Lactic acid bacteria evolution during winemaking: Use of rpoB gene as a target for PCR-DGGE analysis

ARTICLE IN PRESS FOOD MICROBIOLOGY Food Microbiology 23 (2006) 136–145 www.elsevier.com/locate/fm

Lactic acid bacteria evolution during winemaking: Use of rpoB gene as a target for PCR-DGGE analysis Vincent Renouf, Olivier Claisse, Ce´cile Miot-Sertier, Aline Lonvaud-Funel Laboratoire de Microbiologie et de Biotechnologie Applique´e, Faculte´ d’œnologie, UMR œnologie-ampe´lologie, INRA-Universite´ Victor Segalen Bordeaux 2, 351 cours de la liberation, 33405 Talence Cedex, France Received 25 October 2004; received in revised form 18 January 2005; accepted 18 January 2005

Abstract Evolution of the microbial population during winemaking is crucial. Winemakers are more and more attentive to microbial aspects during fermentation. During aging, microbial stabilization is preponderant to avoid development of spoilage yeast and bacteria. Therefore, it is necessary to improve methods to study the evolution of micro-organisms and for early detection of undesirable strain. The aim of this study was to develop a culture-independent method for identifying lactic acid bacteria (LAB) and to monitoring predominant species. The benefits of PCR-DGGE for the analysis of microbial changes during winemaking were clearly demonstrated. Targeting rpoB gene allowed a reliable discrimination of each species. The primers were able to avoid the interspecies heterogeneity problem caused by the use of the 16S rRNA gene. This method was applied to study the influence of different oenological practices on LAB population and their evolution during winemaking. r 2005 Elsevier Ltd. All rights reserved. Keywords: Lactic acid bacteria; PCR-DGGE; rpoB; Winemaking

1. Introduction Lactic acid bacteria (LAB) play an important role in winemaking (Lafon-Lafourcade et al., 1983). They are present on the grape surface and they are able to develop in musts in anaerobic environment (Lonvaud-Funel, 1999). After the alcoholic fermentation, LAB convert malic acid into lactic acid during the malolactic fermentation (MLF) (Lonvaud-Funel and Strasser de Saad, 1982). The beneficial effects of MLF are now established (Davis et al., 1985; Vivas et al., 1995) and wine makers are trying hard to it. They can favour the development of the indigenous flora or use commercial malolactic starters (Gindreau et al., 1997). After MLF Corresponding author. Tel.: +33 5 40 00 64 66; fax: +33 5 56 84 64 68. E-mail address: [email protected] (A. Lonvaud-Funel).

0740-0020/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2005.01.019

and during aging, the microbial stabilization of wine by sulphur dioxide and racking operations are primordial. LABs can spoil wines by producing exopolysaccharides (Walling et al., 2001), biogenic amines (Coton et al., 1998) or 3-hydroxypropionalde´hyde (Claisse and Lonvaud-Funel, 2001) Therefore, the survey of LAB populations is important to favour development of MLF and to avoid contamination by spoilage agents. Plate counting is currently used to monitor the quantitative evolution of LAB populations during winemaking (Lafon-Lafourcade and Joyeux, 1979). The use of commercially available miniaturized identification systems such as API (Biomerieux), DNA–DNA hybridization (Lonvaud-Funel et al., 1991) and sequencing 16 rDNA gene (Kaufman et al., 1997) are currently used for LAB identification. However, these methods need a preliminary cultivation step. In addition to the time necessary for colony growth, the definition of a medium and conditions relevant for all bacteria is not

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feasible. Cultivation-dependent methods can be considered for the complete inventory of bacteria population (Hugenholtz et al., 1998). The use of the polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE) gives an alternative option free of separation and cultivation steps (Temmerman et al., 2004). This method allows the resolution of complex microbial mixture (Muyzer, 1999). DNA fragments differing by single base-pair substitutions could be separated (Fischer and et Lerman, 1983). PCR/DGGE targets DNA fragments with identical sizes. The presence of single copies of the gene is important to avoid having several bands per species are seen in the electrophoresis gel. Ribosomal genes are present in several copies each one having a different sequence (Coenye and Vandamme, 2003). Hence, the choice of 16S rRNA gene is not suitable as a PCR-DGGE target. To avoid this complication, we chose to target the RNA polymerase beta subunit gene rpoB (Dahllof et al., 2000; Da Mota et al., 2004; Rantsiou et al., 2004). However, the use of rpoB presents a taxonomic disavadantage: the database of the sequence is less documented than that of the 16S rRNA gene. Each DGGE band cannot easily be attributed to a species. However, our objective was focused on the microbial changes and their oenological incidence. Thus, the discrimination of each species and their evolution are more important than giving a name to each band. The first step was to find suitable primers. The primers must be present in all the species and delimite variable sequences to separate each species. Then it was important to avoid interspecies differences: for each species only one band should appear in the electrophoresis gel. Duplex DNA problem is bypassed by an attachment of a GCrich DNA sequence (Myers et al., 1985). The last step is to find the most suitable and accurate gradient and the best DGGE condition (temperature, time). After all adjustment stages a comparison between DGGE analysis and conventional bacteria isolation during micro-vinification in the laboratory was made. DGGE sensitivity was revealed to follow dynamic evolution of a mixture of LAB in wine medium. Then, we combined both of these methods to follow LAB dynamics during winemaking in different chateaux. Several LAB species were identified and a database of their rpoB partial sequences was created. The influence of oenological practice on LAB population was clearly exposed.

2. Materials and methods 2.1. Bacterial and yeast strains The four lactic acid bacteria strains and the yeast used for this study are listed in Table 1. LAB were chosen for

137

Table 1 List of strains used in this study Species

Designation

Saccharomyces cerevisiae Oenoccocus oeni Oenoccocus oeni Pediococcus parvulus Lactobacillus hilgardii

522Davis IOEB* 9807 (HDC+) IOEB 8406 (ADI+) IOEB 8801 IOEB 0001

*IOEB: collection of the Faculty of Oenology of Bordeaux.

their ability to grow in wine. The two Oenococcus oeni (O. oeni) strains were chosen for their ability to produce histamine, O. oeni IOEB 9807 (histidine decarboxylase HDC+) or to degrade arginine, O.oeni IOEB 8406 (arginine desaminase ADI+). 2.2. Micro-vinifications Laboratory scale micro-vinifications were set up to study by PCR-rpoB/DGGE microbial dynamic changes. Micro-vinifications were carried out in commercial grape juice at pH 3.5. The juice was inoculated by mixed cultures of Saccharomyces cerevisiae, O. oeni IOEB 9807, O. oeni IOEB 8406, Pediococcus parvulus (P. parvulus) and Lactobacillus hilgardii (L. hilgardii) (105 cells/ml of each species). The fermentations were carried out without agitation in 500 ml flask at 25 1C for 45 days. After homogenization, samples were periodically collected for chemical, microbial analyses and DNA extraction. 2.3. Wine samples We evaluated the influence of different oenological practices by collecting wines samples from three different chateaux localized in different areas of the Bordeaux appellation: Graves (A), Me´doc (B) and Saint-Emilion (C). Samples were collected during the main winemaking steps: harvesting, tank filling and homogenization, alcoholic fermentation, maceration, running off, MLF, racking, and aging. We tried to evaluate the influence of oenological practices (Table 2) on this diversity and dynamic change of LAB population. 2.4. Microbial and chemical analyses Serial dilutions of each sample were used to inoculate plates of MRS agar: (L
1 distilled water) Lactobacilli MRS broth (Difco) 55 g, D-L malic acid (Prolabo) 10 g, agar 20 g, pH 5.0 with NaOH 10 N and YPG agar: (L
1 distilled water) yeast extract (Difco) 10 g, bactopeptone (Difco) 10 g, glucose (Avocado) 20 g, agar 20 g, pH 5.0 with orthophosphoric acid, to count respectively LAB

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Table 2 List of the most important oenological practices differences between the three chaˆteau where samples were collected

Region Vines Harvesting date Sulfur dioxide addition at harvesting Yeast Alcoholic fermentation Maceration Malolactic fermentation pH after MLF

Chaˆteaux A

Chaˆteau B

Chaˆteau C

Graves Merlot 27/08/03 4 g/hl Indigenous flora 6 days Vat 3.7

Me´doc Cabernet-Sauvignon 26/09/03 4 g/hl Levain 522 Davis 8 days Vat 3.85

Saint-Emilion Cabernet-Franc 03/09/03 6 g/hl Levain Actiflore RB2 10 days Barrels 4.0

and total yeast populations. The MRS agar plates were incubated at 25 1C for 5 days in anaerobic conditions using an anaerobic system envelope with palladium catalyst (BBL). YPG agar plates were incubated at 25 1C for 3 days. Epifluorescence (Millet and LonvaudFunel, 2000) was used to estimate viable micro-flora. DNA–DNA hybridization method was used to identify colony (Lonvaud-Funel et al., 1991). Results were given in species percentage. These hybridization experiments were made on whole Petri dishes carrying between 30 and 300 colonies. L-lactic acid was measured using a commercial enzymatic kit (Boerhinger Mannheim).

2.5. DNA extraction A DNA extraction protocol was adapted to wine sample. In total, 10 ml of wine were centrifuged at 10,000 g at room temperature for 10 min. The pellet was washed in 1 ml of Tris 10mM (GenApex)-EDTA 1 mM (GenApex) (TE) buffer. After a second centrifugation (10,000 g for 5 min), the supernatant was discarded and the pellet resuspended in 300 ml of 0.5 mM EDTA pH 8. 300 ml of glass beads were added (+ 0.1 mm) and samples were mixed at maximum speed for 10 min. Then, 300 ml of nuclei lysis (Promega) and 200 ml of protein precipitation solution (Promega) were added and mixed for 20 s. Precipitation of cellular fragments was made on ice for 5 min. After another centrifugation (10,000 g for 3 min), the supernatant containing the DNA was transferred in a new micro-centrifuge tube and 60 mL of a PolyVinyl-Pyrrolidone (PVP) (SigmaAldrich) 10% solution was added. Vortex at high speed for 10 s allowed wine polyphenols precipitations, which are known to inhibit amplification reaction. After centrifugation (10,000 g for 2 min), the supernatant was transferred to a clean 1.5 ml micro-centrifuge tube containing 300 ml of room temperature isopropanol. The tube was gently mixed by inversion until a visible mass of DNA could be seen. After centrifugation (10,000 g for 15 min), 300 ml of room temperature 70% ethanol were added to the pellet before an ultimate stage of

centrifugation (10,000 g for 2 min). Ethanol was carefully sucked up and the tube was dried. In total, 50 ml of pour preparation injectable (PPI) water with 1 ml of RNase (Promega) was used to rehydrate DNA overnight at 4 1C. After rehydratation, DNAs were stored at
20 1C. 2.6. Amplification of the rpoB gene The sequence of each primer was determined after alignment of the sequence of the rpoB gene of different LAB. Primers rpoB1 (50 -ATTGACCACTTGGGTAACCGTCG-30 ), rpoB1o (50 -ATCGATCACTTAGGCAATCGTCG-30 ), rpoB2 (50 -ACGATCACGGGTCAAACCACC-30 ) were used to amplify a region of 336 bp of the rpoB gene. A GC clamp was added to primer rpoB2 (Schffield et al., 1989; Dahllof et al., 2000) to improve DGGE separation (Scheffield et al., 1989). Reactions were carried out in 50 ml volume containing DNA (50 mg/ml), commercial PCR mix 4 mL (Qbiogen), MgCl2 3 mM, 0.1 mM of each primer. After DNA addition, the samples were amplified in a Thermocycler (IQ BioRad). The PCR profile started by an initial touchdown step in which the annealing temperature was lowered from 59 to 45 1C in intervals of 1 1C every cycles and 20 additional cycles were carried out with an annealing of 45 1C. An initial denaturation at 95 1C for 10 min and a final 10 min extension at 72 1C were used. Negative controls, without DNA, were run in all amplifications. A DNA mixture of different known LAB strains was amplified. In total, 10 ml of each PCR products were visualized on a 1.5% agarose gel (Eurobio) electrophoresis in the presence of a 100 bp ladder (Promega) followed by staining with ethidium bromide (Eurobio). 2.7. Analysis of PCR products by DGGE DGGE was performed on a Bio Rad DGGE system. In total, 10 ml of each amplicon were loaded on an acrylamide (QBIOgene) gel containing a denaturant urea (Promega)—formamide (QBIOgene) gradient ranging 25–55%. In total, 10 ml of each PCR products at

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50 ng/ml were run with 4 ml a mixture of glycerol (80%), TE buffer (20%) and bromophenol blue (J.T. Baker). Electrophoresis was run in 1  TAE buffer (Labosi) at constant temperature (60 1C) for 10 min at 20 V and subsequently for 16 h at 85 V. After electrophoresis, the gels were stained for 10 min with SYBR Green I nucleic acid gel strain (Molecular Probes) and photographed under UV light. A mixture of several known LAB DNA amplicons was loaded as marker bands.

Phylogenetic and molecular evolutionary analyses were conduced using MEGA version 2.1 (Kumar et al., 2001). For these analyses, we have excluded the primers, and the sequence comparison was carried on 250 pb. The function of the neighbor-joining (Saitou and Nei, 1987) was selected, phylogenetic distance was calculated according to Kimura’s method and 1000 repetitions were made for bootstrap (Felsentein, 1985).

2.8. Sequence alignment and phylogenetic analysis

3. Results

Each interesting DGGE band was excised with a razor blade, and the small blocks of acrylamide containing the DNA were placed in sterile 1.5 ml micro-centrifuge tube. In total, 100 ml TE buffer were added and DNA was allowed to diffuse out of the gel fragments overnight at 4 1C. Acrylamide residues were pelleted after a low speed centrifugating step. The supernatant was used for reamplification using the primers and reaction conditions described above. After amplification, 10 ml of the PCR products were rerun on a 1.5% agarose gel electrophoresis to confirm the reamplification. Finally, 40 ml were purified using the Qiaquick Kit (Qiagen) and were sent for sequencing (Millegen, France). The sequence targeted with our primers is not known in all LAB species. Hence, we decided to constitute our own data bank in order to complete the existing data. We chose 20 of the most relevant LAB in wine. Then, we amplified the rpoB gene using previously selected primers and sequenced DNA fragment obtained. The sequences mentioned in the results part are deposited in Genbank and their accession numbers are listed in Table 3. Although this method cannot be used on its own as species identification method, it allowed to build a phylogenetic tree and thus giving information on the genetic relatedness of the different species analysed.

3.1. Micro-vinification The results obtained by numeration on MRS agar plates are shown in Fig. 1. Yeast growth and AF preceded LAB growth and MLF as in usual vinifications. L-lactic acid production started after 17 days of culture when LAB population increased, hence corresponding to the beginning of the MLF. As reported in Table 4, the results of DNA–DNA hybridization analyses showed that before the beginning of MLF the predominant species is P. parvulus. Then O. oeni appeared and became predominant during MLF. L. hilgardii was detected only at the end of MLF. The DGGE profiles (Fig. 2) were in accordance with colony hybridization results. At the first steps, the P. parvulus band was the only band that could be seen on the gel. When production of lactic acid began, a band appeared corresponding to O. oeni. Before the end of MLF, L. hilgardii was detected together with O. oeni. When, malic acid was consumed L. hilgardii was dominant. At day 45, O. oeni represented only 5% of the colonies. Nevertheless, O. oeni was still detected by PCR-rpoB/DGGE. 3.2. Winemaking studies The number of bands in the DGGE fingerprints gave an insight of the diversity in practical winemaking.

Species

Origin of reference strains

Accession number of reference sequence

Oenococcus oeni Lactobacillus hilgardii Lactobacillus casei Lactobacillus collinoides Lactobacillus plantarum Pediococcus parvulus Lactobacillus buchneri Gluconobacter oxydans

*ATCC23277 ATCC8290 ATCC334 ATCC27612 ATCC8014 ATCC19371 ATCC11305 ATCC621

AY875845 AY875846 AY875847 AY875848 AY875849 AY875850 AY875851 AY875852

*ATCC: American Type Culture Collection.

CFU/mL

Table 3 Bacterial strains mentioned and accession number of the rpoB sequences derived from this study

1E+10 1E+09 1E+08 1E+07 1E+06 1E+05 1E+04 1E+03 1E+02 1E+01 1E+00

1

0

5

10

2

15

20 25 Days

30

35

40

45

Fig. 1. Saccharomyces cerevisiae (&) and LAB (J) populations during microvinifications. Arrow 1: end of alcoholic fermentation: all sugars (glucose+fructose) were consumed. Arrow 2: beginning of MLF.

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Table 4 DNA–DNA bybridization results in percentage of the species detected on agar plates during the following of the micro-vinification

Pedioccocus parvulus Oenoccocus oeni Lactobacillus hilgardii

3 days (%)

10 days (%)

18 days: beginning of the FML (%)

24 days (%)

45 days (%)

100 0 0

100 0 0

4 96 0

0 10 90

0 5 95

band was visualized on DGGE gel, O. oeni (100% of identity: 100% of identity) was just detected at the end of AF. However, after racking and sulphiting, P. parvulus appeared (100% of identity) along with O. oeni. The detection of P. parvulus coincided with an increase of the LAB population on plates.

4. Discussion

Fig. 2. DGGE profiles of wine samples during laboratory microvinification. The four last lanes correspond to pure species: lane A, Oenoccocus oeni; lane B, Pediococcus parvulus; lane C Lactobacillus higardii; lane D DNA mixture of the three species subjected to PCR amplification and run on DGGE gel.

Fig. 3 shows the results of the analysis for chateau A. The PCR-rpoB/DGGE revealed several bands before the beginning of the alcoholic fermentation. Several species were close to Bacillus mycoides and Lactobacillus casei. Among the species detected at the first step of the winemaking, three were Gram-negative bacteria. O. oeni was only detected when alcoholic fermentation was starting. At this time O. oeni was dominant. For chateau A, MLF was carried out in tanks and the wine was barreled two months after the end of the MLF. As soon as the alcoholic fermentation began, O. oeni was the only LAB, which was detected by PCR-rpoB/ DGGE. Concerning chateau B (Fig. 4), few bacterial species were detected on the grapes at the harvest. They were close to Lactobacillus plantarum (L. plantarum), Lactobacillus collinoides, Lactobacillus buchneri and L. hilgardii. However O. oeni was also identified on the grapes. When the AF started, the number of DGGE bands decreased. Among the bacteria detected on the grapes, species, which are close to L. hilgardii, L. plantarum and O. oeni were revealed during AF. At the end of AF, when glucose and fructose were entirely consumed, O. oeni was the only LAB detected during MLF. After racking and sulphur dioxide addition the intensity of the O. oeni band decreased which correlates well with the decrease in LAB population. Concerning chateau C (Fig. 5), DGGE fingerprint heterogeneity was less important; only two different bands could be seen on DGGE gel during the winemaking. Before AF, no

In this study, the suitability of PCR-rpoB/DGGE to follow LAB evolution was investigated during laboratory micro-vinification and several chateaux winemaking. The amplification of a variable region of the bsubunit RNA polymerase gene followed by DGGE electrophoresis led to a LAB mixture fingerprint of bacteria. The use of the PCR-DGGE approach has been recently developed to study several food ecosystems (Cocolin et al., 2001; Randazzo et al., 2002). However, in some of these studies the PCR target is usually a region of the 16S rRNA gene. Owing to interspecies heterogeneity of the 16S rRNA gene sequence, targeting a region of this gene can lead to the detection of several bands when only one species is present (Coenye and Vandamme, 2003). The choice of the rpoB gene has permitted to avoid this artefact. The comparison of DNA–DNA hybridization and PCR/DGGE during micro-vinifications studies confirmed the PCR/DGGE sensitivity. During the micro-vinifications, the three different species were easily discriminated. The analysis of PCR-rpoB/DGGE profiles hence allowed following the major species evolution in accordance with plates numeration and hybridization. Quantitative and qualitative dynamic changes of LAB populations were clearly shown. DGGE appeared as a sensitive method (a minima population of 102 cfu/ml could be analysed). Moreover, no yeast was detected suggesting that eukaryotic DNA does not interfere during bacterial DNA extraction and rpoB amplification. This limitation was observed with some primers targeting the 16S rRNA gene (Lopez et al., 2003). Therefore, use of enumeration plates and PCR/DGGE seemed to be appropriate for a quantitative and dynamic study of LAB evolution during winemaking. This approach was then used to follow real scale winemaking of three chateaux LAB population could be monitored during winemaking by PCR-rpoB/DGGE.

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Fig. 3. Quantitative (Graphic) and qualitative (DGGE gel and Neighbour-joining tree) evolution of LAB population during winemaking at chateau A.

Sampling had been done to study the consequence of different winemaking practices. The first interesting result was the LAB species diversity on grapes and in fresh musts, before the beginning of AF. However results differed according to the cellar and hence revealing the significance of micro-flora already present on the grape surface. Difference of LAB population between each chateau could be influenced by winegrowing practices and treatments. LAB found in fresh musts are part of the grape micro-flora. A hypothesis to explain the absence of LAB in fresh must, in the case of the chateau C, was the higher level of sulphur dioxide

addition during harvesting than in the other cellars. However, as the level of LAB population on grapes and fresh must are close. It suggests that PCR-rpoB/DGGE was only able to reveal the predominant species. Detection of numerous different species present at low concentrations appeared to be difficult using PCR-rpoB/ DGGE. During AF, production of ethanol and decrease of sugar concentrations is an additional selection pressure, illustrated by a LAB diversity decrease. The most resistant species was O. oeni. In each case, after AF and during MLF, analyses of dominant DGGE bands

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Fig. 4. Quantitative (Graphic) and qualitative (DGGE gel and Neighbour-joining tree) evolution of LAB population during winemaking of chateau B.

revealed that O. oeni was the most resistant bacteria detected in the wine which is a well-known phenomenon (Kunkee, 1991). The importance of pH was revealed. In the most acidic wine (A), O. oeni was alone during MLF and its population decreased rapidly after racking and sulphur dioxide addition. At higher pH, few other species were present during AF (Chateau B) and P. parvulus was detected after racking (Chateau C). In this case, MLF was run in barrels. Moreover, the stage of maceration was longer than for A and B. The

maintenance of a high LAB population (4105 cfu/ml) and the presence of P. parvulus could be a result of a combination of these two parameters. In addition to pH, long macerations leads to the development of the most resistant LAB species and barrels provide convenient conditions (sugar, oxidation) (Alamo et al., 2000; Feuillat et al., 1994) for the multiplication of P. parvulus. Indeed, according to the intensity of DGGE fragments P. parvulus dominated O. oeni in the beginning of aging. Detection of P. parvulus at this step

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Fig. 5. Quantitative (Graphic) and qualitative (DGGE gel) evolution of LAB population during winemaking of chateau C.

of winemaking was interesting since P. parvulus is considered as the most prejudicial LAB species.

5. Conclusion Several methods are used for the identification of LAB (API test, DNA–DNA hybridization, sequencing of 16S rRNA gene). However, none is suitable to study

the complex microbial community of wine. The PCR/ DGGE, which targets a region of rpoB gene is a cultureindependent molecular method allowing a sensitive and fast bacterial analysis. It enables the survey of the most important species during all winemaking steps. It revealed the LAB population diversity on grape surface, in the fresh must and the predominance of O. oeni after the beginning of AF. All DGGE fingerprint fluctuations were correlated with an important event in winemaking

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(beginning of AF, maceration, MLF, racking). Combined with agar plate numeration, PCR/DGGE gave the necessary information on LAB evolution. The detection of spoilage LAB, as P. parvulus which is able to synthesize exocellular polysaccharides (EPS) and to modify wine viscosity (Cerning, 1990; Walling et al., 2005), is another argument for using of this method. However, quantitative data is also needed. Future investigation will focus on the optimization of coupling DGGE to real-time and reverse transcriptase PCR, which may allow the culture-independent quantitative study of viable LAB community.

Acknowledgements The authors wish to thank J.P. Masclef, V. Millet and K. Van Leeuwen for their winemaking assistance and for supplying wine samples. We also thank J. Coulon for her redaction assistance.

References Alamo, M.D., Bernal, J.L., De Nozal, L., Cordoves, C., 2000. Red wine aging in oak barrels: evolution the monosaccharides content. Food Chem. 71, 189–193. Cerning, J., 1990. Exocellular polysaccharides produced by lactic acid bacteria. FEMS Microbiol. Rev. 87, 113–130. Claisse, O., Lonvaud-Funel, A., 2001. De´tection de bacte´ries lactiques produisant du 3-hydroxypropionalde´hyde (pre´curseur d’acrole´ine) a` partr du glycerol par tests mole´culaires. Le Lait. 81, 173–181. Cocolin, L., Manzano, M., Cantoni, C., Comi, G., 2001. Denaturing gradient gel electrophoresis analysis of the 16S rRNA gene V1 region to monitor dynamic changes in the bacterial population during fermentation of italian sausages. Appl. Environ. Microbiol. 67, 5113–5121. Coenye, T., Vandamme, P., 2003. Intragenomic heterogeneity between multiple 16S ribosomal RNA operon in sequenced bacterial genomes. FEMS Microbiol. Lett. 228, 45–48. Coton, E., Rollan, G.C., Bertrand, A., Lonvaud-Funel, A., 1998. Histamine-producing lactic acid bacteria in wines: early detection, frequenc- y and distribution. Am. J. Enol. Vitic. 49, 19–203. Dahllof, I., Baillie, H., Kjelleberg, S., 2000. rpoB-based microbial community analysis avoids limitations inherent in the 16 rRNA gene intraspecies heterogeneity. Appl. Environ. Microbiol. 40, 1333–1338. Da Mota, F.F., Gomes, E.A., Paiva, E., Rosado, A.S., Seldin, L., 2004. Use of the rpoB gene analysis for identification of nitrogenfixing Paenibacillus species as an alternative to the 16 rRNA gene. Lett. Appl. Microbiol. 39, 34–40. Davis, C.R., Wibowo, D., Eschenbruch, R., Lee, T.H., Fleet, G.H., 1985. Practical implications of malolactic fermentation: a review. Am. J. Enol. Vitic. 36, 290–301. Felsentein, J., 1985. Confidence limits on phylodenies: an approach using the bootsrap. Evolution 39, 783–791. Feuillat, F., Keller, R., et Hubert, L., 1994. Structure anatomique et porosite´ du bois de cheˆne rouvre et pe´doncule´. Conse´quence sur l’utilisation en tonnellerie. Rev. Oenol. 20, 26–31. Fischer, S.G., et Lerman, L.S., 1983. DNA fragment differing by single base-pair substitutions are separated in denaturing gradient gels:

correspondence with melting theory. Proc. Natl. Acad. Sci. USA 80, 1579–1583. Gindreau, E., Joyeux, A., De Revel, G., Claisse, O., Lonvaud-Funel, A., 1997. Evaluation de l’e´tablissement de levains malolactiques au sein de la microflore bacte´rienne indige`ne. J. Int. des sciences de la vigne et du vin 31, 197–202. Hugenholtz, P., Goebel, B.M., Race, N.R., 1998. Impact of cultureindependent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180, 4765–4774. Kaufman, P., Pfefferkorn, A., Teuber, M., Meile, L., 1997. Identification and quantification of Bifidocbacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR. Appl. Environ. Microbiol. 63, 1268–1273. Kumar, S., Tanura, K., Jakobsen, I.B., Masatoshi, N., 2001. MEGA2: Molecular Evolutionnary Genetcs Analysis software, Arizona State University, Tempe, Arizona, USA. Kunkee, R.E., 1991. Some roles of malic acid in the malolactic fermentation in winemaking. FEMS Microbiol. Rev. 88, 55–72. Lafon-Lafourcade, S., Joyeux, A., 1979. Techniques simplifie´es pour le de´nombrement et l’identification des micro-organismes vivants dans les mouˆts et les vins. Connaissance de la vigne et du vin 13, 295–310. Lafon-Lafourcade, S., Carre, E., Ribereau-Gayon, P., 1983. Occurrence of lactic acid bacteria during the different stages of vinification and conservation of wines. Appl. Environ. Mircobiol. 46, 876–880. Lonvaud-Funel, A., 1999. Lactic acid bacteria in the quality improvement and depreciation of wine. Antonie Leeuwenhoek 76, 317–331. Lonvaud-Funel, A., Strasser de Saad, A., 1982. Purification and properties of a malolactic enzyme from a strain of Leuconostoc mesenteroides isolated from grapes. Appl. Environ. Microbiol. 43, 357–361. Lonvaud-Funel, A., Joyeux, A., Ledoux, O., 1991. Specific enumeration of lactic acid bacteria in fermenting grape must and wine by colony hybridisation with non-isotopic DNA-probes. J. Appl. Microbiol. 71, 501–508. Lopez, I., Ruiz-Larrea, F., Cocolin, L., Orr, E., Phister, T., Marshall, M., VanderGheynst, J., Mills, D.A., 2003. Design and evaluation of PCR primers for analysis of bacterial populations in wine by denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 69, 6801–6807. Millet,, V., Lonvaud-Funel, A., 2000. The viable but non-cultivable state of wine micro-organisms during storage. Lett. Appl. Microbiol. 30, 136–141. Muyzer, G., 1999. DGGE/TGGE a method for identifying genes from natural ecosystems. Curr. Opinions Microbiol. 2, 317–322. Myers, R.M., Fischer, S.G., Manaitis, T., Lerman, L.S., 1985. Modification of the melting properties of duplex DNA by attachement of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis. Nucl. Acids Res. 13, 3111–3129. Randazzo, C.L., 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. Rantsiou, K., Comi, G., Cocolin, L., 2004. The rpoB gene as a target for PCR-DGGE analysis to follow lactic acid bacterial population dynamics during food fermentations. Food Microbiol. 21, 481–487. Saitou, N., Nei, M., 1987. The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.

ARTICLE IN PRESS V. Renouf et al. / Food Microbiology 23 (2006) 136–145 Schffield, V.C., Cox, D.R., Lerman, L.S., Myers, R.M., 1989. Attachement of a 40-base pair G+C rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Prot. Natl. Acad. Sci. 86, 232–236. Temmerman, R., Huys, G., Swings, J., 2004. Identification of lactic acid bacteria: culture-dependent and culture-independent methods. Trends Food Sci. Technol. 15, 348–359. Vivas, N., Lonvaud-Funel, A., Glories, Y., Augustin, M., 1995. The effect of malolactic fermentation in barrels and in tanks on the

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composition and the quality of red wines. J. Sci. Tech. Tonnell. 1, 65–80. Walling, E., Gindreau, E., Lonvaud-Funel, A., 2001. La biosynthe`se d’exopolysaccharides par des souches de Pediococcus damnosus isole´es du vin: mise au point d’outils mole´culaire de de´tection. Le Lait. 81, 289–300. Walling, E., Gindreau, E., Lonvaud-Funel, A., 2005. A putative glucan synthase gene dps detected in exopolysaccharide producing by Pediococcus damnosus and Oenococcus oeni strains isolated from wine and cider. Int. J. Food Microbiol. 48, 53–62.