Lysis of yeast cells by Oenococcus oeni enzymes

Oenococcus oeni exhibited extracellular β (1→3) glucanase activity. This activity increased when cells were cultivated with glycosidic cell-wall macromolecules. In addition, the culture supernatant of the organism effectively lysed viable or dead cells of Saccharomyces cerevisiae. This lytic activity appeared in the early stationary phase of bacterial growth. Yeast cells at the end of the log phase of growth were the most sensitive. The optimum temperature for lysis of viable yeast cells was 40°C, which is very different from the temperatures observed in enological conditions (15–20°C). Moreover, the rate of the lytic activity was significantly lower in comparison with yeast cell wall-degrading activities previously measured in various other microorganisms. Therefore, yeast cell death that is sometimes observed during the alcoholic fermentation could hardly be attributed to the lytic activity of O. oeni. Journal of Industrial Microbiology & Biotechnology (2000) 25, 193–197. Received 27 December 1999/ Accepted in revised form 14 July 2000     

Effects of yeast proteolytic activity on Oenococcus oeni and malolactic fermentation

Alcoholic fermentation of synthetic must was performed using either Saccharomyces cerevisiae or a mutant Deltapep4, which is deleted for the proteinase A gene. Fermentation with the mutant Deltapep4 resulted in 61% lower levels of free amino acids, and in 62% lower peptide concentrations at the end of alcoholic fermentation than in the control. Qualitative differences in amino acid composition were observed. Changes observed in amino acids in peptides were mainly quantitative. After alcoholic fermentation, each medium was inoculated with Oenococcus oeni. Malolactic fermentation in the medium with the Deltapep4 strain took 10 days longer than the control. This difference may have been due to a difference in the nitrogen composition of the two media. Free amino acids and amino acids in peptides were poorly consumed by O. oeni. Thus, the qualitative aspects of nitrogen composition, which depend in part on yeast metabolism, may be a determinant for the optimal growth of O. oeni in wine.     

Rapid detection of Oenococcus oeni in wine by real-time quantitative PCR

AIMS: To develop a real-time polymerase chain reaction (PCR) method for rapid detection and quantification of Oenococcus oeni in wine samples for monitoring malolactic fermentation. METHODS AND RESULTS: Specific primers and fluorogenic probe targeted to the gene encoding the malolactic enzyme of O. oeni were developed and used in real-time PCR assays in order to quantify genomic DNA either from bacterial pure cultures or wine samples. Conventional CFU countings were also performed. The PCR assay confirmed to be specific for O. oeni species and significantly correlated to the conventional plating method both in pure cultures and wine samples (r = 0.902 and 0.96, respectively). CONCLUSIONS: The DNA extraction from wine and the real-time PCR quantification assay, being performed in ca 6 h and allowing several samples to be concurrently processed, provide useful tools for the rapid and direct detection of O. oeni in wine without the necessity for sample plating. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid quantification of O. oeni by a real-time PCR assay can improve the control of malolactic fermentation in wines allowing prompt corrective measures to regulate the bacterial growth.     

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.     

Malolactic fermentation in wine with high densities of non-proliferating Oenococcus oeni

Five strains of Oenococcus oeni (syn. Leuconostoc oenos) under non-proliferating conditions were assessed for the performance of the malolactic fermentation in wine at various initial pH values, malic acid concentration and densities of cells. We succeeded in inducing the malolactic fermentation after inoculation of high densities of O. oeni G6 even in recalcitrant wines where the traditional malolactic fermentation was inhibited by adverse environmental conditions (low pH and high concentration of malic acid). Optimal degrading conditions in wine, under different physico-chemical environments, were determined in order to achieve rapid depletion of malic acid in red wine. Off-odour compounds were not formed under these conditions, suggesting an attractive alternative for wine production. This revised version was published online in November 2006 with corrections to the Cover Date.     

Oligopeptide assimilation and transport by Oenococcus oeni

Aims: Oenococcus oeni is a slow‐growing wine bacterium with a low growth yield. It thrives better on complex nitrogen sources than on free amino‐acid medium. We aimed to characterize the oligopeptide use of this micro‐organism. Methods and Results: Several peptides of two to eight amino‐acid residues were able to provide essential amino acids. The disappearance of various peptides from extracellular medium was assessed with whole cells. Initial rates of utilization varied with the peptide, and free amino acids were released into the medium. Conclusions: Oenococcus oeni was able to transport the oligopeptides with two to five amino‐acid residues tested and to hydrolyse them further. Significance and Impact of the Study: This study has clear implications for the relationship between wine nitrogen composition and the ability of O. oeni to cope with its environment.     

Arginine and citrulline do not stimulate growth of two Oenococcus oeni strains in wine

Arginine metabolism by wine lactic acid bacteria (LAB) may lead to wine quality degradation. While arginine is essential for growth of the wine relevant LAB Oenococcus oeni , it remains unclear whether it also stimulates its growth. This study evaluated the effect of arginine and citrulline, the partially metabolized intermediate of the arginine deiminase pathway, on the growth of two commercial O. oeni strains in comparison with a Lactobacillus buchneri strain in wine and at wine pH values. Neither arginine nor citrulline increased growth of both O. oeni strains in comparison with the L. buchneri strain. However, arginine and citrulline were partially degraded in all incubations. The extent of citrulline degradation correlated with lower pH values in oenococcal cultivations but with higher pH values in those of the L. buchneri strain. The degradation kinetics of O. oeni and L. buchneri for malic acid and arginine differed and the latter grew in sterile filtered post-malolactic fermentation wine. This study shows that arginine and citrulline did not stimulate growth of the two O. oeni strains studied, and that their physiological role differed among the wine LAB considered. While arginine may play a role in wine microbiological stability, other nutrients should be investigated for their suitability to create a selective ecological advantage for O. oeni strains in wine.     

Inhibitory effect of sulfur dioxide and other stress compounds in wine on the ATPase activity of Oenococcus oeni

Malolactic fermentation (MLF) is carried out by Oenococcus oeni under very harsh conditions. This paper shows that stress compounds in wine such as SO(2), fatty acids and copper have an inhibitory effect on cell growth and MLF duration, and relates this effect to an inhibition of ATPase activity. Of the stress compounds, SO(2) and dodecanoic acid had the strongest effect, decreasing the ATPase specific activity to 37% and 58%, respectively. It can be concluded that ATPase is a good indicator of the physiological state of the cells and their ability to lead MLF.     

Cell surface damage and morphological changes in Oenococcus oeni after freeze-drying and incubation in synthetic wine

The aim of the present study was to evaluate the effects of freeze-drying in the presence of trehalose as a cryoprotectant, followed by incubation in synthetic wine, on surface damage, viability and l-malic acid consumption of the oenological strain Oenococcus oeni UNQOe 73.2. After freeze-drying, no significant differences were observed in the number of viable cells (for both acclimated and non-acclimated cultures) respect to the fresh culture. In contrast, loss of viability was observed after wine incubation for 24 h, being acclimated freeze-dried cells the best conditions for this. After the preservation process, small changes in cell morphology were observed by Atomic Force Microscopy (AFM). The Zeta potential and AFM showed that 24 h of wine incubation was enough to induce several cell surface modifications. Plate count data allowed us to establish that surface damage is an important factor for loss of viability, regardless of the acclimation treatment. Although the number of surviving O. oeni cells decreased dramatically after incubation in synthetic wine for 15 days, the consumption of l-malic acid was higher than 70%, with freeze-dried cells showing a better performance than fresh cultures. These results demonstrate that O. oeni freeze-dried cultures could be applied to direct wine inoculation, to conduct malolactic fermentation, maintaining its technological properties and reducing the time and costs of the winemaking process.     

Production of Oenococcus oeni biomass to induce malolactic fermentation in wine by control of pH and substrate addition

To increase the commercial production of Oenococcus oeni strains to be used for biological deacidification of wines, substrates addition and pH control have been optimized. The highest biomass yield of Oenococcus oeni (Y=6.9 mg mmol–1 sugar) was obtained when 55 mmol glucose l–1 and 30 mmol fructose l–1 were added both to the culture medium, and the pH was controlled at 4.8. Fructose was used as carbon and energy source, but also as electron acceptor improving the ability to reoxidize NAD(P)H.     

Effect of phenolic compounds on the co-metabolism of citric acid and sugars by Oenococcus oeni from wine

AIMS: The goal of this study was to examine the growth of Oenococcus oeni in the presence of phenolic compounds under wine conditions and to see how these compounds affect bacterial metabolism. METHODS AND RESULTS: Phenolic compounds have been added to a basal medium that simulates the composition of wine. Fifty milligrams per litre or more of phenolic compounds stimulated bacterial growth. Oenococcus oeni seemed to use citric acid and trehalose, if they were present, before glucose and fructose. Citrate was completely exhausted in three days and the yield of acetate was higher when phenolic compounds were present. CONCLUSIONS: Phenolic compounds reduced the rate of sugar consumption and enhanced citric acid consumption, increasing the yield of acetic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: This study allows a better knowledge of co-metabolism of citric acid and sugars by O. oeni in the presence of phenolic compounds of wine.     

Influence of fatty acids on the growth of wine microorganisms Saccharomyces cerevisiae and Oenococcus oeni

The effects of fatty acids, extracted during prefermentation grape skin-contact on Saccharomyces cerevisiae and Oenococcus oeni, were studied. The influence of skin-contact on total fatty acid content was evaluated both in Chardonnay must and in synthetic medium. Prior to alcoholic fermentation, the skin-contact contributes to a large enrichment of long-chain fatty acids (C₁₆ to C_18:3). These results induced a positive effect on yeast growth and particularly on cell viability. In the skin-contact fermented media, levels of C₁₂ and especially C₁₀ are lower and macromolecules content higher than in controls. This production of extracellular mannoproteins and the reduction of medium-chain fatty acids in media by S. cerevisiae increased growth of O. oeni. The influence of fatty acids (C₁₀ to C_18:3), in their free and esterified forms, on bacterial growth and on malolactic activity was also examined. Only C₁₀ and C₁₂, especially in their esterified forms, always appeared to be toxic to O. oeni. Received 15 May 1997/ Accepted in revised form 02 December 1997     

The ftsH gene of the wine bacterium Oenococcus oeni is involved in protection against environmental stress

The wine bacterium Oenococcus oeni has to cope with harsh environmental conditions, including an acidic pH, a high alcoholic content, nonoptimal growth temperatures, and growth-inhibitory compounds such as fatty acids, phenolic acids, and tannins. We describe the characterization and cloning of the O. oeni ftsH gene, encoding a protease belonging to the ATP binding cassette protein superfamily. The O. oeni FtsH protein is closest in sequence similarity to the FtsH homologue of Lactococcus lactis. The O. oeni ftsH gene proved to be stress-responsive, since its expression increased at high temperatures or under osmotic shock. O. oeni FtsH protein function was tested in an Escherichia coli ftsH mutant strain, and consistent with the O. oeni ftsH gene expression pattern, the O. oeni FtsH protein provided protection for the E. coli ftsH mutant against heat shock. O. oeni and Bradyrhizobium japonicum FtsH proteins also triggered E. coli resistance to wine toxicity. Genes homologous to O. oeni ftsH were detected in many other lactic acid bacteria found in wine, suggesting that this type of gene constitutes a well-conserved stress-protective molecular device.     

Population dynamics of Oenococcus oeni strains in a new winery and the effect of SO2 and yeast strain

The effects of different yeast starters and SO(2) addition on malolactic fermentation in a new winery were evaluated by a molecular approach in three vintages. Alcoholic fermentations with 40 and 100mgl(-1) SO(2) were carried out, followed by uninoculated malolactic fermentations. Isolated colonies of Oenococcus oeni obtained from samples throughout the vinification were identified and typified by multiplex RAPD-PCR. This made it possible to monitor the population dynamics and follow the proportion of as many as 29 different indigenous strains. In one of the vintages, O. oeni strains were more inhibited when a specific yeast starter was used.     

Anti-Prelog Reduction of Prochiral Carbonyl Compounds by Oenococcus oeni in a Biphasic System

An aqueous-organic biphasic system was established and used with whole cells of Oenococcus oeni to reduce 2-octanone to (R)-2-octanol. The conversion reached 99% when the Tris/borate buffer was increased from 50 mM to 300 mM in the aqueous phase. In addition, the conversion increased as the log P value of the organic solvent changed from 0.5 to 6.6. Under optimized conditions, the conversion of (R)-2-octanol reached 99% from 0.5 M 2-octanone with an optical purity of 99% e.e. The biphasic system allows the anti-Prelog reduction of aliphatic and aromatic ketones to furnish (R)-configurated alcohols in high optical purity as well.     

Molecular and biochemical diversity of Oenococcus oeni strains isolated during spontaneous malolactic fermentation of Malvasia Nera wine

The diversity of indigenous Oenococcus oeni strains was investigated by molecular and biochemical characterization of isolates from Malvasia Nera wine, an economically important red wine of the Salento Region (Apulia, Italy), during spontaneous malolactic fermentation (MLF). A total of 82 isolates of this species, identified by species-specific PCR and 16S rDNA sequence analysis, was molecularly characterized by the amplified fragment length polymorphism (AFLP) technique. Three main groups resulted from cluster analysis and showed intraspecific homology higher than 50%, and a total of seven subgroups, with similarity values ranged from 80% to 98%, were obtained within these groups. Enzymatic activities, such as esterase, β-glucosidase, protease, and the consumption rate of l-malic acid, citric acid, acetaldehyde and arginine were assessed in the representative strains, according to AFLP analysis. The results showed different enzymatic activities and consumption rates of the tested metabolites among the strains. No correlation between molecular and biochemical data was observed. The evidence of biochemical variability observed among Malvasia Nera strains demonstrated that the wine aroma can be modulated depending on the strains involved in MLF. Hence, the heterogeneity existing within natural O. oeni populations represents an interesting ecological source that can be useful for technological purposes.     

Yeast adapted to wine: nitrogen compounds released during induced autolysis in a model wine

As important as the blend of base wines before bottling, one of the most important steps in the champagne-making process is the long ageing on lees. Two yeast strains of Saccharomyces cerevisiae MC001 and MC002, used in champagne wine production, were allowed to autolyse. After 8 days of autolysis, active dry yeasts adapted to wine released 1.7- to 1.8-fold more nitrogen compounds than nonadapted active dry yeast. The nitrogen content (total, proteins, peptides and amino) present in autolysates was measured for yeasts adapted to wine. The composition of free amino acids and amino acids constituting peptides showed no difference between the two strains of yeast used. Studies of intracellular proteolytic activity and release of peptides showed no correlation between these two phenomena. These results indicate that yeasts adapted to wine give results similar to those that occur in wine during ageing. Journal of Industrial Microbiology & Biotechnology (2002) 29, 134–139 doi: 10.1038/sj.jim.7000291 Received 19 December 2001/ Accepted in revised form 14 June 2002