Malolactic fermentation of lactic acid bacteria isolated from southern Brazilian red wine.

The objective was to isolate lactic acid bacteria (LAB) from southern Brazil's wines and investigate their potential as starter cultures for malolactic fermentation (MLF) in Merlot (ME) and Cabernet Sauvignon (CS) wines through the fermentative capacity. The LAB were isolated from CS, ME, and Pinot Noir (PN) wines in the 2016 and 2017 harvests and evaluated for morphological (color and shape of the colonies), genetic, fermentative (increase in pH, acidity reduction, preservation of anthocyanins, decarboxylation of L-malic acid, yield of L-lactic acid, and content of reduced sugars), and sensory characteristics. Four strains were identified as Oenococcus oeni [CS(16)3B1, ME(16)1A1, ME(17)26, and PN(17)65], one as Lactiplantibacillus plantarum [PN(17)75], and one as Paucilactobacillus suebicus [CS(17)5]. Isolates were evaluated in the MLF and compared to a commercial strain (O. oeni), as well as a control (without inoculation and spontaneous MLF), and standard (without MLF). CS(16)3B1 and ME(17)26 isolates finished the MLF for CS and ME wines, respectively, after 35 days, similar to the commercial strain, and CS(17)5 and ME(16)1A1 isolates ended the MLF in 45 days. In the sensory analysis, ME wines with isolated strains received better scores for flavor and overall quality than the control. Compared to the commercial strain, CS(16)3B1 isolate obtained the highest scores for buttery flavor and taste persistence. CS(17)5 isolate received the higher scores for a fruity flavor and overall quality and the lowest for a buttery flavor. The native LAB displayed MLF potential, regardless of the year and grape species from which they were isolated.

Utilization of Oenococcus oeni strains to ferment grape juice: Metabolic activities and beneficial health potential.

This study aimed to investigate the behavior of Oenococcus oeni MS9 and MS46 strains in sterile grape juice (SGJ, pH 4.0) incubated at 30 °C, in terms of growth and glucose, organic acids and total phenolic compounds utilization. In addition, their antimicrobial activity and the changes in antioxidant properties of fermented juice with selected strain were evaluated. Both strains grew without lag period by ~1.40 log CFU/mL at 12 days with maximum growth rates of about 0.019 h-1. After this time the MS9 and MS46 strains counts declined by 0.6 log units and remained unchanged respectively. O. oeni MS46 was evaluated in SGJ for low inoculum size (~104 CFU/mL). In this condition it also grew without lag period by 3.11 ± 0.01 log CFU/mL with a µmax of 0.05 h-1. Glucose and L-malic and citric acids were simultaneously utilized but at different rates and extents, yielding mainly lactic acid with concomitant pH reduction. Acetic acid ranged between 11 and 19 mmol/L. Total phenolic compounds significantly decreased in fermented SGJ with strain MS9 but not MS46. In this last condition, the antioxidant activity increased by 21%. In addition, both O. oeni strains showed antibacterial properties against Escherichia coli 700, Salmonella Typhimurium and Listeria monocytogenes. O. oeni strains, especially MS46, with the ability to growth in SGJ, high malolactic potential and adequate sugars and organic acids profiles from the sensorial viewpoint may be used to ferment grape juice with safer and healthier properties than fresh juice.

l-Malate (-2) Protonation State is Required for Efficient Decarboxylation to l-Lactate by the Malolactic Enzyme of Oenococcus oeni.

Malolactic fermentation (MLF) is responsible for the decarboxylation of l-malic into lactic acid in most red wines and some white wines. It reduces the acidity of wine, improves flavor complexity and microbiological stability. Despite its industrial interest, the MLF mechanism is not fully understood. The objective of this study was to provide new insights into the role of pH on the binding of malic acid to the malolactic enzyme (MLE) of Oenococcus oeni. To this end, sequence similarity networks and phylogenetic analysis were used to generate an MLE homology model, which was further refined by molecular dynamics simulations. The resulting model, together with quantum polarized ligand docking (QPLD), was used to describe the MLE binding pocket and pose of l-malic acid (MAL) and its l-malate (-1) and (-2) protonation states (MAL- and MAL2-, respectively). MAL2- has the lowest ∆Gbinding, followed by MAL- and MAL, with values of -23.8, -19.6, and -14.6 kJ/mol, respectively, consistent with those obtained by isothermal calorimetry thermodynamic (ITC) assays. Furthermore, molecular dynamics and MM/GBSA results suggest that only MAL2- displays an extended open conformation at the binding pocket, satisfying the geometrical requirements for Mn2+ coordination, a critical component of MLE activity. These results are consistent with the intracellular pH conditions of O. oeni cells-ranging from pH 5.8 to 6.1-where the enzymatic decarboxylation of malate occurs.

Relative expression of stress-related genes during acclimation at low temperature of psychrotrophic Oenococcus oeni strains from Patagonian wine.

In the present study, we evaluated the transcriptional response of four stress-related genes in three Oenococcus oeni strains after acclimation at two different temperatures. Gene expression was analyzed at time zero and after 48 h acclimation at 18 and 21 °C. After the acclimation period cells were inoculated into sterile Pinot noir wine and MLF was followed for 25 days to investigate if different acclimation temperatures could influence cell survival and MLF performance. L-malic acid consumption, population survival, and transcriptional behavior were different upon the acclimation temperature. rmlB and hsp20 genes presented a considerable increase in their expression level when strains were acclimated at 18 °C particularly in the psychrotrophic strains UNQOe19 and UNQOe4 isolated from Patagonian Pinot noir wine in comparison with the control strain (ATCC 27310). The increase in rmlB and hsp20 expression could account for the better survival of these strains in Pinot noir in comparison with the control strain. In addition, Patagonian populations acclimated at 18 °C were able to consume a higher percentage of L-malic acid in comparison with cells acclimated at 21 °C. Our results suggest that gene expression analysis of cells acclimated at sub-optimal temperatures could benefit the selection of psychrotrophic strains aimed as starter cultures.

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.

Influence of glycosides on behavior of Oenococcus oeni in wine conditions: growth, substrates and aroma compounds.

Autochthonous Oenococcus oeni strains (MS9, MS20 and MS46) with good malolactic performance and yielding adequate diacetyl levels, were selected to investigate the effect of synthetic and grape glycosides on bacterial growth, substrate utilization and ß-glucosidase (ßGlu), α-arabinofuranosidase (αAra) and α-rhamnopyranosidase (αRha) activities in a wine-like medium containing 6% ethanol, pH 4.0 (WBM). Then, changes in the volatile compounds profile were evaluated at the end of malolactic fermentation (MLF) carried out by the MS46 strain in WBM containing 1 mg L-1 of natural glycoside. All strains grew and efficiently degraded L-malic acid in WBM where ßGlu and αAra activities were found but not αRha. In presence of a synthetic glycoside (eriodictyol 7-O-ß-rutinoside) ßGlu activity was significantly enhanced for two of the cultures tested (MS20 and MS460) while a low αRha activity was induced, presenting MS46 the better performance. Glycosides extracted from fermented grape musts under different conditions allowed maximum growths, L-malic acid utilization rates and glycosidase activities in the MS46 strain. Thus, ßGlu, αAra and αRha activities increased between 30-50 and 3-11% respectively. This indirectly correlated to significant changes in total esters and higher alcohols at the end of MLF, which increased by up to 140 and 30% respectively. Moreover, ethyl and acetate esters formed up to 100-fold than alcohols or esters degraded highlighted the main role of this microorganism in the esters synthesis. Results obtained encourage the potential use of selected indigenous O. oeni strains as a tool to enhance wine complexity through MLF, mainly on highly fruity aroma.

Growth and consumption of L-malic acid in wine-like medium by acclimated and non-acclimated cultures of Patagonian Oenococcus oeni strains.

Five Oenococcus oeni strains, selected from spontaneous malolactic fermentation (MLF) of Patagonic Pinot noir wine, were assessed for their use as MLF starter cultures. After the individual evaluation of tolerance to some stress conditions, usually found in wine (pH, ethanol, SO2, and lysozyme), the behavior of the strains was analyzed in MLO broth with 14 % ethanol and pH 3.5 in order to test for the synergistic effect of high ethanol level and low pH and, finally, in a wine-like medium. Although the five strains were able to grow in MLO broth under low pH and/or high ethanol, they must be acclimated to grow in a wine-like medium. Additionally, glycosidase and tannase activities were evaluated, showing differences among the strains. The potential of the strains to ferment citrate was tested and two of the five strains showed the ability to metabolize this substrate. We did not detect the presence of genes encoding histidine, tyrosine descarboxylase, and putrescine carbamoyltransferase. All the strains tested exhibited good growth capacity and ability to consume L-malic acid in a wine-like medium after cell acclimation, and each of them showed a particular enzyme profile, which might confer different organoleptic properties to the wine.

Antibacterial activity of hen egg white lysozyme modified by heat and enzymatic treatments against oenological lactic acid bacteria and acetic acid bacteria.

The antimicrobial activity of heat-denatured and hydrolyzed hen egg white lysozyme against oenological lactic acid and acetic acid bacteria was investigated. The lysozyme was denatured by heating, and native and heat-denatured lysozymes were hydrolyzed by pepsin. The lytic activity against Micrococcus lysodeikticus of heat-denatured lysozyme decreased with the temperature of the heat treatment, whereas the hydrolyzed lysozyme had no enzymatic activity. Heat-denatured and hydrolyzed lysozyme preparations showed antimicrobial activity against acetic acid bacteria. Lysozyme heated at 90°C exerted potent activity against Acetobacter aceti CIAL-106 and Gluconobacter oxydans CIAL-107 with concentrations required to obtain 50% inhibition of growth (IC50) of 0.089 and 0.013 mg/ml, respectively. This preparation also demonstrated activity against Lactobacillus casei CIAL-52 and Oenococcus oeni CIAL-91 (IC50, 1.37 and 0.45 mg/ml, respectively). The two hydrolysates from native and heat-denatured lysozyme were active against O. oeni CIAL-96 (IC50, 2.77 and 0.3 mg/ml, respectively). The results obtained suggest that thermal and enzymatic treatments increase the antibacterial spectrum of hen egg white lysozyme in relation to oenological microorganisms.

Oenococcus alcoholitolerans sp. nov., a lactic acid bacteria isolated from cachaça and ethanol fermentation processes.

Four strains of lactic acid bacteria isolated from cachaça and alcohol fermentation vats in Brazil were characterised in order to determine their taxonomic position. Phylogenetic analysis revealed that they belong to the genus Oenococcus and should be distinguished from their closest neighbours. The 16S rRNA gene sequence similarity against the type strains of the other two species of the genus was below 94.76 % (Oenococcus kitaharae) and 94.62 % (Oenococcus oeni). The phylogeny based on pheS gene sequences also confirmed the position of the new taxon. DNA-DNA hybridizations based on in silico genome-to-genome comparison, Average Amino Acid Identity, Average Nucleotide Identity and Karlin genomic signature confirmed the novelty of the taxon. Distinctive phenotypic characteristics are the ability to metabolise sucrose but not trehalose. The name Oenococcus alcoholitolerans sp. nov. is proposed for this taxon, with the type strain UFRJ-M7.2.18(T) ( = CBAS474(T) = LMG27599(T)). In addition, we have determined a draft genome sequence of the type strain.

Effect of physicochemical factors on glycerol production by simultaneous cultures of wine micro-organisms using the response surface method.

AIM: To evaluate the effect of temperature, pH and SO2 on growth and glycerol production improvement by Saccharomyces cerevisiae mc2, Kloeckera apiculata mF and Oenococcus oeni X2L using the response surface method (RSM). METHODS AND RESULTS: Multifactorial design of cultures with physicochemical factors variations was performed. The micro-organisms grew in all cultures conditions. Overall, after 6 days yeasts prevailed, especially S. cerevisiae (10(9) CFU ml(-1)), while O. oeni reached 10(7) CFU ml(-1). At initial fixed pH 5·5, metabolic behaviour of cultures showed a temperature-dependent response. Total malate consumption occurred at 26°C, 50 mg l(-1) SO2. Glucose and pentoses utilization was highly modified when varying SO2. Ethanol showed negative interaction with temperature-SO2 relationship. At low SO2, glycerol and acetate production increased when temperature enhanced. Predictive results of RSM indicate that 26°C, 60·24 mg l(-1) SO2 and pH 5·5 were the optimal conditions for glycerol and organic acids synthesis compatible with wine quality. CONCLUSIONS: We propose a predictive condition to improve the performance of mixed cultures for must fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: To optimize the culture conditions to design mixed starters containing autochthonous yeasts and O. oeni strains for winemaking and to obtain products with high glycerol content, low acidity and maintenance of regional characteristics.

Simultaneous and successive inoculations of yeasts and lactic acid bacteria on the fermentation of an unsulfited Tannat grape must.

Interactions between yeasts and lactic acid bacteria are strain specific, and their outcome is expected to change in simultaneous alcoholic--malolactic fermentations from the pattern observed in successive fermentations. One Oenococcus oeni strain Lalvin VP41™ was inoculated with two Saccharomyces cerevisiae strains either simultaneously, three days after the yeast inoculation, or when alcoholic fermentation was close to finish. Early bacterial inoculations with each yeast strain allowed for the growth of the bacterial populations, and the length of malolactic fermentation was reduced to six days. Alcoholic fermentation by Lalvin ICV D80® yeast strain left the highest residual sugar, suggesting a negative effect of the bacterial growth and malolactic activity on its performance. In sequential inoculations the bacterial populations did not show actual growth with either yeast strain. In this strategy, both yeast strains finished the alcoholic fermentations, and malolactic fermentations took longer to finish. Lalvin ICV D80® allowed for higher viability and activity of the bacterial strain than Fermicru UY4® under the three inoculation strategies. This was beneficial for the sequential completion of both fermentations, but negatively affected the completion of alcoholic fermentation by Lalvin ICV D80® in the early bacteria additions. Conversely, Fermicru UY4®, which was rather inhibitory towards the bacteria, favored the timely completion of both fermentations simultaneously. As bacteria in early inoculations with low or no SO2 addition can be expected to multiply and interact with fermenting yeasts, not only are the yeast-bacterium strains combination and time point of the inoculation to be considered, but also the amount of bacteria inoculated.

Glycerol production by Oenococcus oeni during sequential and simultaneous cultures with wine yeast strains.

Growth and fermentation patterns of Saccharomyces cerevisiae, Kloeckera apiculata, and Oenococcus oeni strains cultured in grape juice medium were studied. In pure, sequential and simultaneous cultures, the strains reached the stationary growth phase between 2 and 3 days. Pure and mixed K. apiculata and S. cerevisiae cultures used mainly glucose, producing ethanol, organic acids, and 4.0 and 0.1 mM glycerol, respectively. In sequential cultures, O. oeni achieved about 1 log unit at 3 days using mainly fructose and L-malic acid. Highest sugars consumption was detected in K. apiculata supernatants, lactic acid being the major end-product. 8.0 mM glycerol was found in 6-day culture supernatants. In simultaneous cultures, total sugars and L-malic acid were used at 3 days and 98% of ethanol and glycerol were detected. This study represents the first report of the population dynamics and metabolic behavior of yeasts and O. oeni in sequential and simultaneous cultures and contributes to the selection of indigenous strains to design starter cultures for winemaking, also considering the inclusion of K. apiculata. The sequential inoculation of yeasts and O. oeni would enhance glycerol production, which confers desirable organoleptic characteristics to wines, while organic acids levels would not affect their sensory profile.

Simultaneous and successive inoculations of yeasts and lactic acid bacteria on the fermentation of an unsulfited Tannat grape must

Interactions between yeasts and lactic acid bacteria are strain specific, and their outcome is expected to change in simultaneous alcoholic -malolactic fermentations from the pattern observed in successive fermentations. One Oenococcus oeni strain Lalvin VP41TM was inoculated with two Saccharomyces cerevisiae strains either simultaneously, three days after the yeast inoculation, or when alcoholic fermentation was close to finish. Early bacterial inoculations with each yeast strain allowed for the growth of the bacterial populations, and the length of malolactic fermentation was reduced to six days. Alcoholic fermentation by Lalvin ICV D80® yeast strain left the highest residual sugar, suggesting a negative effect of the bacterial growth and malolactic activity on its performance. In sequential inoculations the bacterial populations did not show actual growth with either yeast strain. In this strategy, both yeast strains finished the alcoholic fermentations, and malolactic fermentations took longer to finish. Lalvin ICV D80® allowed for higher viability and activity of the bacterial strain than Fermicru UY4® under the three inoculation strategies. This was beneficial for the sequential completion of both fermentations, but negatively affected the completion of alcoholic fermentation by Lalvin ICV D80® in the early bacteria additions. Conversely, Fermicru UY4®, which was rather inhibitory towards the bacteria, favored the timely completion of both fermentations simultaneously. As bacteria in early inoculations with low or no SO2 addition can be expected to multiply and interact with fermenting yeasts, not only are the yeast-bacterium strains combination and time point of the inoculation to be considered, but also the amount of bacteria inoculated.

Simultaneous and successive inoculations of yeasts and lactic acid bacteria on the fermentation of an unsulfited Tannat grape must

Interactions between yeasts and lactic acid bacteria are strain specific, and their outcome is expected to change in simultaneous alcoholic -malolactic fermentations from the pattern observed in successive fermentations. One Oenococcus oeni strain Lalvin VP41TM was inoculated with two Saccharomyces cerevisiae strains either simultaneously, three days after the yeast inoculation, or when alcoholic fermentation was close to finish. Early bacterial inoculations with each yeast strain allowed for the growth of the bacterial populations, and the length of malolactic fermentation was reduced to six days. Alcoholic fermentation by Lalvin ICV D80® yeast strain left the highest residual sugar, suggesting a negative effect of the bacterial growth and malolactic activity on its performance. In sequential inoculations the bacterial populations did not show actual growth with either yeast strain. In this strategy, both yeast strains finished the alcoholic fermentations, and malolactic fermentations took longer to finish. Lalvin ICV D80® allowed for higher viability and activity of the bacterial strain than Fermicru UY4® under the three inoculation strategies. This was beneficial for the sequential completion of both fermentations, but negatively affected the completion of alcoholic fermentation by Lalvin ICV D80® in the early bacteria additions. Conversely, Fermicru UY4®, which was rather inhibitory towards the bacteria, favored the timely completion of both fermentations simultaneously. As bacteria in early inoculations with low or no SO2 addition can be expected to multiply and interact with fermenting yeasts, not only are the yeast-bacterium strains combination and time point of the inoculation to be considered, but also the amount of bacteria inoculated.(AU)

A strategy to design efficient fermentation processes for traditional beverages production: prickly pear wine.

This paper describes a methodology to establish an optimal process design for prickly pear wine production that preserves the peculiar and unique traits of traditional products, generating at the same time, technical information for appropriate design of both bioreactor and overall process. The strategy includes alcoholic fermentation optimization by the mixed native culture composed by Pichia fermentans and Saccharomyces cerevisiae, followed by malolactic fermentation optimization by Oenococcus oeni. The optimization criteria were based on multiple output functions: alcohol content, volatile compounds profile, organic acids profile, and compound contents related to color, which were analyzed by spectroscopy-chromatography methods and sensory analysis. The results showed that the mixed culture inoculated into a bioreactor containing prickly pear juice with 20 °Bx of fermentable sugars concentration, processed at a constant temperature of 20 °C for 240 h, leads to a fermented product with 9.93% (v/v) total alcohol content, and significant abundance of volatile compounds, which provide fruity and ethereal aromatic notes, complemented by a lively but not unpleasant acidity. This young wine was further subjected to malolactic fermentation at constant temperature (16 °C) for 192 h, decreasing malic acid, and balancing volatile compounds contents, thus resulting in a product with better aroma and flavor perception, and a velvety feeling of long aftertaste. Repeated assays showed that the process is stable, predictable, controllable, and reproducible. These results were used for process design and spreadsheet construction in order to simulate the process, and properly select and size the equipment required for such process.

Increase in antioxidant and antihypertensive peptides from Argentinean wines by Oenococcus oeni.

Cells from an exponential Oenococcus oeni m1 culture in a grape juice medium were inoculated into a synthetic wine medium (SW) supplemented with a protein and polypeptide fraction (PPF) of high molecular weight (higher than 12,400 Da) obtained from four varietals of Cafayate Argentinean wines. O. oeni maintains viability after 48 h incubation time and enables the increase in extracellular proteolytic activity and the release of low molecular weight peptides by 1.067, 0.397, 0.915 and 0.705 mg N/L in the respective SW supplemented with PPF from Cabernet Sauvignon, Malbec, Tannat and Torrontés wine varietals. After 48 h incubation time, concomitantly with peptide release, an increase in antioxidant and antihypertensive activities was detected in all studied media. The highest increase was detected in the presence of PPF from Cabernet and Tannat wine varietals. Maximum increase in antioxidant activity (366.1 µmol FeSO4/L in the case of ferric reducing antioxidant power and 8.9% in 2,2-diphenyl-1-picrylhydrazyl radical scavenging) was produced by the peptides released from PPF of Cabernet Sauvignon wine. The peptides released from PPF Tannat wine varietal caused the highest increase in antihypertensive activity (56.2% in angiotensin I-converting enzyme inhibitory activity). Oenococcus oeni m1 would provide additional benefits to wine such as an increase in bioactive peptides with multifunctional beneficial activities.

Characterization of wines produced by mixed culture of autochthonous yeasts and Oenococcus oeni from the northwest region of Argentina.

Two autochthonous yeasts from the northwest region of Argentina, Kloeckera apiculata mc1 and Saccharomyces cerevisiae mc2, were used as pure or mixed starter cultures in microvinification trials conducted in Malbec red must. Also, the effect of Oenococcus oeni X(2)L was evaluated. S. cerevisiae mc2 showed adequate growth and fermentative activity in single and composite fermentations, producing standard concentration of ethanol. The amount of esters was higher in fermentations conducted using mixed yeast starters. Independent of the timing of inoculation of O. oeni, this malolactic bacterium completely depleted malic acid. Sensory evaluation indicated that young wines fermented with mixed yeast cultures and sequential inoculation of O. oeni were preferred, achieving the highest scores for positive descriptors and they allowed better control of the sensory quality. Consequently, this study proposes inclusion of autochthonous K. apiculata mc1 as an adjunct culture to S. cerevisiae mc2 during Malbec must fermentation to improve the organoleptic properties of red wines. Furthermore, sequential inoculation of O. oeni X(2)L should be carried out after completion of the alcoholic fermentation to enhance sensory characteristics.

Increase in antioxidant and antihypertensive activity by Oenococcus oeni in a yeast autolysis wine model.

Accelerated autolysis of Saccharomyces cerevisiae mc2 in synthetic wine medium enabled the release of 3.7 mg peptide nitrogen/l, concomitantly with an increase in antioxidant properties (243 micromol FeSO(4)/l in the case of ferric reducing antioxidant power and 0.5% in 2,2-diphenyl-1-picrylhydrazyl radical scavenging) and antihypertensive activity (22% in angiotensin I-converting enzyme inhibitory activity). Sequential inoculation of a proteolytic Oenococcus oeni strain in the synthetic medium after yeast autolysis produced an increase in peptide nitrogen concentration of 1.5 mg/l after 48 h of growth. After this incubation time an improvement in antihypertensive and antioxidant activities was detected. Oenococcus oeni X(2)L could give additional value to wine because of the bioactive peptides with multifunctional beneficial activity released as consequence of its proteolytic activity.

Evidence of distinct populations and specific subpopulations within the species Oenococcus oeni.

Among the lactic acid bacteria (LAB) present in the oenological microbial ecosystem, Oenococcus oeni, an acidophilic lactic acid bacterium, is essential during winemaking. It outclasses all other bacterial species during malolactic fermentation (MLF). Oenological performances, such as malic acid degradation rate and sensorial impact, vary significantly according to the strain. The genetic diversity of the O. oeni species was evaluated using a multilocus sequence typing (MLST) scheme. Seven housekeeping genes were sequenced for a collection of 258 strains that had been isolated all over the world (particularly Burgundy, Champagne, and Aquitaine, France, Chile, South Africa, and Italy) and in several wine types (red wines, white wines, and champagne) and cider. The allelic diversity was high, with an average of 20.7 alleles per locus, many of them being rare alleles. The collection comprised 127 sequence types, suggesting an important genotypic diversity. The neighbor-joining phylogenetic tree constructed from the concatenated sequence of the seven housekeeping genes showed two major phylogenetic groups, named A and B. One unique strain isolated from cider composed a third group, rooting the phylogenetic tree. However, all other strains isolated from cider were in group B. Eight phylogenetic subgroups were statistically differentiated and could be delineated by the analysis of only 32 mutations instead of the 600 mutations observed in the concatenated sequence of the seven housekeeping genes. Interestingly, in group A, several phylogenetic subgroups were composed mostly of strains coming from a precise geographic origin. Three subgroups were identified, composed of strains from Chile, South Africa, and eastern France.

Interaction between Oenococcus oeni and Lactobacillus hilgardii isolated from wine. Modification of available nitrogen and biogenic amine production.

During the mixed culture of Lactobacillus hilgardii 5w, a common spoilage wine bacteria and Oenococcus oeni X(2)L, an amensalistic growth response of the malolactic bacteria was produced due to a competition for nitrogenous nutrients, mainly peptides. Arginine was fully consumed and peptide concentration diminished 60% with respect to both pure cultures at the end of exponential growth. Histamine release increased 34% with respect to L. hilgardii single culture. Under the poor nutritional conditions present during winemaking, L. hilgardii could increase histamine production and adversely affect malolactic fermentation conducted by O. oeni and hence the quality of the final product.