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.     

Identification of pOENI-1 and Related Plasmids in Oenococcus oeni Strains Performing the Malolactic Fermentation in Wine

Plasmids in lactic acid bacteria occasionally confer adaptive advantages improving the growth and behaviour of their host cells. They are often associated to starter cultures used in the food industry and could be a signature of their superiority. Oenococcus oeni is the main lactic acid bacteria species encountered in wine. It performs the malolactic fermentation that occurs in most wines after alcoholic fermentation and contributes to their quality and stability. Industrial O. oeni starters may be used to better control malolactic fermentation. Starters are selected empirically by virtue of their fermentation kinetics and capacity to survive in wine. This study was initiated with the aim to determine whether O. oeni contains plasmids of technological interest. Screening of 11 starters and 33 laboratory strains revealed two closely related plasmids, named pOENI-1 (18.3-kb) and pOENI-1v2 (21.9-kb). Sequence analyses indicate that they use the theta mode of replication, carry genes of maintenance and replication and two genes possibly involved in wine adaptation encoding a predicted sulphite exporter (tauE) and a NADH:flavin oxidoreductase of the old yellow enzyme family (oye). Interestingly, pOENI-1 and pOENI-1v2 were detected only in four strains, but this included three industrial starters. PCR screenings also revealed that tauE is present in six of the 11 starters, being probably inserted in the chromosome of some strains. Microvinification assays performed using strains with and without plasmids did not disclose significant differences of survival in wine or fermentation kinetics. However, analyses of 95 wines at different phases of winemaking showed that strains carrying the plasmids or the genes tauE and oye were predominant during spontaneous malolactic fermentation. Taken together, the results revealed a family of related plasmids associated with industrial starters and indigenous strains performing spontaneous malolactic fermentation that possibly contribute to the technological performance of strains in wine.     

Leuconostoc oenos and malolactic fermentation in wine: a review

This review article summarizes the state of the art on Leuconostoc oenos, the bacteria responsible for malolactic fermentation in wine. Both basic and practical aspects related to the metabolism of this microorganism and malolactic fermentation in general are critically reviewed. The former examines the role of genetics for the identification and classification of L. oenos and energetic mechanisms on solute transport (malic and lactic acid). The latter includes practical information on biomass production, optimal growth conditions and stress factors, which are important in growth optimization of malolactic starter cultures. Extensive data and references on the effect of malolactic fermentation on wine composition and sensory analysis are also included. Received 06 May 1999/ Accepted in revised form 13 July 1999     

Determination of lactic acid bacteria producing biogenic amines in wine by quantitative PCR methods

Aims: To develop rapid methods allowing enumeration of lactic acid bacteria producing biogenic amines in wines and to analyse wine samples by the methods. Methods and Results: Methods based on quantitative PCR targeting bacterial genes involved in histamine, tyramine and putrescine production were developed and applied to detect and quantify the bacteria producing these biogenic amines in wine. Analysis of 102 samples revealed low populations of the targeted bacteria in grape must samples, an increased bacteria biomass in wine samples after alcoholic fermentation, reaching the highest population levels (above 10⁶ cells ml^?1) during spontaneous malolactic fermentation. A minimum of 10³ ml^?1 producing cells was required for production of more than 1 mg l^?1 of biogenic amines. Accumulation of putrescine in wine was correlated with the presence of bacteria carrying an ornithine decarboxylation pathway. Trials of winemaking showed that the use of selected bacteria for inducing malolactic fermentation was efficient to limit the proliferation of undesirable bacteria and the production of biogenic amines. Conclusion: Methods using quantitative PCR are efficient to enumerate biogenic amines‐producing cells in wine. Significance and Impact of the Study: The methods can help to better control and to improve winemaking conditions in order to avoid biogenic amine production.     

Bacterial biodiversity and dynamics during malolactic fermentation of Tempranillo wines as determined by a culture-independent method (PCR-DGGE)

The bacterial population during malolactic fermentation of Tempranillo wine was studied using the polymerase chain reaction-denaturing gradient gel electrophoresis, a culture-independent method successfully used for identification and monitoring of bacterial population in different habitats included food fermentations. The results showed that Oenococcus oeni was the predominant species in the malolactic fermentation of Tempranillo wines, although the presence of Gluconobacter oxydans, Asaia siamensis, Serratia sp., and Enterobacter sp. was also observed. These results were partly coincidental with those obtained from a culture-dependent method, using a selective medium. Therefore, it may be concluded that for a more complete knowledge of the bacterial community present during malolactic fermentation of Tempranillo wine, an approach that combines a culture-independent method and a culture-dependent method would be advisable.     

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.     

Controlling Wine Malolactic Fermentation with Nisin and Nisin-Resistant Strains of Leuconostoc oenos

The polypeptide nisin (100 U/ml) prevented malolactic fermentation in wines by indigenous or intentionally added lactic acid bacteria. Nisin (100 U/ml)-resistant mutants of Leuconostoc oenos were obtained and used with nisin in wine to carry out a pure-culture malolactic fermentation in the presence or absence of other lactic acid bacteria. Nisin degradation by mutants was not observed, and residual nisin was detectable in wines 4 months after it was added. Results indicated that nisin or nisin with resistant bacterial starter cultures can be used to control malolactic fermentation in wines.     

Occurrence of Lactic Acid Bacteria During the Different Stages of Vinification and Conservation of Wines

We showed that the growth of lactic acid bacteria during alcoholic fermentation depends on the composition of the must. We illustrated how the addition of sulfur dioxide to the must before fermentation and the temperature of storage both affect the growth of these bacteria in the wine. Whereas species of Lactobacillus and Leuconostoc mesenteroides were isolated from grapes and must, Leuconostoc oenos was the only species isolated after alcoholic fermentation. This organism was responsible for the malolactic fermentation. Isolates of this species varied in their ability to ferment pentoses and hexoses. The survival of Leuconostoc oenos in wines after malolactic fermentation depended on wine pH, alcohol concentration, SO₂ concentration, and temperature of storage.     

Impact of different malolactic fermentation inoculation scenarios on Riesling wine aroma

During malolactic fermentation (MLF), lactic acid bacteria influence wine aroma and flavour by the production of volatile metabolites and the modification of aroma compounds derived from grapes and yeasts. The present study investigated the impact of different MLF inoculation strategies with two different Oenococcus oeni strains on cool climate Riesling wines and the volatile wine aroma profile. Four different timings were chosen for inoculation with bacteria to conduct MLF in a Riesling must/wine with a high acidity (pH 2.9–3.1). Treatments with simultaneous inoculation showed a reduced total fermentation time (alcoholic and malolactic) compared to the sequential inoculations. No negative impact of simultaneous alcoholic and malolactic fermentation on fermentation success and on the final wine volatile aroma composition was observed. Compared to sequential inoculation, wines with co-inoculation tended to have higher concentrations of ethyl and acetate esters, including acetic acid phenylethylester, acetic acid 3-methylbutylester, butyric acid ethylester, lactic acid ethylester and succinic acid diethylester. Results of this study provide some alternatives to diversify the number of wine styles by safely conducting MLF in low-pH, cool-climate white musts with potential high alcohol content.     

Starter cultures as biocontrol strategy to prevent Brettanomyces bruxellensis proliferation in wine

Brettanomyces bruxellensis is a common and significant wine spoilage microorganism. B. bruxellensis strains generally detain the molecular basis to produce compounds that are detrimental for the organoleptic quality of the wine, including some classes of volatile phenols that derive from the sequential bioconversion of specific hydroxycinnamic acids such as ferulate and p-coumarate. Although B. bruxellensis can be detected at any stage of the winemaking process, it is typically isolated at the end of the alcoholic fermentation (AF), before the staring of the spontaneous malolactic fermentation (MLF) or during barrel aging. For this reason, the endemic diffusion of B. bruxellensis leads to consistent economic losses in the wine industry. Considering the interest in reducing sulfur dioxide use during winemaking, in recent years, biological alternatives, such as the use of tailored selected yeast and bacterial strains inoculated to promote AF and MLF, are actively sought as biocontrol agents to avoid the “Bretta” character in wines. Here, we review the importance of dedicated characterization and selection of starter cultures for AF and MLF in wine, in order to reduce or prevent both growth of B. bruxellensis and its production of volatile phenols in the matrix.

     

Effect of Oleic Acid on Oenococcus oeni Strains and Malolactic Fermentation in Wine

A different capability to assimilate oleic acid from the culture medium has been demonstrated among malolactic Oenococcus oeni strains. Strains possessing higher percentages of oleic acid and its methylated derivative, dihydrosterculic acid, in their fatty acid profile showed higher cell viability and carried out a complete malolactic fermentation after their transfer into a wine lacking oleic acid. Wine supplementation with Tween 80 (polyoxyethylene-sorbitan-mono-oleate) enhanced cell survival of strains with lower capability to assimilate oleic acid and caused cell growth of strains with higher assimilative capacity, suggesting that oleic acid may act in wine as a survival factor for the former strains and as a growth factor for the latter strains. Practical consequences of these findings are also discussed. Received: 24 March 2001 / Accepted: 25 April 2001     

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.     

CONTINUOUS ALCOHOL/MALOLACTIC FERMENTATION OF GRAPE MUST IN A BIOREACTOR SYSTEM USING IMMOBILIZED CELLS

Three cultures immobilized by entrapping within alginate gel beads and packed in near-horizontal acrylic columns (15.0° angle) were used for alcohol/malolactic fermentation of grape must. Immobilized cells of Saccharomyces cerevisiae spp. chablis were placed in the 1st column, S. cerevisiae cells (an alcohol-sucrose-tolerant yeast) in the 2nd and the Lactobacillus delbrueckii cells in the 3rd column. Grape must with different levels of sugar(s), were each fed to the bioreactor columns at dilution rate of 0.74 h⁻¹ and recycled at 37.0C. The percent fermentation efficiency and yield using the 1st and 2nd columns for grape must containing 33.3% sugar(s) were 92.9 and 91.5%, respectively, and the wine had 15.5% alcohol after 23 cycles (∼ 50 h fermentation). The viability of the immobilized yeast cells in the alginate gel-bead was 84%± 4.0. Immobilized Lactobacillus delbrueckii cells were then added to the 3rd column (in series 37.0C) and the three cultures resulted in alcohol/malolactic fermentation of the grape must, evidenced by the high level of alcohol formed and simultaneous transformation of malic to lactic acid. Sensory evaluation of the wine scored high (7.8 ± 2.0 based on a value of 10.0) and indicated the potential of using multiple immobilized cells of two specific yeast cultures and a malolactic Lactobacillus for wine production.     

Purification and characterization of a bacteriocin from an oenological strain of Leuconostoc mesenteroides subsp. cremoris

Malolactic fermentation (MLF), which improves organoleptic properties and biologic stability of some wines, may cause wine spoilage if uncontrolled. Bacteriocins were reported as efficient preservatives to control MLF through their bactericidal effect on malolactic bacteria. Leuconostoc mesenteroides subsp. cremoris W3 isolated from wine produces an inhibitory substance that is bactericidal against malolactic bacteria in model wine medium. Treatment of the culture supernatant of strain W3 with proteases eliminated the inhibitory activity, which proved that it is a true bacteriocin and we tentatively termed it mesentericin W3. The bacteriocin inhibited the growth of food-borne pathogenic bacteria such as Enterococcus faecalis, Listeria monocytogenes, and malolactic bacteria. It was active over a wide pH range and stable to organic solvents and heat. Mesentericin W3 was purified to homogeneity by a pH-mediated cell adsorption–desorption method, cation exchange, hydrophobic interaction, and reverse-phase chromatography. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy (MS) and partial amino acid sequence analysis revealed that mesentericin W3 was identical to mesentericin Y105.     

Immobilization of Oenococcus oeni in lentikats® to develop malolactic fermentation in wines

Entrapment of Oenococcus oeni into a polymeric matrix based on polyvinyl alcohol (PVA) (Lentikats®) was successfully used to get a better development of malolactic fermentation (MLF) in wine. The incubation of immobilized cells in a nutrient medium before starting the MLF, did not improve the degradation of malic acid. In only one day, 100% of conversion of malic acid was achieved using a high concentration of immobilized cells (0.35 g gel/ml of wine with a cell‐loading of 0.25 mg cells/mg of gel). While a low concentration of 0.21 g gel/ml of wine (cell‐loading of 0.25 mg cells/mg of gel) needed 3 days to get a reduction of 40%. The entrapped cells could be reused through six cycles (runs of 3 days), retaining 75% of efficacy for the conversion of malic acid into lactic acid. The immobilized cells in PVA hydrogels gave better performance than free cells because of the increase of the alcohol toleration. Consequently, the inhibitory effect of ethanol for developing MLF could be reduced using immobilized cells into PVA hydrogels. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Control of Flavor Development in Wine during and after Malolactic Fermentation by Oenococcus oeni

During malolactic fermentation in wine by Oenococcus oeni, the degradation of citric acid was delayed compared to the degradation of malic acid. The maximum concentration of diacetyl, an intermediary compound in the citric acid metabolism with a buttery or nutty flavor, coincided with the exhaustion of malic acid in the wine. The maximum concentration of diacetyl obtained during malolactic fermentation was strongly dependent on the oxygen concentration and the redox potential of the wine and, to a lesser extent, on the initial citric acid concentration. The final diacetyl concentration in the wine was also dependent on the concentration of SO₂. Diacetyl combines rather strongly with SO₂ (K_f = 7.2 × 10³ M⁻¹ in 0.1 M malate buffer [pH 3.5] at 30°C). The reaction is exothermic and reversible. If the concentration of SO₂ decreases during storage of the wine, the diacetyl concentration increases again.     

Differential Real-Time PCR Assay for Enumeration of Lactic Acid Bacteria in Wine

Oenococcus oeni is often employed to perform the malolactic fermentation in wine production, while nonoenococcal lactic acid bacteria often contribute to wine spoilage. Two real-time PCR assays were developed to enumerate the total, and nonoenococcal, lactic acid bacterial populations in wine. Used together, these assays can assess the spoilage risk of juice or wine from lactic acid bacteria.     

Cloning the Gene for the Malolactic Fermentation of Wine from Lactobacillus delbrueckii in Escherichia coli and Yeasts

The gene responsible for the malolactic fermentation of wine was cloned from the bacterium Lactobacillus delbrueckii into Escherichia coli and the yeast Saccharomyces cerevisiae. This gene codes for the malolactic enzyme which catalyzes the conversion of l-malate to l-lactate. A genetically engineered yeast strain with this enzymatic capability would be of considerable value to winemakers. L. delbrueckii DNA was cloned in E. coli on the plasmid pBR322, and two E. coll clones able to convert l-malate to l-lactate were selected. Both clones contained the same 5-kilobase segment of L. delbrueckii DNA. The DNA segment was transferred to E. coli-yeast shuttle vectors, and gene expression was analyzed in both hosts by using enzymatic assays for l-lactate and l-malate. When grown nonaerobically for 5 days, E. coli cells harboring the malolactic gene converted about 10% of the l-malate in the medium to l-lactate. The best expression in S. cerevisiae was attained by transfer of the gene to a shuttle vector containing both a yeast 2-mum plasmid and yeast chromosomal origin of DNA replication. When yeast cells harboring this plasmid were grown nonaerobically for 5 days, ca. 1.0% of the l-malate present in the medium was converted to l-lactate. The L. delbrueckii controls grown under these same conditions converted about 25%. A laboratory yeast strain containing the cloned malolactic gene was used to make wine in a trial fermentation, and about 1.5% of the l-malate in the grape must was converted to l-lactate. Increased expression of the malolactic gene in wine yeast will be required for its use in winemaking. This will require an increased understanding of the factors governing the expression of this gene in yeasts.     

High Frequency of Histamine-Producing Bacteria in the Enological Environment and Instability of the Histidine Decarboxylase Production Phenotype

Lactic acid bacteria contribute to wine transformation during malolactic fermentation. They generally improve the sensorial properties of wine, but some strains produce histamine, a toxic substance that causes health issues. Histamine-producing strains belong to species of the genera Oenococcus, Lactobacillus, and Pediococcus. All carry an hdcA gene coding for a histidine decarboxylase that converts histidine into histamine. For this study, a method based on quantitative PCR and targeting hdcA was developed to enumerate these bacteria in wine. This method was efficient for determining populations of 1 to 10⁷ CFU per ml. An analysis of 264 samples collected from 116 wineries of the same region during malolactic fermentation revealed that these bacteria were present in almost all wines and at important levels, exceeding 10³ CFU per ml in 70% of the samples. Histamine occurred at an often important level in wines containing populations of the above-mentioned bacteria. Fifty-four colonies of histamine producers isolated from four wines were characterized at the genetic level. All were strains of Oenococcus oeni that grouped into eight strain types by randomly amplified polymorphic DNA analysis. Some strains were isolated from wines collected in distant wineries. Moreover, hdcA was detected on a large and possibly unstable plasmid in these strains of O. oeni. Taken together, the results suggest that the risk of histamine production exists in almost all wines and is important when the population of histamine-producing bacteria exceeds 10³ per ml. Strains of O. oeni producing histamine are frequent in wine during malolactic fermentation, but they may lose this capacity during subcultures in the laboratory.     

Saccharomyces cerevisiae and Oenococcus oeni immobilized in different layers of a cellulose/starch gel composite for simultaneous alcoholic and malolactic wine fermentations

The production of a two-layer composite biocatalyst for immobilization of two different microorganisms for simultaneous alcoholic and malolactic fermentation (MLF) of wine in the same bioreactor is reported. The biocatalyst consisted of a tubular delignified cellulosic material (DCM) with entrapped Oenococcus oeni cells, covered with starch gel containing the alcohol resistant and cryotolerant strain Saccharomyces cerevisiae AXAZ-1. The biocatalyst was found effective for simultaneous low temperature alcoholic fermentation resulting to conversion of malic acid to lactic acid in 5 days at 10 °C. Improvement of wine quality compared with wine fermented with S. cerevisiae AXAZ-1 immobilized on DCM was attributed to MLF as well as to increased ester formation and lower higher alcohols produced at low fermentation temperatures (10 °C) as shown by GC and headspace SPME GC/MS analysis. Scanning electron microscopy showed that the preparation of a three-layer composite biocatalyst is also possible. The significance of such composite biocatalysts is the feasibility of two or three bioprocesses in the same bioreactor, thus reducing production cost in the food industry