Malolactic Fermentation In Wine Research Articles

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.

CtsR, the Master Regulator of Stress-Response in Oenococcus oeni, Is a Heat Sensor Interacting With ClpL1.

Oenococcus oeni is a lactic acid bacterium responsible for malolactic fermentation of wine. While many stress response mechanisms implemented by O. oeni during wine adaptation have been described, little is known about their regulation. CtsR is the only regulator of stress response genes identified to date in O. oeni. Extensively characterized in Bacillus subtilis, the CtsR repressor is active as a dimer at 37°C and degraded at higher temperatures by a proteolytic mechanism involving two adapter proteins, McsA and McsB, together with the ClpCP complex. The O. oeni genome does not encode orthologs of these adapter proteins and the regulation of CtsR activity remains unknown. In this study, we investigate CtsR function in O. oeni by using antisense RNA silencing in vivo to modulate ctsR gene expression. Inhibition of ctsR gene expression by asRNA leads to a significant loss in cultivability after heat shock (58%) and acid shock (59%) highlighting the key role of CtsR in the O. oeni stress response. Regulation of CtsR activity was studied using a heterologous expression system to demonstrate that O. oeni CtsR controls expression and stress induction of the O. oeni hsp18 gene when produced in a ctsR-deficient B. subtilis strain. Under heat stress conditions, O. oeni CtsR acts as a temperature sensor and is inactivated at growth temperatures above 33°C. Finally, using an E. coli bacterial two-hybrid system, we showed that CtsR and ClpL1 interact, suggesting a key role for ClpL1 in controlling CtsR activity in O. oeni.

Identification of the potential inhibitors of malolactic fermentation in wines

Abstract This exploratory work aims to identify the potential inhibitors of lactic bacterial growth and to propose enological practices to guarantee the occurrence of spontaneous malolactic fermentation (MLF) in wines from traditional and double-pruning management harvests in southeast Brazil. One white wine from a summer harvest and one red wine from a winter harvest that failed to complete MLF were utilized as comparative models to identify inhibitor compounds to lactic bacteria. Wine composition, alcoholic-fermentation temperature and bacterial strain contribute to the success or failure of MLF. Temperatures below 12 °C during alcoholic fermentation decrease lactic bacterial metabolism and may impair the bacteria’s growth after yeast cells lysis. A must pH below 3.2 in a summer harvest impairs bacterial growth, and the association of low pH with a free-SO2 concentration above 10 mg L-1 may inhibit MLF. For grapes with a high sugar content, harvested in the winter cycle, enologists should keep the alcohol content below 15% and control the alcoholic-fermentation temperature.

Production and Antimicrobial Activity of Nisin Under Enological Conditions.

Lactic acid bacteria (LAB) are responsible for the malolactic fermentation of wines, and, therefore, controlling the growth of these bacteria is a key factor for elaborating premium wines. Sulfur dioxide has been traditionally used as an efficient antimicrobial and antioxidant agent, however, nowadays consumers’ demand tends toward a reduction of sulfur dioxide levels in wine and other fermented foods. A previous study of our research group had demonstrated the effectiveness of the bacteriocin nisin to inhibit the growth of enological LAB, and its activity had been tested in culture broths. The aim of this study was to investigate the possibility of controlling the growth of bacteria in wine by the use of nisin in combination with sulfur dioxide, and to study nisin production by the natural producer Lactococcus lactis LM29 under enological conditions. Our results showed that L. lactis LM29 produced nisin in the presence of 2 and 4% ethanol (v/v), while higher concentrations of ethanol fully inhibited the production of nisin. We obtained a nisin enriched active extract (NAE) from the cell-free supernatant of a culture of L. lactis LM29 in MRS broth containing 60% (v/v) sterile grape juice, and the extract was fully active in inhibiting the growth of the enological LAB tested by the microtiter method. Moreover, the nisin concentration of the obtained NAE could actually prevent the formation of an undesirable biofilm of LAB strains. Finally, our results of wine ageing under winery conditions showed that the use of 50 mg/L nisin decreased fourfold the concentration of sulfur dioxide required to prevent LAB growth in the wines.

Fluorescence microscopy to monitor wine malolactic fermentation

Malolactic fermentation (MLF) is a natural and biological deacidification of wines and a required step for making premium red wines. MLF is carried out by lactic acid bacteria (LAB) that are present in the fermenting wines. Currently, real-time control of MLF is an issue of great interest as the classical plate count technique for assessing bacterial populations requires long incubation times that are not compatible with a tight control of MLF. The aim of this study was to apply fluorescence microscopy and the bacteria staining kit Live/Dead BacLight™ to quantify viable LAB populations in red wines undergoing MLF. This method proved to be a fast and reliable culture-independent method to monitor wine MLF. Moreover, comparison of bacterial population data obtained by fluorescence microscopy and classical plate counts of LAB populations allowed discriminating a population of fully active and culturable cells, from total viable cells that include cells in an intermediate unculturable state.

Complete Genome Sequence of Lytic Oenococcus oeni Bacteriophage OE33PA.

Oenococcus oeni is the most common species of lactic acid bacteria associated with malolactic fermentation in wine. Here, we report the genome sequence of the lytic phage OE33PA (vB_OeS_OE33PA). It has a morphotype similar to that of members of the Siphoviridae family, a linear 39,866-bp double-stranded genome with cohesive ends, and 57 predicted open reading frames.

Influence of yeast strains on managing wine acidity using Lactobacillus plantarum

Lactobacillus plantarum has been used for deacidifying wines through malolactic fermentation (MLF), and more recently for acidifying them. The species Oenococcus oeni is mainly responsible for MLF in wines at a pH below 3.5. However, the Lactobacillus and Pediococcus species can carry out this reaction in wines at a higher pH. MLF generally begins once yeasts have completed alcoholic fermentation, but nowadays some winemakers prefer carrying out MLF simultaneously to alcoholic fermentation. The advantages of this strategy are shorter times in which to complete wine vinification and better wine color stabilization. Lactobacillus plantarum is preferred to O. oeni for performing early MLF because it catabolizes hexoses homofermentatively, which prevents acetic acid from increasing. In recent years, wine composition has changed as a result of climate change. High temperatures during the maturation time of grapes give rise to musts with low contents of organic acids, a high pH and high sugar contents, which give wines with low acidity, a high ethanol content and a faded color. Although several physical or chemical procedures can increase acidity, it can also be achieved by using microorganisms capable of transforming sugars into acids (biological acidification); e.g. lactic acid bacteria (LAB). The advantages of this procedure are increased wine acidity and reduced ethanol content. When early MLF or biological acidification is desired, yeasts and bacteria must develop together in grape must. In this work, we studied the consequences of the interactions of different combinations of two strains of L. plantarum and six commercial strains of Saccharomyces cerevisiae during white grape must fermentation. We demonstrated that managing wine acidity is possible by co-inoculating L. plantarum and S. cerevisiae. MLF was performed with any LAB/yeast combination, but the degree of biological acidification depended on the combination. LAB growth was more negatively affected by yeast than vice versa. The best combination to manage wine acidity was L. plantarum E4608 and Uvaferm VRB®.

Acetaldehyde released by Lactobacillus plantarum enhances accumulation of pyranoanthocyanins in wine during malolactic fermentation

This study investigated the evolution of acetaldehyde and pyranoanthocyanins in wine during malolactic fermentation, and further evaluated the correlation between acetaldehyde and pyranoanthocyanins. Cabernet Gernischt wine after alcoholic fermentation was inoculated with four lactic acid bacteria strains. Malolactic fermentation kinetics and wine characteristics were compared. Results showed these strains exhibited different kinetics on wine malolactic fermentation. Wine with Lactobacillus plantarum had lower reducing sugar, total acid, and yellowness. Lactobacillus plantarum elevated the level of acetaldehyde in wine model medium and wine during malolactic fermentation. Malolactic fermentation using Lactobacillus plantarum significantly increased the concentration of pyranoanthocyanins, whereas O. oeni strain reduced the level of pyranoanthocyanins in wine. Polymerized anthocyanins percentage in wine was significantly enhanced after fermentation with Lactobacillus plantarum. Principal component analysis indicated that the characteristics of these strains inoculated wines after malolactic fermentation were segregated. The findings from this study could provide useful information on the wine color improvement through malolactic fermentation with suitable lactic acid bacteria strains.

Effect of pH on malolactic fermentation in southern Italian wines

This study investigated the influence of pH on malolactic fermentation (MLF) in white wines (adjusted to pH 3.2, 3.4 and 3.8, respectively) from Falanghina grapes and red wines (adjusted to pH 3.4 and 3.8, respectively) from Tintilia grapes. The wines were inoculated with Oenococcus oeni and Lactobacillus plantarum strains, and a mix of them (50:50), in red Tintilia only. The time required to complete MLF in wines from white Falanghina grapes at pH 3.4 and 3.8 was lower with O. oeni, while MLF did not occur at pH 3.2. In red Tintilia, MLF was always completed within 35 days; at high pH (3.8) a significant increase in histamine was detected, while the decrease in citric acid concentration caused an increase in volatile acidity. Sensorial analysis showed an enhancement of red berry and spicy notes in red Tintilia at pH 3.8. PCA on white Falanghina showed that wines at pH 3.2 were located on the negative side of PC1 with higher scores for dry vegetable, sulphide, violet and toasted attributes. Wines at pH 3.4 and 3.8 were located on the positive side of the PC1 with butter and apple attributes. L. plantarum enhanced floral notes in white Falanghina wines and showed a good organoleptic impact on red Tintilia wines, which sensorial intensity was improved by a commercial mix (50:50) of O. oeni and L. plantarum.

Design and implementation of an ultrasonic sensor for rapid monitoring of industrial malolactic fermentation of wines

ABSTRACTUltrasound is an emerging technology that can be applied to monitor food processes. However, ultrasonic techniques are usually limited to research activities within a laboratory environment and they are not extensively used in industrial processes. The aim of this paper is to describe a novel ultrasonic sensor designed to monitor physical–chemical changes that occur in wines stored in industrial tanks. Essentially, the sensor consists of an ultrasonic transducer in contact with a buffer rod, mounted inside a stainless steel tube section. This structure allows the ultrasonic sensor to be directly installed in stainless steel tanks of an industrial plant. The operating principle of this design is based on the measurement of ultrasonic velocity of propagation. To test its proper operation, the sensor has been used to measure changes of concentration in aqueous samples and to monitor the progress of a malolactic fermentation of red wines in various commercial wineries. Results show the feasibility of using this sensor for monitoring malolactic fermentations in red wines placed in industrial tanks.

Microbial Resources and Innovation in the Wine Production Sector

Microbial starter cultures represent a fundamental level of innovation in the wine sector. Selected yeast strains are routinely used to achieve the needed biomass preparation to accelerate and steer alcoholic fermentation in grape must. The use of starter cultures to induce malolactic fermentation in wine relies on the characterisation and propagation of suitable strains of lactic acid bacteria. Furthermore, the selection of new strains, the renewal of management of microbial resources and new technologies allow continuous improvements in oenology, which may increase the beneficial aspects of wine. In this review, with the aim to stimulate microbial-driven, consumer-oriented advances in the oenological sector, we propose an overview of recent trends in this field that are reported by following the classical separation into ‘product innovation’ and ‘process innovation’. Hence, we shall highlight i) the possible positive innovative impacts of microbial resources on the safety and the sensorial and functional properties of wine (product innovation) and ii) the potential microbial-based improvements allowing the reduction of time/costs and the environmental impacts associated with winemaking (process innovation).

EVALUATION OF LACTIC ACID BACTERIA GROWTH DURING AUTOCHTHONOUS ALBANIAN KALLMET WINE PRODUCTION WITH SPONTANEOUS AND INOCULATED FERMENTATIONS

The grape used in wine making has many wild microorganisms like lactic acid bacteria, yeast, acetic acid bacteria. During the alcoholic fermentation, the evaluation of these microorganisms depends on their activity. There is an interaction between yeast and lactic acid bacteria during this period of wine making. In this study, we have made wine from the autochthonous Albanian grape Kallmet variety using the spontaneous fermentation and inoculated fermentation with the yeast Saccharomyces bayannus. Yeasts carry out the alcohol fermentation, and lactic acid bacteria make malolactic fermentation in wine. With this fermentation, lactic acid bacteria convert malic acid to lactic acid, reducing the acidity of the wine and create a microbiological stability. During the alcoholic fermentation, the evaluation of lactic acid bacteria is not required. The aim of our study is to evaluate the first quantity of lactic acid bacteria to Kallmet grape, their performance during the two fermentations, spontaneous and inoculated fermentations.

Silica-alginate-encapsulated bacteria to enhance malolactic fermentation performance in a stressful environment

Background and Aims Beginning and completion of malolactic fermentation (MLF) are complicated because of the harsh environment of wine. Encapsulation of lactic acid bacteria could be an attractive strategy to improve their activity and stability. This work evaluates the fermentative capacity of Oenococcus oeni encapsulated into silica–alginate (Si–Al) and alginate capsules under adverse winemaking conditions (high alcohol concentration, low pH and low temperature). Mechanical and chemical stability of capsules was also monitored. Methods and Results Silica–alginate capsules showed higher malolactic activity and better mechanical and chemical stability than untreated alginate capsules. Their capacity for bacteria retention was similar to that of alginate capsules. Ethanol, pH and fermentation temperature had great influence on fermentative behaviour of free and encapsulated bacteria. Under extreme winemaking conditions, such as high ethanol concentration (13–16%), low pH (3.0–3.3) and low fermentation temperature (13–15°C), the highest consumption of malic acid was achieved using Si–Al capsules. Free and encapsulated bacteria showed similar fermentative profiles in the presence of variable concentration of malic acid and nitrogen in wine. Conclusions Encapsulated O. oeni in Si–Al gels enhanced MLF in wines with a high concentration of ethanol and low pH and at low fermentation temperature. Moreover, inclusion of an inorganic network of silicates significantly improved the mechanical robustness and chemical stability of alginate capsules. Significance of the Study The application of Si–Al-encapsulated bacteria is an interesting strategy to improve MLF in warm climates where wines have higher ethanol concentration and in cool climates with higher acidity and low fermentation temperature.

Molecular Cloning, Expression and Characterization of Oenococcus oeni Priming Glycosyltransferases.

Oenococcus oeni is the main bacterial species that drives malolactic fermentation in wine. Most O. oeni strains produce capsular exopolysaccharides (EPS) that may contribute to protect them in the wine hostile environment. In O. oeni genome sequences, several genes are predicted to encode priming glycosyltransferases (pGTs). These enzymes are essential for EPS formation as they catalyze the first biosynthetic step through the formation of a phosphoanhydride bond between a hexose-1-phosphate and a lipid carrier undecaprenyl phosphate. In many microorganisms, mutations abolishing the pGT activity also abolish the EPS formation. We first made an in silico analysis of all the genes encoding putative pGT over 50 distinct O. oeni genome sequences. Two polyisoprenyl-phosphate-hexose-1-phosphate transferases, WoaA and WobA, and a glycosyltransferase (It3) were particularly examined for their topology and amino acid sequence. Several isoforms of these enzymes were then expressed in E. coli, and their substrate specificity was examined in vitro. The substrate specificity varied depending on the protein isoform examined, and several mutations were shown to abolish WobA activity but not EPS synthesis. Further analysis of woaA and wobA gene expression levels suggests that WoaA could replace the deficient WobA and maintain EPS formation.

Control of Malolactic Fermentation in Wine. A Review

Malolactic fermentation (MLF) is conducted by lactic acid bacteria (LAB) and refers to the decarboxylation of Lmalate to L-lactate. This secondary fermentation is difficult to control and is mainly driven by Oenococcus oeni. Uncontrolled MLF, especially in wines with a high pH, which are typical of warmer viticultural regions, may render the wine unpalatable or even cause spoilage. In this review we focus on wine compounds and emphasise factors that affect the growth of 0. oeni and MLF, and discuss practical applications. We also explore alternative technologies that may enable better control over MLF.

Genome-Scale Reconstruction of the Metabolic Network in Oenococcus oeni to Assess Wine Malolactic Fermentation.

Oenococcus oeni is the main responsible agent for malolactic fermentation in wine, an unpredictable and erratic process in winemaking. To address this, we have constructed and exhaustively curated the first genome-scale metabolic model of Oenococcus oeni, comprising 660 reactions, 536 metabolites and 454 genes. In silico experiments revealed that nutritional requirements are predicted with an accuracy of 93%, while 14 amino acids were found to be essential for the growth of this bacterial species. When the model was applied to determine the non-growth associated maintenance, results showed that O. oeni grown at 12% ethanol concentration spent 30 times more ATP to stay alive than in the absence of ethanol. Most of this ATP is employed for extruding protons outside of the cell. A positive relationship was also found between specific consumption rates of fructose, amino acids, oxygen, and malic acid and the specific production rates of erythritol, lactate, and acetate, according to the ethanol content of the medium. The metabolic model reconstructed here represents a unique tool to predict the successful completion of wine malolactic fermentation carried out either by different strains of Oenococcus oeni, as well as at any particular physico-chemical composition of wine. It will also allow the development of consortium metabolic models that could be applied to winemaking to simulate and understand the interactions between O. oeni and other microorganisms that share this ecological niche.

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.

Phage-host interactions analysis of newly characterized Oenococcus oeni bacteriophages: Implications for malolactic fermentation in wine

Nowadays, only few phages infecting Oenococcus oeni, the principal lactic acid bacteria (LAB) species responsible for malolactic fermentation (MLF) in wine, have been characterized. In the present study, to better understanding the factors affecting the lytic activity of Oenococcus phages, fifteen O. oeni bacteriophages have been studied in detail, both with molecular and microbiological methods. No correlations were found between genome sizes, type of integrase genes, or morphology and the lytic activity of the 15 tested phages. Interestingly, though phage attack in a wine at the end of alcoholic fermentation seems not to be a problem, it can indeed represent a risk factor for MLF when the alcohol content is low, feature that may be a key point for choosing the appropriate time for malolactic starter inoculation. Additionally, it was observed that some phages genomes bear 2 or 3 types of integrase genes, which point to horizontal gene transfer between O. oeni bacteriophages.

Use of starter cultures ofLactobacillusto induce malolactic fermentation in wine

Background and Aims Oenococcus oeni is the lactic acid bacteria species that best adapts to harsh wine conditions. This species is currently the main component of the malolactic starter cultures used in winemaking. Other species of lactic acid bacteria, however, such as Lactobacillus, can also conduct the malolactic fermentation, especially in low acidity wines. This study aimed to identify suitable Lactobacillus strains and inoculation methods to undertake the malolactic fermentation in wines with pH > 3.5. Methods and Results Six Lactobacillus strains of species L. mali (E4634), L. paracasei (E4539, E4541), L. plantarum (E4538, E4608) and L. satsumensis (E4555) were selected for their good growth performance and high malolactic activity in grape must, although these characteristics differed among them. Freeze-dried starter cultures were obtained for strains E4538, E4608, E4555 and E4634. These starter cultures showed a high rate of malic acid consumption in grape must. Inoculation of the Lactobacillus strains in grape must, prior to fermentative yeasts, was selected as the best inoculation strategy to promote the malolactic fermentation. Conclusions Inoculation of the starter cultures of the selected Lactobacillus strains before yeast inoculation in grape must effectively allows malolactic fermentation in wines. Significance of the Study Inoculation of Lactobacillus strains in grape must, prior to fermentative yeasts, is an effective alternative to O. oeni to undertake malolactic fermentation in wines of pH > 3.5.

Bacterial Inoculation Strategies for the Achievement of Malolactic Fermentation in High-alcohol Wines

The purpose of this work was to study the induction of malolactic fermentation (MLF) in a wine that does not often support malic acid deacidification because of its high alcohol content. Amarone wine, known for its high alcohol content, served as a model. Simultaneous and sequential alcoholic and malolactic fermentation (AF/MLF) were conducted by direct inoculation of bacteria, which resulted in successful MLF in wines containing approximately 16% (v/v) alcohol. At higher ethanol contents, stuck MLF occurred because of growth inhibition. To overcome this technological problem, the performance of bacteria was tested in wine containing approximately 17% (v/v) ethanol using a starter preparation consisting of cells acclimatised in a wine-water solution (1:1) for 24 h and 48 h respectively. Total l-malic acid depletion was recorded when the bacterial cells that had been acclimatised for 48 h were inoculated simultaneously with yeast to conduct AF. The method by which the bacterial cultures are prepared and the time of inoculation affects the efficacy of MLF in high-alcohol wines. The inoculation of yeasts with acclimatised bacteria before AF seems to be a valid strategy to obtain complete MLF in high-alcohol wines.