Malolactic Fermentation In Wine Research Articles

Characterization of malolactic fermentation by Lactiplantibacillus plantarum in red grape must

Malolactic fermentation (MLF) is a desirable biotransformation in red and some high-acidity white wines. This process involves the decarboxylation of l-malic acid into l-lactic acid by lactic acid bacteria (LAB). Among them, Oenococcus oeni and Lactiplantibacillus plantarum are the most common, being the last usually used as starter to perform MLF in wine. Compared with O. oeni, L. plantarum is less tolerant to wine conditions such as pH and ethanol; thus, it is preferable to inoculate it in must. The current literature has not yet addressed the impact of some parameters on the inoculation of this bacterium in grape must. Therefore, the aim of this work was to characterize the effect of the inoculum concentration, pH and ethanol tolerance, as well as the inoculation strategy, on the MLF performance of L. plantarum in grape must. We observed that a population < 108 CFU/mL could compromise MLF performance. Additionally, L. plantarum was able to survive at low pH values, such as 2.50, but in the presence of ethanol or a fermenting yeast, it could tolerate pH values in the range of 3.00–3.25. Finally, coinoculation or sequential inoculation with S. cerevisiae allows for the completion of MLF before the end of alcoholic fermentation.

Impact of indigenous Oenococcus oeni and Lactiplantibacillus plantarum species co-culture on Cabernet Sauvignon wine malolactic fermentation: Kinetic parameters, color and aroma

Malolactic fermentation (MLF) is a crucial process to enhance wine quality, and the utilization of indigenous microorganisms has the potential to enhance wine characteristics distinct to a region. Here, the MLF performance of five indigenous Oenococcus oeni strains and six synthetic microbial communities (SynComs), were comparatively evaluated in Cabernet Sauvignon wine. In terms of malate metabolism rate and wine aroma diversity, the strain of O. oeni Oe114–46 demonstrated comparable MLF performance to the commercial strain of O. oeni Oe450 PreAc. Furthermore, the corresponding SynComs (Oe144–46/LpXJ25) exhibited improved fermentation properties, leading to increased viable cell counts of both species, more rapid and thorough MLF, and increased concentrations of important aroma compounds, such as linalool, 4-terpinenol, α-terpineol, diethyl succinate, and ethyl lactate. These findings highlight the remarkable MLF performance of indigenous O. oeni and O. oeni-L. plantarum microbial communities, emphasizing their immense potential in improving MLF efficiency and wine quality.

Application of white wine lees for promoting lactic acid bacteria growth and malolactic fermentation in wine

In the context of ecological transition, the use of wine by-products for industrial applications is a major challenge. Wine lees, the second wine by-product in terms of quantity, represent a source of nutrients that can be used for stimulating the growth of microorganisms. Here, white wine lees were used as a stimulating agent for the growth of wine lactic acid bacteria (LAB) and to promote wine malolactic fermentation (MLF) driven out by Oenococcus oeni. By adding freeze-dried wine lees to wines under different conditions – including different wine lees at different concentrations and different O. oeni strains at various initial populations - it was observed that wine lees can enhance the growth of LAB and reduce the duration of MLF. The chemical composition of wines was also evaluated, proving that wine lees do not compromise the quality of the wines. In addition, wine lees did not seem to promote the growth of spoilage microorganisms like as Brettanomyces bruxellensis. Altogether, this work reports the possibility of recovering the lees of white wine to obtain a product favoring the MLF of red wines. More general, we propose a recycling strategy of wine by-products to obtain new products for winemaking.

Influence of pH on Oenococcus oeni metabolism: Can the slowdown of citrate consumption improve its acid tolerance?

Oenococcus oeni is the lactic acid bacteria most suited to carry out malolactic fermentation in wine, converting L-malic acid into L-lactic acid and carbon dioxide, thereby deacidifying wines. Indeed, wine is a harsh environment for microbial growth, partly because of its low pH. By metabolizing citrate, O. oeni maintains its homeostasis under acid conditions. Indeed, citrate consumption activates the proton motive force, helps to maintain intracellular pH, and enhances bacterial growth when it is co-metabolized with sugars. In addition, citrate metabolism is responsible for diacetyl production, an aromatic compound which bestows a buttery character to wine. However, an inhibitory effect of citrate on O. oeni growth at low pH has been highlighted in recent years. In order to understand how citrate metabolism can be linked to the acid tolerance of this bacterium, consumption of citrate was investigated in eleven O. oeni strains. In addition, malate and sugar consumptions were also monitored, as they can be impacted by citrate metabolism. This experiment highlighted the huge diversity of metabolisms between strains depending on their origin. It also showed the capacity of O. oeni to de novo metabolize certain end-products such as L-lactate and mannitol, a phenomenon never before demonstrated. It also enabled drawing hypotheses concerning the two positive effects that the slowing down of citrate metabolism could have on biomass production and malolactic fermentation occurring under low pH conditions.

Evaluation of the Addition of Yeast Mannoprotein to Oenococcus oeni Starter Cultures to Improve Wine Malolactic Fermentation

Malolactic fermentation (MLF) in wine is driven by the lactic acid bacterium Oenococcus oeni in most cases. Although this bacterium is resistant to wine stress conditions, it often faces difficulties completing MLF. Previous studies indicate that yeast mannoproteins may improve O. oeni growth and survival in wine. However, very little is known about this topic. This study evaluated the effect of the addition of mannoprotein extracts to culture media on O. oeni growth and its survival to stress conditions and MLF performance. Three commercial mannoprotein extracts were characterized in terms of polysaccharide and protein richness and were used for O. oeni culture media supplementation. The addition of mannoprotein extracts improved the survival of the two evaluated O. oeni strains, PSU-1 and VP41, after acid shock (pH 3.2) in comparison to that of the control. The transcriptional response of four genes involved in mannose metabolism was different depending on the strain, indicating the complexity of sugar metabolism in O. oeni. PSU-1 cells grown with two of the mannoprotein extracts performed faster MLF compared with the control condition, indicating that mannoprotein addition may improve the performance of O. oeni starter cultures, although this effect depends on the strain.

Validation of a routine HPLC method for added fumaric acid determination in wines

Nowadays, grape berries mature earlier due to climate change. Higher sugar contents are observed, whereas lower ones in organic acids, leading to increased pH values. Those biochemical changes have direct consequences on the balance and quality of wines made from such grapes. Chemical acidification, commonly used to compensate for the lack of acidity in musts or wines, includes tartaric, malic, lactic, and citric acids. Fumaric acid (FA), naturally present in the grape berries at low concentrations and already authorized in the member states of the OIV to inhibit malolactic fermentation in wines, seems to be a promising alternative to those acids to lower the pH. However, the evolution of FA levels added at bottling and its impact on wine quality during its conservation have not yet been studied. Therefore, the aim of the present study was to develop and validate a simple method using liquid chromatography coupled with a diode array detector, which can be used routinely for the determination of the added fumaric acid in wines. The proposed and validated method uses a ProntoSIL C-18 analytical column and an isocratic elution with water acidified at 0.1% formic acid. The run time, including column cleaning with acetonitrile and re-equilibration, was 40 minutes.

Effects of inoculation timing and mixed fermentation with Pichia fermentans on Oenococcus oeni viability, fermentation duration and aroma production during wine malolactic fermentation

There has been a growing interest in developing co-inoculum of Oenococcus oeni and Saccharomyces cerevisiae/non-Saccharomyces for simultaneous malolactic fermentation (MLF) and alcoholic fermentation (AF) of wines. This study sought to elucidate the effects of two crucial factors (inoculation timing and paired yeast) on the fermentation performance and aroma production of Merlot wine. O. oeni used for MLF was concurrently or sequentially inoculated with two yeast cultures (i.e., single S. cerevisiae and mixed S. cerevisiae /Pichia fermentans H5Y-28) used for AF. Inoculation timing determined the overall vinification duration, and conditioned the production of principle higher alcohols, terpene and O. oeni-mediated volatiles. In contrast, paired yeast improved O. oeni viability, and showed significant effect on aromatic esters and volatile fatty acids. Possibly due to lower ethanol stress, co-inoculum allowed O. oeni to initiate MLF during AF, resulting in 45% reduction of total fermentation time. Meanwhile, O. oeni growth was stimulated by P. fermentans, with 1.7-fold of the maximum population higher than that in co-fermentation without P. fermentans. Such stimulation of O. oeni growth also occurred in sequential fermentation where P. fermentans had been replaced by S. cerevisiae. Only in sequential inoculum, P. fermentans induced high levels of 3-methylbutyl acetate, ethyl 3-methylbutanoate, ethyl hexanoate and ethyl octanoate, which may result in enhanced fresh fruity trait of wines. These findings suggested a positive effect of P. fermentans H5Y-28 on O. oeni and MLF. This work provides an alternative approach to improve wine MLF and aroma outcomes using friendly non-Saccharomyces yeast with appropriate inoculation strategy.

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.

Use of Yeast Mannoproteins by Oenococcus oeni during Malolactic Fermentation under Different Oenological Conditions.

Oenococcus oeni is the main agent of malolactic fermentation in wine. This fermentation takes place after alcoholic fermentation, in a low nutrient medium where ethanol and other inhibitor compounds are present. In addition, some yeast-derived compounds such as mannoproteins can be stimulatory for O. oeni. The mannoprotein concentration in wine depends on the fermenting yeasts, and non-Saccharomyces in particular can increase it. As a result of the hydrolytic activity of O. oeni, these macromolecules can be degraded, and the released mannose can be taken up and used as an energy source by the bacterium. Here we look at mannoprotein consumption and the expression of four O. oeni genes related to mannose uptake (manA, manB, ptsI, and ptsH) in a wine-like medium supplemented with mannoproteins and in natural wines fermented with different yeasts. We observe a general gene upregulation in response to wine-like conditions and different consumption patterns in the studied media. O. oeni was able to consume mannoproteins in all the wines. This consumption was notably higher in natural wines, especially in T. delbrueckii and S. cerevisiae 3D wines, which presented the highest mannoprotein levels. Regardless of the general upregulation, it seems that mannoprotein degradation is more closely related to the fermenting medium.

Distribution of Prophages in the Oenococcus oeni Species.

Oenococcus oeni is the most exploited lactic acid bacterium in the wine industry and drives the malolactic fermentation of wines. Although prophage-like sequences have been identified in the species, many are not characterized, and a global view of their integration and distribution amongst strains is currently lacking. In this work, we analyzed the complete genomes of 231 strains for the occurrence of prophages, and analyzed their size and positions of insertion. Our data show the limited variation in the number of prophages in O. oeni genomes, and that six sites of insertion within the bacterial genome are being used for site-specific recombination. Prophage diversity patterns varied significantly for different host lineages, and environmental niches. Overall, the findings highlight the pervasive presence of prophages in the O. oeni species, their role as a major source of within-species bacterial diversity and drivers of horizontal gene transfer. Our data also have implications for enhanced understanding of the prophage recombination events which occurred during evolution of O. oeni, as well as the potential of prophages in influencing the fitness of these bacteria in their distinct niches.

Altered Metabolic Strategies: Elaborate Mechanisms Adopted by Oenococcus oeni in Response to Acid Stress.

Oenococcus oeni plays a key role in inducing malolactic fermentation in wine. Acid stress is often encountered under wine conditions. However, the lack of systematic studies of acid resistance mechanisms limits the downstream fermentation applications. In this study, the acid responses of O. oeni were investigated by combining transcriptome, metabolome, and genome-scale metabolic modeling approaches. Metabolite profiling highlighted the decreased abundance of nucleotides under acid stress. The gene-metabolite bipartite network showed negative correlations between nucleotides and genes involved in ribosome assembly, translation, and post-translational processes, suggesting that stringent response could be activated under acid stress. Genome-scale metabolic modeling revealed marked flux rerouting, including reallocation of pyruvate, attenuation of glycolysis, utilization of carbon sources other than glucose, and enhancement of nucleotide salvage and the arginine deiminase pathway. This study provided novel insights into the acid responses of O. oeni, which will be useful for designing strategies to address acid stress in wine malolactic fermentation.

Characterization of the First Virulent Phage Infecting Oenococcus oeni, the Queen of the Cellars.

There has been little exploration of how phages contribute to the diversity of the bacterial community associated with winemaking and may impact fermentations and product quality. Prophages of Oenococcus oeni, the most common species of lactic acid bacteria (LAB) associated with malolactic fermentation of wine, have been described, but no data is available regarding phages of O. oeni with true virulent lifestyles. The current study reports on the incidence and characterization of the first group of virulent oenophages named Vinitor, isolated from the enological environment. Vinitor phages are morphologically very similar to siphoviruses infecting other LAB. Although widespread during winemaking, they are more abundant in musts than temperate oenophages. We obtained the complete genomic sequences of phages Vinitor162 and Vinitor27, isolated from white and red wines, respectively. The assembled genomes shared 97.6% nucleotide identity and belong to the same species. Coupled with phylogenetic analysis, our study revealed that the genomes of Vinitor phages are architecturally mosaics and represent unique combinations of modules amongst LAB infecting-phages. Our data also provide some clues to possible evolutionary connections between Vinitor and (pro)phages associated to epiphytic and insect-related bacteria.

Evaluating the effect of using non-Saccharomyces on Oenococcus oeni and wine malolactic fermentation

Interest in using non-Saccharomyces yeasts in winemaking has increased in recent years due to their ability to improve wine quality. However, little information has been published regarding the possible effect on malolactic fermentation (MLF), carried out mostly by Oenococcus oeni. The aim of this paper is therefore to evaluate the effect of the most representative non-Saccharomyces species on O. oeni and wine MLF. Different strains of Torulaspora delbrueckii, Metschnikowia pulcherrima, Hanseniaspora uvarum, Hanseniaspora vineae and Starmerella bacillaris (syn. Candida zemplinina) were used in sequential alcoholic fermentation with Saccharomyces cerevisiae. The resulting wines were inoculated with four O. oeni strains. The action of non-Saccharomyces affected the final wine composition and the later role of O. oeni. Some of its strains could not perform MLF in H. uvarum wine due to high SO2 concentrations. In some cases, MLF was inhibited in wines inoculated with S. bacillaris. All the H. uvarum and H. vineae strains notably increased acetic acid concentrations, thus threatening wine quality. The best conditions for MLF were provided by some T. delbruecckii and M. pulcherrima strains, which showed increased concentrations of mannoproteins – compounds described as MLF activators –, no production of SO2, and low consumption of L-malic acid. In conclusion, non-Saccharomyces yeasts have diverse effects on O. oeni and MLF depending on the species, with T. delbrueckii and M. pulcherrima being those that showed the best compatibility with MLF development.

Homology analysis of 35 β-glucosidases in Oenococcus oeni and biochemical characterization of a novel β-glucosidase BGL0224

β-Glucosidases play an important role in food industry. Oenococcus oeni are typical lactic acid bacteria that initiate malolactic fermentation of wines. 35 β-glucosidases from O. oeni were selected and their conserved domains and evolutionary relationships were further explored in this study. The homology analysis results indicated that 35 β-glucosidases were basically derived from GH1 and GH3 family. A novel β-glucosidase was successfully expressed and characterized. The recombinant protein, referred to as BGL0224, consisted of a total 480 amino acids with an apparent molecular weight of 55.15 kDa and was classified as GH1 family. It achieved the highest activity at pH 5.0 and 50 °C. The activity and stability were significantly increased when 12% ethanol was supplemented to the enzyme. Using p-NPG as substrate, the Km, Vmax and Kcat of BGL0224 were 0.34 mM, 382.81 U/mg and 351.88 s−1, respectively. In all, BGL0224 has good application prospects in food industry.

Mechanism analysis of combined acid-and-ethanol shock on Oenococcus oeni using RNA-Seq

Acid-and-ethanol tolerance plays an important role in the cell viability of Oenococcus oeni and affects the enological characteristics of malolactic fermentation in wine. To reveal the mechanism of the response to acid-and-ethanol in O. oeni, we analyzed the changes to its gene expression profile after acid-and-ethanol shock for the first time using RNA-Seq. Some physiological indicators related to this stress response were also characterized. Bioinformatic and physiological analyses showed that the O. oeni strengthened the biosynthesis of peptidoglycan as a response to ethanol toxicity, and the cell membranes altered their fatty acid compositions for keeping the acidic H+ outside when the cells were shocked with acid-and-ethanol. When H+ entered the cytoplasm, the F0F1-ATPase system began discharging H+ (producing ATP), and the cells increased their expression of recN and mutT genes for minimizing the DNA damage. The cells also used two-component systems to communicate between same-species cells for improving the overall population survival rate. Genes encoding spermidine and putrescine transport were also upregulated to increase resistance to the acid-and-ethanol environment. This study fills important gaps in the current understanding of the bacterial acid-and-ethanol stress response mechanism, and it may be beneficial to screen more robust O. oeni strains in future works.

Effect of the inoculation strategy of non-Saccharomyces yeasts on wine malolactic fermentation

Interest in some non-Saccharomyces yeasts has increased recently, because they have been associated with an improvement in wine quality. Nevertheless, little attention has been paid to the effect that the use of these yeasts may have on malolactic fermentation (MLF). In this study, the strains Torulaspora delbrueckii Biodiva and Metschnikowia pulcherrima Flavia were evaluated by co-inoculation and sequential fermentation with S. cerevisiae QA23. A fermentation with S. cerevisiae as a single starter was also performed as a control, then MLF was performed inoculating Oenococcus oeni PSU-1 in all wines. Finally, the wines obtained after alcoholic fermentation and MLF were characterised. The results of the coinoculated fermentations were similar to those of the S. cerevisiae control fermentations. Nevertheless, significant differences were observed in sequential fermentations in terms of lower content of acetic, L-malic and succinic acids. These differences were particularly noticeable in fermentations carried out with T. delbrueckii.

Modifications of Phenolic Compounds, Biogenic Amines, and Volatile Compounds in Cabernet Gernishct Wine through Malolactic Fermentation by Lactobacillus plantarum and Oenococcus oeni

Malolactic fermentation is a vital red wine-making process to enhance the sensory quality. The objective of this study is to elucidate the starter cultures’ role in modifying phenolic compounds, biogenic amines, and volatile compounds after red wine malolactic fermentation. We initiated the malolactic fermentation in Cabernet Gernishct wine by using two Oenococcus oeni and two Lactobacillus plantarum strains. Results showed that after malolactic fermentation, wines experienced a content decrease of total flavanols and total flavonols, accompanied by the accumulation of phenolic acids. The Lactobacillus plantarum strains, compared to Oenococcus oeni, exhibited a prevention against the accumulation of biogenic amines. The malolactic fermentation increased the total esters and modified the aromatic features compared to the unfermented wine. The Lactobacillus plantarum strains retained more aromas than the Oenococcus oeni strains did. Principal component analysis revealed that different strains could distinctly alter the wine characteristics being investigated in this study. These indicated that Lactobacillus plantarum could serve as a better alternative starter for conducting red wine malolactic fermentation.

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

ABSTRACTFumaric 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.

Ethanol‐tolerant lactic acid bacteria strains as a basis for efficient malolactic fermentation in wine: evaluation of experimentally evolved lactic acid bacteria and winery isolates

Background and Aims Reliable malolactic fermentation (MLF) is essential for process efficiency and spoilage prevention in wine. This study extends previous research in our laboratory, aimed at the development and selection of new bacterial strains for reliable MLF in wine, focusing on ethanol-tolerant lactic acid bacteria (LAB) strains. Sensory differences of seven LAB strains were assessed, including two commercial strains, two ethanol-tolerant strains derived from directed evolution and three isolates from a high ethanol Grenache. Methods and Results In this study, the performance of 30 LAB strains was first assessed in fermented chemically defined grape juice media. Seven of the best performing strains were then tested in small-scale (5 L) fermentations in Shiraz and Shiraz-Grenache blend wines. All wines were evaluated with a sensory panel using free choice profiling. Conclusions Despite significantly different MLF kinetics between the strains there were no strain-specific differences on the final wines. The choice of LAB strain did not adversely change the sensory properties of either wine. Significance of the Study These findings provide reassurance that the efficient LAB strains (G71 and G55) and the modified directed evolution strains do not compromise the sensory properties of wines despite their marked MLF benefits.

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.