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.

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.

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.

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.

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.

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.

Production of melatonin and other tryptophan derivatives by Oenococcus oeni under winery and laboratory scale.

Malolactic fermentation (MLF) in Valtellina Superiore DOCG red wine was monitored in 4 cellars and the final products were analysed to determine the content of melatonin (MEL) and other tryptophan (TRP) derivatives, including tryptophan ethyl ester (TEE) and MEL isomers (MISs), and to isolate predominant O. oeni strains. MEL and TEE significantly increased in wines after MLF from two cellars out of four. Six strains were isolated during the MLF of red wines and under laboratory scale, in rich and synthetic wine cultural media, together with other four O. oeni strains able to trigger the MLF. Results showed that the presence of stressful growth factors, like ethanol and acid pH, has a pivotal role in triggering the release of TEE by oenococci. Indeed, all the strains became capable to produce also MEL and MISs, together with TEE. under harsh growth conditions, as in a synthetic wine medium. The production of these compounds was strain-dependent and a maximum amount of 0.0078 ±â€¯0.0023 ngT/mL (UMB472) and 619.85 ±â€¯196.16 ngT/mL (UMB436) of MEL and TEE was obtained, respectively. In particular, different MISs were detected under oenological and laboratory scale suggesting that other factors (i.e. technological and/or physico-chemical) could affect the synthesis of TRP derivatives.

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.

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.

Influence of sugars and pH on the citrate metabolism of different lactic acid bacteria strains in a synthetic wine matrix.

AIMS: This study investigated the influence of sugars (glucose and fructose) and pH on the gene expression of citE (citrate lyase ß-subunit) and the subsequent formation of metabolites associated with citrate metabolism. METHODS AND RESULTS: Different levels of glucose (2·5, 50 and 115 g l-1 ), fructose (2·5, 50 and 115 g l-1 ) and pH (3·0, 3·5, 4·0 and 5·0) were evaluated for their effect on citE expression in four different lactic acid bacteria strains. Two Oenococcus oeni strains and two Lactobacillus plantarum strains were used, of which one strain of each species screened positive for the citE gene. Among the factors tested, fructose had the biggest influence on the relative expression of citE in O. oeni. In addition, the citrate-positive strains produced high concentrations of diacetyl and acetoin. CONCLUSIONS: This study gives an overview of how sugar, pH and different lactic acid bacteria strains influence citE gene expression and the formation of metabolites associated with citrate metabolism closely linked to malolactic fermentation (MLF). SIGNIFICANCE AND IMPACT OF THE STUDY: These results can be used to make informed decisions regarding MLF when aiming to create a wine with a buttery aroma or not.

Effect of mixed species alcoholic fermentation on growth and malolactic activity of lactic acid bacteria.

In recent years, there is an increasing interest from the winemaking industry for the use of mixed fermentations with Starmerella bacillaris (synonym Candida zemplinina) and Saccharomyces cerevisiae, due to their ability to modulate metabolites of oenological interest. The current study was carried out to elucidate the effect of this fermentation protocol on the growth and malolactic activity of lactic acid bacteria (LAB) used for malolactic fermentation (MLF) and on the chemical and volatile profile of Nebbiolo wines and their chromatic characteristics. To this end, two LAB species, namely Lactobacillus plantarum and Oenococcus oeni, were inoculated at the beginning and at the end of the alcoholic fermentation (AF) performed by pure and mixed yeast using the abovementioned yeasts. The different yeast inoculation protocols and the combination of species tested influenced greatly the interactions and behavior of the inoculated yeasts and LAB during AF and MLF. For both LAB species, inoculation timing was critical to how rapidly MLF started and finished. Fermentation inoculated with L. plantarum, at the beginning of the AF, completed MLF faster than those inoculated with O. oeni. The presence of Starm. bacillaris in mixed fermentation promoted LAB growth and activity, in particular, O. oeni. Furthermore, LAB species choice had a greater impact on the volatile and chromatic profile of the wines than inoculation time. These findings reveal new knowledge about the importance of LAB species choice and inoculation time to ensure fast MLF completion and to improve wine characteristics in mixed fermentation with Starm. bacillaris and S. cerevisiae.

Measures to improve wine malolactic fermentation.

This review focuses on the considerable amount of research that has been directed towards the improvement of efficiency and reliability of malolactic fermentation (MLF), which is important in winemaking. From this large body of work, it is clear that reliable MLF is essential for process efficiency and prevention of spoilage in the final product. Impediments to successful MLF in wine, the impact of grape and wine ecology and how this may affect MLF outcome are discussed. Further focus is given to how MLF success may be enhanced, via alternative inoculation strategies, MLF progress sensing technologies and the use of different bacterial species. An update of how this information may be used to enhance and improve sensory outcomes through metabolite production during MLF and suggestions for future research priorities for the field are also provided.

Increased ethanol tolerance associated with the pntAB locus of Oenococcus oeni and Lactobacillus buchneri.

Lactobacillus buchneri and Oenococcus oeni are two unique ethanol-tolerant Gram-positive bacteria species. Genome comparison analyses revealed that L. buchneri and O. oeni possess a pntAB locus that was absent in almost all other lactic acid bacteria (LAB) genomes. Our hypothesis is that the pntAB locus contributes to the ethanol tolerance trait of these two distinct ethanol-tolerant organisms. The pntAB locus, consisting of the pntA and pntB genes, codes for NADP(H) transhydrogenase subunits. This membrane-bound transhydrogenase catalyzes the reduction of NADP+ and is known as an important enzyme in maintaining cellular redox balance. In this study, the transhydrogenase operon from L. buchneri NRRL B-30929 and O. oeni PSU-1 were cloned and analyzed. The LbpntB shared 71.0% identity with the O. oeni (OopntB). The entire pntAB locus was expressed in Lactococcus lactis ssp. lactis IL1403 resulting in an increased tolerance to ethanol (6%), butanol (1.8%) and isopropanol (1.8%) when compared to the control strain. However, the recombinant E. coli cells carrying the entire pntAB locus did not show any improved ethanol tolerance. Independent expression of OopntB and LbpntB in recombinant E. coli BL21(DE3)pLysS host demonstrated higher tolerance to ethanol when compared with a control E. coli BL21(DE3)pLysS strain carrying pET28b vector. Ethanol tolerance comparison of E. coli strains carrying LbpntB and OopntB showed that LbpntB conferred higher ethanol tolerance (4.5%) and resulted in greater biomass, while the OopntB conferred lower ethanol tolerance (4.0%) resulted lower biomass. Therefore, the pntB gene from L. buchneri is a better choice in generating higher ethanol tolerance. This is the first study to uncover the role of pntAB locus on ethanol tolerance.

Identification of multiple-derived peptides produced by Saccharomyces cerevisiae involved in malolactic fermentation inhibition.

An oenological strain of Saccharomyces cerevisiae was previously shown to produce a 5-10 kDa peptidic fraction responsible for the inhibition of malolactic fermentation (MLF). In the present study, we aim to further purify the anti-MLF peptides of this fraction. The yeast fermented synthetic grape juice medium was fractionated by ammonium sulfate precipitation combined with ultrafiltration. The 5-10 kDa fraction recovered at a saturation degree of 60%-80% was the only fraction that inhibited both the bacterial growth and the malate consumption in vivo. It also inhibited the malolactic enzyme activity in vitro at a pH range between 3.5 and 6.7. Therefore, it was purified by both anion and cation exchange chromatography. The eluates that inhibited the malolactic enzyme activity in vitro were migrated on Tricine SDS-PAGE and the protein bands were excised and sequenced by LC-MS/MS. The sequencing revealed nine peptides originating from eight proteins of S. cerevisiae. Two GAPDH cationic fragments of 0.9 and 1.373 kDa having a pI of 10.5 and 11 respectively, Wtm2p and Utr2p anionic fragments of 2.42 kDa with a pI of 3.5 and 4 respectively were thought to contribute the most to the MLF inhibition.

Adaptation of two groups of Oenococcus oeni strains to red and white wines: the role of acidity and phenolic compounds.

AIMS: Oenococcus oeni is the lactic acid bacteria species which is the most adapted to wine. Recently, two groups of strains that form two genetic lineages were described in red and white Burgundy wines. The aim of this study was to analyse the phenotypes of these strains in order to determine how they have adapted specifically to either red or white wine. METHODS AND RESULTS: Four strains from each group were tested in grape must and in wines to evaluate their tolerance to pH and to phenolic compound content. White wine strains proved to be the most tolerant to low pH, both in grape must and in wine, whereas they were inhibited by the presence of grape tannins in wine. Red wine strains were more sensitive to acidity, but very resistant to phenolic compounds. CONCLUSIONS: The results suggest that pH and phenolic compounds drive strain selection at several stages of wine production. SIGNIFICANCE AND IMPACT OF THE STUDY: Although it is well known that O. oeni is well adapted to wine, this study shows that strains of some genetic lineages within this species have evolved to adapt better than others to specific types of wines.

Ethanol stress in Oenococcus oeni: transcriptional response and complex physiological mechanisms.

Oenococcus oeni is the dominant species able to cope with a hostile environment of wines, comprising cumulative effects of low pH, high ethanol and SO2 content, nonoptimal growth temperatures and growth inhibitory compounds. Ethanol tolerance is a crucial feature for the activity of O. oeni cells in wine because ethanol acts as a disordering agent of its cell membrane and negatively affects metabolic activity; it damages the membrane integrity, decreases cell viability and, as other stress conditions, delays the start of malolactic fermentation with a consequent alteration of wine quality. The cell wall, cytoplasmic membrane and metabolic pathways are the main sites involved in physiological changes aimed to ensure an adequate adaptive response to ethanol stress and to face the oxidative damage caused by increasing production of reactive oxygen species. Improving our understanding of the cellular impact of ethanol toxicity and how the cell responds to ethanol stress can facilitate the development of strategies to enhance microbial ethanol tolerance; this allows to perform a multidisciplinary endeavour requiring not only an ecological study of the spontaneous process but also the characterization of useful technological and physiological features of the predominant strains in order to select those with the highest potential for industrial applications.

Directed evolution of Oenococcus oeni strains for more efficient malolactic fermentation in a multi-stressor wine environment.

High concentrations of ethanol, low pH, the presence of sulfur dioxide and some polyphenols have been reported to inhibit Oenococcus oeni growth, thereby negatively affecting malolactic fermentation (MLF) of wine. In order to generate superior O. oeni strains that can conduct more efficient MLF, despite these multiple stressors, a continuous culture approach was designed to directly evolve an existing ethanol tolerant O. oeni strain, A90. The strain was grown for ∼350 generations in a red wine-like environment with increasing levels of stressors. Three strains were selected from screening experiments based on their completion of fermentation in a synthetic wine/wine blend with 15.1% (v/v) ethanol, 26 mg/L SO2 at pH 3.35 within 160 h, while the parent strain fermented no more than two thirds of l-malic acid in this medium. These superior strains also fermented faster and/or had a larger population in four different wines. A reduced or equivalent amount of the undesirable volatile, acetic acid, was produced by the optimised strains compared to a commercial strain in Mouvedre and Merlot wines. These findings demonstrate the feasibility of using directed evolution as a tool to generate more efficient MLF starters tailored for wines with multiple stressors.

Non-anthocyanin polyphenolic transformation by native yeast and bacteria co-inoculation strategy during vinification.

BACKGROUND: Co-inoculation has been adapted by many wine-producing countries because it enhances the success of malolactic fermentation and reduces the fermentation cost, as well as time. However, wine phenolics have been sparsely highlighted during co-inoculation, even though polyphenols are an important parameter affecting wine colour, astringency and aroma. In the present study, we investigated the impact of co-inoculation on non-anthocyanin polyphenol profile for two different grape varieties. RESULTS: Co-inoculation of native yeast strain (AAV2) along with Oenococcus oeni was adapted for Cabernet Sauvignon and Shiraz wine. It was observed that the co-inoculation had minimal yet significant impact on the phenolic composition of wines for both the grape varieties. Color loss, as well as fruity aroma development, was observed in co-inoculated wines. The wines were on a par with the commercial wine, as well as wines without malolactic fermentation, in terms of phenolic compounds and overall organoleptic acceptance. Principal component analysis and hierarchical cluster analysis further suggested that the varietal influence on phenolic composition was dominating compared to inoculation strategies. Among the varieties, the inoculation strategies have significantly influenced the Cabernet wines compared to Shiraz wines. CONCLUSION: The results of the present study demonstrate that the phenolic compounds are not drastically affected by metabolic activities of malolactic bacteria during co-inoculation and, hence, are equally suitable for wine fermentation. © 2017 Society of Chemical Industry.

Oenococcus oeni: Queen of the cellar, nightmare of geneticists.

Oenococcus oeni is a wine-associated lactic acid bacterium (LAB) responsible mostly for wine malolactic fermentation (MLF). This fastidious bacterium (auxotrophic for many amino acids and slow growing) possesses remarkable adaptability to harsh physicochemical conditions and can reprogramme its metabolic pathways to enhance its survival in wine. Thus, O. oeni is an instructive bacterial model for investigating stress response mechanisms in LAB. However, the lack of appropriate techniques to modify the O. oeni genome has hampered molecular studies of this species. The application of recent advances in molecular genetics promises to provide a better understanding of the regulation of stress responses in this species in the future.