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Problems can often arise at the beginning of the second fermentation (prise de mousse) of sparkling wines, such as no start, a long lag period or slow fermentation. These problems are generally associated with yeast stress when inoculated in a base wine with high ethanol content and low pH. However, few studies focus on sulphites, which are often added to base wines to prevent malolactic fermentation, microbiological instability, and wine oxidation. This study aimed to evaluate the joint effect of ethanol and sulfur dioxide on yeasts during the second fermentation. For this purpose, yeasts (Saccharomyces cerevisiae EC1118) were subjected to ethanol, sulfur dioxide and ethanol/sulfur dioxide at the beginning of fermentation, and their vitality and viability, as well as the accumulation of intracellular reactive oxygen species and intracellular pH, were evaluated by flow cytometry. Furthermore, the expression of genes involved in sulfur transport and metabolism was determined. The results showed high mortality, ROS accumulation and intracellular pH reduction in fermentations with both ethanol and sulfur dioxide. The negative effect of ethanol, sulfur dioxide and ethanol/sulfur dioxide on yeasts was found to be dose-dependent and high in those commonly found in some base wines. Cells treated with ethanol/sulfur dioxide showed over-expression of genes involved in sulphite transport (SUL1 and SUL2), efflux pump (SSU1 and FZF1) and metabolism of sulfur amino acids (MET14). Altogether, our data indicate that ethanol and sulfur dioxide have a synergistic effect on yeasts, which may be the root cause of the problems encountered at the beginning of the second fermentation of sparkling wines, and should thus be seriously taken into consideration by winemakers.

This work aims to develop a new enzymatic method for analysing fumaric acid in wines. Fumaric acid is a very effective lactic acid bacteria inhibitor that is widely used in the food industry as an additive (E297). Using it to inhibit malolactic fermentation has recently been authorized by the International Organisation of Vine and Wine (OIV). However, the official analytical method for fumaric acid involves the classical HPLC method for organic acids, which is overly complicated for use in wineries. This research proposes a very simple and efficient enzymatic method that allows the simultaneous determination of L-malic acid and fumaric acid. The method uses a commercial enzymatic kit for L-malic acid and adds a supplementary step in which the fumarase enzyme is added to transform fumaric acid into L-malic acid. The results obtained show that this method could be used in different media (synthetic solution, white wine, red wine and white grape juice). This article also proposes a modification of the enzymatic method to be applied when the L-malic acid concentration of the sample is very high.

The yeast Lachancea thermotolerans can produce lactic acid during alcoholic fermentation (AF) and thereby acidify wines with insufficient acidity. However, little is known about the impact of L. thermotolerans on Oenococcus oeni, the primary lactic acid bacterium used in malolactic fermentation (MLF). This study explored the impact of sequential cultures of L. thermotolerans and Saccharomyces cerevisiae on MLF performance in white and red wines. Four L. thermotolerans strains were tested in Sauvignon blanc with sequential S. cerevisiae inoculation, compared to an S. cerevisiae control and the initially un-inoculated treatments. The L. thermotolerans wines showed large differences in acidification, and progression of MLF depended on lactic acid production, even at controlled pH. The highest and lowest lactic acid producing strains were tested further in Merlot fermentations with both co-inoculated and sequentially inoculated O. oeni. The low lactic acid producing strain enabled successful MLF, even when this failed in the S. cerevisiae treatment, with dramatically quicker malic acid depletion in O. oeni co-inoculation than in sequential inoculation. In contrast, a high lactic acid producing strain inhibited MLF irrespective of the O. oeni inoculation strategy. In a follow-up experiment, increasing concentrations of exogenously added lactic acid slowed MLF and reduced O. oeni growth across different matrices, with 6 g/L of lactic acid completely inhibiting MLF. The results confirm the inhibitory effect of lactic acid on O. oeni while highlighting the potential of some L. thermotolerans strains to promote MLF and the others to inhibit it.

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.

Aim: The high-throughput sequencing methods have revolutionized the study of the microbiota in different matrices including those of the grapevine production chain. DNA extraction is a crucial step in the sample processing. In this study, we compared different DNA purification methods and two primer sets for 16S rRNA gene metabarcoding to evaluate the best protocol to explore the wine microbiota by metabarcoding.Methods and results: We collected a wine from Barbera grapes after malolactic fermentation previously inoculated by Oenococcus oeni starter. The same sample was used to evaluate the best performing protocol to study the wine microbiota. DNA was purified using nine different methods and then amplified for the 16S rRNA gene with two primer sets (according to Illumina or Earth Microbiome Project protocols). The obtained amplicons were then sequenced in a single sequencing session on an Illumina MiSeq. We evaluated the best protocol considering DNA concentration and purity, alpha (Observed species) and beta diversity from metabarcoding analysis.The sequencing generated 36,031,756 reads in total. Although no statistically significant difference was observed between purification methods or primer sets, better results were obtained with phenol-chloroform DNA purification combined to Earth Microbiome Project primers.Metabarcoding was able to highlight the domination of the inoculum, O. oeni, representing the main species of the analyzed wine microbiota.Conclusion: Our data show that, for the tested wine, metabarcoding output is more influenced by the primer set than by the DNA purification method. Moreover, the metabarcoding detected that O. oeni represents the main species, evidencing the domination of the inoculum done with lyophilized commercial preparation of this species. Other lactic acid bacteria are present at a much lower abundance.Significance and impact of the study: This is the first report applying the 16S rRNA gene metabarcoding to study the microbiota of wine. For this reason, the evaluation of alternative methods for DNA processing is essential for future research using this innovative methodology.

Aim: The aim of this study was to evaluate the microbial population present in red grape must and wine using polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE).Methods and results: Red wine from the cultivars Pinotage and Merlot were produced and samples taken throughout alcoholic and malolactic fermentation (MLF). PCR fragments were resolved by DGGE and unique fingerprints were obtained for the bacteria and yeasts present in the wines. Lactobacillus plantarum, Enterobacter sakazakii (Cronobacter sp.) and Pantoea agglomerans were present in the Pinotage during both alcoholic and MLF, and in both inoculated and spontaneous fermentations. E. sakazakii (Cronobacter sp.) and P. agglomerans were also observed in the Merlot wines during alcoholic fermentation as well as MLF. Saccharomyces cerevisiae was the most dominant yeast observed in Pinotage, and was the only yeast observed in Merlot. This yeast was observed until the end of MLF.Conclusions: Results showed that the microbial flora that participates in the winemaking process is more diverse than commonly thought.Significance and impact of the study: This method may serve as an alternative to conventional microbiological methods for the identification of the microbial species in red grape must and wine.

Aims: This study aimed at acquiring knowledge of the evolution of anthocyanins from grape to wine and the possibility of deducing the varietal origin of a wine from its anthocyanin profile.Methods and results: The anthocyanin « fingerprint », or profile, of a series of autochthonous Sicilian accessions and their respective young wines was determined by HPLC-DAD in 2008. Data were evaluated by taking into account the evolution of the percentages of side-ring dioxygenated and tri-oxygenated anthocyanins, the ratio between acetylated and p-coumaroylated derivatives, and the principal component analysis (PCA) results. From grape to the end of alcoholic fermentation, the percentages of 3-glucosides of cyanidin, peonidin, delphinidin and p-coumaroylglucosides decreased, whereas those of malvidin-3-glucoside, acetylated derivatives and, in some cases, petunidin-3-glucoside increased. In many but not all wines, after malolactic fermentation, the percentages of p-coumaroylated derivatives decreased further and those of cyanidin- 3-glucoside and acetylated derivatives increased ; less variation was observed in malvidin-3-glucoside values.Conclusions: From grapes to young wines, the anthocyanin profiles varied mainly because of the activity of polyphenol oxidase (PPO), yeasts and lactic acid bacteria. However, they were substantially retained in wines from varieties in which tri-oxygenated side-ring anthocyanins prevailed, but were dramatically different from the respective grapes in wines from varieties rich in di-oxygenated anthocyanins, or in which the di-oxygenated forms prevailed over the tri-oxygenated ones. PCA analysis confirmed these findings.Significance and impact of the study: Only in varieties in which the anthocyanin profile of grapes is characterized by low percentages of dioxygenated side-ring forms is it possible to gain information about the varietal origin of a young wine by comparing the profiles of both grape and wine. However, great care is required.

Oenococcus oeni is the bacterium most often associated with spontaneous malolactic fermentation (MLF) of wine. During MLF, malic acid is transformed into lactic acid and several metabolites are modified, modulating wine’s total acidity and improving its sensory properties. Previous works have suggested that certain genetic groups of O. oeni strains are associated to different kinds of products. In the present study we have spotted two groups of strains isolated mainly from Burgundy wines, one associated to red wines and the other to white wines. Sequencing 14 genomes of red and white wine strains revealed that they share a common ancestor that probably colonised two different substrates –red and white wine-associated environments–, diverging over time and disseminating to various regions. Their capacity to perform MLF and modify the volatile profile of wine was determined by fermenting a chardonnay wine and analysing its volatile fraction with a non-targeted metabolomics approach by GC-MS. The strains had a different impact on the volatile composition depending on their group of origin. These results show for the first time a correspondence between the product of origin of the strains and the volatile profile of the wines they produce. Furthermore, the genetic features that might be implied in these different phenotypes are examined.

Six malolactic starters were experimented. The objective was an evaluation of the capacity of each one to settle in different wines. First we obtained the genomie DNA fingerprinting with Not I a rare cutting enzyme after pulse field gel electrophoresis (PFGE). Previous results with Leuconostoc oenos strains of our collection showed that this enzyme was the most appropriated for identification at the strain level. However, we did not obtain six different patterns, three starters showed the same. Therefore Apa I and Sfi I patterns were also established. Contrary to the expected results A, B and C finally had the same fingerprinting with the three enzymes. Another investigation was conduced using the Southem hybridization of EcoRI restricted DNA with the φMC10 prophage DNA as probe. Our again all three starters showed the same pattern. They probably were prepared with the same strain. Wines in vat or barrels were inoculated just after racking. Malolactic fermentation had already begun in some of them before inoculation. However malic acid was degraded more quickly in all the inoculated wines than in the controls. The PFGE patterns were obtained from bacteria isolated from wines at the end of malolactic fermentation. In 15 cases of 21, the pattern of the starter appeared alone or superimposed with other bacteria. This proved that, even in the worst conditions of this experimentation, where the indigenous microflora was very abundant and active malolactic starters could survive and predominate. The influence of these starters on the sensorial quality of wines needs to be studied.

The red wine« Appellation d'Origine Naoussa de qualité supérieure » stemming from the « Xynomavro » vine cultivated in this wine-growing region of western Macedonia (Greece) needs the advantages coming of malolatic fermentation. This wine, in spite of its incontestable qualities has a strong astringent and a high level of acidity, the malic acid contributing essentially to these two characters.Because of the interest malolactic fermentation presents for this region, we have collaborated with the wine-producters to insure, at first, the generally admitted conditions which favour the evolution of malolactic fermentation, then to study it.The latter includes : a) the control of the conversion of malic acid into lactic acid by chromatography paper and b) the enumeration, isolation and identification of lactic acid bacteria.During this two-year-long research, 187 samples have been taken from 50 different tanks during 12 samplings. The isolation of lactic acid bacteria was done by plating dilutions of wine samples and their identification with the API system and other physico-chemical and enzymatic methods.Data processing has been done by computer and given a strain group distribution according to their similarity.The results show that the homofermentative lactic acid bacteria - mainly Lactobacillus plantarum - are more numerous than the heterofermentative ones before the malolactic fermentation. They decrease during the fermentative process and disappear with its completion.The heterofermentative lactic aeid baeteria, whose main specie is Leuconostoc oenos evolves inversely.A low increase of Pediococcus has been observed too after the beginning of malolactic fermentation. A statistical analysis confirms the main points of this research.The application of malolactic fermentation becomes more efficient the second year; it is favoured in press-wines and for temperatures kept at 20-25°C; the bacteria population is also increased.The persued reduction of total acidity in wines, which underwent malolactic fermentation, was considerable.

Aims: The aim of this study was to develop a colorimetric test to determine Oenococcus carbohydrate consumption profile.Methods and results: A semi-defined growth medium which enabled efficient bacterial growth and medium acidification in the presence of glucose was developed. The inoculum size and the presence of citrate in the medium were optimized. Acidification of the medium was revealed by adding bromocresol green and was shown to be perfectly correlated with D-glucose, D-fructose, L-arabinose, D-xylose, L-rhamnose, D-cellobiose or D-trehalose consumption by 23 distinct O. oeni strains and one O. kitaharae strain (confirmed with HPLC analysis).Significance and impact of the study: Efficient bacterial growth is essential before malic acid degradation occurs in wine. The method developed here will facilitate the comparison of numerous O. oeni strains and their ability to grow by using the various carbohydrates present in wine prior to malolactic fermentation.

The MEXTAR® software is a mathematical tool able to predict the physicochemical state of a wine. Solving rigorously physicochemical equilibria, including complexation ones, it enables to simulate various operations endured by a wine: tartaric precipitations, acid addition, acid removal, malolactic fermentation.Using only classical chemical analytic determinations, namely, pH, total acidity, tartaric acid and potassium content, MEXTAR® computes the physicochemical state of a wine before and after the endured operation: pH and total acidity, tartaric salts supersaturation and the associated saturation temperature taking into account both the ionic activity coefficients and the complexed fraction of the involved ions, amount of tartaric salts that can be precipitated to obtain either a supersaturation chosen by the user at a given temperature or a chosen final saturation temperature, required amount of acidification or desacification chemical substances to reach a chosen pH or total acidity.The article describes the theoretical background upon which MEXTAR® has been developed and details the computation procedures associated to the simulation of the operations described above. Iterative processes are used to determine the various parameters enounced above. They aim in particular at the verification of the electroneutrality of the sample. However, at first electroneutrality is rarely verified because the wine chemical analytical determination is never exhaustive. Therefore, we introduce the concept of «vinic acid» to compensate the negative ion deficit of usual wine chemical analytical determination. This «vinic acid» is describe as a diacid with a fixed second dissociation constant (pK2 = 5) and a first dissociation constant determined automatically by MEXTAR®. This enables MEXTAR® to simulate accurately experimental tartaric precipitations for several samples. Thanks to the predicting capacity of MEXTAR®, the correlation between the total polyphenol content (IPT) in red wines and the difficulty to stabilise high IPT wine with respect to tartaric salts is confirmed. Finally, malolactic fermentations are also well simulated. A validation of MEXTAR® for the acid addition or removal has yet to be done when reliable experimental data are available.

This study presents a rapid and specific microscopie technique for detecting and identifying populations of lactic acid bacteria in musts, wines, and inoculum starter cultures. Through the use of fluorescent antibodies, this procedure can be performed in less than two hours, and it is effective with Leuconostoc, Pediococcus and Lactobacillus concentrations as small as 102 cells/ml. Implementation of this technique will assist winemakers in controlling malolactic fermentations and in preventing lactic acid bacterial spoilage.

Weak malolactic fermentations in wine making are often attributed to the medium constituents such as ethanol and SO2. However, some authors report that the interactions between Saccharomyces cerevisiae and Leuconostoc oenos could be responsible too.The aim of this work is to characterize the influence of S. cerevisiae on the growth of L. oenos. First, a qualitative study was performed on solid medium containing 8 g/l of L-malic acid. In this case, L. oenas was mixed to M.R.S agar and poured into Petri plate, then S. cerevisiae was seeded in rings on the agar surface. After incubation, the coculture result in an inhibition of L. oenos growth by S. cerevisiae.In order to quantify this phenomenon, a culture of L. oenas was carried out on liquid medium prefermented by S. cerevisiae (supplied with M.R.S nutrients) and was compared with a control on M.R.S medium. Both contained 8 g/l of L-malic acid. This technique allows to separate the microorganisms in order to avoid competitions between the strains.The prefermented medium affects L. oenos growth and the malolactic fermentation, but the results depended on the pH and the temperature. Globally, low pH (3,4) and high temperature (30°C) conditions increased the inhibitory effect of the prefermented medium. This medium contains inhibitory molecules for L. oenas. In agreement with some authors, those molecules could be the medium chain fatty acids (C8 to C12) released in the medium by S. cerevisiae because of their pH-dependent toxicity. However, those results may be different with other couples of strains of S. cerevisiae and L. oenos.

A considerable malolactic fermentation can be obtained by adding to musts some commercial active dry yeasts. An addition of thiamine may increase this phenomenon. When addition of Schizosaccharomyces pombe takes place at the end of alcoholic fermentation consequences of the metabolism of this yeast strain about organoleptic properties of wine are considerably decreased. With a well-advised utilization of these yeasts it is possible to obtain a considerable deacidification of wines.

Affected grape bunches contain both shrivelled and non-shrivelled berries. The juice harvest from the former is less than one-half that from the letter. The wine obtained from shrivelled grapes is low in alcohol and highly acid. The alcoholic fermentation occurs with difficulty and the malolactic fermentation does not occur naturally. The wine is unharmonious and unbalanced. In addition, it as a lesser amount of higher alcohols and of higher esters.

L'addition au vin d'écorces de levure (0,2 g par litre) permet une stimulation de la fermentation malolactique. Leur action s'exerce en augmentant la population bactérienne et surtout en retardant et atténuant la phase de déclin. Le traitement du vin par des polysaccharides (alginates, polysaccharides extraits de vin) ou par l'extrait de levure accelere aussi le processus de dégradation de l'acide malique. Ces adjuvants agissent probablement en limitant l'action inhibitrice de certains métabolites levuriens tels les acides gras.+++Addition of yeast ghosts to wine (0,2 g per liter) stimulates the malolactic fermentation. The bacterial population is increased furthermore the declin phase is delayed. Addition of polysaccharides to wine (alginate, polysaccharides extracted from wine) or yeast extract also accelerate the malic acid degradation. These additions probably limit the inhibition by some products of the yeast metabolism like fatty acids.

La durée d'activité très limitée dans le temps des protéases du raisin est confirmée en vinification en blanc, mais une nouvelle activité protéolytique peut être caractérisée à 37 °C et à pH 3,7 dans le moût au cours de la fermentation alcoolique.Cette activité doit être attribuée à des protéases exocellulaires libérées dans le milieu des levures. Parmi les souches essayées S. bayanus a une activité protéolytique plus importante que S. cerevisiae, ce qui se traduit par l'obtention de vins plus riches en azote soluble, donc plus stables du point de vue protéique et vraisemblablement d'une meilleure fermentescibilité malolactique.+++The short time of activity of grape proteolytic enzymes is confirmed in fermentation off skins. However, a new proteolytic activity has been characterized at 37 °C and at pH 3.7, occuring in alcoholic fermentation of the must.This activity comes from exocellular proteolytic enzymes released by the yeasts in the medium. Among tested strains S. bayanus has a proteolytic activity greater than S. cerevisiae; so the soluble nitrogen content of wines is higher. According to this, proteolytic stability is better as, probably, malolactic fermentation capabilities.

L'aboutissement logique des études sur les bactéries de la fermentation malolactique est la maîtrise de cette transformation. On peut espérer la contrôler grâce à une bonne connaissance des exigences bactériennes, à la condition bien entendu que la microflore spontanée ait les qualités requises. Or cette microflore naturelle est variée et dispersée en de multiples espèces qui ne semblent pas uniformément répandues. Dans ces conditions, on peut craindre que certains vins conservent de l'acide malique par insuffisance quantitative ou qualitative de l'ensemencement naturel.

Les lipides extraits des membranes de bactéries lactiques du vin sont séparés par chromatographie sur couche mince en phospholipides, glycolipides et lipides neutres. Le pourcentage de répartition de chaque classe varie, non seulement en fonction des souches, mais aussi selon leurs conditions de culture.+++Lactic and bacteria of wines are inhibited in fermenting must. Éthanol, fatty acids and other unknow yeast metabolites are responsible for the loss of viability and malolactic activity. Prior results suggest that they induce an alteration of the plasma membrane. Owing to their nature, we assume the lipid constituants to be the target of the inhibitors. So, we have undertaken the study of the membrane lipids of two strains, L. plantarum and L. oenos.After extraction, the samples are analysed by HPTLC. Comparing the strains, the patterns reveal small differences in the distribution of phospholipids, glycolipids and neutral lipids.Some culture factors were investigated for their influence. Within the cell cycle, the distribution of the various classes changes according to the period of incubation. If ethanol is added in the culture medium, neutral lipids decrease by 25 to 50 per cent and glycolipids increase by 30 per cent depending on the age of the culture. Phospholipids remain quite constant.Further researchs are in progress to test the influence of others factors. These results might explain the loss of viability of the bacteria varies when they are added to wine, as well as the variable response to yeast antagonism according to the strains.

Stimulation of malolactic fermentation can be obtained by adding to wine an industrial biomass of L. oenos. Before use, deepfreeze bacteria need to be reactivated according to some special conditions which have been studied. Yeasts concerned in alcoholic fermentation have an influence on malolactic fermentation capacity of wines ; this fact is in connection with some differences observed in wine technology. Among the strains of commercial dry yeasts, the influence of S. cerevisiae is lower than that of S. bayanus.

Les interactions entre levures et bactéries lactiques sont déterminées par la nature des souches en présence, leur population et la constitution chimique du moût et du vin. Normalement, pendant la fermentation, les bactéries lactiques sont inhibées par les produits du métabolisme levurien. Mais dans certaines conditions, la croissance précoce des bactéries provoque une chute de la viabilité des levures et, par conséquent, un ralentissement ou un arrêt de la dégradation des sucres. Tous les mécanismes mis en jeu ne sont pas connus.+++ln wine-making, lactic acid bacteria succeed to yeasts to promote the malolactic fermentation (MLF), after the completion of the alcoholic fermentation. Yeasts are better adapted than bacteria to growth in grape must. So their development easily starts and the alcoholic fermentation triggers soon after the harvest. In the same time, the natural bacterial inoculum decreases, due to an antagonism between these two microorganisms. ln addition to ethanol, fatty acids synthesized by actively growing yeasts exert their toxic effect against lactic acid bacteria. At the end of the alcoholic fermentation, the remaining bacterial population (104 to 103 cell.p.ml) begins growing. It is mainly constitued by Leuconostoc oenos more resistant than lactobacilli. At that time, autolysis products from decline phase yeasts act as bacterial growth factors. Then, when the bacterial population is large enough (about 106 cell.p.ml) MLF happens. So, normally, MLF follows alcoholic fermentation within a few days. But in some conditions, the interactions between yeasts and bacteria do not work in this way. If the weather is particularly hot and dry during the last weeks of the maturation, the pH of the must may be high and generally sulfiting is lowered as there are no rotten grapes. Growing conditions are better than usually for bacteria that, in such a case, compete with yeasts. The bacterial population reach a sufficient level that can actually increase the yeast decline rate. Hydrolase activities of bacteria against yeast cell walls may occur. That leads to a fermentation stuck. These antagonism effects between yeasts and lactic acid bacteria in fermenting must control the wine-making process. Their extent vary with must composition even with both yeast and bacteria strains. Further research is needed to clear up involved machanisms.

Cette note présente les premiers résultats obtenus pour reproduire artificiellement et de manière utile technologiquement, les conditions naturelles optimales précédemment mentionnées qui favorisent la fermentation malolactique.

L'addition d'écorces de levure ou de lies fraîches dans un milieu alcoolique entraîne une augmentation de la teneur en acides gras libres suivie d'une diminution progressive; les lies fraîches ne cèdent pratiquement pas d'acide palmitoléique, inhibiteur de l'activité malolactique.La teneur en acides gras libres augmente au cours de la macération des raisins rouges, mais elle est peu affectée par la macération préfermentaire des raisins blancs.Par ailleurs, les teneurs des vins en acides gras libres varient en fonction du cépage.+++This work deals with the free latty acids (C12 to C18:3) content of different mediums; these substances seem to have an activity on the alcoholic and the malotactic fermentations.ln a first step we have studied the influence of the addition to the medium of yeast ghosts and fresh yeast cells (lees).We have also determined free fatty acids in red wines (Merlot, Cabernet Sauvignon and Malbec) in function of the time of maceration (0 to 10 days) and in white wines (Semillon and Sauvignon blanc) in function of skin contact time (0 to 6 hours) and SO2 which have been added, or not, to the medium belore skin contact.The free fatty acids were extracted from 20 ml of wine by three times 10 ml of dichloromethane ; the organic phase was concentrated under vacuum to 1 ml. The extract was analysed by gas chromatography with a capillary CP WAX 57 column from Chrompack (10 m x 0,22 mm); the chromotograph was equiped with a spitless system injector.The addition of yeast ghosts and fresh lees into an alcoholic medium causes a temporary increase of the free fatty acids followed by a constant decrease. However, the fresh lees do not give palmitoleic acid which is an inhibitor of the malolactic activity.The time of maceration plays a positive part on the free fatty acids content of the wine; this is more important for the red wines than for the white ones.The free fatty acids content of the wines changes according to the grape variety.

A method is described, by wich the malolactic fermentation is induced through direct inoculation with an industrial preparation of L. plantarum (CHL2). When inoculated into must, this homofermentative lactobacillus metabolizes very little sugar, and is unable to produce acetic acid. The inoculated population of bacteria has a high malolactic activity even at low pH values, which is closely related to cell viability. The decarboxylation of malic acid starts immediately after inoculation and proceeds at a high rate during the first days of alcoholic fermentation. As the alcohol concentration rises, the lactobacilli are gradually eliminated, so inoculation with the malolactic culture has to be done very early in the vinification process, in order to achieve a complete malolactic fermentation. At the end of alcoholic fermentation, the malolactic fermentation is finished, and the bacterial inoculum has been completely killed of.The efficiency of this new process is demonstrated in laboratory experiments and field trials with different types of must and different vinification procedures. The advantages of the method, which the winemakers would particularly appreciate, include the direct inoculation mode and considerable gain in terms of production time.