A. Lonvaud-funel - Academia.edu (original) (raw)

Papers by A. Lonvaud-funel

Le Centre pour la Communication Scientifique Directe - HAL - Diderot, Jun 19, 2003

Le Centre pour la Communication Scientifique Directe - HAL - Université de Nantes, 1994

Bulletin de l'OIV, 1999

Les amines biogenes sont des constituants frequents des aliments et boissons fermentes. Elles son... more Les amines biogenes sont des constituants frequents des aliments et boissons fermentes. Elles sont produites lors de la decarboxylation biochimique d'acides amines, notamment par les bacteries lactiques. Leur presence ne peut etre toleree qu'a faible concentration en raison de leurs effets indesirables sur la sante. L'histamine, le diaminobutane et la tyramine sont les plus couramment rencontres dans les vins ou des bacteries lactiques se sont multipliees. L'activite histidine decarboxylase, produisant l'histamine, a ete etudiee chez Leuconostoc oenos (Oenococcus oeni), l'enzyme purifiee et le gene qui code la proteine clone et sequence. Le resultat pratique est l'isolement d'une sonde d'ADN et la definition de sequences oligonucleotidiques specifiques pour la detection des souches impliquees. Les vins dans lesquels ces souches sont detectees contiennent aussi la plupart du temps d'autres amines, donc des bacteries capables de decarboxyler les autres acides amines car l'histidine decarboxylase est specifique. Des souches productrices de tyramine et de diaminobutane (decarboxylation de la tyrosine et de l'ornithine) existent donc. La capacite a former les amines est liee a la souche bacterienne et non pas a l'espece. Des souches de Leuconostoc oenos et d'autres especes ont ce caractere; leur repartition dans les chais de vinification est tres variable. Par ailleurs, la composition du mout de raisin et le mode de vinification, notamment le contact plus ou moins prolonge avec les lies de levure, interviennent egalement sur la teneur finale en amines.

Sciences Des Aliments, May 23, 1987

Bulletin De L Oiv, 1998

Les bacteries lactiques sont fortement impliquees en oenologie tout au long de l'elaboration ... more Les bacteries lactiques sont fortement impliquees en oenologie tout au long de l'elaboration du vin. Une assez grande diversite d'especes est habituellement presente sur le raisin et dans le mout. Le milieu, par la suite, devient plus selectif a cause de la fermentation alcoolique et seules persistent des especes et des souches capables de resister spontanement, ou de s'adapter. Elles sont alors responsables de la fermentation malolactique mais aussi d'alterations du vin. Les methodes de la biologie moleculaire sont des outils d'investigation qui trouvent leur interet essentiellement dans le controle de la microflore bacterienne et, specialement, dans la detection des bacteries d'alteration. L'isolement de genes ou de fragments d'ADN necessaires a la synthese d'enzymes impliquees dans les metabolismes indesirables fournit les bases a l'elaboration de sondes et d'amorces de PCR specifiques. Ces memes outils constituent egalement une aide a la selection et a la caracterisation des souches pour la production des levains malolactiques.

Progress in Industrial Microbiology, 2002

Publisher Summary This chapter discusses the role of lactic acid bacteria in winemaking. The clas... more Publisher Summary This chapter discusses the role of lactic acid bacteria in winemaking. The classification of enological lactic acid bacteria was established by using the conventional methods, which focus on phenotypes. According to morphological, biochemical and physiological properties, wine and grape lactic acid bacteria can be classified as lactobacilli and cocci , being either obligate and facultative heterofermentative or homofermentative. Since lactic acid bacteria are necessary to obtain malolactic fermentation, winemakers must find a solution for minimizing amine levels, even if indigenous bacteria possess decarboxylating activities. Today, a solution is the use of selected malolactic starters. O. oeni strains, free of undesirable activities, are produced and lyophilized in concentrated populations. They are massively added to wine to complete malolactic fermentation. They normally overcome the indigenous bacteria and seem to eliminate them by competition. Then after malolactic fermentation, wine is readily stabilized by sulfur dioxide, which eliminates the malolactic starter and the residual wild-type strains. It needs to know more about the ecology of micro-organisms living on grapes, how they survive on cellar equipment and how they grow in wines. Such studies were almost impossible in the past. Now the new molecular methods are opening new horizons for studying wine micro-organisms, particularly the lactic acid bacteria.

Journal of Applied Bacteriology, 1994

A. LONVAUD-FUNEL A N D A. JOYEUX. 1994. Populations of Leuconostoccenos were harvested from wines... more A. LONVAUD-FUNEL A N D A. JOYEUX. 1994. Populations of Leuconostoccenos were harvested from wines containing a relatively high concentration of biogenic amines. Cultivation of the biomass in synthetic media and wine showed that it consisted of histamine-producing strains. Histamine levels after culture depended on the quantity of precursor available and on the presence of yeast lees, which certainly enriched the medium in histidine. Ethanol and pH, which control bacterial growth rate and total population, were also significant factors : pH and low ethanol concentration enhanced histamine production. Strain Leuc. @nos 9204 was isolated and studied since it retained its ability to produce histamine after several transfers. I n synthetic medium this strain produced large amounts of histamine especially in the poorest nutritional conditions (no glucose, no L-malic acid). These results clearly demonstrate that Leuc. @nos involved in wine-making might play a role in biogenic amine production. T h e vinification method might also influence the final amine concentration in wine.

Research in Microbiology, 2001

The Lactobacillus genus has been shown to be associated with the dental carious process, but litt... more The Lactobacillus genus has been shown to be associated with the dental carious process, but little is known about the species related to the decay, although Lactobacillus rhamnosus is suspected to be the most implicated species. Conventional identification methods based on biochemical criteria lead to ambiguous results, since the Lactobacillus species found in saliva are phenotypically close. To clarify the role of this genus in the evolution of carious disease, this work aimed to find a rapid and reliable method for identifying the L. rhamnosus species. Methods based on hybridization with DNA probes and DNA amplification by PCR were used. The dominant salivary Lactobacillus species (reference strains from the ATCC) were selected for this purpose as well as some wild strains isolated from children's saliva. DNA profiling using semirandom polymorphic DNA amplification (semi-RAPD) generated specific patterns for L. rhamnosus ATCC 7469. The profiles of all L. rhamnosus strains tested were similar and could be grouped; these strains shared four common fragments. Wild strains first identified with classic methods shared common patterns with the L. rhamnosus species and could be reclassified. One fragment of the profile was purified, cloned, used as a probe and found to be specific to the L. rhamnosus species. These results may help to localize this species within its ecological niche and to elucidate the progression of the carious process.  2001 Éditions scientifiques et médicales Elsevier SAS Lactobacillus rhamnosus / dental carious process / DNA profiling / nucleotide probe * Correspondence and reprints.

Journal of Agricultural and Food Chemistry

The polysaccharide content of wine is generally assumed to originate from grapes and yeasts, inde... more The polysaccharide content of wine is generally assumed to originate from grapes and yeasts, independent of bacterial metabolism, except for the action of certain spoilage species. This study shows that malolactic fermentation (MLF) significantly modifies the soluble polysaccharide (SP) concentration of various red Bordeaux wines. Wines with the highest initial SP concentration go on to present decreased SP concentration, whereas those with the lowest initial SP concentration rather go on to have a higher SP concentration after MLF. These tendencies were observed whatever the Oenococcus oeni strain (indigenous or starter) used for MLF. Neutral and charged SPs were affected, but to a degree that depended on the microorganisms driving the MLF. The SP modifications were directly linked to bacterial development, because non MLF controls did not present any significant change of SP concentration.

FEMS Microbiology Letters, 2001

Biogenic amines have undesirable physiological effects when absorbed at too high a concentration.... more Biogenic amines have undesirable physiological effects when absorbed at too high a concentration. Several kinds of food and beverages contain biogenic amines. Lactic acid bacteria can decarboxylate amino acids. Since winemaking involves the growth of lactic acid bacteria for malolactic fermentation, biogenic amines may occur. However, not all bacterial strains carry these activities. In the same wine-producing area, some wines may contain very low amounts of biogenic amines while others may have relatively large quantities. It is now possible to detect the presence of undesirable histamine-producing strains by PCR test or DNA probe based on the presence of the gene encoding histidine decarboxylase. Other strains have the ornithine and/or tyrosine decarboxylase. When biogenic amine-producing strains are present, the winemaker is encouraged to inoculate selected malolactic starters to replace the indigenous microflora.

FEMS Microbiology Letters, 1999

Malolactic fermentation by Oenococcus oeni is a crucial step in wine-making. Oe. oeni phages are ... more Malolactic fermentation by Oenococcus oeni is a crucial step in wine-making. Oe. oeni phages are thought to be responsible for fermentation failures, yet they have received little attention. After a molecular analysis concerning the phage P10MC integration system, this paper focuses on the lytic system. The attP (phage attachment site)-flanking region has been cloned and sequenced. The 1296-bp lysin gene (Lys) was identified in this region. The deduced amino acid sequence showed classical structural features of phage lysins, and this gene product expressed in Escherichia coli had a lytic activity against Oe. oeni. Downstream of Lys, a second ORF was present (P163). According to its amino acid sequence and the location of its gene, the product could be the P10MC holin. This study shows that the genomic organization of phage P10MC attP-flanking regions is very similar to that of other lactic acid bacteriophages.

Annals of the New York Academy of Sciences, 1988

Le Centre pour la Communication Scientifique Directe - HAL - Diderot, Jun 15, 1995

Applied and environmental microbiology, 1982

An enzymatic complex able to transform l-malate to l-lactate was obtained from a Leuconostoc mese... more An enzymatic complex able to transform l-malate to l-lactate was obtained from a Leuconostoc mesenteroides strain isolated from grapes. The molecular weight was about 235,000, the isoelectric point was at pH 4.35, and the optimal pH for activity was 5.75. The malolactic activity followed a sequential pattern concerning the involved substrates. At pH values substantially different from the optimum, a positive cooperativity between malate molecules was observed. Oxamate, fructose-1, 6-diphosphate, and l-lactate acted as noncompetitive inhibitors, whereas succinate, citrate, and tartrate isomers produced a competitive inhibition.

International journal of systematic bacteriology, 1999

DNA sequences covering 36% of the mle gene that encodes the malolactic enzyme were determined for... more DNA sequences covering 36% of the mle gene that encodes the malolactic enzyme were determined for 13 strains of lactic acid bacteria, representing Pediococcus, Leuconostoc, Lactobacillus and Oenococcus genera. The sequences were aligned with the corresponding region of mleS in Lactococcus lactis. The phylogenetic distance matrix tree of all mle sequences was compared with the 16S rRNA phylogenetic tree. The analysis showed that the mle fragment evolved more rapidly than the 16S gene and differently. Pediococcus and Lactobacillus species were intermixed in the 16S rRNA tree whereas they were separated in the mle tree. Leuconostoc mesenteroides and Oenococcus oeni were distinct from other species in the 16S rRNA tree, whereas they were intermixed with Lactobacillus species and Lactococcus lactis in the mle tree. The amino acid sequences deduced from partial mle genes were aligned with 22 malic enzyme sequences and the corresponding phylogenetic tree was constructed. Malic and malolact...

International journal of systematic bacteriology, 1999

Conventional phenotypic methods lead to misidentification of the lactic acid bacteria Lactobacill... more Conventional phenotypic methods lead to misidentification of the lactic acid bacteria Lactobacillus hilgardii and Lactobacillus brevis. Random amplified polymorphic DNA (RAPD) and repetitive element PCR (REP-PCR) techniques were developed for a molecular study of these two species. The taxonomic relationships were confirmed by analysis of the ribosomal operon. Amplified DNA fragments were chosen to isolate L. hilgardii-specific probes. In addition to rapid molecular methods for identification of L. hilgardii, these results convincingly proved that some strains first identified as L. brevis must be reclassified as L. hilgardii. The data clearly showed that these molecular methods are more efficient than phenotypic or biochemical studies for bacterial identification at the species level.

Research in microbiology, 1997

Leuconostoc oenos is involved in malolactic fermentation occurring during wine-making. An increas... more Leuconostoc oenos is involved in malolactic fermentation occurring during wine-making. An increasing number of wines are being inoculated with malolactic starters to control the process, and the identification and differentiation of selected strains are now indispensable both for quality control of production and for commercial purposes. In the present work we evaluated the potential use of the intergenic regions of three L. oenos strains for their differentiation. The three 16S/23S rRNA intergenic spacers were amplified in vitro by PCR, and sequences were compared. The spacer sequence was highly conserved in all strains. Inside this spacer, a tRNA-Ala gene containing an 18-bp sequence stretch which is conserved in all tRNA genes was discovered. This sequence, together with random primers, was used for characterization of ten L. oenos strains by PCR.

Le Centre pour la Communication Scientifique Directe - HAL - Diderot, Jun 19, 2003

Le Centre pour la Communication Scientifique Directe - HAL - Université de Nantes, 1994

Bulletin de l'OIV, 1999

Les amines biogenes sont des constituants frequents des aliments et boissons fermentes. Elles son... more Les amines biogenes sont des constituants frequents des aliments et boissons fermentes. Elles sont produites lors de la decarboxylation biochimique d'acides amines, notamment par les bacteries lactiques. Leur presence ne peut etre toleree qu'a faible concentration en raison de leurs effets indesirables sur la sante. L'histamine, le diaminobutane et la tyramine sont les plus couramment rencontres dans les vins ou des bacteries lactiques se sont multipliees. L'activite histidine decarboxylase, produisant l'histamine, a ete etudiee chez Leuconostoc oenos (Oenococcus oeni), l'enzyme purifiee et le gene qui code la proteine clone et sequence. Le resultat pratique est l'isolement d'une sonde d'ADN et la definition de sequences oligonucleotidiques specifiques pour la detection des souches impliquees. Les vins dans lesquels ces souches sont detectees contiennent aussi la plupart du temps d'autres amines, donc des bacteries capables de decarboxyler les autres acides amines car l'histidine decarboxylase est specifique. Des souches productrices de tyramine et de diaminobutane (decarboxylation de la tyrosine et de l'ornithine) existent donc. La capacite a former les amines est liee a la souche bacterienne et non pas a l'espece. Des souches de Leuconostoc oenos et d'autres especes ont ce caractere; leur repartition dans les chais de vinification est tres variable. Par ailleurs, la composition du mout de raisin et le mode de vinification, notamment le contact plus ou moins prolonge avec les lies de levure, interviennent egalement sur la teneur finale en amines.

Sciences Des Aliments, May 23, 1987

Bulletin De L Oiv, 1998

Les bacteries lactiques sont fortement impliquees en oenologie tout au long de l'elaboration ... more Les bacteries lactiques sont fortement impliquees en oenologie tout au long de l'elaboration du vin. Une assez grande diversite d'especes est habituellement presente sur le raisin et dans le mout. Le milieu, par la suite, devient plus selectif a cause de la fermentation alcoolique et seules persistent des especes et des souches capables de resister spontanement, ou de s'adapter. Elles sont alors responsables de la fermentation malolactique mais aussi d'alterations du vin. Les methodes de la biologie moleculaire sont des outils d'investigation qui trouvent leur interet essentiellement dans le controle de la microflore bacterienne et, specialement, dans la detection des bacteries d'alteration. L'isolement de genes ou de fragments d'ADN necessaires a la synthese d'enzymes impliquees dans les metabolismes indesirables fournit les bases a l'elaboration de sondes et d'amorces de PCR specifiques. Ces memes outils constituent egalement une aide a la selection et a la caracterisation des souches pour la production des levains malolactiques.

Progress in Industrial Microbiology, 2002

Publisher Summary This chapter discusses the role of lactic acid bacteria in winemaking. The clas... more Publisher Summary This chapter discusses the role of lactic acid bacteria in winemaking. The classification of enological lactic acid bacteria was established by using the conventional methods, which focus on phenotypes. According to morphological, biochemical and physiological properties, wine and grape lactic acid bacteria can be classified as lactobacilli and cocci , being either obligate and facultative heterofermentative or homofermentative. Since lactic acid bacteria are necessary to obtain malolactic fermentation, winemakers must find a solution for minimizing amine levels, even if indigenous bacteria possess decarboxylating activities. Today, a solution is the use of selected malolactic starters. O. oeni strains, free of undesirable activities, are produced and lyophilized in concentrated populations. They are massively added to wine to complete malolactic fermentation. They normally overcome the indigenous bacteria and seem to eliminate them by competition. Then after malolactic fermentation, wine is readily stabilized by sulfur dioxide, which eliminates the malolactic starter and the residual wild-type strains. It needs to know more about the ecology of micro-organisms living on grapes, how they survive on cellar equipment and how they grow in wines. Such studies were almost impossible in the past. Now the new molecular methods are opening new horizons for studying wine micro-organisms, particularly the lactic acid bacteria.

Journal of Applied Bacteriology, 1994

A. LONVAUD-FUNEL A N D A. JOYEUX. 1994. Populations of Leuconostoccenos were harvested from wines... more A. LONVAUD-FUNEL A N D A. JOYEUX. 1994. Populations of Leuconostoccenos were harvested from wines containing a relatively high concentration of biogenic amines. Cultivation of the biomass in synthetic media and wine showed that it consisted of histamine-producing strains. Histamine levels after culture depended on the quantity of precursor available and on the presence of yeast lees, which certainly enriched the medium in histidine. Ethanol and pH, which control bacterial growth rate and total population, were also significant factors : pH and low ethanol concentration enhanced histamine production. Strain Leuc. @nos 9204 was isolated and studied since it retained its ability to produce histamine after several transfers. I n synthetic medium this strain produced large amounts of histamine especially in the poorest nutritional conditions (no glucose, no L-malic acid). These results clearly demonstrate that Leuc. @nos involved in wine-making might play a role in biogenic amine production. T h e vinification method might also influence the final amine concentration in wine.

Research in Microbiology, 2001

The Lactobacillus genus has been shown to be associated with the dental carious process, but litt... more The Lactobacillus genus has been shown to be associated with the dental carious process, but little is known about the species related to the decay, although Lactobacillus rhamnosus is suspected to be the most implicated species. Conventional identification methods based on biochemical criteria lead to ambiguous results, since the Lactobacillus species found in saliva are phenotypically close. To clarify the role of this genus in the evolution of carious disease, this work aimed to find a rapid and reliable method for identifying the L. rhamnosus species. Methods based on hybridization with DNA probes and DNA amplification by PCR were used. The dominant salivary Lactobacillus species (reference strains from the ATCC) were selected for this purpose as well as some wild strains isolated from children's saliva. DNA profiling using semirandom polymorphic DNA amplification (semi-RAPD) generated specific patterns for L. rhamnosus ATCC 7469. The profiles of all L. rhamnosus strains tested were similar and could be grouped; these strains shared four common fragments. Wild strains first identified with classic methods shared common patterns with the L. rhamnosus species and could be reclassified. One fragment of the profile was purified, cloned, used as a probe and found to be specific to the L. rhamnosus species. These results may help to localize this species within its ecological niche and to elucidate the progression of the carious process.  2001 Éditions scientifiques et médicales Elsevier SAS Lactobacillus rhamnosus / dental carious process / DNA profiling / nucleotide probe * Correspondence and reprints.

Journal of Agricultural and Food Chemistry

The polysaccharide content of wine is generally assumed to originate from grapes and yeasts, inde... more The polysaccharide content of wine is generally assumed to originate from grapes and yeasts, independent of bacterial metabolism, except for the action of certain spoilage species. This study shows that malolactic fermentation (MLF) significantly modifies the soluble polysaccharide (SP) concentration of various red Bordeaux wines. Wines with the highest initial SP concentration go on to present decreased SP concentration, whereas those with the lowest initial SP concentration rather go on to have a higher SP concentration after MLF. These tendencies were observed whatever the Oenococcus oeni strain (indigenous or starter) used for MLF. Neutral and charged SPs were affected, but to a degree that depended on the microorganisms driving the MLF. The SP modifications were directly linked to bacterial development, because non MLF controls did not present any significant change of SP concentration.

FEMS Microbiology Letters, 2001

Biogenic amines have undesirable physiological effects when absorbed at too high a concentration.... more Biogenic amines have undesirable physiological effects when absorbed at too high a concentration. Several kinds of food and beverages contain biogenic amines. Lactic acid bacteria can decarboxylate amino acids. Since winemaking involves the growth of lactic acid bacteria for malolactic fermentation, biogenic amines may occur. However, not all bacterial strains carry these activities. In the same wine-producing area, some wines may contain very low amounts of biogenic amines while others may have relatively large quantities. It is now possible to detect the presence of undesirable histamine-producing strains by PCR test or DNA probe based on the presence of the gene encoding histidine decarboxylase. Other strains have the ornithine and/or tyrosine decarboxylase. When biogenic amine-producing strains are present, the winemaker is encouraged to inoculate selected malolactic starters to replace the indigenous microflora.

FEMS Microbiology Letters, 1999

Malolactic fermentation by Oenococcus oeni is a crucial step in wine-making. Oe. oeni phages are ... more Malolactic fermentation by Oenococcus oeni is a crucial step in wine-making. Oe. oeni phages are thought to be responsible for fermentation failures, yet they have received little attention. After a molecular analysis concerning the phage P10MC integration system, this paper focuses on the lytic system. The attP (phage attachment site)-flanking region has been cloned and sequenced. The 1296-bp lysin gene (Lys) was identified in this region. The deduced amino acid sequence showed classical structural features of phage lysins, and this gene product expressed in Escherichia coli had a lytic activity against Oe. oeni. Downstream of Lys, a second ORF was present (P163). According to its amino acid sequence and the location of its gene, the product could be the P10MC holin. This study shows that the genomic organization of phage P10MC attP-flanking regions is very similar to that of other lactic acid bacteriophages.

Annals of the New York Academy of Sciences, 1988

Le Centre pour la Communication Scientifique Directe - HAL - Diderot, Jun 15, 1995

Applied and environmental microbiology, 1982

An enzymatic complex able to transform l-malate to l-lactate was obtained from a Leuconostoc mese... more An enzymatic complex able to transform l-malate to l-lactate was obtained from a Leuconostoc mesenteroides strain isolated from grapes. The molecular weight was about 235,000, the isoelectric point was at pH 4.35, and the optimal pH for activity was 5.75. The malolactic activity followed a sequential pattern concerning the involved substrates. At pH values substantially different from the optimum, a positive cooperativity between malate molecules was observed. Oxamate, fructose-1, 6-diphosphate, and l-lactate acted as noncompetitive inhibitors, whereas succinate, citrate, and tartrate isomers produced a competitive inhibition.

International journal of systematic bacteriology, 1999

DNA sequences covering 36% of the mle gene that encodes the malolactic enzyme were determined for... more DNA sequences covering 36% of the mle gene that encodes the malolactic enzyme were determined for 13 strains of lactic acid bacteria, representing Pediococcus, Leuconostoc, Lactobacillus and Oenococcus genera. The sequences were aligned with the corresponding region of mleS in Lactococcus lactis. The phylogenetic distance matrix tree of all mle sequences was compared with the 16S rRNA phylogenetic tree. The analysis showed that the mle fragment evolved more rapidly than the 16S gene and differently. Pediococcus and Lactobacillus species were intermixed in the 16S rRNA tree whereas they were separated in the mle tree. Leuconostoc mesenteroides and Oenococcus oeni were distinct from other species in the 16S rRNA tree, whereas they were intermixed with Lactobacillus species and Lactococcus lactis in the mle tree. The amino acid sequences deduced from partial mle genes were aligned with 22 malic enzyme sequences and the corresponding phylogenetic tree was constructed. Malic and malolact...

International journal of systematic bacteriology, 1999

Conventional phenotypic methods lead to misidentification of the lactic acid bacteria Lactobacill... more Conventional phenotypic methods lead to misidentification of the lactic acid bacteria Lactobacillus hilgardii and Lactobacillus brevis. Random amplified polymorphic DNA (RAPD) and repetitive element PCR (REP-PCR) techniques were developed for a molecular study of these two species. The taxonomic relationships were confirmed by analysis of the ribosomal operon. Amplified DNA fragments were chosen to isolate L. hilgardii-specific probes. In addition to rapid molecular methods for identification of L. hilgardii, these results convincingly proved that some strains first identified as L. brevis must be reclassified as L. hilgardii. The data clearly showed that these molecular methods are more efficient than phenotypic or biochemical studies for bacterial identification at the species level.

Research in microbiology, 1997

Leuconostoc oenos is involved in malolactic fermentation occurring during wine-making. An increas... more Leuconostoc oenos is involved in malolactic fermentation occurring during wine-making. An increasing number of wines are being inoculated with malolactic starters to control the process, and the identification and differentiation of selected strains are now indispensable both for quality control of production and for commercial purposes. In the present work we evaluated the potential use of the intergenic regions of three L. oenos strains for their differentiation. The three 16S/23S rRNA intergenic spacers were amplified in vitro by PCR, and sequences were compared. The spacer sequence was highly conserved in all strains. Inside this spacer, a tRNA-Ala gene containing an 18-bp sequence stretch which is conserved in all tRNA genes was discovered. This sequence, together with random primers, was used for characterization of ten L. oenos strains by PCR.