is usually found in fermented vegetables[25]; Lb. coryniformis is easily detected in fermentedvegetable products [31] such as silage [39]; Lb. curvatus is

ARTICLE IN PRESSA. Corsetti et al. / Systematic and Applied Microbiology 30 (2007) 561–571 569

reported to be one of the main micro-organismsresponsible for kimchi fermentation, a traditionalKorean fermented vegetable food [38].

On the other hand, some potentially pathogenic LABwere also identified. Strains of L. garvieae were isolatedfrom several samples. Such a species was originallyisolated from cows with mastitis [10,28] and, although itrepresents a cause of infection in humans and animals[22,23], it has been reported to be associated with foodproducts such as Moroccan soft white cheese [46] andItalian fermented sausages [47]. In one wheat sample,the species A. viridans, which is reported to be aninfectious LAB, was detected. It is associated withanimal [18] and human (Forster et al., unpublishedresults, accession number DQ402378) septicemia.

The biodiversity of LAB collected from wheat grainsis barely represented by 11 species belonging to fivegenera. To avoid underestimation of culturable species,LAB were collected before and after enrichmentprocedures. Furthermore, plating was performed onfour media specific for LAB, including two mediacommonly used for the isolation of LAB fromsourdough environments, such as SDB-containing mal-tose and SFM-containing glucose, maltose and fructose– sugars that together with sucrose, represent the mainsoluble carbohydrates of flour [40]. The stressingnutritional conditions of the external teguments ofwheat kernels could be restrictive to most LAB, knownas nutritionally fastidious micro-organisms [30]. An-other explanation to the low LAB biodiversity and tothe lack of correspondence between grain/flour andsourdough LAB communities could be that typicalsourdough LAB are present in raw materials as dormant(non-cultivable) flora and, for this reason, only detect-able by means of culture-independent tools.

The RAPD-PCR (Fig. 1) and phylogenetic tree(Fig. 2) showed a random distribution of LAB speciesisolated from T. durum cultivated throughout Italy.

Furthermore, our findings demonstrated that, exceptLb. curvatus which, together with Lactobacillus brevis

was reported to be one of the more dominantlactobacilli of Portuguese sourdough [48], LAB speciesisolated from durum wheat grains are different fromthose commonly associated with sourdough fermenta-tion such as Lactobacillus sanfranciscensis, Lb. brevis,Lactobacillus plantarum [14], thus confirming that thematrix composition and the fermentation parametersplay a defining role in the selection of LAB [17]. Asrecently shown by Corsetti et al. [16], LAB (E. faecium

and P. pentosaceus) from wheat grains determine therapid acidification of dough and prepare the environ-ment for the growth of the LAB responsible for thematuration of dough, such as Lb. sanfranciscensis.Further studies based on culture-independent methods,e.g. multiplex PCR and/or PCR-DGGE strategies asdeveloped by our group to detect typical sourdough

Lactobacillus spp. [49,50], are warranted in orderto have a more complete view on the non-cultiv-able lactobacilli eventually populating sourdough rawmaterials.

Acknowledgment

The authors wish to thank Prof. Michele Pisante(Dipartimento di Scienze degli Alimenti, Sezione diAgronomia e Produzioni Vegetali, Universita degliStudi di Teramo, Teramo, Italy) for providing thewheat samples.

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