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.     

Microbiology of the malolactic fermentation: Molecular aspects

Abstract Malolactic fermentation conducted by lactic acid bacteria follows alcoholic fermentation during winemaking, and several positive effects make it indispensable for most wines. Research has focused on the growth and physiology of lactic acid bacteria in wine; resulting in the design of malolactic starter cultures. Future work on these starters will concentrate on aromatic changes as additional criteria for strain selection. Although the main features of the malolactic enzyme and its gene are known, the detailed mechanism of the malolactic reaction remains unclear. Cloning and expression of this activity in enological strains of Saccharomyces cereuisiae might be one of the next most important advances in the control of malic acid degradation in wine.     

The degradation of malic acid by high density cell suspensions of Leuconostoc oenos

Washed cell suspensions of Leuconostoc oenos catalysed the degradation of L-malic acid to L-lactic acid. Cell suspensions of 10₁₀ cfu ml_-1 degraded 90–95% of the malic acid in a buffer assay system and in wine within 30 min. A reaction time of 6 h was needed to obtain the same extent of degradation with suspensions of 10₉ cfu ml_-1. With a reaction period of 6 h and an initial malic acid concentration of 3 g 1_-1, reaction variables of pH 2.5-4.0, temperature 10–30°C, ethanol up to 15%, and L-lactic acid up to 4 g 1_-1 did not decrease the degradation of malic acid to below 90–95%. Total SO₂ at 100 mg 1_-1 decreased the degradation of malic acid to 80%. The degradation (%) of malic acid was decreased when the concentration of malic acid was decreased below 2 g 1_-1. The results indicate the prospect of using high densities of Leuc. oenos cells in membrane bioreactor systems for the rapid, continuous, deacidification of wine.     

Continuous malolactic fermentation in red wine using free Oenococcus oeni

Malolactic fermentation (MLF) of wine in continuous culture was obtained by using Oenococcus oeni (formerly Leuconostoc oenos). The maximum malic acid degradation in our bioreactor system was reached at a dilution rate of 0.016h^–1, and 92–95% of the malic acid (3.9–4.0g/l) was converted to lactic acid and CO₂.     

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.     

A mathematical model of the link between growth and L-malic acid consumption for five strains of Oenococcus oeni

In winemaking, after the alcoholic fermentation of red wines and some white wines, L-malic acid must be converted into L-lactic acid to reduce the acidity. This malolactic fermentation (MLF) is usually carried out by the lactic acid bacteria Oenococcus oeni. Depending on the level of process control, selected O. oeni is inoculated or the natural microbiota of the cellar is used. This study considers the link between growth and MLF for five strains of O. oeni species. The kinetics of growth and L-malic acid consumption were followed in modified MRS medium (20 °C, pH 3.5, and 10 % ethanol) in anaerobic conditions. A large variability was found among the strains for both their growth and their consumption of L-malic acid. There was no direct link between biomass productivities and consumption of L-malic acid among strains but there was a link of proportionality between the specific growth of a strain and its specific consumption of L-malic acid. Experiments with and without malic acid clearly demonstrated that malic acid consumption improved the growth of strains. This link was quantified by a mathematical model comparing the intrinsic malic acid consumption capacity of the strains.     

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.     

Lactic acid bacteria of wines: stimulation of growth and malolactic fermentation

After the appearance of “Etudes sur le vin” by Pasteur, in enology lactic acid bacteria have been considered as deteriorating agents for more than 50 years. About 1920, Ferré in Burgundy and Ribéreau-Gayon in Bordeaux demonstrated the enological importance of the transformation of malic to lactic acid. This notion is now generally accepted in most vinicultural areas. Malolactic fermentation is encouraged, especially for red wines, for two reasons: a) it eliminates the taste of malic acid and lowers the acidity of the wine, b) it assures the biological stability of wines conserved with a minimum of sulphurous anhydride. In traditional vinification, malolactic fermentation is the result of bacterial growth. It is spontaneous, that means induced by the endogenous lactic acid bacteria of grapes and winery equipment. In the must, yeasts and bacteria develop simultaneously; in the antagonism between yeasts and bacteria the bacterial population is more often becoming dominant than being suppressed. The grapes are sulphited so that bacterial growth occurs only after complete exhaustion of sugars by the yeasts. Consequently, alteration of the wine, as a result of sugar fermentation by the bacteria, is prevented. In a well-controlled vinification lactic acid bacteria can complete their growth cycle in the wine. Wine, however, is a poor culture medium and the bacteria multiply under restricted nutritional, physical and chemical conditions. As a consequence, malolactic fermentation is difficult to control in practice, in spite of all the research done for more than 30 years. For a long time, one has tried to stimulate malolactic fermentation by inoculating wine with bacteria. Until now, the problem has been to determine the biomass of bacteria, sufficient for fermentation to take place as well as the quality required. The desired physiological state of the bacteria in the inoculum is also not known.     

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.     

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.     

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.     

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.     

The metabolism of sugar and malic acid by Leuconostoc oenos: effect of malic acid, pH and aeration conditions

The co-metabolism of sugars by Leuconostoc oenos was studied under different environmental conditions. Under aerobic conditions, growth and sugar metabolism were poorer than under CO₂ or N₂ atmosphere and acetic acid accumulated to a larger extent. Glycerol was found in the aerobic cultures while erythritol was detected under N₂ or CO₂. When medium conditions make growth difficult (low pH, aerobic conditions, low nutrients), sugars were only slightly metabolized and growth was very slow while malic acid was rapidly and completely degraded, leading to an increase in the y _ATP. Aeration effects on the malic acid degradation rate depended on the nutrients and carbon source in the medium. Malic acid clearly stimulated bacterial growth, allowing an increase in the molar growth yields and ATP production. The results suggest that under adverse conditions cells are not able to grow and malic degradation supplies additional energy production.     

Inhibition of bacterial growth and malolactic fermentation in wine by bacteriophage

Bacteriophage present in wine can attack bacterial starter cultures and inhibit the malolactic fermentation. The possibility of starter culture failure due to phage attack was studied in a commercial dry red wine of pH 3·23, inoculated with a multiple strain starter culture. During two stages of malolactic fermentation, bacterial growth and malate degradation in the wine were inhibited. A phage capable of lysing isolates of Leuconostoc oenos was isolated from the wine. The isolated phage had an icosahedral head of 42–45 nm diameter and a flexible, regularly cross-striated tail 197–207 nm long with a small baseplate. The results confirm that phage can attack bacterial starter cultures in wine at low pH.     

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.     

The inhibitory activity of wine yeast starters on malolactic bacteria

Malolactic fermentation is a process that is influenced by various factors that can inhibit the growth of the malolactic bacteria. Inhibitory metabolites produced by yeast may have an important role in the correct development of malolactic fermentation. For these reasons, we have investigated the effects of such metabolites on the growth of malolactic bacteria under different environmental conditions, to aid in our understanding of the significance of these interactions in the wine-making environment. Our screening methods to detect interactions between yeast and malolactic bacteria showed a variable and wide diffusion of yeast inhibitory activity on the growth of the malolactic bacteria. However, this first approach to determine this inhibitory activity of yeast gave an overestimation when compared to the results obtained under actual wine-making conditions. The evaluation of malic acid consumption indicated that under inhibitory conditions a partial L-malic acid degradation was seen, indicating that the malolactic activity continued without bacterial growth. However, these yeast-inhibiting effects in addition to other environmental factors could cause a complete failure of malolactic fermentation.     

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.     

Impact of winemaking practices on arginine and citrulline metabolism during and after malolactic fermentation

AIMS: To study arginine degradation and carcinogenic ethyl carbamate precursor citrulline formation during and after malolactic fermentation (MLF). METHODS AND RESULTS: MLF was induced in white wine with two commercial Oenococcus oeni strains under different winemaking conditions regarding the type of alcoholic fermentation (spontaneous, induced) and the lees management (racked, on lees). Arginine degradation and citrulline formation did not occur during malic acid degradation in any treatment. In five of the six treatments in which arginine degradation took place, it occurred 3 weeks after malic acid depletion and significant amounts of citrulline were formed. Presence of yeast lees in wines led to increased citrulline formation. Conclusions: This study suggests that arginine metabolism is inhibited in oenococci at low pH values (< 3.5) and that in the postalcoholic fermentation phase, citrulline formation from arginine degradation can be avoided if MLF is induced by pure cultures of O. oeni with inhibition of the bacterial biomass after malic acid depletion. Residual yeast lees in the wine have been identified as a significant risk factor for increased citrulline formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Conclusions drawn from this study allow reducing the risk of carcinogenic ethyl carbamate formation from citrulline excretion by wine lactic acid bacteria.     

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.