Oenococcus

Numerous articles have been published documenting the enological importance ofmalolactic fermentation (Davis et al., 1985 Kunkee, 1967 Lonvaud-Funel, 1999 Van Vuuren and Dicks, 1993 Wibowo et al., 1985). During this 

1 Proposed model for energy generation (ATP) by Oenococcus oeni through conversion of malic acid to lactic acid and carbon dioxide. (Adapted from Poolman et al., 1991 and with permission of the Journal of Bacteriology.) 

MLF most commonly occurs after alcoholic fermentation but may occur simultaneously with the primary fermentation. Because relying on natural microflora can be unpredictable and difficult to control, starter cultures of pure strains of bacteria have been developed (Henick-Kling, 1993 Krieger et al., 1993 Kunkee et al., 1964 Nielsen et al., 1996 Pilone, 1995). Although selected strains of Lactobacillus can be inoculated, O. oeni is the primary species to conduct MLF because of acid tolerance and the flavor profile produced (Guzzo et al., 1994 Krieger et al., 1993 Kunkee et al., 1964 Liu, 2002 Nielsen et al., 1996 Wibowo et al., 1985). 

Exactly when to inoculate O. oeni during vinification is a point of contention among researchers and enologists. Some have argued that starter 

Aside from deacidification, MLF may also influence the sensory qualities of a wine by the production of many flavor and aroma compounds (Davis et al., 1985, 1988 Henick-Kling, 1993 Kunkee, 1967 Rankine, 1977). However, there is still debate regarding the contribution of MLF to the sensory properties of a wine. Early work by Kunkee et al. (1964) and Rankine (1972) indicated that MLF may not have a measurable effect on the sensory properties of a wine. On the other hand, many other studies have shown that MLF causes significant changes in wine aroma and flavor (Boido et al., 2002 Delaquis-Pascal et al., 2000 De Revel et al., 1999 Gambaro et al., 2001 Henick-Kling, 1995 Laurent et al., 1994 Maicas et al., 1999 McDaniel et al., 1987 Nielsen and Richelieu, 1999). 

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Bacterial cells of Oenococcus oeni incubated for 48 h with three azo dyes (Fast red, Fast orange, and Methanil yellow) gave rise to decolorization due to adsorption, from 68% with Fast red to 30% with Fast orange and Methanil yellow [41]. Ozdemir et al. [44] observed a 93.9% decolorization of Acid Black 210 within 24 h by Vibrio harveyi TEMS1, a bioluminescent bacterium isolated from coastal seawater in Turkey. After extraction in methanol of biomass, the major part of the decolorized dye was recovered, indicating that decolorization was mainly due to... 

El Ahwany AMD (2008) Decolorization of Fast red by metabolizing cells of Oenococcus oeni ML34. World J Microbiol Biotechnol 24 1521-1527... 

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. 

Vidal, M. T., Poblet, M., Constanti, M., and Bordons, A. (2001). Inhibitory effect of copper and dichlofluanid on Oenococcus oeni and malolactic fermentation. Am.. Enol. Vitic. 52, 223-229. 

M. Esti, G. Volpe, L. Micheli, E. Delibato, D. Compagnone, D. Moscone and G. Palleschi, Electrochemical biosensors for monitoring malolactic fermentation in red wine using two strains of Oenococcus oeni, Anal. Chim. Acta, 513(1) (2004) 357-364. 

Usually, after alcoholic fermentation, the wine undergoes malolactic fermentation, induced primarily by Oenococcus oeni. Not only can this lactic acid bacterium convert L-malic acid into L-lactic acid but also it is involved in many other transformations fundamental to Amarone quality. 

Beltramo, C., Desroche, N., Tourdot-Marechal, R., Grandvalet, C., and Guzzo, J. (2006). Realtime PCR for characterizing the stress response of Oenococcus oeni in a wine-like medium. Res. Microbiol. 157, 267-274. 

Bon, E., Delaherche, A., Bilhere, E., De Daruvar, A., Lonvaud-Funel, A., and Le Marrec, C. (2009). Oenococcus oeni genome plasticity is associated with fitness. Appl. Environ. Microbiol. 75, 2079-2090. 

Borneman, A. R., Bartowsky, E. J., McCarthy, J., and Chambers, P. J. (2010). Genotypic diversity in Oenococcus oeni by high-density microarray comparative genome hybridization and whole genome sequencing. Appl. Microbiol. Biotechnol. 86, 681-691. 

Guerrini, S., Bastianini, A., Blaiotta, G., Granchi, L., Moschetti, G., Coppola, S., Romano, P., and Vincenzini, M. (2003). Phenotypic and genotypic characterization of Oenococcus oeni strains isolated from Italian wines. Int. J. Food Microbiol. 83,1-14. 

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. 

Zapparoli, G., Reguant, C., Bordons, A., Torriani, S., and Dellaglio, F. (2000). Genomic DNA fingerprinting of Oenococcus oeni strains by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA-PCR. Curr. Microbiol. 40,351-355. 

Alegri, G. E., Lopez, I. J., Ruiz, J. I., Saenz, J., Fernandez, E., Zarazaga, M., Dizy, M., Torres, C., and Ruiz-Larrea, F. 2004. High tolerance of wild Lactobacillus planta-rum and Oenococcus oeni strains to lyophilisation and stress environmental conditions of acid pH and ethanol. FEMS Microbiol. Lett., 230, 53-61. 

Richter, H., Hamann, I., and Unden, G. 2003. Use of the mannitol pathway in fructose fermentation of Oenococcus oeni due to limiting redox regeneration capacity of the ethanol pathway. Arch. Microbiol., 179,227-233. 

Wagner, N., Tran, Q. H., Richter, H., Selzer, P. M., and Unden, G. 2005. Pyruvate fermentation by Oenococcus oeni and Leuconostoc mesenteroid.es and role of pyruvate dehydrogenase in anaerobic fermentation. Appl. Environ. Microbiol., 71, 4966-4971. 

Several heterofermentative LAB belonging to the genera Lactobacillus, Leu-conostoc, and Oenococcus can produce mannitol from fructose effectively (Saha, 2003). In addition to mannitol, these bacteria may produce lactic acid, acetic acid, carbon dioxide, and ethanol. The process is based on the ability of the LAB to use fructose as an electron acceptor and reduce it to mannitol with the participation of the enzyme mannitol 2-dehydrogenase (EC 1.1.1.38). 

Malolactic fermentation (MLF) in wine is by definition the enzymatic conversion of L-malic acid to L-lactic acid, a secondary process which usually follows primary (alcoholic) fermentation of wine but may also occur concurrently. This reduction of malic acid to lactic acid is not a true fermentation, but rather an enzymatic reaction performed by lactic acid bacteria (LAB) after their exponential growth phase. MLF is mainly performed by Oenococcus oeni, a species that can withstand the low pFi (<3.5), high ethanol (>10 vol.%) and high SO2 levels (50 mg/L) found in wine. More resistant strains of Lactobacillus, Leuconostoc and Pediococcus can also grow in wine and contribute to MLF especially if the wine pH exceeds 3.5 (Davis et al. 1986 Wibowo et al. 1985). The most important benefits of MLF are the deacidification of high acid wines mainly produced in cool climates, LAB contribute to wine flavour and aroma complexify and improve microbial sfabilify (Lonvaud-Funel 1999 Moreno-Arribas and Polo 2005). 

The LAB from grape, musts or wine belong to two families representing three genera. Lactobacillaceae are represented by the genus Lactobacillus, and Strepto-coccaceae are represented by Oenococcus and Pediococcus. 

Oenococcus oeni is described as a Gram-positive non-mobile coccus and frequently occurs in pairs and chains of different sizes (Fig. 2.2). 

Oenococcus is a facultative acidophilic anaerobe and grows at pH 4.8 with temperatures between 18 °C and 30 °C. It requires a rich medium supplemented with tomato juice or grape juice, and its growth is not inhibited in the presence of 10% ethanol. Glucose is fermented in lactic acid, carbon dioxide, acetic acid and ethanol (it is a heterofermenter). It converts malate into lactate and CO2 in the presence of fermentable carbohydrate. 

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). 

Fig. 2.2 Oenococcus oeni cells observed under optical microscope (CRA - Centro di ricerca per TEnologia)...

Of gas production is observed, the strain is a heterofermenter and this can be confirmed by analysing the presence of ethanol and acetic acid (Oenococcus, Lactobacillus)... 

Alexandre, H., Costello, P.J., Remize, R, Guzzo, J., Guilloux-Benatier, M. (2004). Saccha-romyces cerevisiae-Oenococcus oeni interactions in wine current knowledge and perspectives. Int. J. Food. Microbiol, 93, 141-154. 

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. 

Comitini, F., Ferretti, R., Clementi, F., Mannazzu, 1., Ciani, M. (2005). Interactions between Saccharomyces cerevisiae and malolactic bacteria preliminary characterization of a yeast proteinaceous compound(s) active against Oenococcus oeni.J. Appl. Microbiol., 99, 105-111. 

DTncecco, N., Bartowsky, E,L, Kassara, S., Lante, A., SpettoU, P. Henschke, P.A. (2004). Release of glycosidically bound flavour compounds of Chardonnay by Oenococcus oeni during malolactic fermentation. Pood Microbiol, 21, 257-265. 

De las Rivas, B., Marcobal, A., Munoz, R. (2003). Allelic diversity and population structure in Oenococcus oeni as determined from sequence analysis of housekeeping genes. Appl. Environ. 

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. 

Grimaldi, A., McLean, H., Jiranek, V. (2000). Identification and partial characterization of gly-cosidic activities of commercial strains of the lactic acid bacterium Oenococcus oeni. Am. J. Enol. Vitic., 51, 362-369. 

Guilloux-Benatier, M., Pageault, O., Man, A., Feuillat, M. (2000). Lysis of yeast cells by Oenococcus oeni enzymes. J. Ind. Microbiol. Biotech., 25, 193-197. 

Hernandez, T., Estrella, I., Perez-Gordo, M., Alegria, E-G., Tenorio, C., Ruiz-Larrea, R, Moreno-Arribas, M.V. (2007). Contribution of Oenococcus oeni and Lactobacillus plantarum to the non anthocyanin phenolic composicion of red wine during malolactic fermentation. J. Agric. Food Chem., 55, 5260-5266. 

Leitao, M.C., Teixeira, H.C., Barreto Crespo, M.T., San Romao, N.V. (2000). Biogenic amine ocurrence in wine. Amino acid decarboxylase and proteolytic activities expression by Oenococcus oeni. J. Agric. Food Chem., 48, 2780-2784. 

Malaciino, P, Zapparoli, G., Torrioni, S., Dell aglio, F. (2003). Adaptation in Amarone wine of indigenous Oenococcus oeni strains differentiated by pulsed-field gel electrophoresis. Ann. 

Manca de Nadra, M.C., Farias, M., Moreno-Anibas, M.V., Pueyo, E., Polo, M.C. (1999). A proteolytic effect of Oenococcus oeni on the nitrogenous macromolecular fraction of red wine. FEMS Microbiol. Lett., 174, 41-47. 

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