A mutant GH3 family ß-glucosidase from Oenococcus oeni exhibits superior adaptation to wine stresses and potential for improving wine aroma and phenolic profiles.

In this study, we conducted a comprehensive investigation into a GH3 family ß-glucosidase (BGL) from the wild-type strain of Oenococcus oeni and its mutated counterpart from the acid-tolerant mutant strain. Our analysis revealed the mutant BGL's remarkable capacity to adapt to wine-related stress conditions, including heightened tolerance to low pH, elevated ethanol concentrations, and metal ions. Additionally, the mutant BGL exhibited superior hydrolytic activity towards various substrates. Through de novo modeling, we identified specific amino acid mutations responsible for its resilience to low pH and high ethanol environments. In simulated wine conditions, the mutant BGL outperformed both wild-type and commercial BGLs, efficiently releasing terpene and phenolic aglycones from glycosides in wine grapes. These findings not only expand our understanding of O. oeni BGLs but also highlight their potential in enhancing wine production. The mutant BGL's enhanced adaptation to wine stress conditions opens promising avenue for improving wine quality and flavor.

Succinic acid production by wine yeasts and the influence of GABA and glutamic acid.

As a consequence of alcoholic fermentation (AF) in wine, several compounds are released by yeasts, and some of them are linked to the general quality and mouthfeel perceptions in wine. However, others, such as succinic acid, act as inhibitors, mainly of malolactic fermentation. Succinic acid is produced by non-Saccharomyces and Saccharomyces yeasts during the initial stages of AF, and the presence of some amino acids such as γ-aminobutyric acid (GABA) and glutamic acid can increase the concentration of succinic acid. However, the influence of these amino acids on succinic acid production has been studied very little to date. In this work, we studied the production of succinic acid by different strains of non-Saccharomyces and Saccharomyces yeasts during AF in synthetic must, and the influence of the addition of GABA or glutamic acid or a combination of both. The results showed that succinic acid can be produced by non-Saccharomyces yeasts with values in the range of 0.2-0.4 g/L. Moreover, the addition of GABA or glutamic acid can increase the concentration of succinic acid produced by some strains to almost 100 mg/L more than the control, while other strains produce less. Consequently, higher succinic acid production by non-Saccharomyces yeast in coinoculated fermentations with S. cerevisiae strains could represent a risk of inhibiting Oenococcus oeni and therefore the MLF.

Protein content of the Oenococcus oeni extracellular vesicles-enriched fraction.

Malolactic fermentation is essential for the quality of red wines and some other wine styles. Spontaneous malolactic fermentation is often driven by Oenococcus oeni, and commercial starters for this purpose are also often of this species. The increasing number of microbial species and inoculation strategies in winemaking has prompted a growing interest in microbial interactions during wine fermentation. Among other interaction mechanisms, extracellular vesicles have been hypothesized to play a role in this context. Extracellular vesicles have already been described and analysed for several wine yeast species. In this work, the production of extracellular vesicles by O. oeni is reported for the first time. The protein content of these extracellular vesicles is also characterised. It shows differences and similarities with the recently described protein content of Lactiplantibacillus plantarum, a bacterial species also capable of performing malolactic fermentation of wine (and used sometimes as an alternative starter). This work further contributes to the development of the field of extracellular vesicles in food biotechnology.

Comparative functional analysis of malate metabolism genes in Oenococcus oeni and Lactiplantibacillus plantarum at low pH and their roles in acid stress response.

Oenococcus oeni and Lactiplantibacillus plantarum are major wine-associated lactic acid bacteria that positively influence wine by carrying out malolactic fermentation. O. oeni is the most widely used commercial starter in winemaking because of its fast and efficient malate metabolism capacity under harsh wine conditions. To date, very little is known about the specific molecular mechanism underlying the differences in malate metabolism between O. oeni and L. plantarum under harsh wine conditions. Therefore, in this study, the functions of genes encoding malic enzyme (ME) and malolactic enzyme (MLE) under acid stress in O. oeni and L. plantarum, previously described to have the ability to direct malate metabolism, were comparatively verified through genetic manipulation in L. plantarum. Results showed that the MLE was the only enzyme responsible for direct malate metabolism under acid stress in O. oeni and L. plantarum. In addition, the MLEs in O. oeni and L. plantarum were positively related to acid tolerance by metabolizing malate and increasing the medium pH. Furthermore, the MLE in O. oeni exhibited significantly higher malate metabolism activity than that in L. plantarum under acid stress.

Biological management of acidity in wine industry: A review.

Climate change is generating several problems in wine technology. One of the main ones is lack of acidity and difficulties performing malolactic fermentation to stabilize wines before bottling. Among the different available acidity management technologies, such as direct acid addition, ion exchange resins, electro-membrane treatments, or vineyard management, the microbiological option is reliable and deeply studied. The main approach is the increase in malic acid content because of the metabolism of specific Saccharomyces strains and to increase lactic acid because of the metabolism of Lachancea genus. Other non-Saccharomyces yeasts, such as Starmerella bacillaris or Candida stellata can also acidify significantly because of the production of pyruvic or succinic acid. Wine industry needs the removal of malic acid in most red wines before bottling to achieve wine stability. Oenococus oeni performs the malolactic fermentation of red wines on most conditions because of the metabolization of malic acid into lactic acid. However, modern oenology challenges such as high ethanol concentrations, high pH or low levels of malic acid have made researchers to look for other options to reduce potential risks of deviation. Other wine-related microorganisms able to de-acidify malic acid have appeared as interesting alternatives for specific difficult scenarios. Lactiplantibacillus plantarum and Schizosaccharomyces genus make up nowadays the main studied alternatives.

The production of preconditioned freeze-dried Oenococcus oeni primes its metabolism to withstand environmental stresses encountered upon inoculation into wine.

Oenococcus oeni is the most resistant lactic acid bacteria species to the environmental stresses encountered in wine, particularly the acidity, presence of ethanol and phenolic compounds. Indigenous strains develop spontaneously following the yeast-driven alcoholic fermentation and may perform the malolactic fermentation whereby improving taste, aroma, and the microbial stability of wine. However, spontaneous fermentation is sometimes delayed, prolonged or incomplete. In order to better control its timing and quality, O. oeni strains are selected and developed to be used as malolactic starters. They are prepared under proprietary manufacturing processes to survive direct inoculation and are predominantly provided as freeze-dried preparations. In this study, we have investigated the physiological and molecular alterations occurring in O. oeni cells prepared by an industrial process that consists of preconditioning protocols and freeze-drying, and compared them to the same strain grown in a grape juice medium. We found that compared to cultured cells, the industrial production process improved survival under extreme conditions, i. e. at low pH or high tannin concentrations. In contrast, cultured cells resumed active growth more quickly and strongly than freeze-dried preparations in standard pH wines. A proteomic analysis showed that during the industrial production most non-essential metabolic processes are shut down and components of the general and the stringent stress response are upregulated. The presence of major components of the stress response facilitates protein homeostasis and physiological changes that further ensure the integrity of cells.

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.

Analysis of Transcriptomic Response to SO2 by Oenococcus oeni Growing in Continuous Culture.

To successfully complete malolactic fermentation (MLF), Oenococcus oeni must overcome wine stress conditions of low pH, high ethanol, and the presence of SO2. Failure to complete MLF may result in detrimental effects to the quality and stability of the resulting wines. Research efforts to date have focused on elucidating the mechanisms and genetic features that confer the ability to withstand low pH and high ethanol concentrations on O. oeni; however, the responses to SO2 stress are less well defined. This study focused on characterizing the transcriptional response of O. oeni to SO2 challenge during cultivation in a continuous system at wine-like pH (3.5). This experimental design allowed the precise discrimination of transcriptional changes linked to SO2 stress from responses associated with growth stage and cultivation parameters. Differential gene expression analysis revealed major transcriptional changes following SO2 exposure and suggested that this compound primarily interacts with intracellular proteins, DNA, and the cell envelope of O. oeni. The molecular chaperone hsp20, which has a demonstrated function in the heat, ethanol, and acid stress response, was highly upregulated, confirming its additional role in the response of this species to SO2 stress. This work also reports the first nanopore-based complete genome assemblies for O. oeni. IMPORTANCE Malolactic fermentation is an indispensable step in the elaboration of most wines and is generally performed by Oenococcus oeni, a Gram-positive heterofermentative lactic acid bacterium species. While O. oeni is tolerant to many of the wine stresses, including low pH and high ethanol concentrations, it has high sensitivity to SO2, an antiseptic and antioxidant compound regularly used in winemaking. Understanding the physiological changes induced in O. oeni by SO2 stress is essential for the development of more robust starter cultures and methods for their use. This study describes the main transcriptional changes induced by SO2 stress in the wine bacterium O. oeni and provides foundational understanding on how this compound interacts with the cellular components and the induced protective mechanisms of this species.

Wine aroma profile modification by Oenococcus oeni strains from Rioja Alavesa region: selection of potential malolactic starters.

Previously six selected Oenococcus oeni strains (P2A, P3A, P3G, P5A, P5C and P7B) have been submitted to further characterization in order to clarify their potential as malolactic starters. Laboratory scale vinifications gave an insight of the most vigorous strains: both P2A and P3A strains were able to conclude malolactic fermentation (MLF) in less than 15 days. The remaining strains showed good viability and were able to successfully finish MLF in the established analysis time, except for the strain P5A, which viability was totally lost after inoculation. Also spontaneous fermentation was not initiated. None of the strains was biogenic amine producer; however, P5C strain significantly increased the concentration of volatile phenol-precursor hydroxycinnamic acids after MLF. Regarding the evolution of wine aromatic compounds, main changes were detected for both ethyl and acetate esters after MLF; however, key aromatic compounds including alcohols, terpenes or acids were also found to significantly increase. Principal component analysis classified the strains in two distinct groups, each one correlated with different key volatile compounds. P2A, P3A, P3G and P5C strains were mainly linked to esters, while P7B and the commercial strain Viniflora OENOS showed higher score for diverse compounds as hexanoic acid, ß-damascenone, linalool or 2-phenylethanol. These results confirmed the specific impact of each strain on wine aroma profile, which could lead to the production of wines with individual characteristics, in which the reliability and safety of MLF is also ensured.

Exploring the catalytic mechanism of a novel ß-glucosidase BGL0224 from Oenococcus oeni SD-2a: Kinetics, spectroscopic and molecular simulation.

The ß-glucosidase derived from microorganisms has attracted worldwide interest for their industrial applications, but studies on ß-glucosidases from Oenococcus oeni are rare. In this paper, catalytic mechanism of a novel ß-glucosidase BGL0224 of Oenococcus oeni SD-2a was explored for the first time by kinetic parameters determination, fluorescence spectroscopy and quenching mechanism analysis, molecular dynamics simulation. The results indicated that BGL0224 had universal catalytic effect on different types of glycoside substrates, but the catalytic efficiencies were different. Fluorescence quenching analysis results suggested that the quenching processes between BGL0224 and seven kinds of substrates were predominated by the static quenching mechanism. A reasonable three-dimensional model of BGL0224 was obtained using the crystal structure of E.coli BglA as a template. The analysis results of molecular simulation (RMSD, Rg, RMSF and hydrogen bonding) showed that the composite system 'BGL0224-pNPG' was very stable after 40 ns. The catalytic process of BGL0224 acting on 'p-Nitrophenyl ß-d-glucopyranoside' conformed to the double displacement mechanism. Two glutamic acid residues 'Glu178 and Glu377' played a vital role in the whole catalytic process. Overall, this study gave specific insights on the catalytic mechanism of BGL0224, which was of great significance for developing its potential applications in food industry.

Simulated lees of different yeast species modify the performance of malolactic fermentation by Oenococcus oeni in wine-like medium.

The use of non-Saccharomyces yeast together with S. cerevisiae in winemaking is a current trend. Apart from the organoleptic modulation of the wine, the composition of the resulting yeast lees is different and may thus impact malolactic fermentation (MLF). Yeasts of Saccharomyces cerevisiae, Torulaspora delbrueckii and Metschnikowia pulcherrima were inactivated and added to a synthetic wine. Three different strains of Oenococcus oeni were inoculated and MLF was monitored. Non-Saccharomyces lees, especially from some strains of T. delbrueckii, showed higher compatibility with some O. oeni strains, with a shorter MLF and a maintained bacterial cell viability. The supplementation of lees increased nitrogen compounds available by O. oeni. A lower mannoprotein consumption was related with longer MLF. Amino acid assimilation by O. oeni was strain specific. There may be many other compounds regulating these yeast lees-O. oeni interactions apart from the well-known mannoproteins and amino acids. This is the first study of MLF with different O. oeni strains in the presence of S. cerevisiae and non-Saccharomyces yeast lees to report a strain-specific interaction between them.

Combinatorial analysis of population dynamics, metabolite levels and malolactic fermentation in Saccharomyces cerevisiae/ Lachancea thermotolerans mixed fermentations.

The outcome of co- or sequential inoculation of Lachancea thermotolerans in winemaking remains unpredictable due to a lack of integrated data regarding the impact of grape juice composition on L. thermotolerans fermentation behaviour. Here, we investigate the impact of nitrogen composition on fermentation characteristics and aroma compound production in grape juice sequentially inoculated with commercial L. thermotolerans and S. cerevisiae strains. Subsequently, all treatments were subjected to malolactic fermentation (MLF) using two commercial strains of Oenococcus oeni. Addition of amino acids led to faster growth for S. cerevisiae fermentations, compared to the nitrogen-equivalent addition of diammonium phosphate (DAP). L. thermotolerans persistence in the mixed fermentations was significantly higher following DAP addition, with higher glycerol and lactic acid production. Interestingly, the lower total Nitrogen content in DAP-treated musts compared to other treatments did not alter the subsequent growth of S. cerevisiae. MLF was more similar between musts fermented with L. thermotolerans, regardless of nutrient regime, whereas significant differences in MLF completion times were observed for different nitrogen treatments in S. cerevisiae fermentations. Collectively, the data present an integrated view of the impact of nitrogen treatment on multispecies co-inoculation (growth kinetics and aromatic outcomes) and the downstream impact on MLF.

Impact of changes in wine composition produced by non-Saccharomyces on malolactic fermentation.

Non-Saccharomyces yeasts have increasingly been used in vinification recently. This is particularly true of Torulaspora delbrueckii and Metschnikowia pulcherrima, which are inoculated before S. cerevisiae, to complete a sequential alcoholic fermentation. This paper aims to study the effects of these two non-Saccharomyces yeasts on malolactic fermentation (MLF) carried out by two strains of Oenococcus oeni, under cellar conditions. Oenological parameters, and volatile and phenolic compounds were analysed in wines. The wines were tasted, and the microorganisms identified. In general, non-Saccharomyces created more MLF friendly conditions, largely because of lower concentrations of SO2 and medium chain fatty acids. The most favourable results were observed in wines inoculated with T. delbrueckii, that seemed to promote the development of O. oeni and improve MLF performance.

Dynamics of changes in organic acids, sugars and phenolic compounds and antioxidant activity of sea buckthorn and sea buckthorn-apple juices during malolactic fermentation.

Sea buckthorn (Hippophaë rhamnoides L.) berries have high biological value as a rich source of phenolic compounds, fatty acids and vitamins A, C, E. Due to the high organic acid content and sour taste, the fruits are rarely used in juice production. Therefore, the study aimed to determine the metabolic activity of Lactobacillus plantarum, Lactobacillus plantarum subsp. argentoratensis and Oenococcus oeni strains along with the dynamics of changes in organic acids, sugars, phenolic compounds, and antioxidant activity during 72-h fermentation of 100% sea buckthorn and mixed with apple (1:1) juices. The strongest malolactic conversion was in mixed juices (to 75.0%). The most efficient strains were L. plantarum DSM 10492, 20174 and 6872. L. plantarum strains caused an increase in flavonols and antioxidant activity of sea buckthorn-apple juices. The results can be used to select conditions and strains in industrial-scale fermentation, to produce novel sea buckthorn products and increase their consumption.

Effect of Saccharomyces cerevisiae, Torulaspora delbrueckii and malolactic fermentation on fermentation kinetics and sensory property of black raspberry wines.

Alcoholic fermentation (AF) and malolactic fermentation (MLF) both have significant influence on the production of black raspberry wine. In this study, three microbes associated with AF and MLF including S. cerevisiae, T. delbrueckii and O. oeni were used to investigate their combined effect on basic compositional, volatile and sensory property of black raspberry wine, and four fermentation trials including single S. cerevisiae inoculation plus spontaneous MLF (BSU) and controlled MLF with O. oeni (BSO), sequential culture of T. delbrueckii and S. cerevisiae plus spontaneous MLF (BTSU) and controlled MLF (BTSO) were tested and compared. Fermentation results showed MLF in BSU, BSO and BTSO were successful, with respective period of 40, 25 and 23 days, whereas a stuck MLF occurred in BTSU. Volatile compounds were determined by HS-GC-IMS method, with a total of 45 aromas identified. BTSO was distinguished by a significant higher signal intensity of many fruity esters and a lower production of several alcohols and terpenes, which was in agreement with its perception result of strong 'fruity' and slight note of 'solvent' and 'herbaceous' during quantitative descriptive analysis. On the contrary, BSU was found to reinforce the synthesis of most detected volatiles, resulting in the enhancement of both beneficial and off-flavour compounds, therefore scoring lower in the 'global aroma' descriptor. Principal component analysis showed BSU and BSO were similar in the volatile composition, whereas BTSO was quite different. Overall, BTSO had greater potential to be used in the production of black raspberry wine.

Evaluating the influence of simultaneous inoculation of SiO2-alginate encapsulated bacteria and yeasts on volatiles, amino acids, biogenic amines and sensory profile of red wine with lysozyme addition.

The influence of the timing of inoculation (sequential and simultaneous alcoholic fermentation (AF)/malolactic fermentation (MLF)) on the chemical and sensory properties of red wines was studied. The impact of the encapsulation of Oenococcus oeni into SiO2-alginate hydrogel (Si-ALG) and the addition of lysozyme in wines inoculated with encapsulated bacteria were also analysed. There was a significant influence of the timing of inoculation on the volatile composition of the wines just as on the amino acid and biogenic amine content. The wines produced by simultaneous AF/MLF showed the highest contents of some volatile compounds, such as ethyl esters and terpenes, as well as amino acids and tyramine. Bacterial encapsulation affected the volatile and amino acid profile of the wines, while the biogenic amine composition was not modified. The chemical composition of the wines was not altered by the presence of lysozyme. A trained panel did not perceive substantial differences between treatments.

Processivity of dextransucrases synthesizing very-high-molar-mass dextran is mediated by sugar-binding pockets in domain V.

The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (∼109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10-13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OKΔ1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis.

The influence of different strains of Oenococcus oeni malolactic bacteria on profile of organic acids and phenolic compounds of red wine cultivars Rondo and Regent growing in a cold region.

Wines produced from grapes cultivated in cool climate areas are characterized by high levels of organic acids. One method to correct this is malolactic fermentation (MLF). The aim of this study was to determine the effectiveness of different strains of Oenococcus oeni bacteria (Viniflora CH11, Viniflora CH16, Viniflora CH35, Viniflora Oenos, SIHA LACT Oeno) during the biological acidity reduction process. Red wine from Rondo and Regent cultivars was obtained by ethanol fermentation of the pulp, at 20 °C for 14 days. The profile of organic acids was examined with a particular focus on changes in the content of l-malic and l-lactic acids. Additionally, the impact on profile and quantity of phenolic compounds and antioxidant capacity was measured. The results showed that MLF had a positive influence on content of organic acids through the reduction of l-malic acid content with a simultaneous increase of the amount of l-lactic acid. The best effect was obtained with the CH11 and CH35 bacterial strains. The biological acidity reduction process had no significant (P > 0.05) impact on phenolic content or antioxidant capacity. However, the wine making process (ethanol fermentation, maturation) contributed to the reduction of polyphenols and in consequence lower antioxidant capacity of the final tested wines. PRACTICAL APPLICATION: The present study provides useful information on the impact of different Oenococcus oeni bacterial strains on MLF in red wines, reduction of l-malic to l-lactic acid, and stability of phenolic compounds during MLF and the maturation period. Also, this article provides information about phenolic compounds and antioxidant capacity during malolactic fermentation and maturity of red wines made from hybrids of Vitis vinifera such as Rondo and Regent cultivars.

Adsorption and biotransformation of anthocyanin glucosides and quercetin glycosides by Oenococcus oeni and Lactobacillus plantarum in model wine solution.

BACKGROUND: Anthocyanins and flavonols play a significant role in contributing to wine color and mouthfeel, and the interaction of malolactic fermentation with these compounds is not well known. Here we investigated the adsorption of these compounds by Oenococcus oeni and Lactobacillus plantarum. RESULTS: Delphinidin-3-glucoside (D3G) was adsorbed the most, followed by malvidin-3-glucoside (M3G) and peonidin-3-glucoside (P3G) for both the bacterial species, while flavonols were not adsorbed. An increase in ß-glycosidase activity suggested that this enzyme breaks down the anthocyanin glucosides, providing sugars for growth. An average decline of approximately 65% in enzyme activity in the presence of substantial residual sugar was observed. The specific metabolic rates were found to be dependent on the class of anthocyanin and species / strain of the bacteria. Selective adsorption of anthocyanins and not the flavonol glycosides suggest that electrostatic interactions mediate the adsorption. Further, a breakdown of anthocyanins resulted in phloroglucinol aldehyde from the flavonoid A-ring and corresponding phenolic acids from the B-ring, i.e., gallic acid for D3G, syringic acid for M3G, and vanillic acid for P3G. CONCLUSIONS: The breakdown and adsorption of the anthocyanin glucosides can help explain the color loss and aroma changes, such as the appearance of syringic and vanillic acid, associated with malolactic fermentation. © 2019 Society of Chemical Industry.

Use of fumaric acid to control pH and inhibit malolactic fermentation in wines.

Fumaric acid is an additive allowed by the Codex Alimentarius and under evaluation by the International Organisation of Vine and Wine (OIV) that can be used for wine acidification but also to inhibit malolactic fermentation (MLF). The use of 300-900 mg/L of fumaric acid can inhibit MLF in red wines decreasing pH by 0.2 units or more depending on the buffer capacity. When MLF was running with populations of either 7 or 8 log CFU/mL strain alpha (Oenococcus oeni) the application of 600 mg/L of fumaric acid stopped the process for more than 50 days and cells were undetected in specific media. In triangular tastings, fumaric acid was not detected at 300-600 mg/L (p < .05). In subsequent preference tests, some tasters perceived more acidity and body. Fumaric acid is a useful technological additive to improve wine microbiological stability and freshness, also allowing reduction of SO2 levels.