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Annals of Microbiology, 54 (1), 31-42 (2004)

Microbial population changes during sourdough fermentation monitored by DGGE analysis of 16S and 26S rRNA gene fragments

V. GATTO, S. TORRIANI*

Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Strada Le Grazie 15, 37134 Verona, Italy

Abstract ­ In this study, the microbial population dynamics were monitored during a traditional sourdough fermentation process using an integrated approach, including PCRDGGE and conventional culturing procedures. Total DNA was extracted directly from samples taken at intervals during 24 h of fermentation using a rapid protocol. Partial 16S and 26S rDNA sequences from bacteria and yeasts, respectively, were amplified using two new pairs of primers and the products obtained were separated using denaturing gradient gel electrophoresis (DGGE). DGGE profiles indicated changes in the lactic acid bacteria (LAB) population structure. Sequencing of purified rDNA amplicons revealed that Lactobacillus sanfranciscensis, Lactobacillus brevis, Lactobacillus arizonensis-Lactobacillus plantarum group and Lactobacillus kimchii-Lactobacillus paralimentarius prevailed throughout fermentation. A close relative of the strain CS1, proposed to be a new species associated with Italian sourdoughs, was also detected. The yeast population was represented only by Saccharomyces cerevisiae species. Conventional culture-dependent methods partially reflected PCR-DGGE results, as a lower diversity in LAB species was detected. The present approach proved to be effective in giving a reliable overview of the microbial components of sourdough in a short time. Key words: sourdough, PCR-DGGE, microbial dynamics, biodiversity, lactic acid bacteria, yeasts.

INTRODUCTION The development of molecular techniques based on sequence variability in ribosomal RNA genes (rDNA) has led to an improved understanding of the natural microbial populations present in a variety of food-associated ecosystems (Giraffa and Neviani, 2001). Among these, denaturing gradient gel electrophoresis (DGGE) of PCR-amplified rDNA fragments has become increasingly popular among microbial ecologists because of its rapidity and reliability

*Corresponding author. Phone: +39-0458027921; Fax: +39-0458027051; E-mail: [email protected]

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(Muyzer, 1999). In this culture-independent approach, total microbial DNA is directly extracted from the sample of interest, and amplified using conserved primers that bracket variable regions of rDNA. This produces amplicons of the same length, but with differing sequences each of which are specific for a given species. DGGE allows electrophoretic resolution of these amplicons on polyacrylamide gels with a gradient of denaturing agents, producing a "molecular fingerprint" of the species present in the samples (Muyzer et al., 1998). Recently, a number of studies have provided evidences that PCR-DGGE has a great potential in profiling the microbial diversity and changes occurring in traditional fermented foods (ben Omar and Ampe, 2000; Randazzo et al., 2002; Ercolini et al., 2002, 2003; Miambi et al., 2003). These studies have also underlined the necessity to apply an integrated approach of culture-dependent and culture-independent methods in order to reduce the potential bias of the different techniques and hence obtaining a more complete picture of the microbial assemblage responsible for food fermentation. The use of sourdough is a common practice in traditional bread-making processes, because it influences the final organoleptic and structural attributes of the baked products, thus enhancing the overall quality and extending its shelf-life (Hammes and Gänzle, 1998). The predominant microorganisms of sourdough consist essentially of yeasts and lactic acid bacteria (LAB). More than 23 species of yeasts, mostly species belonging to the genera Saccharomyces and Candida and 43 species of LAB, mainly species of the genera Lactobacillus, have been detected in this ecosystem (Ottogalli et al., 1996; Meroth et al., 2003). Several endogenous factors and process parameters including type of cereal flour used, dough yield, temperature and fermentation time greatly influence the number and composition of the species recovered as well as the LAB/yeast ratio (Gobbetti et al., 1994; Hammes and Gänzle, 1998). Moreover, due to the artisan and region dependent handling, traditional sourdoughs harbour unique microbial consortia (De Vuyst et al., 2002). So far, only one study has used the PCR-DGGE technique to investigate the bacterial population of rye sourdoughs produced under different ecological conditions by using commercial starter cultures (Meroth et al., 2003). Specific community profiles correlated well with certain conditions and the PCR-DGGE results were consistent with those obtained with culturing. In the present research, we monitored changes in both yeast and LAB populations at the species level during sourdough fermentation process by an integrated approach, including PCR-DGGE and conventional culturing procedures. Traditional wheat sourdough was manufactured without the addition of baker's yeast and using a home-made sourdough starter continuously propagated for more than 20 years in the Centre of Italy (Molise region) to produce typical local bread.

MATERIALS AND METHODS Microbial strains and culture conditions. Reference strains used in this study were: (i) bacteria: Enterococcus faecalis LMG 7937T (BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie, Universiteit Gent, Belgium), Enterococcus faecium LMG 11423T, Lactobacillus acidophilus LMG 9433T,

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Lactobacillus arizonensis ATCC BAA-171T (ATCC, American Type Culture Collection, Manassas, VA, USA), Lactobacillus brevis LMG 7944T, Lactobacillus buchneri LMG 6892T, Lactobacillus casei ATCC 334, Lactobacillus delbrueckii subsp. lactis LMG 7942T, Lactobacillus fermentum LMG 6902T, Lactobacillus fructivorans LMG 9201T, Lactobacillus paraplantarum LMG 16673T, Lactobacillus plantarum LMG 6907T, Lactobacillus pentosus LMG 10755T, Lactobacillus pontis LMG 14187T, Lactobacillus reuteri LMG 9213T Lactobacillus rhamnosus LMG 6400T, Lactobacillus sanfranciscensis LMG 7946T, Lactococcus lactis subsp. lactis LMG 6890T, Leuconostoc mesenteroides LMG 6893T, Pediococcus acidilactici LMG 11384T, Pediococcus pentosaceus LMG 11488T; (ii) yeasts: Brettanomyces bruxellensis MUCL 27700T (BCCM/MUCL AgroIndustrial Fungi and Yeast Collection, Université Catholique de Louvain, Louvain-La-Neuve, Belgium), Candida humilis DBVPG 7219 (Dipartimento Biologia Vegetale Perugia, Italy), Candida milleri MUCL 38021, Pichia membranifaciens MUCL 90193, Saccharomyces barnettii CBS 5648T (Centraalbureau voor Schimmelcultures, Baarn, The Netherlands), Saccharomyces bayanus CBS 380T, Saccharomyces castelli CBS 4309T, Saccharomyces cerevisiae CBS 1171T, Saccharomyces dairensis CBS 421T, Saccharomyces exiguus CBS 379T, Saccharomyces kluyveri CBS 3082T, Saccharomyces paradoxus CBS 432T, Saccharomyces pastorianus CBS 1538T, Saccharomyces servazzii CBS 4311T, Saccharomyces unisporus CBS 398T. Enterococci and LAB were grown at 30 °C for 24 h in MRS broth (Oxoid, Milan, Italy), except for L. pontis and L. sanfranciscensis that were grown in MRS5 broth (Meroth et al., 2003). Yeasts were grown in YPD broth (2% bactopeptone extract, 1% yeast extract, and 2% glucose) at 28 °C for 16-18 h. Sourdough fermentation and sampling. A duplicate sourdough fermentation batch was prepared by mixing soft wheat flour and tap-water (dough yield 190) and inoculated (25% wt/wt) with a sample of wheat sourdough drawn from household scale fermentation prepared in the Centre of Italy (Molise region). Sourdough fermentations were conducted at 28 °C for 24 h. Samples were taken at one h intervals from 0 to 10 h and after 24 h of fermentation and the pH, total titrable acidity (TTA), microbial composition by standard plate count and by PCR-DGGE were determined. Microbiological analysis and determination of pH and TTA. LAB and yeast cell numbers were estimated after 72 h at 28 °C on MRS5 agar containing 0.1 g/L cycloheximide and YPD agar containing 0.1 g/L chloramphenicol, respectively. Representative colonies of LAB and yeasts were selected on the basis of their morphology from plates prepared after 5, 10 and 24 h of fermentation, and picked for further analysis. The pH value of each sourdough samples was recorded by adding 225 mL of distilled water to 25 g of sample, while the acidity was titrated using 0.1 M NaOH to a final pH of 8.5. The TTA value was expressed in mL of 0.1 M NaOH. DNA extraction from pure cultures and sourdough samples. Genomic DNA was extracted from pure cultures by the procedures described by Marmur (1961) for bacteria and by Cocolin et al. (2000) for yeasts. For the extraction of total DNA from sourdough, each sample was initially

Ann. Microbiol., 54 (1), 31-42 (2004) 33

TABLE 1 ­ Developed PCR-DGGE primers Primera Sequence (5'-3') Position Target gene 16S rDNA 26S rDNA PCR product size (bp) 218 214

HDA6-f-GC L1395-r LIEV-f-GC LIEV-r

a f,

AAA CCG GAG GAA GGT GGG GA CCC GGG AAC GTA TTC ACC G TTG TTG AAA GGG AAG GG CAT TAC GCC AGC ATC CT

1164b 1364b 346c 544c

b Escherichia

forward; r, reverse; GC, a 40-bp GC clamp (CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG G) was attached to the 5' end of the primer; coli numbering; c Saccharomyces cerevisiae numbering.

diluted 1:10 with a lysis solution (0.1 N NaOH, 1% SDS) and homogenised for 3 min. The preparation was allowed to settle for 30 min at room temperature, and a 1 mL sample was centrifuged at 5,500 x g for 2 min. The aqueous phase was removed, added with 1 M NaClO4 and nucleic acids were first purified by chloroform extraction and hence precipitated with ethanol. Primer design and PCR conditions. Two sets of newly designed primers were used for PCR amplification (Table 1). Construction of these primers was carried out using the CLUSTALX program. The melting temperature of the PCR fragments was predicted using the WinMelt software (Bio-Rad, Richmond, Calif.). PCRs were performed with a GeneAmp PCR System 2400 thermal cycler (Perkin Elmer). For the amplification of bacterial DNA, the reaction mixture (50 µL) contained 1x PCR buffer, 2.5 mM MgCl2, 200 µM dNTP, 0.2 µM each primer (HDA6-f-GC/L1395-r), 1.25 U of Taq polymerase and template DNA (2 µL). After an initial incubation at 94 °C for 5 min, 35 cycles of the following cycle were used: 94 °C for 20 s, 58 °C for 20 s, and 72 °C for 40 s. A final extension at 72 °C for 7 min was performed. The PCR mixture used for the amplification of yeast DNA was the same as for bacterial DNA except for the concentrations of MgCl2 and primers (LIEV-f-GC/LIEV-r), 1.5 mM and 0.5 µM, respectively. The amplification program was 94 °C for 5 min, 35 cycles of 94 °C for 20 s, 51 °C for 30 s, and 72 °C for 40 s, and an elongation step at 72 °C for 7 min. Species-specific PCR protocols previously published were applied for the identification of the following microorganisms: L. brevis (Guarneri et al., 2001), L. plantarum group (Torriani et al., 2001), L. sanfranciscensis (Zapparoli and Torriani, 1997) and S. cerevisiae (Torriani et al., 2004). DGGE analysis. PCR products were analysed by using a DCode apparatus (Bio-Rad). Samples (30 µL) were applied to 8% (w/v) polyacrylamide gels (acrylamide/bisacrylamide, 37.5:1) in 1× TAE buffer (2 M Tris base, 1 M glacial acetic acid, 50 mM EDTA, pH 8.0). Optimal separation of the PCR fragments was achieved with a 30 to 60% urea-formamide denaturant gradient (100% denaturing solution contained 40% (v/v) formamide and 7.0 M urea) increasing in the direction of electrophoresis. Gels were electrophoresed for 5 h at 60 °C with a

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constant voltage of 130 V, stained with ethidium bromide, rinsed in distilled water and photographed under UV illumination. Sequencing of DGGE fragments. The DGGE bands to be sequenced were excised from gels and purified as described by ben Omar and Ampe (2000). Sequencing of fragments was performed at the Centro Genoma VegetaleENEA CR Casaccia (Rome, Italy) using the primers L1395-r and LIEV-r for bacteria and yeasts, respectively. The thus obtained partial 16S and 26S rDNA sequences were compared with the sequences present in the GenBank using the Blastn program in order to determine their closest relatives.

RESULTS AND DISCUSSION Classical counts, pH and TTA Results concerning microbial counts, pH and TTA of the prepared sourdough are given in Fig. 1. LAB and yeast populations increased from 6.4 to 9.1 and from 7 to 9.5 log CFU/g, respectively, during 24 h of fermentation. Hence, a high level of yeasts was found throughout the fermentation even though no baker's yeast was added. pH and TTA value changes could easily be correlated with LAB evolution: due to the acidifying activity of LAB, pH values of sourdough dropped from 6.08 to 3.5 at the end of the fermentation and the relative final TTA was 12.8. These values are similar to those reported in literature (Röcken and Voysey, 1995). The morphological analysis of the colonies grown on MRS5 and YPD agar plates revealed a diversity among LAB, while only one colony type was observed for yeasts in all the samples. A total of 15 LAB and six yeast isolates were selected from the agar plates for the subsequent identification by molecular methods, as discussed below.

Time (h)

FIG. 1 ­ Evolution of microorganisms, pH and TAA in wheat sourdough samples during 24 h fermentation. Results are the means of two repetitions.

Ann. Microbiol., 54 (1), 31-42 (2004)

pH and TTA (mL of 1 M NaOH)

Microbial counts (log CFU/g)

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Microbial population fingerprinting by PCR-DGGE Information on the microbial diversity and changes at the species level during the sourdough fermentation process was also achieved by extracting DNA directly from samples, followed by the amplification of 16S rDNA and 26S rDNA regions and DGGE analysis. Preliminarily, each phase of the PCR-DGGE analysis was optimised (Fig. 2 and 3). Total DNA was extracted from the sourdough samples by the simplified procedure described in the Material and Methods section. This protocol requires only about 90 min to be completed and was suitable for obtaining PCR-quality DNA. Two new couples of primers were developed to amplify regions of the 16S and 26S rDNA (Table 1). PCR primers HDA6-f and L1395-r were used for the amplification of 218 bp of the 16S rDNA of bacteria. These primers were designed on the variable V6-V8 region of 16S rDNA to differentiate the species of LAB most frequently associated with sourdough. The primers LIEV-f and LIEV-r were constructed in order to amplify a 214 bp portion of the 26S rDNA of yeasts. These primers, covering most of the D2 hypervariable domain, have the potential to differentiate different genera and species of yeasts present in sourdough. The specificity of the primers, firstly analysed in silico, was evaluated using purified DNA from the panel of microbial species listed in Material and Methods. The PCR amplicons of the analysed microorganisms containing the 40-bp CG clamp attached to the forward primers (HDA6-f and LIEV-f) showed a melting behaviour suitable for DGGE. All of the bacterial species considered could be differentiated according to the migration distances of their respective 16S rDNA fragments in a 30 to 60% denaturing gradient (data not shown, apart

FIG. 2 ­ DGGE analysis of PCR-amplified 26S rDNA fragments obtained with primers LIEV-f-GC and LIEV-r and DNA from the reference yeast strains. Lane 1, Candida milleri MUCL 38021; 2, Brettanomyces bruxellensis MUCL 27700T; 3, Pichia membranifaciens MUCL 90193; 4, Saccharomyces barnettii CBS 5648T; 5, Saccharomyces dairensis CBS 421T; 6, Saccharomyces unisporus CBS 398T; 7, Candida humilis DBVPG 7219; 8, Saccharomyces exiguus CBS 379T; 9, Saccharomyces servazzi CBS 4311T; 10, Saccharomyces castellii CBS 4309T; 11, Saccharomyces kluyveri CBS 3082T; 12, Saccharomyces paradoxus CBS 432T; 13, Saccharomyces cerevisiae CBS 1171T; 14, Saccharomyces pastorianus CBS 1538T; 15, Saccharomyces bayanus CBS 380T.

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FIG. 3 ­ DGGE analysis of PCR-amplified 16S rDNA fragments obtained with primers HDA6-f-GC and L1395-r and DNA from sourdough samples taken over the 24 h fermentation period. Lanes 1 through 10 and lane 24 indicate the time of fermentation sampling (h). Lane R, reference strains: a, Lactobacillus sanfranciscensis; b, Lactobacillus buchneri LMG 6892T; c, Lactobacillus fructivorans LMG 9201T; d, Lactobacillus brevis LMG 7944T; e, Lactobacillus reuteri LMG 9213T; f, Lactobacillus pontis LMG 14187T; g, Lactobacillus acidophilus LMG 9433T. Lanes A, B and C, the DGGE profiles of individual isolates obtained from agar plates. Some bands (bands 1 through 11) were excised, purified, sequenced and subjected to a Blastn analysis for identification. See Table 1 and text for species correspondence.

from the species reported in Fig. 3, lane R), with the exception of L. casei, which co-migrated with L. rhamnosus. Remarkably, the amplicons from L. sanfranciscensis and L. pontis could be distinguished, a goal that has not been achieved with the primers HAD2 and L1 recently designed by Meroth et al. (2003). The DGGE profiles of all the strains were composed of a single band except for P. acidilactici and L. delbrueckii subsp. lactis which presented two bands. As regards the yeasts, the new primer set gave PCR amplicons that allowed a differentiation at species level by DGGE. Previously reported sets of primers targeting 18S rDNA or others targeting 26S rDNA regions allowed differentiation of yeasts only at genus level (Hernán-Gómez et al., 2000; Cocolin et al., 2000). The DGGE profiles obtained for reference cultures of yeasts are depicted in Fig. 2. Optimal separation of PCR fragments was achieved with a 30 to 60% denaturant gradient. As shown, most of the species considered in this study generated a single specific band and can be distinguished on the basis of the different migration distances in the DGGE gel. Bacterial population DGGE analysis of the amplified 16S rDNA fragments obtained from the samples collected throughout the 24 h fermentation process provided the fingerprint shown in Fig. 3. Up to eight bands (indicated with progressive numbers in Fig. 3) were detected on the polyacrylamide gel. The number and the intensity of visible bands varied among samples with fermentation time and these

Ann. Microbiol., 54 (1), 31-42 (2004) 37

changes could be related to qualitative and semi-quantitative shift of the bacterial composition. To identify the bacterial species present in the samples, the bands were excised from the gel, re-amplified and run again on a denaturing gel to confirm their position relative to that in the original sourdough sample. Purification and sequencing of faint band 2 did not prove successful. The closest relatives of the sequences analysed and the percent of their similarity are reported in Table 2. Most of the sequences retrieved corresponded to portions of 16S rDNA of LAB; exceptions are the sequence of band 1, which showed 98% similarity with Erwinia amylovora, and the sequence of band 6 which had a 99% identity with a portion of the mitochondrial 18S rDNA of Triticum aestivum. Erwinia amylovora is a common Gram-negative epiphyte, unable to utilise starch, which can explain its detection only in the first hours of

TABLE 2 ­ Identification of bands in DGGE profiles of the bacterial population Banda 1 3 4a, 4b Lenght Accession no. Closest relative (bp) (DGGE band) 173 178 178 AJ583434 AJ583435 AJ583436 Erwinia amylovora Lactobacillus sanfranciscensis Lactobacillus sp. ACA-DC 3411 t1 Lactobacillus brevis Accession no. Similarity (homologs) (%) AF141892 LSA422037 LBR422039 AF515220 98 100 100 98 99

5

178

AJ583437

Lactobacillus paraplantarum AF516754 Lactobacillus pentosus AF516755 AF093757 AJ306297 AL935260 D79211 Triticum aestivum 18S rDNA Lactobacillus kimchii Lactobacillus paralimentarius Lactobacillus sp. CS1 Lactobacillus manihotivorans Lactobacillus sp. ACA-DC 3411 t1 Lactobacillus brevis Lactobacillus arizonensis Lactobacillus plantarum Lactobacillus pentosus Z14078 AF183558 AJ422036 AJ564009 AF000163 AJ422039 AF515220 AF516754 AF516755 AF093757 AJ306297 AL935260 D79211 AJ422037

6 7 8 9b

184 175 175 175

AJ583443 AJ583438 AJ583439 AJ583440

99 99 100 95 100 98 99

10b

178

AJ583441

11b

178

AJ583442

Lactobacillus sanfranciscensis

100

a Numbers correspond to bands extracted from the DGGE gel shown in Fig. 3. We are not able to obtain sequence from band 2; b band generated from individual cultures isolated from MRS5 plates.

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sourdough fermentation. Presumably, unfavorable conditions due to the lactic fermentation opposed its permanence in this ecosystem. In silico primer specificity analysis with HDA6-f and L1395-r confirmed the amplification of the mitochondrial plant DNA. This amplification was reduced during fermentation in concurrence with the substantial increase of the microbial cells. The sequence of the most intense and persistent band (band 4, a and b) showed a similarity of 100% to the strain ACA-DC 3411 t1, referred as L. brevis-like (De Vuyst et al., 2002). This strain, recently isolated from Greek traditional wheat sourdough, could not be identified properly and might be a new sourdough species. Band 5 was also clearly visible throughout the entire fermentation process. Its sequence corresponded to those of several LAB species, including L. arizonensis and the species of L. plantarum group (L. plantarum, L. paraplantarum and L. pentosus), which are hardly distinguishable because of the high homology of their 16S rDNA sequences. Bands 7 and 8 were less intense but were visible during the whole fermentation. The closest relatives corresponding to band 7 were two phylogenetically highly related species, L. kimchii and L. paralimentarius. Recent DNA hybridisation data raised the question if these taxa are two distinct species (De Vuyst et al., 2002). Band 8 showed 100% similarity with the strain CS1, belonging to a new species of Lactobacillus originated from sourdough (Corsetti et al., unpublished data). The key sourdough species L. sanfranciscensis corresponded to band 3, which was detected after 3 h of fermentation and remained visible until the end of fermentation. L. brevis, L. sanfranciscensis and L. plantarum are the species most frequently isolated from traditional sourdoughs produced in different geographical areas (De Vuyst et al., 2002). PCR-DGGE analysis was also performed on DNA extracted from 15 bacterial isolates randomly sampled from agar plates. Bands corresponding to the species L. brevis-like, L. arizonensis-L. plantarum group and L. sanfranciscensis were recognised (Fig. 3, bands 9, 10, and 11; Table 2). Yeast population DGGE fingerprints obtained by analysing the amplified 26S rDNA fragments with primers LIEV-f-GC and LIEV-r are shown in Fig. 4. Surprisingly, no diversity was found in yeast population. In fact, only one band of increasing intensity throughout fermentation process was visible in the gel. Two very faint bands can also be observed in the sample taken after 3 h; however, these bands were not clearly visible in fingerprint replicates obtained at the same sampling time, thus they may represent PCR artefacts. The band was compared with those obtained from the reference strains, and was hence identified as S. cerevisiae. The same DGGE profile was obtained for the yeast cultures isolated from YPD plates (Fig. 4, lane D). Identification of the microbial isolates by species-specific PCR In order to obtain a rapid and reliable identification of the microbial isolates chosen from the agar plates and to validate the PCR-DGGE results, the speciesspecific PCR assays reported in the Materials and Methods section were carried out. The outcomes of these assays (data not shown) can be summarised as follows: the species-specific PCR developed by Zapparoli and Torriani (1997) identified five out of 15 isolates as L. sanfranciscensis. This was in

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FIG. 4 ­ DGGE analysis of PCR-amplified 26S rDNA fragments obtained with primers LIEV-f-GC and LIEV-r and DNA from sourdough samples taken over the 24 h fermentation period. Lanes 1 through 10 and lane 24 indicate the time of fermentation sampling (h). Lanes R and D, the DGGE profiles of Saccharomyces cerevisiae CBS 1171T and of an individual isolate obtained from a YPD plate, respectively.

agreement with the PCR-DGGE results. The species-specific PCR published by Guarneri et al. (2001) identified seven isolates as L. brevis, supporting again the PCR-DGGE data. In fact, this result was predictable as the sequences of the 16S rDNA amplified products of L. brevis and of Lactobacillus sp. ACA-DC 3411 t1, compared in silico, were identical. The remaining three isolates were definitely allotted to the species L. paraplantarum by the multiplex PCR assay with primers targeted to the recA gene (Torriani et al., 2001). This coding gene has proven to be more suitable than 16S rDNA to distinguish closely related species such as L. plantarum, L. paraplantarum and L. pentosus (Torriani et al., 2001). Finally, using the multiplex PCR recently developed to differentiate members of Saccharomyces sensu strictu complex (Torriani et al., 2004) all of the six yeast isolates were identified as S. cerevisiae, since they gave the typical 1710 bp fragment. CONCLUSIONS The diversity and changes of the microbial populations during the fermentation of a traditional wheat sourdough were monitored by using an integrated approach. The overall results indicate the presence of a complex association of LAB species, which includes several obligate or facultative heterofermentative lactobacilli frequently recovered in artisanal sourdoughs from different European areas, in particular L. sanfranciscensis, L. brevis and L. paraplantarum. From an ecological point of view, the presence of close relatives of the strain CS1, proposed to be a new species associated with Italian sourdoughs is interesting. In contrast with the variability found in LAB, the yeasts population was uniform and represented only by the species S. cerevisiae. The peculiar asso40 V. GATTO and S. TORRIANI

ciation of yeasts and LAB species detected in this study resulted in a prompt and intense acidification of the dough. More investigations are needed to evaluate the influence of this microbiota on the reological and organoleptic properties of wheat dough. Our results confirmed that PCR-DGGE is a rapid, economic and efficient method to study the species diversity of the dominant components of the ecosystem throughout fermentation. In fact, the composition of the microbial population of multiple samples can be compared simultaneously in a short time through the analysis of patterns on one single gel. A semiquantitative evaluation of the abundance of a species within the population was also feasible on the basis of the relative intensity of a PCR fragment in the DGGE gel. In addition, preparation of reference ladders composed by the main species found in sourdough will avoid excision and sequencing of DGGE bands and speed up once more future analysis. On the other hand, data from conventional culture-dependent method in combination with molecular identification procedures complemented PCRDGGE identification results and provided evidence of the viability of the microbial group detected. In fact, in some cases, closely related organisms such as L. arizonensis-L. plantarum group, can not be accurately identified due to the similarity of their 16S rDNA sequences. One possibility to overcome the above problem is the use of supplementary primer pairs targeting more discriminating regions in rRNA genes or even other functional genes. In conclusion, the present approach proved to be effective in giving a reliable overview of the microbial components of wheat sourdough produced in the Centre of Italy and their changes over time. This method could be used for the investigation of the microbial diversity in order to understand better its role in traditional fermentation processes. Acknowledgements This research was partly supported by a grant from the Ministry of University, Scientific and Technological Research (MIUR), Rome, Italy. V. Gatto is a recipient of a grant from the European Social Fund (FSE) 2001, objective 3 - measure D4.

References ben Omar N., Ampe F. (2000). Microbial community dynamics during production of the Mexican fermented maize dough pozol. Appl. Environ. Microbiol., 66: 3664- 3673. Cocolin L., Bisson L.F., Mills D.A. (2000). Direct profiling of the yeast dynamics in wine fermentation. FEMS Microbiol. Lett., 189: 81-87. De Vuyst L., Schrijvers V., Paramithiotis S., Hoste B., Vancanneyt M., Swings J., Kalantzopoulos G., Tsakalidou E., Messens W. (2002). The biodiversity of lactic acid bacteria in Greek traditional wheat sourdoughs is reflected in both composition and metabolite formation. Appl. Environ. Microbiol., 68: 6059-6069. Ercolini D., Blaiotta G., Moschetti G., Coppola S. (2002). Evaluation of PCR-DGGE analysis for molecular typing of cheeses. Ann. Microbiol., 52: 81-87. Ercolini D., Hill P-J-, Dodd C.E.R. (2003). Bacterial community structure and location in Stilton cheese. Appl. Environ. Microbiol., 69: 3540-3548. Giraffa G., Neviani E. (2001). DNA-based, culture independent strategies for evaluating microbial communities in food-associated ecosystems. Int. J. Food Microbiol., 67: 19-43.

Ann. Microbiol., 54 (1), 31-42 (2004)

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Gobbetti M., Corsetti A., Rossi J., La Rosa F., De Vincenzi S. (1994). Identification and clustering of lactic acid bacteria and yeasts from wheat sourdoughs of central Italy. Ital. J. Food Sci., 1: 85-94. Guarneri T., Rossetti L., Giraffa G. (2001). Rapid identification of Lactobacillus brevis using the polymerase chain reaction. Lett. Appl. Microbiol., 33: 377-381. Hammes W. P., Gänzle M. G. (1998). Sourdough bread and related products. In: Wood B.J.B., Ed., Microbiology of Fermented Foods. Vol. 1. Blackie Academic & Professional, London, United Kingdom, pp. 199-216. Hernán-Gómez S., Espinosa J.C., Ubeda J.F. (2000). Characterization of wine yeasts by temperature gradient gel electrophoresis (TGGE). FEMS Microbiol. Lett., 193: 45-50. Marmur J. (1961). A procedure for the isolation of DNA from microorganisms. J. Mol. Biol., 3: 208-218. Meroth C.B., Walter J., Hertel C., Brandt M.J., Hammes W.P. (2003). Monitoring the bacterial population dynamics in sourdough fermentation processes by using PCRdenaturing gradient gel electrophoresis. Appl. Environ. Microbiol., 69: 475-482. Miambi E., Guyot J.P., Ampe F. (2003). Identification, isolation and quantification of representative bacteria from fermented cassava dough using an integrated approach of culture-dependent and culture-independent methods. Int. J. Food Microbiol., 82: 111-120. Muyzer G., Brinkhoff T., Nubel U., Santegoeds C., Schafer H., Wawer C. (1998). Denaturing gradient gel electrophoresis (DGGE) in microbial ecology. Mol. Microb. Ecol. Manual, Vol. 3.4.4, pp. 1-27. Muyzer G. (1999). DGGE/TGGE a method for identifying genes from natural ecosystems. Curr. Opin. Microbiol., 2: 317-322. Ottogalli G., Galli A., Foschino R. (1996). Italian bakery products obtained with sourdough: characterization of the typical microflora. Adv. Food Sci., 18: 131-144. Randazzo C., Torriani S., Akkermans A.D.L., de Vos W.M., Vaughan E.E. (2002). Diversity, dynamics, and activity of bacterial communities during production of an artisanal Sicilian cheese as evaluated by 16S rRNA analysis. Appl. Environ. Microbiol., 68: 1882-1892. Röcken W., Voysey P. A. (1995). Sourdough fermentation in bread making. J. Appl. Bacteriol. (Symp. Suppl.), 79: 38S-48S. Torriani S., Felis G.E., Dellaglio F. (2001). Differentiation of Lactobacillus plantarum, L. pentosus and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers. Appl. Environ. Microbiol., 67: 34503454. Torriani S., Zapparoli G., Malacrinò P., Suzzi G., Dellaglio F., (2004). Rapid identification and differentiation of Saccharomyces cerevisiae and Saccharomyces bayanus and their hybrids by multiplex PCR. Lett. Appl. Microbiol., 38: 239-244. Zapparoli G., Torriani S. (1997). Rapid identification and detection of Lactobacillus sanfrancisco in sourdoughs by species-specific PCR with 16S rRNA-targeted primers. Syst. Appl. Microbiol., 20: 640-644.

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04 GATTO documento
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