CH662819A5 - BACTERIAL COMPOSITION FOR INDUCING MALOLACTIC FERMENTATION BY A STRAIN OF MICROORGANISMS OR ITS MUTANTS, METHOD FOR TREATING WINE USING THE SAME AND WINE THUS TREATED. - Google Patents

Description

The present invention relates to a bacterial composition for inducing malolactic fermentation by a strain of microorganisms, or its mutants, a process for treating wine using this composition and the wine thus treated.

The composition is characterized by the characterizing part of claim 1, the process by the characterizing part of claim 8 and the wine by claim 11.

In general, the invention relates to the fermentation processes as well as the ingredients and compounds which are used there. The invention relates to a new composition of microbiological origin useful in the fermentation processes, in particular in the production of wine and methods for its preparation and its use in fermentation.

Non-sparkling wines are prepared by fermentation of the fruit juice sugars. After completion of the primary fermentation, many secondary fermentations can occur. Most of these secondary fermentations are undesirable and affect the wine. All are undesirable when they occur after bottling.

The most common secondary fermentation is malolactic fermentation in which microorganisms cause conversion of malic acid to lactic acid, the sugars being transformed into acids and other organic or mineral compounds. The malolactic fermentation must be completed before bottling the wine or prevented with a very high degree of security. If it occurs after bottling, the release of gas and the development of bacteria alter the product.

One can, to avoid malolactic fermentation, for example, filter to eliminate the organisms that cause it and promote it or pasteurize the wine to deactivate microorganisms. Both are expensive, reduce the quality of the wine, for example by giving it unwanted flavors, and in some cases they fail. Malolactic fermentation is considered to be very desirable in red wines and in certain white wines. It causes a natural decrease in acidity and improves the organoleptic qualities.

It is therefore preferred to promote and ensure the completion of the malolactic fermentation before bottling by adding to the wine microorganisms producing the malolactic fermentation. A superior and stable product is thus obtained.

The phenomena of malolactic fermentation and the importance of this fermentation in winemaking are well known. In many wine regions, native microflora can cause natural malolactic fermentation. However, it is common for fermentative organisms not to start quickly and there is always a risk that unwanted organisms will become established and cause unwanted side effects.

To a certain extent, a controlled malolactic fermentation was carried out before bottling by using added cultures. However, the cultures of microorganisms that have been used for this purpose lack certain characteristics, including ease of use and vigor of culture. The cultures previously used must be added to the wine at a pH of more than 3.3 and, during and after the addition, the wine must be maintained at a temperature of 18 to 22 ° C. These two compositions may not always be possible to obtain or desirable for obtaining superior quality wines.

In many cases, the pH of the wine, when it is desired to induce malolactic fermentation, is less than 3.3 and it is particularly undesirable to adjust the pH at this stage. The temperature for storing wine in the tanks before bottling is preferably much lower than 18 ° C, for example close to 10 ° C. In addition, it is obviously desirable in industrial practice to carry out the malolactic fermentation as quickly as possible to increase the production rate and reduce the storage time.

The licensee has isolated a new strain of microorganisms here called Leuconostoc oenos B-44-40 which, surprisingly, effectively induces malolactic fermentation when added to wine. The new strain is more vigorous than the bacterial strains of Leuconostoc previously known and used for this purpose. In addition, it energetically causes malolactic fermentation at temperatures below 18 ° C and at least as low as 10 ° C and at low pH below 3.3, for example lowering to about 3.0.

In addition, antimicrobial resistant mutants of Leuconostoc oenos B-44-40, ATCC 31668, hereinafter called Leuconostoc Bio Logicals 44-40 ATCC 31689, 31690, 31691, 31692, 31693, were prepared in biologically form. viable, in combination with a biologically acceptable support, which are also effective in inducing malolactic fermentation, under the same conditions and which, moreover, allow monitoring and quality control of the fermentation process.

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The support is preferably constituted by remains of the media used for the biological development of the microorganism.

The media residues preferably include glutamic acid and / or its biologically acceptable salts.

The microorganism and glutamic acid and / or its salts are preferably in a lyophilized form, glutamic acid and / or its salts acting as cryoprotectors of the lyophilized microorganism to maintain it in a biologically active form.

The composition is preferably mixed with an at least equal weight of a fluid material in particles suitable in biology to increase the volume and promote the dispersion of the composition in an aqueous medium.

The media remains preferably contain an acid which behaves like a cryoprotective in the subsequent lyophilization of the cells, this acid being chosen from a-ketoglutamic acid, L-malic acid, DL-malic acid, acid L-aspartic, DL-a-aminopimelic acid, a-methyl L-glutamic acid, malonic acid, N-acetyl L-glutamic acid, N-dimethyl L-glutamic acid that, N-dimethyl L-aspartic acid, L-cysteic acid, cys-tein, DL-threonine, acetylglycine, DL-pyrrolidone-5 carboxylic acid-2, acid D-glutamic, DL-glutamic acid, L-glutamic acid, aminomalonic acid, tartronic acid and lysine.

Unexpectedly, these cryoprotectants have been found to ensure the viability of the cultures after lyophilization, so that the composition can be used reliably to induce malolactic fermentation by addition to wine, at the appropriate stage of vinification. Glutamic acid or a salt thereof can be added as such or formed in situ before or during the culture of the microorganism, for example by hydrolysis or chemical reaction of a proteinaceous material or the like or by hydrolysis of proteins or peptides containing glutamic acid in the culture medium.

The new bacterial strain here called Leuconostoc oenos B-44-40 ATCC 31668 has been isolated from wine samples that have undergone or undergone extensive malolactic fermentation, producing good quality wine with no aftertaste or other signs of deterioration. It is cultivated and isolated using generally accepted microbiological culture techniques. Mixed cultures from a wine sample are grown on a substrate containing lactic acid, glucose or the like to select and isolate the fast growing cultures. The exact taxonomic identity of culture is not currently certain, due to the well-known difficulties of classification and definitions that exist in this field. The bacterial strain B-44-40 certainly belongs to the family of lactic acid producing bacteria. There remains some uncertainty as to whether it should be considered to belong to the genus Leuconostoc species oenos or to the genus Pediococci. In all cases, the reservations concerning the taxonomy of the bacterial strains B-44-40 and Bio Logicals 44-40 apply to the rest of this description.

The taxonomic description of B-44-40 will now be made.

Taxonomy

B-44-40 was classified as a Leuconostoc oenos based on the following characteristics and their similarity to the characteristics of this strain described by Garvie (Garvie, EI Leuconostoc Oenos SP. Nov. J. Gen Microbiol, 48, 431 -438, 1967). Gram staining: positive Catalase presence: negative Production of gas from glucose Production of D-lactic acid from glucose Production of L-lactic acid from L-malate Catabolism of citric acid in the presence of glucose Catabolism of L-malate in the presence of glucose No production of dextran from sucrose Production of ammonia and urea cycle intermediates from arginine during malolactic fermentation in wine

Production of mannitol from fructose Growth on the following compounds as sole sources of carbon: D-glucose, D-fructose, D-ribose, trehalose, cellobiose, es-culine, salicin, maltose, D-mannose 5 No culture on the following compounds as sole carbon sources: D-deoxyribose, D-xylose, L-arabinose, L-rhamnose, sucrose, lactose, raffinose, glycerol, D-mannitol, L-malic acid, citric acid, fumaric acid

Cell morphology: diplococci (cocci in pairs) with a single tetrad in two planes, formation of short chains and aggregates, size 0.5-0.7 µm, culture and conversion of malate to lactate up to pH 3; no culture or very weak culture at pH 2.9; culture and conversion of malate to lactate in the presence of free SO 2 up to 25 ppm, total SO 2 up to 100 ppm; no culture for a content 15 of free SO 2 greater than 50 ppm and a content of total SO 2 greater than 200 ppm; culture and conversion of malate to lactate for an alcohol concentration of 16% by volume

Absolutely requires oleic fatty acids. A viable sample of strain B-44-40 has been deposited in National Research Council sou-20, Ottawa, Canada, where it has been given reference number 2477. It is there maintained in a viable form. Another deposit was made on July 15, 1980 at the American Type Culture Collection (12301 Parklawn Drive, ROCKVILLE, Maryland 20852) where it was given the reference number ATCC 31688. 25 As described in more detail below, according to a preferred embodiment of the invention, antibiotic resistant mutants of B-44-40 are used. These strains were called Bio Logicals 44-40 and remarks similar to those concerning strain B-44-40 apply to the classification and nomenclature of these mutants. The taxonomy of useful mutants was essentially the same as that of B-44-40 except of course the additional characteristic of resistance to at least one selected antibiotic.

Viable samples of antibiotic resistant strains B-44-40 were deposited on July 15, 1980 at the American Type Culture Collection, where they are maintained in a viable form, under the following reference numbers:

rifamycin B-44-40 resistant, Rl-50 - ATCC 31689 rifamycin B-44-40 resistant, Rl-100 - ATCC 31690 erythromycin B-44-40 resistant, RI-25 - ATCC 31691 40 erythromycin resistant B-44-40, R2-25 - ATCC 31692 erythromycin resistant B-44-40, Rl-40 - ATCC 31693 The antibiotic resistant mutants of B-44-40 called Bio Logicals 44-40 are prepared from the parent strain B-44-40 by mutation, then culture of the mutants obtained in a culture medium fa-45 promoting growth containing a selected antibiotic. By repeated selections and classifications or dilutions and cultures, a mutant strain is isolated and cultivated, the identifying characteristic of which is viability in the presence of the chosen antibiotic.

The methods used to cause mutation of the parent strain are generally the conventional methods known to produce this effect in microbiological culture techniques. Any method which causes the parent strain to undergo chemical modification or cleavage and recombination of the DNA chains of cellular chromosomes can be adopted, either randomly or in a directed fashion. For example, the sample may be subjected to the action of ultraviolet light or X-rays or other forms of radiation, or chemical mutagens or selectively cultivate spontaneous mutants.

Almost any known antibiotic or any combination of antibiotics can be used to cultivate and isolate the antibiotic mutants. Mention may be made, as suitable antibiotics useful in such a process, of the antibiotics commonly known and which are easy to obtain, consisting of rifamycin, streptovaricin, streptolydigine, tetracycline, Chlortetracycline, gentamy-65 cine, ampicillin and aminoglycosidic antibiotics such as streptomycin, spectinomycin and other macrole antibiotics such as erythromycin and the like, and particularly preferred are rifamycin and erythromycin. The use of these antibiotics

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or one of their combinations makes it possible to produce and isolate different mutants called Bio Logicals 44-40 which all respond to the taxonomy previously described and which moreover have the characteristic of being viable in the presence of the antibiotic chosen or of the combination of antibiotics.

Resistance to a specific antibiotic or to a combination of antibiotics constitutes a distinctive characteristic of the Bio Logicals 44-40 ATCC 31688 strains which makes it possible to easily detect their presence in fermented liquids and which therefore constitutes a practical means for quality control. and process monitoring. The risks that a sample of wine chosen at random contains a strain of microorganisms resistant to a specific antibiotic as a result of natural processes are so low (around 1/107) that they are in practice negligible. In the case of a strain of microorganisms resistant to two specific antibiotics, the risks are correspondingly lower (1/1014). Therefore, if the culture of a sample of wine in a medium containing a selected antibiotic leads to the development of an antibiotic resistant microorganism, this development constitutes proof of the addition of this antibiotic resistant microorganism during winemaking. Additional tests to characterize the culture are unnecessary. This allows for quality control testing and investigation of this microorganism with a degree of precision and certainty rarely seen in the fermentation of natural products. The use of the micro-organism can be detected with certainty so that it can be established that it is the cause of specific effects in wine. Fermented natural products normally result from a complex series of microbiological processes that use a large number of different microorganisms, some of which are unknown and unidentified, so it is normally difficult to attribute any specific effect or characteristic. from the product to any specific process or organism. The use of bacteria resistant to antibiotics also allows the labeling of wines, to ensure production standards and authenticity.

The lyophilized compositions of microorganisms which have been previously suggested or which have been proposed for use in inducing malolactic fermentation have had unpredictable efficacy.

Although some samples of compositions have given satisfactory results (with less vigor than would be desired and with the difficult conditions of use previously exposed), other samples having apparently the same composition have been found to be completely incapable of initiating the desired malolactic fermentation. This appears to be due to accidental destruction of viable microorganisms during lyophilization. According to another characteristic, the microorganism used to initiate the malolactic fermentation is cultivated, during at least part of its culture cycle, in a medium containing glutamic acid or other related molecules mentioned above. Unexpectedly, the glutamic acid / glutamate residues from the culture medium behave like cryoprotectants so that the cultures remain in a biologically viable form ready for use. This applies to strains B-44-40 and Bio Logicals 44-40. Consequently, the lyophilized compositions according to the invention are reliable and regularly initiate and cause an energetic malolactic fermentation under a wide range of conditions, in contrast to the lyophilized compositions previously known.

As indicated, the cryoprotector consisting of glutamic acid / glutamate can be formed, at least partially, in situ in the fermentation broth where B-44-40 or Bio Logicals 44-40 is cultivated or by the hydrolysis of materials protein before their addition to the culture medium. This occurs when proteins, peptides or protein hydrolyzates containing reasonable amounts of glutamic acid-forming precursors are used as the culture medium for microorganisms.

It should be noted in this regard that it is not satisfactory to carry out the cultures in the absence of glutamic acid / glutamate and then to add glutamic acid or glutamates to the product obtained before lyophilization. This procedure does not ensure the regular production of a viable and effective lyophilized composition. A molecule similar to glutamic acid or glutamates must be present as a residue from a culture medium in order to obtain a regular effective cryoprotective effect. It is unclear why this is so. However, it seems that the reason for this phenomenon is linked to the presence in the cryoprotector of a group producing a hydrogen bond to form a mutual intracellular relationship between the molecules present in the cytoplasm of the microorganism and the molecules of glutamic acid or glutamate transported from the culture medium.

When using the parent strain B-44-40 to initiate malolactic fermentation, glutamic acid / glutamate must of course be present during the culture. When using a Bio Logi-15 cals 44-40 mutant, glutamic acid / glutamate must be present during the culture of the mutant.

The cryoprotector is preferably added to the culture medium at the start of the growth process of the microorganism and in the form of glutamic acid. As the medium generally contains various mineral salts to promote growth, a significant amount, if not all, of the glutamic acid is transformed into glutamates in the medium. Appropriate amounts of glutamic acid which are added to the culture medium to obtain sufficient cryoprotection of the final product are between approximately 0.1 and approximately 5% (by weight by volume), preferably between approximately 0.5 and about 2%.

With the above condition regarding the presence of glutamate or another cryoprotective above, the culture medium of B-44-40 and Bio Logicals 44-40 is generally conventional and can be easily designed, adjusted and optimized by those skilled in the art. Basically, the medium is an aqueous liquid containing mineral salts such as potassium and magnesium phosphate and manganese sulfate, ferrous sulfate, etc., a substrate for the microorganism such as malic acid, glucose, amino acids, vitamins, fatty acids and buffers suitable for biology to allow adjustment of the pH to a value leading to optimal growth normally between 3.0 and 7.0. The buffer substance must make it possible to maintain a constant pH in the medium. Cultures are carried out practically anaerobically or 40 microaerophilically.

After the culture, the microorganisms are collected in the medium according to conventional techniques, for example by centrifugation and / or filtration, then the mass is lyophilized. This lyophilization is also carried out according to conventional techniques which consist in submitting the mass to low temperatures in a low pressure environment. The product obtained is a fluid substance in powder or in particles which can be kept dry, preferably in stoppered containers, for extended periods of time, before adding it to wine during vinification.

n / a The amounts of lyophilized composition which are added to the wine to cause the malolactic fermentation have no particular limitation, but the effective amounts are very small. When expressed by the number of viable microorganisms, the appropriate amounts can be as low as 100 viable organisms 55 per ml of wine. The practical amounts are between approximately 10 2 and 10 8 and preferably between 10 6 and 10 7 viable microorganisms per ml. In other words, 1 g of composition is enough to cause the malolactic fermentation of 2,500 to 25,000 liters of wine. In practice, it is therefore preferred to mix the lyophili-60 composition with an inert solid support which is suitable in biology so as to dilute it and to obtain quantities of material which are easy to measure in order to carry out the addition. Suitable carriers are diatomaceous earth and other forms of safe clay, cellulose powder, silicates, and the like. A mixture containing 1 to 65% by weight of lyophilized composition is suitable and is practical.

As previously indicated, the cultures of the invention are more vigorous and act under a wider range of conditions to induce malolactic fermentation than the micro

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previously indicated organizations. Regarding vigor, B-44-40 causes significantly faster conversions of malate under similar conditions than any of the known micro-organisms Lact. Delbruchii, Ped. Cerevisiae, Lact. Buchneri, Leu. Oenos ML-34, Leu. Oenos P-6, Lact. Hildigardii and Lact. Prevo; for example, in direct comparative trials with Leuconostoc Oenos ML-34 with Rogosa medium at pH 5.5 at room temperature, B-44-40 produces a maximum number of bacteria in four days, while ML-34 produces its maximum number of bacteria (significantly lower) after 13 days.

The extension of the pH range in which the malolactic fermentation can be carried out up to a pH of 3.0 is a very important practical advantage. No other cultures are known which allow malolactic fermentation to be carried out at a pH below 3.2. At the time of bottling, when it is desirable to carry out malolactic fermentation, many wines have a pH below 3.2. These wines can increase the pH as they age in the cellar, but it is undesirable to wait until this process has started to start the malolactic fermentation, due to the risk of undesirable side effects resulting from natural organisms that can contaminate wine. The possibility of starting malolactic fermentation at a pH of 3.0 with microorganisms is an important advantage which further reduces the risk of wine deterioration.

Likewise, the possibility of operating at lower temperatures up to about 10 ° C with microorganisms is an important advantage. It is a desirable temperature for the wine cellars at which one can perform as many vinification operations, after primary fermentation, as desired. The other known microorganisms used for malolactic fermentation only act effectively at a temperature of at least 18 ° C, which is undesirable.

It is sometimes observed that the lyophilized composition of microorganisms has difficulties in spontaneously initiating malolactic fermentation in certain types of wine. The reason for this difficulty may be that wine has a poor balance of nutrients and acidity for this purpose. In this case, it is possible, to further ensure the malolactic fermentation with the composition, at the start add the lyophilized organism to a seeding broth containing GMT medium adjusted to pH 4-7. This makes it easier for the microorganism to return from its lyophilized state to a metabolic state of maximum activity for introduction into suboptional analog media. After having thus incubated for a period of 8 to 72 h, for example, an initial amount of between approximately 1 × 108 and approximately 1 × 109 bacterial cells per milliliter of medium, the entire broth can be added to the wine and the malolactic fermentation then begins without difficulty.

The invention is illustrated by the following nonlimiting examples.

Example 1:

Selection of strains

Randomly isolated strains are prepared from a variety of natural sources. Wine samples are first passed through a sterile filter with 0.65 µm pores to remove yeast and then through a sterile filter with 0.45 µm pores which is placed on a box of Rogosa medium (Difco) and incubated at 30 ° C until distinct colonies appear. Generally, one obtains only one strain isolated from a sample, this strain always being constituted by the predominant organism. If possible, the strains are isolated while the wines undergo a malolactic fermentation. This is done to be sure that the isolated organisms are the predominant organisms responsible for the malolactic fermentation.

The bacteria called B-44-40 was thus isolated from a California Chardonnay wine.

Example 2:

Enrichment and selection of strains

Cell mass is measured with a Klett Sum-merson colorimeter. A red filter No. 66 is used with the wine and a green filter No. 54 with the synthetic media. The screw-cap test tubes measuring 10 x 100 mm in Kimex or Pyrex containing 5 ml of wine are vigorously shaken for optical determination of the cell mass. The cells are grown at 22 ° C unless otherwise indicated. All the selected strains are introduced as previously described in test tubes under variable conditions of alcohol concentration, pH and temperature.

Example 3:

Fortification based on alcohol

Groups of six tubes (10 x 100 mm screw cap) are filled with 5 ml of a wine sample. 95% pure ethanol is added to the tubes to obtain final alcohol concentrations of 13.0%, 14.0%, 14.5%, 15.0%, 15.5% and 16.0% (in volume). Each microorganism is placed in a set of test tubes at an initial cell concentration of between 5 × 10 4 and 5 × 10 5 cells / milliliter and the growth is measured as previously described. We choose organisms with a high tolerance towards alcohol.

B-44-40 bacteria develop well at alcohol concentrations of 16% by volume and are capable of carrying out malolactic fermentation in all the wines studied at this alcohol concentration.

Example 4:

Enrichment as a function of pH

The conditions for enrichment as a function of pH are the same as for enrichment as a function of the alcohol concentration except that, in the various tubes, the pH and not the alcohol content varies. PH values of 3.0, 3.3 and 3.7 are used. The strains are selected according to the development at a low pH.

B-44-40 bacteria grow at pH 3.0 and allow malolactic fermentation to take place at this pH. B-44-40 bacteria grow better at low pH than the other strains studied.

Example 5:

Tolerance to temperature

The enrichment conditions as a function of the temperature are the same as the enrichment conditions as a function of the alcohol concentration, except that the temperature and not the alcohol concentration varies. The temperatures studied are 10 C, 20 ° C and 30 ° C. We select the strains that develop at low temperature.

B-44-40 bacteria develop better at low temperature (10 ° C) than all the other strains studied and they allow malolactic fermentation to be carried out at this temperature.

Example 6:

Selection based on malate metabolism

The conditions for selection according to the conversion of malate are those described for normal growth conditions in wine. After inoculation, the malate content of the samples is measured daily. The malate content is determined by spectrometry with Boehringer Mannheim reagents and procedures. Among all the strains studied, the strain B-44-40 allows the completion of a malolactic fermentation before all the other strains.

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Example 7:

Antibiotic resistant strains

Variable amounts of the antibiotic Rifampin (rifamycin) or the antibiotic erythromycin are added to boxes of nutrient medium such as Rogosa medium. The antibiotic content of the boxes varies from 10 (ig / ml to 100 (ig / ml. Rifampin and erythromycin are in the purest form available. B-44-40 bacteria are cultivated in GMT media (described below)

up to the maximum density. 0.2 ml of culture liquid is spread on each dish (approximately 2 × 10 8 cells) and then the dishes are incubated at 30 ° C. until separate colonies can be observed. Colonies that develop normally (compared to untreated B-44-40 bacteria) on dishes containing the antibiotics are considered to resist the corresponding concentration of antibiotic. Resistant colonies are placed on separate dishes for isolation. These colonies are subjected to new comparative tests with normal B-44-40 bacteria not resistant to antibiotics, that is to say the parent strain, to ensure that they resist the indicated antibiotic concentrations well. The following strains are obtained:

1. Rif-Rl-50 has a resistance to Rifampin at 50 [ig / ml; first isolation;

2. Rif-Rl-100 has a resistance to Rifampin at 100 Hg / ml; first isolation;

3. Ery-Rl-25 has resistance to erythromycin at 25 µg / ml; first isolation;

4. Ery-R2-25 exhibits erythromycin resistance at 25 µg / ml; second isolation;

5. Ery-Rl-40 has resistance to erythromycin at 40 [ig / ml; first isolation.

Example 8:

Cryogenic protection

The high survival of bacteria during lyophilization is based on the use of glutamic acid and other compounds containing a group forming a hydrogen bond and two acid groups. Several characteristics of the culture and lyophilization processes are essential for obtaining high survival of the bacteria, namely appropriate culture media, the pH of the culture media containing the cryogenic mixture, the percentage of cryogenic mixture used and the growth phase when cells are collected and lyophilized.

B-44-40 bacteria are grown in GMT medium prepared as follows.

A mixture of 1.0% yeast extract, 0.25% Tween 80.0.068% monopotassium phosphate, 0.057% magnesium sulfate heptahydrate, 0.012% sodium sulfate is autoclaved together for 15 min at 121 ° C. manganese monohydrate, 0.003% ferrous sulfate heptahydrate and 1% sodium citrate. After autoclaving, 1% of previously sterilized glucose is added. The cryogenic mixture used consists of 1% sodium glutamate, 0.1% malate and 0.05% cysteine. This mixture is added to the media after autoclaving as a filter sterilized mixture. The pH of the mixture obtained is adjusted to 4.5 with glacial acetic acid. The bacterial cells are cultured until a density of between 1 x 109 and 2 x 109 cells / milliliter is reached and the cells are then collected and lyophilized. Before lyophilization, the cells are quickly frozen at -80 ° C and placed in the lyophilization apparatus in the frozen state, ensuring that they do not thaw.

Example 9:

Addition of freeze-dried bacteria to wine

For best results, the wine should be seeded during or shortly after the completion of primary fermentation. No special wine treatment is necessary. The lyophilized bacterial culture is placed directly in the wine and it is recommended that the inoculum corresponds to 106 cells / milliliter. The bacterial culture is combined with a special dispersant, for example diatomaceous earth, to facilitate its rapid and homogeneous mixing in the wine to be treated.

The preparation of B-44-40 bacteria is designed so that they can be used in wines with a pH below 3.3 and at temperatures below 15 ° C. After inoculation of the wine, the malate content can be monitored by simple chromatography.

When all the malate has been consumed, the lees which may have accumulated during secondary fermentation can be separated. B-44-40 or the antibiotic resistant strains Bio Logicals 44-40 can be used as bacteria. In the latter case, if the user wishes to identify the presence of bacteria in a given wine, he can determine the absolute need for oleic acid or the antibiotic resistance marker of the strain, in addition to the morphological aspect.

Of course, various modifications can be made by those skilled in the art to the devices or methods which have just been described only by way of nonlimiting examples without departing from the scope of the invention.

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Claims (11)

662,819 1. Bacterial composition to induce malolactic fermentation, characterized in that it consists of the strain of microorganisms Leuconostoc oenos B-44-40, ATCC 31668, or its antibiotic resistant mutants Bio Logicals 44-40, ATCC 31689, 31690, 31691, 31692.31693 in a biologically viable form, in association with a biologically acceptable carrier. 2. Composition according to claim 1, characterized in that the support consists of remains of the media used for the biological development of the microorganism. 2 3. Composition according to claim 2, characterized in that the medium residues comprise glutamic acid and / or its biologically acceptable salts. 4. Composition according to claim 3, characterized in that the microorganism and glutamic acid and / or its salts are in a lyophilized form, glutamic acid and / or its salts acting as cryoprotectors of the lyophilized microorganism to keep it in a biologically active form. 5. Composition according to claim 4, characterized in that the microorganism is a strain of Leuconostoc oenos antibiotic-resistant Bio Logicals 44-40, ATCC 31688. 6. Composition according to claim 5, characterized in that it is a mixture with an at least equal weight of a fluid material in particles suitable in biology to increase the volume and promote the dispersion of the composition in an aqueous medium. 7. Composition according to Claim 2, characterized in that the medium residues contain an acid which behaves like a cryoprotective in the subsequent lyophilization of the cells, this acid being chosen from a-ketoglutamic acid, L-malic acid , DL-malic acid, L-aspartic acid, DL-a-aminopiméli-que acid, a-methyl L-glutamic acid, malonic acid, N-acetyl L-glutamic acid , N-dimethyl L-glutamic acid, N-dimethyl L-aspartic acid, L-cysteic acid, cysteine, DL-threo-nine, acetylglycine, DL-pyrrolidone-5 acid carboxylic-2, D-glutamic acid, DL-glutamic acid, L-glutamic acid, aminomalonic acid, tartronic acid and lysine. 8. Process for treating wine to induce a rapid controlled malolactic fermentation, characterized in that it consists in adding to the wine a composition according to claim I. 9. Method according to claim 8, characterized in that it comprises the addition of 105 to 107 viable microorganisms of the composition per milliliter of wine. 10. Method according to claim 8, characterized in that the addition to the wine is carried out at a temperature between 8 ° C and 18 ° C and at a pH below 3.3. 11. Wine stabilized against malolactic fermentation, prepared according to the process of claim 8.