A numerical taxonomic study of Leuconostoc oenos strains from wine

Phenotypic data of 108 tests conducted on 70 malolactic bacteria, including 54 presumptive Leuconostoc oenos strains, five presumptive pediococci isolated from wine and 11 reference strains, were analysed by numerical taxonomic techniques. Using the simple matching coefficient, 58 strains were grouped in six clusters at the 87% S level. Cell wall analysis of the interpeptide bridges and morphology was also used to differentiate between the strains. No L. oenos reference strains were found to group in any cluster. The hypothetical median organism (HMO) of cluster A was related at only the 63% S level to the L. oenos type strain and it is proposed that these strains be regarded as 'L. gracile'. The majority of the L. oenos strains (35) grouped together at above the 91% S level in cluster B, with the HMO of this cluster related at the 75% S level to the L. oenos type strain. Results indicate that the majority of L. oenos strains included in this study are closely related, but more research is needed to justify the separation of these strains into more than one species.     

Cellular fatty acid composition of Leuconostoc oenos

The cellular fatty acid composition of 70 lactic acid bacteria was examined by capillary gas chromatography. Fifty-four Leuconostoc oenos strains, including three reference, type strains from the other Leuconostoc spp., nine Pediococcus spp. and two Lactobacillus spp. were studied. Eighteen fatty acids were determined, of which 10 were identified by gas chromatography-mass spectrometry. The relative percentages of the 18 fatty acids of the Leuconostoc strains were analyzed numerically and grouped using the unweighted pair-group method. Results show that four clusters could be defined at r = 0.920, with five strains unassigned. The major fatty acids of the Leuc. oenos strains were found to be palmitic acid (C16:0), palmitoleic acid (C16:1–9), oleic acid (C18: 1–9), vaccenic acid (C18: 1–11), dihyrosterculic acid (C19-cyclopropane-9) and lactobacillic acid (C19-cyclopropane-11). It was mainly on the basis of the amounts of oleic acid and the C19-cyclopropane fatty acids that the strains of Leuc. oenos could be distinguished from each other. This is the first report of the occurrence of dihydrosterculic acid in lactic acid bacteria. For the majority of Leuc. oenos strains, the result obtained with the cellular fatty acid analysis confirmed the phenotypic relationships.     

Plasmids in Leuconostoc oenos

A new procedure was used to isolate 11 plasmids from eight Leuconostoc oenos strains. Plasmid DNA was not detected in 34 other strains of this species. Plasmid sizes ranged from 2.47 to 4.61 kilobase pairs. This is the first report of extrachromosomal elements in L. oenos.     

Inter-strain relationships among wine leuconostocs and their divergence from other Leuconostoc species, as revealed by low frequency restriction fragment analysis of genomic DNA

Thirty Leuconostoc oenos strains, representing 28 different isolates, were distributed into 20 genomic groups according to PFGE patterns of restriction digests. The 8 bp-specific enzymes Sfi I, Not I and Asc I cleaved the Leuc. oenos DNA in a mean of 17, 11 and four fragments respectively and Sma I produced more than 50 fragments per genome. The strain differentiating capacity of the four enzymes was similar; only two related genomic groups failed to be distinguished by Asc I or Not I. Genomic relationships between Leuc. oenos strains were quantified by numerical analysis of Not I and Sfi I banding patterns. More than half of the strains, including the starters ML34 and PSU-1, formed a major cluster. The average size of the Leuc. oenos genome was estimated as 1.86 Mb. Although similar values were obtained for the genomes of Leuc. mesenteroides, Leuc. pseudomesenteroides, Leuc. gelidum and Leuc. citreum, a significant divergence between wine and non-wine species was inferred from comparisons of genome cleavage frequencies, determined with five different enzymes.     

Occurrence and properties of bacteriophages of Leuconostoc oenos in Australian wines

Bacteriophages specific for Leuconostoc oenos were isolated from four red wines undergoing malolactic fermentation in one winery. Bacteriophages were not found in samples of 16 other wines. The morphology of the phages was examined by electron microscopy. The phages did not lyse all strains of L. oenos, and susceptibility correlated to some extent with the colony morphology of the strain. Phage survived in wines at pH values greater than 3.5 but was inactivated in wines of lower pH and by the addition of sulfur dioxide or bentonite. Phage did not affect the growth of a sensitive strain of L. oenos in filter-sterilized wine.     

Occurrence and properties of bacteriophages of Leuconostoc oenos in Australian wines.

Bacteriophages specific for Leuconostoc oenos were isolated from four red wines undergoing malolactic fermentation in one winery. Bacteriophages were not found in samples of 16 other wines. The morphology of the phages was examined by electron microscopy. The phages did not lyse all strains of L. oenos, and susceptibility correlated to some extent with the colony morphology of the strain. Phage survived in wines at pH values greater than 3.5 but was inactivated in wines of lower pH and by the addition of sulfur dioxide or bentonite. Phage did not affect the growth of a sensitive strain of L. oenos in filter-sterilized wine.     

Characterization of temperate bacteriophages of Leuconostoc oenos and evidence for two prophage attachment sites in the genome of starter strain PSU-1

The close relatedness between 17 Leuconostoc oenos bacteriophages, induced with mitomycin C from strains isolated in different geographic regions, was inferred from their morphology, DNA homology and protein composition. The genome of all the phages had cohesive end termini and ranged in size from 36.4 to 40.9 kb. According to the restriction patterns obtained by digestion with five enzymes, the phages were divided in six groups. Lysogenization of a spontaneous phage-cured derivative of Leuc. oenos strain PSU-1 was achieved with 16 phages and the analysis of the lysogens showed that the phage DNA integrates in the host chromosome in one or two sites. The att B loci were located on the macrorestriction Asc I and Not I fragments of the recipient strain. A survey of Leuc. oenos strains with a phage DNA probe confirmed the lysogenic nature of several, but not all of the original phage hosts. These results are discussed in the light of evidence for the instability of some lysogenic PSU-1 derivatives.     

Factors affecting the induction of malolactic fermentation in red wines with Leuconostoc oenos

Malolactic fermentation was induced in red wines by inoculation with several strains of Leuconostoc oenos . The progress of Malolactic Fermentation was monitored by following the kinetics of bacterial growth and degradation of malic acid. These kinetics varied significantly depending on the strain of Leuc. oenos inoculated, the strain of Saccharomyces cerevisiae used to conduct the alcoholic fermentation, and the wine properties of pH and concentrations of ethanol and sulphur dioxide. Rapid, predictable malolactic fermentation was achieved by inoculating a high density (> 10⁶ cfu/ml) of Leuc. oenos , whereby malic acid degradation was not connected to the growth of the bacterial cells. Wines after malolactic fermentation were not bacteriologically stable and supported the growth of Leuc. oenos inoculated into the wines.     

Malolactic fermentation in Chardonnay: growth and sensory effects of commercial strains of Leuconostoc oenos

Samples of fermenting Chardonnay juice were inoculated with five commercial cultures of Leuconostoc oenos to promote malolactic fermentation. Controls were not inoculated with malolactic starter cultures; one was held under the same conditions as the juice inoculated with malolactic starter cultures and the other was held under conditions in which malolactic fermentation was inhibited. Bacterial growth and chemical composition of the wines were monitored for eight weeks after the wines were inoculated with the yeast starter culture. The five strains of L. oenos differed in growth kinetics and rates of malic acid degradation. Significant differences were detected among the finished wines subjected to sensory evaluation.     

Identification of lactic acid bacteria isolated from Portuguese wines and musts by SDS-PAGE

Thirty-two strains were isolated from spoiled port wines, from musts and from various styles of young, Northeastern Portuguese red table wines that had undergone spontaneous malolactic fermentation. Comparison of their SDS-PAGE whole-cell protein patterns with an SDS-PAGE database of lactic acid bacteria indicated that the isolates were members of the species Leuconostoc oenos or Lactobacillus paracasei subsp. paracasei. The latter were found in low acidity table wines and in port wine. The isolation of Lactobacillus paracasei strains from wines indicates the importance of using known strains for wine deacidification because spontaneous malolactic fermentation of table wines can occur from an indigenous flora, adapted to the particular composition of the wine.     

Formation of histamine and tyramine by lactic acid bacteria in decarboxylase assay medium

A modified decarboxylase assay medium (DCA medium) was used for studying the production of biogenic amines by Leuconostoc oenos DSM 20252 and two strains of Lactobacillus buchneri (Lb14 and St2A). The DCA medium contained histidine, lysine, ornithine and tyrosine as precursors of the respective biogenic amines. Under the experimental conditions both strains of Lact. buchneri produced > 90% of the maximum amount of histamine within 24 h. Only tyramine was produced by Leuc. oenos DSM 20252. accountine for 88% of the maximum theoretical amount within 24 h.     

Transposons Tn916 and Tn925 can transfer from Enterococcus faecalis to Leuconostoc oenos

Abstract The streptococcal transposons Tn916 and Tn925 were transferred to several strains of Leuconostoc (Ln.) oenos using the filter mating method. The insertion of both transposons into the chromosome occurred at different sites. Transconjugants of Ln. oenos carrying Tn916 could serve as donors in mating experiments with Lactococcus lactis LM2301. Further analysis of L. lactis LM2301 transconjugants showed that the insertion of the transposon Tn916 into the chromosome was site-specific. These studies establish a basis for the initiation of genetic studies in this Leuconostoc species since there are no efficient conjugal or transformation systems previously described for this microorganism.     

Using specific polyclonal antibodies to study the malolactic enzyme from Leuconostoc oenos and other lactic acid bacteria

Specific polyclonal antibodies directed against the malolactic enzyme of Leuconostoc oenos were obtained. Despite the homologies between the malolactic enzymes from Leuc. oenos and Lactococcus lactis , no immunological relationship was detected with the L. lactis malolactic enzyme, suggesting differences in their structural organization. The use of the antiserum also demonstrated that the problem of heterologous expression occurring in the recombinant Escherichia coli strain (Labarre et al. 1996a) resulted in a low synthesis of the malolactic enzyme from Leuc. oenos . Moreover, a small amount of the protein was found to be peripherally associated to the membrane of Leuc. oenos.     

Nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases in homofermentative and heterofermentative lactic acid bacteria

Three homofermentative (Lactobacillus plantarum B38, L. plantarum B33, Pediococcus pentosaceus B30) and three heterofermentative (Leuconostoc mesenteroides 39, L. oenos B70, Lactobacillus brevis) lactic acid bacteria were examined for the presence or absence of nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent d- and l-lactate dehydrogenases. Two of the six strains investigated, P. pentosaceus and L. oenos, did not exhibit an NAD-independent enzyme activity capable of reducing dichlorophenol indophenol. The pH optima of the lactic dehydrogenases were determined. The NAD-dependent enzymes from homofermentative strains exhibited optima at pH 7.8 to 8.8, whereas values from 9.0 to 10.0 were noted for these enzymes from heterofermentative organisms. The optima for the NAD-independent enzymes were between 5.8 and 6.6. The apparent Michaelis-Menten constants determined for both NAD and the substrates demonstrated the existence of a greater affinity for d- than l-lactic acid. A comparison of the specific NAD-dependent and NAD-independent lactate dehydrogenase activities revealed a direct correlation of the d/l ratios of these activities with the type of lactic acid produced during the growth of the organism.     

Lysogeny in Leuconostoc oenos.

Thirty strains of Leuconostoc oenos were exposed to mitomycin C to induce lysogenic bacteriophages. Lysis curves typical for lysogenic strains were obtained with 19 strains. Indicator strans were found for 17 of these phages. Five were characterized by electron microscopy, lytic spectrum, molecular masses of the proteins, sequencing of five N-terminal amino acids of the two major proteins and DNA analysis (restriction patterns, cross hybridization). The results revealed a very close relationship between the phages. Hybridization experiments between the DNAs of the temperate phages and the appropriate lysogenic strains revealed phage-related sequences in the DNA of the lysogenic strain.     

A note on the occurrence of Leuconostoc oenos as a spoilage organism in canned mango juice

E thiraj , S. & S uresh , E.R. 1985. A note on the occurrence of Leuconostoc oenos as a spoilage organism in canned mango juice. Journal of Applied -Bacteriology 59 , 239–242.
A strain of Leuconostoc oenos was isolated from a blown can of mango juice. Various tests to identify and characterize the bacterium suggested that it could be a strain of L. oenos . This is the first report of L. oenos as a spoilage organism in fruit products other than wine.     

A note on the occurrence of Leuconostoc oenos as a spoilage organism in canned mango juice

A strain of Leuconostoc oenos was isolated from a blown can of mango juice. Various tests to identify and characterize the bacterium suggested that it could be a strain of L. oenos. This is the first report of L. oenos as a spoilage organism in fruit products other than wine.     

Inhibition of malolactic fermentation by cryotolerant yeasts

White wines produced by some cryotolerant strains of Saccharomyces cerevisiae are more resistant to malolactic fermentation than those produced by normal strains: e.g. for two months of storage, the wines, inoculated with Leuconostoc oenos or Lactobacillus plantarum, were fully stabilized with levels of 51-65 mg total SO 2 /l and 5.70-5.75 g titratable acidity/l. The use of these yeasts in wine-making can decrease the quantities of sulfites added to stabilize wines.     

Antibacterial polypeptides of Lactobacillus species

Twelve of 79 strains of the genus Lactobacillus , mainly isolated from plants or fermenting material, were found to inhibit at least one of the nine indicator strains of the species Lact. brevis, Pediococcus damnosus and Leucanostoc oenos . The antimicrobial activities from Lact. brevis B 37 and Lact. casei B 80 were caused by polypeptides detectable in the culture liquids. They are bacteriocins with a narrow antimicrobial spectrum. Brevicin 37 from Lact. brevis B 37 was active against many lactic acid bacteria and Nocardia corallina , whereas caseicin 80 from Lact. casei B 80 inhibits only one other strain of Lact. casei . Brevicin 37 is stable at 121C, caseicin 80 is inactivated above 60C, and both are inactivated under alkaline conditions.     

Comparison of Leuconostoc oenos Strains by Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis of chromosomal DNA digested with NotI or SfiI was used to differentiate individual strains of Leuconostoc oenos. L. oenos isolates with 13 different restriction digest patterns were detected in New Zealand wines undergoing malolactic fermentation. The average genome size was estimated to be 1,800 kb.