Leuconostoc oenos

Lonvaud-Funel, A. and Joyeux, A. (1994). Histamine production by wine lactic acid bacteria isolation of a histamine-producing strain of Leuconostoc oenos, J. Appl. Bacterial., 11, 401. 

Practical and fundamental aspects of malo-lactic fermentation are given. Conditions which winemakers can use for better control of the fermentation, including detailed procedures for inoculation with Leuconostoc oenos ML 34 and for inhibition with fumaric acid, are presented. New information on the role of malic acid decarboxylation in bacterial metabolism and on the enzymatics of malic acid decarboxylation are reviewed. The malic acid decarboxylation seems to involve two pathways a direct decarboxylation of malic to lactic acid with NAD as a coenzyme and a concurrent but small oxidative decarboxylation to pyruvic acid and NADH. How these pathways can bring about the marked stimulation of bacterial growth rate by the malo-lactic reaction and their negligible effect on growth yield are discussed. 

Figure I. Photomicrographs of Leuconostoc oenos ML 34 grown on a grape juice medium for use as starter cultures.

In Leuconostoc oenos ML 34, we have shown oxaloacetic acid decarboxylation manometrically (6, 7, 8). We were also able to demonstate fluorometrically the enzymatic production of reduced NAD with malic acid as a substrate, but, of course, were unable to do so with oxaloacetic acid since no NADH could be formed from this substrate. It is likely that this oxaloacetic acid decarboxylation activity, as in Lactobacillus plantarum, is distinct from the activity causing the malic-lactic transition. It is also possible that oxaloacetic acid decarboxylation is caused by a malic enzyme. However, there is no verified NAD dependent malic oxidoreductase (decarboxylating) enzyme which does so (12). For example, Macrae (31) isolated a malic enzyme from cauliflower bud mitochondria which showed no activity with oxaloacetic acid. Similarly, Saz (32) isolated a malic enzyme from Ascaris lumbricoides which is also inactive toward oxaloacetic acid. True, the Enzyme Commission (12) lists an enzyme described as L-malate NAD oxidoreductase (decarboxylating) (E.C. 1.1.1.38) which is said to be capable of decarboxylating oxaloacetic acid, but its description dates back to the studies of Ochoa and his group, and we now feel this listing may be improper. 

Ochoas group reported that in their malic enzyme, Co2+ could replace the Mn2+ requirement, but that Mg2+ was considerably less effective. Macrae (31) reported that malic enzyme from cauliflower bud mitochondria has an absolute requirement for either Mn2+, Co2+, or Mg2+. Morenzoni (6) has shown that the NADH producing activity of Leuconostoc oenos exhibited an absolute specificity for Mn2+ Co2+ and Mg2+ could not substitute, nor could Fe3+, Zn2+, or Cu+. Furthermore, Cu2+ inhibits this activity as well as the malo-lactic acivity. 

Ochoa reported that malic enzyme from L. plantarum was NAD and not NADP specific. The malic enzyme of cauliflower bud mitochondria (31) is NAD and NADP specific, with NAD being the preferred cofactor. Both the malo-lactic activity and NADH producing activity of the Leuconostoc oenos system (6,7, 8) was strictly NAD specific. Nicotinamide-adenine dinucleotide phosphate, flavin adenine dinucleotide, and flavin mononucleotide could not substitute in either of these activities. 

Antohi (34) has reported several possible isozyme structures of a malic acid decarboxylating enzyme in Bacillus subtilis, and Peak (35) has reported the same in Euglena gracilis this must be kept in mind for the Leuconostoc oenos system. It is possible that the enzymatic activities that we have reported (6,7,8) may be the result of isozyme interactions of the same protein. 

In summary, the current concept (6, 7, 8) of malic acid utilization in the Leuconostoc oenos ML 34 system involves two separate enzyme activities (located on the same protein) which act simultaneously on malic acid. One activity, which we call the malo-lactic activity, catalyzes the reaction,... 

This system appears to be somewhat novel, and it is expected that malic acid utilization in malo-lactic bacteria other than Leuconostoc oenos may occur through this mechanism. 

Malolactic fermentation (MLF) is an important secondary fermentation that occurs in many wines generally about 2-3 weeks after completion of the alcoholic fermentation. Lactic acid bacteria, principally Oenococcus oeni (formerly Leuconostoc oenos) are responsible for this fermentation. 

Le Jeune, C. and Fonvaud-Funel, A. (1997). Sequence of DNA 16S/23S spacer region of Leuconostoc oenos (Oenococcus oeni) Application to strain differentiation. Res. Microbiol. 148, 79-86. 

Zavaleta, A. I., Martinez-Murcia, A. J., and Rodriguez-Valera, F. (1996). 16S-23S rDNA intergenic sequences indicate that Leuconostoc oenos is phylogenetically homogeneous. Microbiology 142, 2105-2114. 

It is well known that a malo-lactic fermentation generally does not occur in North Coast white wines because of high S02 levels, low pH, high acidity, and cool storage temperatures, all conditions inhibiting the activity of Leuconostoc oenos, the common lactic acid bacteria used in the North Coast region. Some work has been done promoting malo-lactic fermentation in... 

Leuconostoc oenos (ML-34) was isolated from California wines and it has become the most popular organism to be used in induced fermentations. Myers (21) reports successful use of a frozen culture of the strain PSU-1 introduced at the start of the alcoholic fermentation. He finds that, in most cases, the malo-lactic fermentation parallels the alcoholic fermentation and both come to completion at about the same time. PSU-1 is a strain of Leuconostoc oenos similar to the ML-34 and was first reported on by Beelman et al. (22). 

Pimentel, M. S., Silva, M. H., Cortes, I., and Faia, A. M. 1994. Growth and metabolism of sugar and acids of Leuconostoc oenos under different conditions of temperature and pH. J. Appl. Bacteriol., 76, 42 18. 

Salou, P., Divies, C., and Cardona, R. 1994. Growth and energetics of Leuconostoc oenos during cometabolism of glucose with citrate or fructose. App. Environ. Microbiol., 60,1459-1466. 

Wine bacteria belonging to the genus Oenococcus were previously classified as Leuconostoc oenos by Garvie (1967) and were the only acidophilic members of the genus Leuconostoc. Later, phylogenetic studies revealed that L. oenos represented a distinct subline separate from other Leuconostoc spp. (Martmez-Murcia et al. 1993), and this bacterium was, finally, assigned to a new genus Oenococcus (Dicks et al. 1995). 

Bartkowsky, E.J., Henschke, P.A. (1999). Use of polymerase chain reaction for specific detection of the MLF bacterium Oenococcus oeni (formerly Leuconostoc oenos) in grape juice and wine samples. Aus. J. Grape Wine Res., 5, 39-44. 

Coton, E., Rollan, G.C., Lonvaud-Eunel, A. (1998). Histidine carboxylase of Leuconostoc oenos 9204 purification, kinetic properties, cloning and nucleotide sequence of the hdc gene. J. Appl. Microbiol, 84, 143-151. 

Dicks, L.M., Dell aglio, F, Collins, M.D. (1995). Proposal to reclassify Leuconostoc oenos as Oenococcus oeni. Int. J. System BacterioL, 45, 395-397. 

King, S.W., Beelman, R.B. (1986). Metabolic interactions between Saccharomyces cerevisiae and Leuconostoc oenos in a model juice/wine system. Am. J. Enol. Vitic., 37, 53-60. 

Labarre, C., Guzzo, J., Cervin, J. R, Divies, C. (1996). Cloning and characterization of the genes encoding the malolactic enzyme and the malate permease of Leuconostoc oenos. Appl. Environ. Microbiol, 62, 1274-1282. 

Liu, S.-Q., Pritchard, G.G., Hardman, M.J., PUone, G.J. (1994). Citrulline production and ethyl carbamate (urethane) precursor formation from arginine degradation by wine lactic acid bacteria Leuconostoc oenos and Lactobacillus buchneri. Am. J. Enol. Vitic., 45, 235-242. 

Lonvaud-Eunel, A., Joyeux, A. (1993) Antagonism between lactic acid bacteria of wines inhibition of Leuconostoc oenos by Lactobacillus planlarwn and Pediococcus pentosaceus. Food Microbiol, 10, 411 19. 

Manca de Nadra, M.C., Farias, M., Moreno-Ariibas, M.V., Pueyo, E., Polo, M.C. (1997). Proteolytic activity of Leuconostoc oenos Effect on proteins and polypeptides from white wine. FEMS Microbiol. Lett., 750,135-139. 

Naouri, P., Chagnaud, P., Amaud, A., Galzy, P. (1990). Purification and properties of a malolactic enzyme from Leuconostoc oenos ATCC 23278. J. Basic Microbiol, 30, 577-585. 

Nielsen, J.C., Prahl, C., Lonvaud-Runel, A. (1996). MLR in wine by direct inoculation with freeze-dried Leuconostoc oenos cultures. Am. J. Enol. Vitic., 47, 42-48. 

Poblet-lcart, M., Bordons, A., Lonvaud-Runel, A. (1998). Lysogeny of Oenococcus oeni (syn. Leuconostoc oenos) and study of their induced bacteriophages. Curr. Microbiol, 36, 365-369. 

Sozzi, X, Gnaegi, F., D Amico, N., Hose, H. (1982). Difficultes de fermentation malolactique du vin dues a des bacteriophages de Leuconostoc oenos. Revue Suisse Vitic. Arboric. Hortic., 14, 17-23. 

Veiga-da-Cunha, M., Santos, H., van Schaftingen, E. (1993). Pathway and regulation of erythri-tol formation in Leuconostoc oenos. J. Bacterial, 175, 3941-3948. 

Guilloux-Benatier, M., Son, H.S., Bouhier, M. Feuillat, M. (1993). Activites enzymatiques gly-cosidases et peptidases chez Leuconostoc oenos au cours de la croissance bacterienne. Influence des macromolecules des levures. Vitis, 32, 51-57. 

Lactic acid bacteria, including the typical "wine lactic acid bacteria" Leuconostoc oenos (85, 90), can produce ethyl and vinyl derivatives by hydroxycinnamic acid metabolism (91) although, the minimal concentration produced in red wines by Leuconostoc oenos is insignificant compared to the odor threshold (85, 87). 

Martineau, B., and Henick-Kling, T. (1995) Formation and degradation of diacetyl in wine during alcoholic fermentation with Saccharomyces cerevisiae Strain EC 1118 and malolactic fermentation with Leuconostoc oenos Strain MCW, Am.. Enol. Vitic., 46, 442-448. 

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