Cognac distillation wines host specific Oenococcus oeni strains representing a novel genetic lineage.

Investigating Oenococcus oeni citrate locus expression: Role of citR and impact on acid tolerance.

Wine fermentation and flavor development are mediated by complex microbial interactions. Previous studies have shown that coinoculation with Oenococcus oeni can alter acid composition and aroma, yet the underlying transcriptional responses of S. cerevisiae and the metabolic strategies adopted by both species remain poorly understood. In this study, we performed mixed fermentation of S. cerevisiae and O. oeni, dividing the process into 6 representative stages for stage-specific analysis. Comparative evaluation with pure S. cerevisiae fermentation revealed that the presence of O. oeni reshaped both the acid profile and aromatic complexity of wine, while also influencing the growth kinetics of S. cerevisiae. Time-resolved transcriptomics demonstrated that O. oeni markedly altered the transcriptional dynamics of S. cerevisiae through changing the magnitude of transcriptional level while incurring opposite regulatory patterns in a small set of genes involved in stress responses and sulfur metabolism. Through the integration of a community genome-scale metabolic model with stage-resolved transcriptomic constraints, we resolved dynamic flux distributions of the mixed fermentation (MF) system and further revealed the metabolic impact of O. oeni on S. cerevisiae and MF. O. oeni exhibited low competitiveness for glucose but actively utilized fructose to generate energy through the phosphoketolase pathway. Also, it showed reversible reaction fluxes of two reactions involved in acetate formation (ACKr/PTAr), thereby impacting acetate levels. In addition, metabolic interactions including amino acid (e.g., arginine, serine, threonine) and mannitol exchange were identified, reflecting both cooperative and competitive features of mixed fermentation. Collectively, this work provides the first comprehensive depiction of the transcriptional and metabolic interplay between S. cerevisiae and O. oeni during mixed fermentation, offering mechanistic insights into microbial cooperation and strategies for the rational design of wine fermentation and flavor quality.

External stress conditions can induce disturbances in the internal environment of cells, sufficient for protein misfolding or membrane disorganization. To cope with this phenomenon, cells have developed several strategies, including the production of heat stress proteins (HSPs). These include small heat shock proteins (sHSPs). sHSPs share a characteristic three-dimensional architecture, with a central α-crystalline domain (ACD) flanked by two terminal domains. This structural organization enables them to form a variety of oligomeric complexes, giving these proteins their function. As large oligomers, all sHSPs act as chaperones. Conversely, some of them adopt a lipochaperone role when present in dimeric form. While the role of the ACD domain in oligomerization and the mechanism of action of sHSPs has been studied extensively, the functions of the terminal domains, in particular the N-terminal domain, remain poorly explored. In this context, the role of the N-terminal domain of sHSP Lo18, produced by the bacteria Oenococcus oeni, was investigated. To assess its importance, a truncated protein, depleted of the first 27 amino acids, was compared with the wild-type protein. Analyses focused on the oligomeric structure, the ability to prevent protein aggregation, and the ability to maintain membrane fluidity. In parallel, the involvement of the N-terminal domain in the interaction between Lo18 and membranes was examined by RP-HPLC. The results reveal that Lo18's N-terminal domain plays a decisive role, both in the formation and stabilization of oligomeric structures, and in interaction with protein and lipid substrates, essential parameters in chaperone and lipochaperone activity.

Effects of exogenous proline on malolactic fermentation by Oenococcus oeni and on volatiles, phenols, and antioxidant properties of Cabernet Sauvignon wine.

ABSTRACTThis study aimed to assess the potential of Lentilactobacillus hilgardii as a novel candidate for malolactic fermentation (MLF) in winemaking, through comparative genomics and experimental validation, in direct comparison with Oenococcus oeni. We performed a pangenome analysis on 16 L. hilgardii and 7 O. oeni strains to explore their genetic diversity, focusing on wine‐related traits. Functional predictions were generated using genome‐scale metabolic models (ModelSEED/KBase), including in silico co‐inoculation with Saccharomyces cerevisiae EC1118 and post‐alcoholic fermentation simulations. The reference strains L. hilgardii DSM 20176 and O. oeni DSM 20252 were experimentally tested for MLF performance in a synthetic wine‐like medium at 25°C and 10°C. Core‐genome comparison revealed that 67.9% of the malolactic enzyme sequence is conserved between the two species, with comparable docking affinity to L‐malic acid. L. hilgardii harboured unique enzymes with potential oenological interest (phenolic acid decarboxylase, mannitol dehydrogenase, glucosidase) and distinctive stress‐related proteins (YaaA, HrcA, ASP23), suggesting improved tolerance to oxidative, temperature, and alkaline stresses. Notably, L. hilgardii showed genomic potential to degrade putrescine, arginine, and ornithine, precursors of ethyl carbamate. Experimentally, L. hilgardii reduced L‐malic acid from 2.5 g/L to < 0.1 g/L within 12 days at 10°C, while O. oeni showed no MLF activity at this temperature. At 25°C, both strains completed MLF within 6–7 days. L. hilgardii also consumed > 80% of residual fructose at 10°C, whereas O. oeni showed minimal utilisation. Our results demonstrate that L. hilgardii combines a favourable genomic repertoire for wine adaptation with superior MLF performance at low temperature, suggesting its potential as an alternative to O. oeni in cool‐climate winemaking. This work provides the first genome‐scale comparative and functional evaluation of L. hilgardii in the winemaking context, highlighting its technological promise to improve fermentation reliability, reduce spoilage risk, and expand the biodiversity of malolactic starters.

Different composition of mannoprotein extracts and their beneficial effects on Oenococcus oeni and wine malolactic fermentation.

Effects of the addition of fumaric acid on the spontaneous alcoholic fermentation of white grape must.

This study deals with the influence of various oenological preparations on malolactic fermentation. The influence of chitosan, fumaric acid, a tannin-based (Estaan) oenological preparation and medium-chain fatty acids (MCFAs) was investigated, along with a new preparation based on a combination of selected hydroxycinnamic acids and MCFAs. Growth curves were obtained using Oenococcus oeni, Lactobacillus brevis and Lactobacillus plantarum bacteria. Experimental work was also carried out on microsamples of wine, where individual inhibitors were added to wine inoculated with O. oeni culture and an HPLC analysis was performed to measure malic acid levels. Fumaric acid had the strongest inhibitory effect on L. plantarum at a dose of 2.5 g∙L−1, while chitosan had the strongest effect on O. oeni at a dose of 2.5 mg∙L−1. P-coumaric acid in combination with MCFAs (0.4 g∙L−1 of p-coumaric acid + 10 mg∙L−1 MCFAs) and Mix (0.4 g∙L−1 of p-coumaric acid + 0.4 g∙L−1 of ferulic acid + 10 mg∙L−1 MCFA) had the strongest inhibitory effects on O. oeni and L. brevis. Finally, MCFAs had the strongest inhibitory effect on L. brevis at a dose of 1000 mg∙L−1, and Estaan had the strongest effect on L. plantarum at a dose of 25 g∙L−1.

The increasing prevalence of wildfires presents a growing risk to wine production through the development of smoke-taint, a sensory defect in wine caused by volatile phenols absorbed by grapevines during smoke exposure. In grapes and wine, these volatile phenols are often present in glycosylated forms that can be hydrolyzed during fermentation, releasing undesirable smoky aromas. This study investigated the glycosidase activity of diverse Saccharomyces cerevisiae and Oenococcus oeni strains to evaluate their roles in modulating smoke-taint-associated glycosides during wine fermentation. Mini-scale alcoholic and malolactic fermentations were conducted in synthetic media enriched with flavor extracts from smoke-exposed grapes using reverse osmosis. LC–MS profiling revealed strain-dependent hydrolysis of glycosides, linked to smoke-taint. Notably, S. cerevisiae strains UCD514 and UCD525, and O. oeni strain UCD199, showed the highest glycosidase activity. Principal component analysis further confirmed that individual microbial strains had distinct metabolic impacts on glycoside profiles. This study highlights a wide range of glycosides that can be hydrolyzed by wine yeast and bacteria. These findings demonstrate the dual potential of microbial glycosidase activity to mitigate smoke-taint while enhancing wine aroma. In addition, the results help distinguish smoke-taint-associated glycosides that are resistant to microbial hydrolysis from those that are readily cleaved, enabling targeted removal of released aroma compounds through downstream filtration approaches.

Malolactic fermentation (MLF) is a bioprocess driven by lactic acid bacteria (LAB), which is desired in red and highly acidic white wines. Among all LAB, Oenococcus oeni is the main species in wine, followed by Lactiplantibacillus plantarum. The harsh conditions found in wine—not only due to the low nutrient concentration but also the presence of antimicrobial compounds such as ethanol, high acidity, SO2, and polyphenols—can compromise MLF performance. In recent years, the use of certain non-Saccharomyces yeasts, such as Torulaspora delbrueckii or Metschnikowia pulcherrima, as starter cultures for alcoholic fermentation, has emerged as a promising strategy to improve MLF. In this study, we evaluated the effect of four different fractions from a T. delbrueckii strain on MLF performance. First, the positive impact of this strain as a starter culture on O. oeni growth was confirmed; then, yeast-derived compounds were tested in different wines. Two fractions showed the most promising results in reducing MLF duration: the inactivated yeast fraction and the autolysate fraction. Those enhanced bacterial viability and promoted mannoprotein consumption. These findings highlight the potential of T. delbrueckii-derived compounds as enological tools to support MLF under restrictive wine conditions.

From spontaneous apple fermentations to Calvados: Insights into its aromatic complexity.

Interaction effect among key volatile compounds on the aroma profile of chardonnay wine co-inoculated with Saccharomyces cerevisiae and Oenococcus oeni.

Updated insights into esterified Flavan-3-ols in grapevine and wine: Origin, transformation, and taste response of epicatechin-3-O-vanillate and-3-O-gallate.

Controlling the speed of malolactic fermentation in red wine is an important challenge to produce certain short-rotation wines, like primeur style wines, for entry-level market segments. This study shows the possibility of inducing the adhesion and biofilm formation of Oenococcus oeni Vitilactic F© and Saccharomyces cerevisiae 522D©, in a low-nutrient medium, on Nylon© carriers in a continuous flow 250 mL bioreactor. The biofilm formation medium was then replaced by fermentation media (grape must for co-alcoholic and malolactic fermentations with O. oeni and S. cerevisiae biofilms) or wine (with O. oeni biofilms only) and the progress of malolactic fermentation was monitored: over periods of three to four weeks under a continuous regime, stable conversion speeds for L-malic acid of 0.53 g/L/24 h (malolactic fermentation in wine medium) and of 2.04 g/L/24 h (co-fermentations fermenting grape must medium) are reached. O. oeni biofilms on Nylon© carriers were also transferred in wine for four successive batch fermentations: in these conditions, L-malic acid conversion speed was 0.35 g/L/24 h. These biofilm implementation systems could be the first step towards perfectly controlled industrial malolactic fermentation processes.

Oenococcus oeni is the predominant species of lactic acid bacteria in wine, where it carries out malolactic fermentation (MLF), which helps to ensure and preserve the quality of the wine. Today, existing combinations of grape varieties, soil composition, fluctuating climatic parameters, and specific technical processes implemented by wineries lead to incredibly varied wine compositions that pose challenges for spontaneous MLF. Commercial starter cultures have been developed for use as inoculants. However, their effectiveness in ensuring consistent and reliable MLF is also limited in modern wines. The selection process must therefore adapt to these new challenges, which means expanding current portfolios by selecting more robust bacteria from wines that are more varied in terms of grape varieties and chemical constraints. We have assembled a set of 21 wines produced in Europe from different grape varieties, with varying and, in some cases, extreme ethanol contents, total polyphenolic indices, and pH levels. The isolation and MLVA typing of 385 dominant colonies were combined with whole-genome sequencing of 48 representative strains, and we observed several strains with unique accessory genomic content. Different selective pressures led to the formation of groups of genetically related individuals, particularly in white and rosé wines with moderate ethanol content. However, cohabiting strains with contrasting genetic profiles were also observed in some red wines. Our data highlight the complexity of the factors involved in population heterogeneity and raise the possibility that this phenomenon may increase fitness through diversification of strategies or division of labor in specific production environments.

Oenococcus oeni is the predominant lactic acid bacteria species in wine, where it performs the malolactic fermentation, which helps to secure and preserve wine quality. Here, we describe the morphological, biological, and genomic characterization of siphophage Krappator X27, a strictly lytic phage that was previously isolated from Merlot wines. Several aspects of the life cycle of the phage were investigated using the sensitive strain IOEBS277 under optimal growth conditions. X27 has a large burst size (149 Plaque Forming unit [PFU] per infected cell) and targets industrially relevant strains of O. oeni used for production of wines. X27 also shows lytic activity against its host in red and white grape juice media. The phage genome consists of 41,633 nucleotides. Structure prediction of the viral adhesion devices reveals elongated multi-domain machinery containing carbohydrate-binding modules likely involved in host recognition and binding. Genome-based phylogeny revealed that X27 has less than 30% intergenomic similarities with its most similar phages and represents a new species in a yet unassigned genus, for which we propose the name « Krappavirus ». Aiming to expand the available collection of genomes in this genus, we sequenced two independently isolated phage homologs from Merlot wines. Modular protein evolution may play a role in the diversification of this specific phage lineage, and naturally occurring domain combinations were found in three crucial proteins of known function: the small terminase subunit, the endolysin, and the replisome organizer.IMPORTANCEOenococcus oeni is commonly used for wine and cider production. Characterizing strictly lytic oenophages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur along the fermentation process.

Abstract The experiment dealt with the different procedures for malic acid degradation in red wine using the malolactic fermentation process, its influence on qualitative parameters and the formation of undesirable acetic acid in wine in the vintages of 2020 and 2021. We used the ‘Cabernet Sauvignon’ variety, active dry Saccharomyces cerevisiae yeast and pure culture of the Oenococcus oeni malolactic bacteria. We found that malolactic fermentation by noble, selected cultures can take place simultaneously when fermentable carbohydrates are present without significantly increasing acetic acid levels. All inoculation variations reached final acetic acid values of 0.807–0.820 g/L in 2020 and 0.753–0.780 g/L in 2021. The control variations without inoculation had the highest acetic acid values, which were 0.897 g/L (2020) and 0.803 g/L (2021) at the end of fermentation. In 2021, control variant proved inadequate, leaving 0.53 g/L of malic acid in the wine. The duration of fermentation was shortened by simultaneous co-inoculation of yeast and malolactic bacteria without affecting unwanted acetic acid. During this process, it is, in addition to monitoring acetic acid, extremely important to control the pH value, which must be lower than 3.5. The alternative of using malolactic fermentation as a technology involving the co-inoculation of yeast and malolactic bacteria starter culture without a negative impact on the wine’s quality proved to be suitable and effective.

Proteomic insight into the beneficial effect of mannoproteins on Oenococcus oeni in wine malolactic fermentation.

Koshu wine, produced from the indigenous Japanese grape Vitis vinifera L. cv. Koshu exhibits a lower pH than other white wines, hindering malolactic fermentation (MLF) by lactic acid bacteria (LAB). Here, we aimed to isolate LAB strains capable of performing MLF under these challenging conditions to improve wine quality. Sixty-four Oenococcus oeni and one Lactobacillus hilgardii strain were isolated from Koshu grapes and wines that had undergone spontaneous MLF. MLF activity was assessed under varying pH, SO2, and ethanol conditions in modified basal medium (BM) and Koshu model wine media. Expression of stress-related genes was analyzed using real-time PCR. Carbon source utilization was evaluated via API 50CH assays. All isolates degraded malic acid and produced lactic acid at 15 °C and pH 3.2 in BM without reducing sugars. Seven strains, all identified as O. oeni, demonstrated MLF activity at pH 3.0 in modified BM lacking added reducing sugars or tomato juice. Six wine-derived strains tolerated up to 12% ethanol, whereas the grape-derived strain was inhibited at 10%. In a synthetic Koshu wine model (13% ethanol, pH 3.0), wine-derived isolates exhibited higher MLF activity than commercial starter strains. In high-performing strains, mleA was upregulated, and most isolates preferred fructose, arabinose, and ribose over glucose. These findings suggest that indigenous O. oeni strains from Koshu wine possess unique stress tolerance and metabolic traits, making them promising candidates for region-specific MLF starter cultures that could enhance Koshu wine quality and terroir expression.