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.     

Cloning and characterization of the genes encoding the malolactic enzyme and the malate permease of Leuconostoc oenos.

Using degenerated primers from conserved regions of the protein sequences of malic enzymes, we amplified a 324-bp DNA fragment by PCR from Leuconostoc oenos and used this fragment as a probe for screening a Leuconostoc oenos genomic bank. Of the 2,990 clones in the genomic bank examined, 7 with overlapping fragments were isolated by performing colony hybridization experiments. Sequencing 3,453 bp from overlapping fragments revealed two open reading frames that were 1,623 and 942 nucleotides long and were followed by a putative terminator structure. The first deduced protein (molecular weight, 59,118) is very similar (level of similarity, 66%) to the malolactic enzyme of Lactococcus lactis; as in several malic enzymes, highly conserved protein regions are present. The synthesis of a protein with an apparent molecular mass of 60 kDa was highlighted by the results of labelling experiments performed with Escherichia coli minicells. The gene was expressed in E. coli and Saccharomyces cerevisiae and conferred "malolactic activity" to these species. The second open reading frame encodes a putative 34,190-Da protein which has the characteristics of a carrier protein and may have 10 membrane-spanning segments organized around a central hydrophilic core. Energy-dependent L-[14C]malate transport was observed with E. coli dicarboxylic acid transport-deficient mutants carrying the malate permease-expressing vector. Our results suggest that in Leuconostoc oenos the genes that encode the malolactic enzyme and a malate carrier protein are organized in a cluster.     

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.     

Specific enumeration of lactic acid bacteria in fermenting grape must and wine by colony hybridization with non-isotopic DNA probes

A. LONVAUD-FUNEL, A. JOYEUX AND O. LEDOUX. 1991. Total DNA extracted from lactic acid bacteria commonly found in musts and wines was randomly labelled with digoxigenin. It was assayed for the detection of several species by dot-blot hybridization. The method proved to be specific as there was no cross-hybridization between most of the species belonging to the genera Leuconostoc, Pediococcus and Lactobacillus , homofermentative and heterofermentative ( Lact. plantarum, Lact. casei, Leuc. mesenteroides, Leuc. oenos, Ped. damnosus, Ped. pentosaceus ). However, it failed for some Lact. brevis strains which strongly hybridized with Lact. hilgardii.
Colony hybridization was performed directly on plates soon after enumeration. Eight probes of the most common species were used; it was possible to follow the evolution of each species during the vinification of two red wines. According to the phase of alcoholic fermentation, then malolactic fermentation, the predominance or regression of bacilli and cocci could be established.     

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.     

A selective medium for the isolation of malolactic mutants of Leuconostoc oenos

We have developed a selective medium for the isolation of Leuconostoc oenos mutants defective in malolactic fermentation. Forty per cent of colonies isolated directly on selective plates after UV mutagenesis had lost their ability to degrade malate. None of the tested mutants showed any detectable malolactic activity and all lacked a protein band corresponding in size to that of the malolactic enzyme. The availability of such mutants provides a valuable tool both for physiological and genetic research on malolactic fermentation.     

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.     

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.     

Bacteriophages induced by mitomycin C treatment of Leuconostoc oenos strains from Portuguese wines

Twenty-two Leuconostoc oenos strains from Portuguese wines and seven strains from other sources were induced with mitomycin C. Bacteriophages were detected in the supernatants of 19 different cultures. Analysis of their host-range and plating efficiencies suggests that phage typing could be useful for strain identification in Leuc. oenos.     

Characterization of strains of Leuconostoc mesenteroides by analysis of soluble whole-cell protein pattern, DNA fingerprinting and restriction of ribosomal DNA

Of 215 leuconostocs isolated from field grass, natural whey cultures and water-buffalo milk, 178 were identified as Leuconostoc mesenteroides ssp. mesenteroides while 37 strains could not be identified Biochemical characterization allowed seven groups to be defined. Representative strains of each group and different habitat and nine reference strains were selected for further analyses. Protein profiles appeared suitable for species discrimination, but did not differentiate between the three subspecies of Leuc. mesenteroides. The technique also showed some differences among equivocal strains. DNA fingerprinting for most strains of Leuc. mesenteroides ssp. mesenteroides examined showed a different restriction pattern from that of the type strain. Ribotyping was not useful for discriminating species and subspecies of the genus Leuconostoc: Leuc. mesenteroides ssp. mesenteroides and ssp. dextranicum showed the same ribopattern as Leuc. lactis while Leuc. mesenteroides ssp. cremoris exhibited a pattern distinct from all the other species examined. On the basis of ARDRA-PCR, two main groups could be distinguished: the larger group included Leuc. mesenteroides, Leuc. lactis, Leuc. pseudomesenteroides and some unidentifiable strains; the second one included Leuc. citreum, Leuc. fallax, Weissella paramesenteroides and some unidentified strains.     

Functional expression in Saccharomyces cerevisiae of the Lactococcus lactis mleS gene encoding the malolactic enzyme

Abstract Malolactic fermentation, a crucial step in winemaking, results mostly in degradation by lactic acid bacteria of L-malic acid into L-lactic acid. This direct decarboxylation is catalysed by the malolactic enzyme. Recently we, and others, have cloned the mleS gene of Lactococcus lactis encoding malolactic enzyme. Heterologous expression of mleS in Saccha-romyces cerevisiae was tested to perform simultaneously alcoholic and malolactic fermentations by yeast. mleS gene was cloned in a yeast multicopy vector under a strong promoter. Malolactic activity was present in crude extracts of recombinant yeasts. Malic acid degradation was tested during alcoholic fermentation in synthetic media and must. Yeasts expressing the mleS gene actually produced L-lactate from L-malate; nevertheless malate degradation was far from complete.     

Malolactic fermentation of wine: study of the influence of some physico-chemical factors by experimental design assays

The ability of selected lactic acid bacteria to carry out malolactic fermentation depends on the level of numerous wine characteristics. A Hadamard's experimental matrix was used to determine the main effects of 11 physico-chemical factors on malolactic activity of three Leuconostoc œnos strains and one Lactobacillus plantarum strain. Ethanol had the greatest inhibitory effect on the achievement of malolactic fermentation for all Leuc. œnos strains. An inhibitory effect of the L-malic acid was also found in the operating conditions. These strains show different degrees of sensitivity to pH. One of these strains was inhibited by SO₂. Malolactic activity of the Lact. plantarum strain is mainly affected by a low pH, and this strain is often less efficient than Leuc. œnos strains. This methodology could be used for the selection of strains for malolactic starters. Further work is in progress using factorial design in order to determine the interactions between influential factors.     

Cloning and sequence analysis of the gene encoding Lactococcus lactis malolactic enzyme: Relationships with malic enzymes

Abstract Malolactic enzyme is the key enzyme in the degradation of L-malic acid by lactic acid bacteria. Using degenerated primers designed from the first 20 N-terminal amino acid sequence of lactococcal malolactic enzyme, a 60-bp DNA fragment containing part of the mleS gene was amplified from Lactococcus lactis in a polymerase chain reaction. This specific probe was used to isolate two contiguous fragments covering the gene as a whole. The 1.9-kb region sequenced contains an open reading frame of 1623 bp, coding a putative protein of 540 amino acids. The deduced amino acid sequence reveals that lactococcal putative protein (Mlep) is highly homologous to the malic enzyme of other organisms. Expression of the mleS gene in Escherichia coli results in malolactic activity.     

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.     

Medium for Screening Leuconostoc oenos Strains Defective in Malolactic Fermentation

A new sensitive medium was developed to screen and isolate mutagenic Leuconostoc oenos strains defective in malolactic fermentation. The essential components of the medium included fructose (22 mM), l-malic acid (74.6 mM), bromocresol green (as pH indicator), and cellulose powder. The wild-type colonies turned blue, but defective malolactic colonies gave an acid reaction and remained yellow-green.     

乳酸乳球菌苹果酸-乳酸酶基因的克隆

用酚抽提的方法提取乳酸乳球菌的基因组DNA,利用PCR方法从乳酸菌的基因组DNA中扩增出含有苹果酸-乳酸酶基因(malolactic enzyme gene,mle)的约1.6kb的DNA片断,用1%的琼脂糖凝胶分离扩增的片断,用试剂盒回收目的基因。将回收的目的基因与pGEM-T载体连接构建mle-T载体并转化大肠杆菌DH5a,挑取阳性克隆(白色菌落),酶切鉴定并测序。SalI酶切mle-T,回收mle DNA片断,与表达载体pET-28a载体连接,构建细菌Escherichia coli表达载体。     

Sugar and organic acid metabolism in mixed cultures of Pediococcus pentosaceus and Leuconostoc oenos isolated from wine

Pediococcus pentosaceus 12p and Leuconostoc oenos X₂L isolated from Argentinian wine were examined for growth and changes in the concentrations of glucose, fructose, sucrose and mannitol and malic, citric, acetic and lactic acids in pure and mixed cultures. In mixed cultures a mutualistic growth response and a change in the balance of end-products of sugar and organic acid metabolism were observed. The production of mannitol and acetic acid was lower while D(-) and L(+) lactic acids were detected in higher levels than in pure cultures. Malic and citric acids were metabolized simultaneously, but the amount of citric acid consumed was lower than in pure culture of Leuc. oenos.     

Mixed culture of Lactobacillus hilgardii and Leuconostoc oenos isolated from Argentine wine

Growth, sugar and organic acid metabolism of Lactobacillus hilgardii X₁B and Leuconostoc oenos X₂L isolated from Argentinian wine were examined in pure and mixed cultures. In mixed culture Leuc. oenos X₂L did not grow, no viable cells were detected after 24 h, but the consumption of glucose and fructose by Lact. hilgardii was higher. An increase of mannitol and acetic acid production was detected during the early stages of growth. Over 12 h, higher levels of d (-) lactic acid and slightly increased levels of l (+) lactic acid were observed. Citric and malic acid were simultaneously metabolized, but a slight increase in citric acid consumption was observed in mixed culture.