FR2485037A1 - NOVEL BACTERIAL STRAIN AND ITS MUTANTS USEFUL FOR STABILIZING WINE, PROCESS FOR OBTAINING SUCH MUTANTS, COMPOSITION CONTAINING SAME OR ITS MUTANTS, METHOD FOR STABILIZING WINE USING THIS COMPOSITION AND WINE THUS STABILIZED - Google Patents

Abstract

Malolactic fermentation in wine is induced and vigorously performed by [lacuna] to wine during or after the primary fermentation new lactic acid bacteria which are called B-44-40 herein, or the antibiotic-resistant mutants thereof which are called Bio-Logicals B-44-40. In a preferred embodiment, the cultures are prepared and added to the wine in freeze-dried form, the cultures having been prepared by cultivation in a medium which contains glutamic acid or similar acid as cryoprotective. The cultures induce malolactic fermentation at lower temperatures and lower pH conditions than all other cultures described or tested.

Description

The present invention relates to a new bacterial strain and its mutants useful for stabilizing wine, a process for obtaining these mutants, a composition containing this strain or its mutants, a process for stabilizing wine using this composition and the wine thus obtained.

 In general, the invention relates to the fermentation processes as well as the ingredients and compounds which are used there. More particularly 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 of these procedures 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 fermenting organisms not to start quickly and there is always a risk that unwanted organisms will become established and cause unpleasant side effects.

 To a certain extent, a controlled malolactic fermentation was carried out before bottling by using added cultures. However, the cultures of microorganisms which have been used for this purpose lack certain characteristics in particular the ease of use and the 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 kept at a temperature of 18 to 22 OC. These two conditions may not always be possible or desirable for obtaining wines of superior quality.

 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 180 ° 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 Applicant has isolated a new bacterium here called Leuconostoc B-44-40 which, surprisingly, effectively induces malolactic fermentation when added to wine. The new bacterium is more vigorous than the bacterial strains of Leuconostoc previously known and used for this purpose. In addition, it vigorously causes the malolactic fermentation at temperatures below 180 ° C. and at least as low as 10 ° C. and at low pH below 3.3, for example lowering to around 3.0.

In addition, antibiotic-resistant mutants of
Leuconostoc B-44-40 hereinafter called Leuconostoc Bio Logicals 44-40, which are also effective in inducing malolactic fermentation, under the same conditions and which moreover allow monitoring and quality control of the process fermentation.

The invention also relates to these new bacteria in a lyophilized form with as an cryoprotective acid (or a corresponding metal salt) chosen from L-glutamic acid, DL-glutamic acid, L-aspartic acid, acid DL-a-aminopimelic, a-methyl L-glutamic acid, malonic acid, acid
DL-malic, L-malic acid, a-ketoglutaric acid, acid
N-acetyl L-glutamic, N-dimethyl L-glutamic acid, acid
N-dimethyl L-aspartic, L-cysteic acid, cysteine, DLthreonine, acetylglycine, DL-pyrrolidone-5-carboxylic-2 acid, aminomalonic acid, tartronic acid and lysine. Among this group, glutamic acid is preferred. Unexpectedly, these cryoprotectants have been found to ensure the viability of cultures after lyophilization, so that the composition can be used reliably to induce malolactic fermentation by addition to wine. , at the appropriate stage of winemaking. 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
B-44-40 has been isolated from wine samples that have undergone or are undergoing 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 which 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.

The bacterium B-44-40 was classified as a Leuconostoc oenos based on the following characteristics and their similarity with 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
Gas production 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 dextran production from sucrose
Production of ammonia and intermediates of the urea cycle 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, esculin, salicin, maltose, D-mannose
No culture on the following compounds as sole sources of carbon: D-deoxyribose, D-xylose, L-arabinose, L-rhamnose, sucrose, lactose, raffinose, glycerol, D-mannitol, L-malic acid, citric acid, acid fumaric
Cell morphology: diplococci (cocci in pairs with a single tetrad in two planes, formation of short chains and aggregates, size 0.5-0.7 pm, 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 free SO 2 content greater than 50 ppm and a content in total SO2 greater than 200 ppm; culture and conversion of malate to lactate for an alcohol concentration of 16% by volume
Absolutely requires oleic type fatty acids.

A viable sample of strain B-44-40 has been deposited in the National Research Council's strain bank, Ottawa, Canada, where it has been assigned the reference number 2477. It is maintained there in a viable form. Another deposit was made at llAmerican Type Culture
Collection where it received the reference number ATCC 31688.

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 regarding the 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 have been deposited in the American Type Culture Collection, where they are maintained in a viable form, under the following reference numbers
resistant to rifamycin B-44-40, R1-50 - ATCC 31689
resistant to rifamycin B-44-40, Rl-100 - ATCC 31690
Erythromycin B-44-40 resistant, R1-25 - ATCC 31691
resistant to erythromycin B-44-40, R2-25 - ATCC 31692
resistant to erythromycin B-44-40, R1-40 - ATCC 31693
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 growth-promoting culture medium containing a selected antibiotic. By selection and classification or repeated dilutions and cultures, a mutant strain is isolated and cultivated, the identifying characteristic of which is viability in the presence of the selected antibiotic.

 The methods used to cause the mutation of the parent strain are generally the conventional methods known to produce this effect in microbiological culture techniques. One can adopt any method causing in the parent strain a chemical modification or a cleavage and a recombination of DNA chains of cellular chromosomes, in a random or directed manner. For example, the sample can be subjected to the action of ultraviolet light or X-rays or other forms of radiation, or to chemical mutagens, or to selectively cultivate spontaneous mutants.

Almost any known antibiotic or any combination of antibiotics can be used to cultivate and isolate the antibiotic resistant mutants. As suitable antibiotics useful in such a process, commonly known and readily available antibiotics consisting of rifamycin, streptovaricin, streptolydigine, tetracycline, chlortetracycline, gentamycin, ampicillin and aminoglycoside antibiotics such as streptomycin, spectinomycin and other macrolide antibiotics such as erythromycin and the like and rifamycin and erythromycin are particularly preferred. The use of these antibiotics 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 chosen antibiotic or combination of antibiotics.

Resistance to a specific antibiotic or to a combination of antibiotics is a distinguishing characteristic of Bio Logicals 44-40 strains which makes it possible to easily detect their presence in fermented liquids and which therefore constitutes a practical means for controlling the quality and process monitoring. The risks that a random sample of wine contains a strain of microorganisms resistant to a specific antibiotic as a result of natural processes are
So low (around 1/107) that in practice they are 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 soothsayer sample 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 the winemaking. Additional tests to characterize the culture are unnecessary. This allows quality control testing and investigation of this microorganism with a degree of precision and certainty rarely observed in the fermentation of natural products. The use of the microorganism can be detected with certainty so much 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 that it is normally difficult to attribute any specific effect or characteristic to them. product to any specific process or organism. The use of bacteria resistant to antibiotics according to the invention 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 Malolactic fermentation desired. This seems to be due to an accidental destruction of viable microorganisms during lyophilization.

According to another characteristic of the invention, 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 aforementioned related molecules.

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 in a wide range of conditions, unlike 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 is cultivated or
Bio Logicals 44-40 or by the hydrolysg of proteinaceous materials before their addition to the culture medium. This occurs when proteins, peptides or protein hydrolysates 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 for a regular effective cryoprotective effect to be obtained. 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 an intracellular mutual relationship between the molecules present in the cytoplasm of the microorganism and the glutamic acid or glutamate molecules transported from the culture medium.

 When using the parent strain s-44-40 to initiate malolactic fermentation, glutamic acid / glutamate must of course be present during the culture. When using a Bio Logicals 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 quantity, if not all, of the glutamic acid is transformed into glutamates in the medium. The 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 per volume), preferably between approximately 0.5 and approximately 2%.

 With the above condition regarding the presence of glutamate or another cryoprotectant mentioned 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 the 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 buffer substances suitable in 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 allow maintain a constant pH in the medium. Cultures are carried out practically under anaerobic or microaerophilia.

 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 subjecting 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 mouthful containers, for extended periods of time, before adding it to wine during vinification.

 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 per ml of wine. The practical amounts are between about 102 and 108 and preferably between 106 and 10 viable microorganisms per ml. In other words, 1 g of composition is sufficient to cause the malolactic fermentation of 2,500 to 25,000 liters of wine. In practice, it is therefore preferable to mix the lyophilized composition with an inert solid support suitable in biology so as to dilute it and obtain quantities of material which are easy to measure for carrying 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 5% 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 microorganisms previously indicated. Regarding vigor, B-44-40 causes significantly faster conversions of malate under similar conditions than any of the known microorganisms Lact. Delbruchii, Ped. Cerevisiaes
The act. 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 which can contaminate the wine. The possibility of starting malolactic fermentation at a pH of 3.0 with the microorganisms of the invention is an important advantage which further reduces the risk of wine deterioration.

Likewise, the possibility of operating at lower temperatures down to about 10 ° C. with the microorganisms of the invention 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 act effectively only at a temperature of at least 180C, which is undesirable.

 It is sometimes found that the lyophilized composition of microorganisms of the invention has difficulties in spontaneously initiating malolactic fermentation in certain types of wines. The reason for this difficulty may be that the wine has for this purpose a poor balance of nutrients and acidity. 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 incubated for a period of 8 to 72 h, for example, a initial quantity of between approximately 1 × 10 8 and approximately 9 1 × 10 bacterial cells per milliliter of medium, all of the 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 having pores of 0.65 µm to remove the yeasts and then through a sterile filter having pores of 0.45 µm which are placed on a bundle of Rogosa medium (Difco) and incubated at 30 "C until distinct colonies appear. Generally, only one strain is obtained 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 ensure that the isolated organisms are the predominant organisms responsible for the malolactic fermentation.

The bacterium 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 colorimeter
Klett Summerson. A red filter no. 66 is used with the wine and a green filter no. 54 with the synthetic media. The test tubes with screw cap measuring 10 x 100 mm are vigorously shaken in
Kimex or Pyrex containing 5 ml of wine for optical determination of cell mass. The cells are grown at 220C 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 - ENRICHMENT AS A FUNCTION OF ALCOHOL
Groups of six tubes (with screw cap measuring 10 x 100 mm) are filled with 5 ml of a wine sample. 95% pure methanol 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 wines studied at this alcohol concentration.

EXAMPLE 4 - ENRICHMENT AS A FUNCTION OF THE 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 fold 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 develop at pH 3.0 and allow malolactic fermentation to be carried out at this pH. B-44-40 slaughterhouses develop better at a low pH than the other strains studied.

EXAMPLE 5 - TEMPERATURE TOLERANCE
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 100 ° C., 20 ° C. and 300 ° C. The strains which develop at low temperature are selected.

 B-44-40 bacteria develop better at low temperature (100C) than all the other strains studied and they allow malolactic fermentation to be carried out at this temperature.

EXAMPLE 6 - SELECTION BASED ON THE ESTABOLIST OF MALATE
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.

EXAMPLE 7 ANTIBIOTIC RESISTANT STRAINS
Has boxes of nutrient medium such as
Rogosa, variable amounts of the antibiotic Rifampin (rifamycin) or the antibiotic erythromycin are added. The antibiotic content of the boxes varies from 10 / ug / ml to 100 / ug / ml. Rifampin and erythromycin are in the purest form available.

B-44-40 bacteria are grown in GMT media (described below) to maximum density. We spread on each box
8 0.2 ml of culture liquid (approximately 2 × 10 cells), 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 antibiotic concentration. 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 make sure that they resist the indicated antibiotic concentrations well. The following strains are obtained
1. Rif-Rl-50 has resistance to Rifampin
50 / ugJml; first isolation;
2. Rif-RI-100 has resistance to Rifampin a
100 µg / ml; first isolation;
3. Ery-Rl-25 has resistance to erythromycin
at 25 g / ml; first isolation;
4. Ery-R2-25 shows resistance to erythromycin
at 25 rg / ml; second isolation;
5. Ery-Rl-40 has resistance to erythromycin
at 40% g / 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 suitable 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 medium
GMT 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% OS cysteine. This mixture is added to the media after autoclaving in the form of a mixture sterilized by filtration. 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 × 10 9 and 2 × 10 9 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, taking care that they do not thaw.

EXAMPLE 9 ADDITION OF LYOPHILIZED WINE BACTERIA
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 lrinoculum correspond 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 150C. After inoculation of the wine, one can follow the content of mate 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 processes which have just been described solely by way of nonlimiting examples without departing from the scope of the invention.

Claims (16)

1. Composition for inducing malolactic fermentation characterized in that it consists of the strain of microorganisms Leuconostoc B-44-40 or its antibiotic-resistant mutants Bio Logicals 4440, in a biologically viable form in combination with a biologically acceptable carrier. 2. Composition according to claim 1, characterized in that the support suitable for biology consists of remains of the media used for the biological development of the microorganism. 3. Composition according to claim 9 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 for maintain it in a biologically active form. 5. Composition according to claim 4 characterized in that the microorganism is a strain of Leuconostoc antibiotic resistant Bio Logicals 44-40. 6. Composition according to claim 5, characterized in that it is 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. 7. Composition according to Claim 2, characterized in that the medium remains contain an acid which behaves like a cryoprotective in the subsequent lyophilization of the cells, this acid being chosen from a-ketoglutamic acid, Malic acid, l DL-malic acid, L-aspartic acid, DL- -aminopimelic 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-threonine, acetylglycine, DL-pyrrolidone-5 carboxylic-2 acid, l D-glutamic acid, DL-glutamic acid, Itacid L-glutamic acid, aminomalonic acid, tartronic acid and lysine. 8. Process for preparing new bacteria producing antibio-resistant lactic acid of the genus Leuconostoc or Pediococcus, constituting the Bio Logicals strains 44-40 characterized in that it consists in cultivating bacteria of the strain B-4440 in a medium promoting growth under essentially anaerobic conditions, causing the mutation of this strain B-44-40, cultivating the mutants obtained in the presence of antibiotic and recovering an antibiotic-resistant mutant strain Bio Logicals 44-40 in the culture medium of the mutants . 9. Method according to claim 8, characterized in that the bacterial mutants of B-44-40 are cultured in a growth-promoting medium containing glutamic acid and / or its salts suitable in biology. 10. Method according to claim 9, characterized in that it comprises a stage of recovery from the medium promoting growth, after the stage of culture of the mutants, of a biologically viable bacterial strain of Bio Logicals B-44-40 in combination with residual glutamic acid and / or glutamate and lyophilization of the recovered composition. 11. The method of claim 8, characterized in that it further comprises a subsequent stage of intimate mixing of the lyophilized composition in a dry form, with an at least equal weight of a fluid support in biologically acceptable particles. 12. New biologically viable lactic acid producing bacteria of the genus Leuconostoc or Pediococci constituting the Bio Logicals 44-40 strains, these strains resistant at least to one antibiotic and being capable of inducing malolactic fermentation in wine at temperatures below 180C and at a pH below 3.3. 13. 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 any one of claims 1, 4 or 6. 14. Method according to claim 13, characterized in that it comprises the addition of approximately 105 μ to viable microorganisms of the composition per milliliter of wine. 15. The method of claim 13, characterized in that the addition to the wine is carried out at a temperature between 80C and 18 "C and at a pH below 3.3. 16. Wine stabilized against malolactic fermentation characterized in that it contains bacteria producing lactic acid biologically viable of the genus Leuconostoc or Pediococcus constituting a strain Bio Logicals 44-40, this strain resistant to at least one antibiotic and being more capable of inducing malolactic fermentation in wine.