DE3124404A1 - Composition for promoting malolactic fermentation - Google Patents

Description

DESCRIPTION

The invention relates to fermentation processes and ingredients and compounds suitable for these methods. The invention particularly relates to a new microbiological agent Origin for use in fermentation processes and especially in winemaking. The invention relates to Furthermore, the process for the production of the new agent and its use in fermentation.

Still wines are made by fermenting sugars in fruit juices. After the primary fermentation is complete, a number of secondary fermentations can take place. Most of them are undesirable and spoil the wine. All of these processes are undesirable if they occur after filling in Bottles are made.

The most common secondary fermentation is called "malolactic acid" ermentation ", in which microorganisms cause the conversion of malic acid to lactic acid. This also converts sugar converted into acids and other organic or inorganic compounds. The malo-lactic acid fermentation must either before the bottling of the wine be completed or prevented with a very high degree of security. If after filling in Bottles then lead to gas formation and bacterial growth to spoil the product.

Malo-lactic acid fermentation can be prevented, for example by filtering the wine to remove therefrom the organisms that cause and promote this fermentation. But you can also pasteurize the wine to remove the microorganism to deactivate. All of these procedures are expensive and degrade the quality of the wine, for example by adverse flavor effects are introduced. There are also cases

where these methods fail utterly. The malo-lactic acid fermentation is considered highly desirable in red wine and some white wines. It leads to a natural reduction the activity, and it leads to an improvement in the taste properties.

It is thus preferred to complete the malolactic acid fermentation To promote and ensure prior to bottling by promoting malolactic acid fermentation There are microorganisms in the wine. This results in a more superior and stable product.

The phenomenon of malolactic acid fermentation and its importance in the treatment of wine are widely recognized. Indigenous microflora can be found in many wine-producing areas lead to natural malolactic acid fermentation. However, the fermentation organisms often do not start immediately, and there is always the risk that the "wrong" organisms will naturalize themselves, which leads to undesirable side effects.

To a certain extent, controlled malolactic acid fermentation is already possible before filling by using added cultures. The cultures of microorganisms, however, which have been used for this purpose lack several distinctive properties, in particular the ease of use and vitality the culture. The cultures used hitherto had to be added to the wine at a pH value above 3.3. While and after the addition, the wine should be kept at a temperature of 18-22 ° C. Both conditions are in -procedures for the production of top quality wines not always feasible or desirable.

In many cases, by the time it is desired to induce malolactic fermentation, the pH of the wine is lower than 3.3, and it is extremely undesirable to go into this-

Adjust the pH value in this stage. The storage temperature of the wine in vats before filling are preferably considerably lower than 18 ° C, for example, about 10 ⁰ C. Furthermore, it is clearly desirable in the technical realization that Malo-lactic acid fermentation as quickly as possible
to increase the output and the storage time
to diminish.

New bacteria, which are referred to herein as Leuconostoc B 44-40, have now been isolated which surprisingly do so
are effective in inducing malolactic acid fermentation when added to wine. The new bacteria are considerably more viable than those known and described for this purpose
Leuconostoc bacterial strains. In addition, the new bacteria act violently to contribute to malolactic acid fermentation
Temperatures below 18 ⁰ C and at least as low as
1O ⁰ C and at low pH values of less than 3.3, for example down to about 3.0.

Antibiotic-resistant mutations of Leuconostoc B 44-40 were also produced, hereinafter referred to as Leuconostoc Bio Logicals 44-40 are designated. These are equally effective in malo-lactic acid fermentation under the same conditions to induce, and they also give the opportunity to follow the process and perform quality control.

The invention therefore also relates to these new bacteria in freeze-dried or lyophilized form, wherein
select an acid (or a metal salt thereof) as a cryoprotectant
of the group L-glutamic acid, DL-glutamic acid, L-aspartic acid,
DL-qC -aminopimelic acid, o £ -Methyl-L-glutamic acid, malonic acid,
DL-malic acid, L-malic acid, "^! - ketoglutaric acid, N-acetyl-L-glutamic acid, N-dimethyl-L-glutamic acid, N-dimethyl-L-aspartic acid, L-cysteic acid, cysteine, DL-threonine, acetylglycine, DL-5-pyrrolidone-2-carboxylic acid, aminomalonic acid, tartaronic acid and ly-

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sin is used. Glutamic acid is a representative and preferred substance in this group. It was surprising found that these cryoprotectants ensure the viability of cultures after freeze-drying, so that the agent can be reliably used to prevent malolactic acid fermentation when added to wine in a to induce appropriate level of wine production. The glutamic acid or the glutamate salt can be added as such or in situ before or during the cultivation of the microorganism are formed, for example by hydrolysis or chemical reaction of proteinaceous substances or the like or by hydrolysis of glutamic acid-containing proteins or peptides in the culture medium.

The new strain of bacteria referred to herein as Leuconostoc B 44-40 is isolated from wine samples that have undergone rapid and thorough malo-lactic acid fermentation or just go through to a desired wine product without developing any off-flavor or other evidence of spoilage to surrender. The bacterial strain is grown and grown according to generally accepted microbiological cultivation techniques cultivated, with the mixed cultures obtained from a wine tasting on a substrate containing malic acid, glucose, or the like, grows to produce cultures with rapid growth characteristics on it to select and isolate. The correct nomenclature for this culture is currently not entirely clear what the lack of agreement among the relevant professionals as to the proper classification and definitions in this area is due. The bacterial strain B 44-40 certainly belongs to the lactic acid-producing bacteria family. In terms of the question of whether the strain as belonging to the genus Leuconostoc, species oenus or belongs to the genus Pediococcus, there is still a certain uncertainty. The references to the Bacterial strains B 44-40 and Bio Logicals 44-40 should be understood in this way that they are subject to the above reservation with regard to the clature.

A taxonomic description of bacteria B 44-40 is given below:

Taxonomy

B 44-40 is classified as a Leuconostoc oenus due to the following characteristics and the similarity to the corresponding characteristics of this tribe, that of Garvie (Garvie, E.I. Leuconostoc Oenos SP. Nov. J. Gen Microbiol, 48: 431-438 1967).

Gram staining: positive;

Catalase presence: negative;

Gas production of glucose;

Generation of D-lactic acid from glucose; Generation of L-lactic acid from L-malate;

Dissimilation of citric acid in the presence of glucose;

Dissimilation of L-malate in the presence of glucose; no dextran production from sucrose;

Formation of ammonia and cyclic urea intermediates from arginine during malolactic acid fermentation in wine;

Mannitol formation from fructose;

Growth on the following carbon-only sources: D-glucose, D-fructose, D-ribose, trehalose, cellobiose, esculin, salicin, maltose, D-mannose;

no growth on the following carbon-only sources: D-deoxyribose, D-xylose, L-arabinose, L-rhamnose, sucrose, Lactose, raffinose, glycerin, D-mannitol, L-malic acid, citric acid, fumaric acid;

Cell morphology:

Diplococci (cocci in pairs) with unique biplanar tetrad formation, short chain and aggregate size 0.5 to 0.7 μΐη;

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Growth and conversion of malate to lactate down to pH 3; no growth or very little growth at pH 2.9; Growth and conversion of malate to lactate in the presence of free SO ₂ up to 25 ppm, total SO «up to 100 ppm; no growth with free SO2 above 50 ppm, total SO2 above 200 ppm; Growth and conversion of malate to lactate at an alcohol concentration of 16% (volume);
absolute requirement for oleic fatty acids.

A viable sample of the B-44-40 strain has been deposited with the depository National Research Council, Ottawa, Canada with deposit number 2477. The tribe will be more viable there Form kept. Another deposit was made with the American Type Culture Collection under the deposit number ATCC 31688.

As will be explained in more detail below, in a preferred Embodiment of the present invention opposite Antibiotic resistant mutations of B-44-40 used. These strains are called Bio Logicals 44-40. With regard to the Classification and nomenclature apply similar considerations as in the case of the strain B-44-40. The taxonomy of the usable Mutant is essentially the same as indicated for strain B-44-40, with the exception of the other, of course Characteristic of resistance to at least one selected antibiotic.

Viable specimens of antibiotic resistant strains B-44-40 have been deposited with the American Type Culture Collection, where they are kept in viable form. You have received the following deposit numbers:

Rifamycin Resistant Strain B-44-40, R1-50 - ATCC 31689 Rifamycin resistant strain B-44-40, R1-100 - ATCC 31

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Erythromycin Resistant Strain B-44-40, R1-25 - ATCC 31691 Erythromycin-resistant strain B-44-40, R2-25 - ATCC 31692 Erythromycin-resistant strain B-44-40, R1-40 - ATCC 31693

The antibiotic-resistant mutants of B-44-40, called Bio Logicals 44-40, are derived from the parent strain B-44-40 produced by inducing a mutation. Thereafter, the resulting mutants are in a growth promoting Culture medium containing a selected antibiotic is grown. A mutant strain is isolated and grown by repeated selections and cloning or dilution and breeding, the identifying characteristic of viability in the presence of the selected antibiotic Has.

The mutation of the parent strain is generally effected according to standard methods that are known to bring about this effect in microbiological cultivation techniques. So one can use the starting culture by any means or measure that cause a chemical modification. One can also split and recombine the DNA chains of the chromosomes of the cell in any or directional manner. This can be done, for example, by exposing the sample to ultraviolet light or exposure to X-rays or some other form of radiation or chemical mutagens, or that one mutants formed spontaneously are cultured selectively.

Virtually any known antibiotic or combination can be used of antibiotics can be used for the purpose of cultivating and isolating the antibiotic-resistant mutant. The commonly known and readily available antibiotics rifamycin, streptovaricin, streptolydigin, tetracycline, chlortetracycline, Gentamicin, ampicillin, aminoglycoside antibiotics such as streptomycin and spectinomycin, and other macrolide antibiotics, such as erythromycin and the like are preferred examples of suitable antibiotics for use in this process, wherein

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Rifamycin and erythromycin are particularly preferred. The usage The individual different antibiotics or combinations of antibiotics leads to the formation and isolation of one different variant or mutant of Bio Logicals 44-40. All of them have the taxonomy described above, but all of them have the additional characteristic of viability in the presence of a selected antibiotic or agent selected combination of antibiotics.

The resistance to a specific antibiotic or one Combination of antibiotics represents a pronounced characterizing Characteristic for the strain Bio Logicals 44-40. This property enables its immediate and easy detection Presence in the fermentation liquids. This enables quality control and process monitoring in practice. The chances that any selected wine sample contains a strain of a microorganism which is opposed to a particular Antibiotic resistant. Is, as a result, one in nature the process taking place are so small (about 1:10) that they can be neglected in practice. In the case of a microorganism strain, who is resistant to two special antibiotics, the chances are even lower (1:10) Therefore, when cultivating a wine sample in a medium containing a selected antibiotic, opposed to the growth of one If antibiotic-resistant microorganism leads, then this is effective evidence for the addition of such an opposite Antibiotic resistant microorganism during manufacture. Further tests to characterize the culture are not necessary. This enables the operator to conduct quality control tests and can conduct studies on this microorganism with a degree of precision that can be achieved by fermentation is rarely found in natural products. The application of the microorganism can be determined with certainty, so that his responsibility for producing special effects in the wine can be determined. Fermented natural v products are usually the result of performing a

complex series of microbiological processes, being large in number used by various microorganisms, some of which are unknown and unidentified, so it is Usually it is difficult to find a special effect or a special characteristic property of the product on any one specific process or organism. The inventive The use of antibiotic-resistant bacteria thus continues to provide a way of making wines to ensure product standard and authenticity.

In the case of freeze-dried microorganism preparations that have hitherto been used to induce malolactic acid fermentation suggested or offered, the effectiveness could not be predicted. While some samples of such preparations are satisfactory have worked (with less vitality than would be desired and in uncomfortable conditions of use, such as described herein), it has been found for other samples with approximately the same composition that these in no way are able to initiate the desired malolactic acid fermentation. This is believed to be due to inevitable destruction of viable microorganisms during the freeze-drying process.

According to a further feature of the present invention, the microorganism involved in the initiation of malolactic acid fermentation is used, during at least part of its growth cycle, grown in a growth medium, the glutamic acid or contains any of the aforesaid related molecules. Unexpectedly, it was found that such glutamic acid Glutamate residues from the culture medium act as cryoprotectants, so that the cultures are in a biologically viable form, which are ready for use can be preserved. This applies to both the B-44-40 culture and the Bio Logicals culture 44-40 to. As a result, the freeze-dried according to the invention

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Neten preparations are effective in a reliable and constant way to the malo-lactic acid fermentation in a violent manner and at to promote a wide range of conditions, which is in contrast to the previously known freeze-dried culture preparations stands.

As mentioned above, the glutamic acid / glutamate cryoprotectant at least partially in situ in the permeability broth in which B-44-40 or Bio Logicals 44-40, or by hydrolysis of proteinaceous materials before adding to the Culture medium are formed. This occurs when protein, peptides or as a substrate for growing the microorganisms Protein hydrolysates containing significant amounts of glutamic acid-forming precursors can be used.

In this regard, it should be noted that it is not satisfactory is to grow cultures in the absence of glutamic acid or glutamates and then the glutamic acid or Add glutamate to the resulting product before freeze drying. Such a. Procedure is unsatisfactory to ensure a consistently viable and effective freeze-dried preparation. A glutamic acid / glutamate-like one Molecule must be present as a residue of a culture medium in order to be consistently effective as a cryoprotectant. The reason for this is not yet fully understood. It is believed, however, that this is due to the presence of a hydrogen bond generating group in the cryoprotectant, creating an intracellular interrelationship between the Molecules in the cytoplasm of the microorganisms and the glutamic acid / glutamate molecules, which are transported by the culture medium is formed.

When the parent strain B-44-40 is used for malo-lactic acid fermentation to initiate, then naturally the glutamic acid / glutamate must be present during its cultivation be. If a mutant Bio Logicals 44-40 is to be used, then the glutamic acid / glutamate must be used during growth the mutant may be present.

The cryoprotectant is preferably added to the culture medium Beginning of the microorganism growth process and added in the form of glutamic acid. Since, as is conventionally the case, The medium usually contains various mineral salts to promote the growth of the culture will be a great part, though not all of the glutamic acid is converted to glutamate salts in the medium. Appropriate amounts of glutamic acid to add to the culture medium given in order to obtain sufficient cryoprotection for the end product are about 0.1 to about 5% (weight / Volume), with preferred amounts being about 0.5 to about 2%.

Apart from the above requirement regarding the presence of glutamate or another of the aforementioned cryoprotectants, is the culture medium for the growth of B-44-40 and Bio Logicals 44-40 is generally conventional. It can easily be properly adjusted and optimized by those skilled in the art. Essentially the medium is .an aqueous liquid containing mineral salts such as potassium phosphate, magnesium and manganese sulfate, iron (II) sulfate etc., substrates for the microorganism such as malic acid, glucose, amino acids, vitamins, fatty acids and biologically acceptable bare buffering agents to control the pH to a value for the optimal growth rate, usually in the range from 3.0 to 7.0, to allow. The buffer component should be such that it has a steady pH in maintains the medium. The cultivated varieties become anaerobic or carried out microaerobically.

After growing, cultures are removed from the medium using standard procedures, for example by centrifugation and / or filtration, and the mass is then freeze-dried. Again, this is done according to standard procedures, with the mass in an environment of low pressure and low temperatures is exposed. The resulting product is a powdery or particulate free flowing substance that is responsible for this is able to dry under, preferably enclosed

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Conditions to be stored for extended periods of time to be added to wine during its production.

The amounts of the freeze-dried preparation added to the wine to induce malolactic fermentation are not particularly critical, but the effective amounts are actually very low. Expressed in terms of the number of viable Microorganisms, suitable amounts can be as little as 100 viable organisms per milliliter of wine. For

2 8 amounts suitable in practice are, for example, about 10 to 10,

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preferably 10 to 10 viable microorganisms per milliliter. In other words, one gram of the preparation is sufficient to undergo malolactic acid fermentation in 2,500 to 25,000 liters To induce wine. In practice it is therefore preferred to use the freeze-dried preparation with an inert, biologically acceptable one to mix solid carrier for dilution -. to easily to give measurable amounts of material for addition. Suitable carriers of this type are, for example, diatomaceous earth and others Forms of harmless clays, powdered cellulose, silicates and similar materials. A mixture of 1 to 5% by weight of the freeze-dried culture preparation is suitable and is expedient used.

As mentioned above, the cultures of the invention are more viable, and they operate over a wider range of conditions in the induction of malolactic acid fermentation as microorganisms described so far. Regarding the severity B-44-40 shows significantly higher rates of malate conversion under similar conditions as the known organisms Lact. delbruchii, Ped. cerevisiae, Lact. buchneri; Leu. Oenos ML-34; Leu. Oenos PSÜ-1; Lact. hildigardii; and lact. Prevo. For example, in tests against Leuconostoc Oenos ML-34 under Using a Rogosa medium and a pH of 5.5 at room temperature the strain B-44-40 has a maximum number of bacteria in four days, while ML-34 produces its (significantly lower) Maximum number of bacteria only developed after thirteen days.

The extension of the range of pH at which malo-lactic acid fermentation can be made, down to a pH value of 3.0, is a very significant advantage in practice. No other culture has been reported to be capable of malo-lactic acid fermeritation in one Carry out a pH of less than 3.2. At the time of bottling, if desired, malo-lactic acid fermentation many wines have a pH below 3.2. The pH of these wines can change with aging increase in the cellar, but it is inexpedient to wait for this process to begin, for malolactic acid fermentation perform. This is at the risk of undesirable Due to side reactions caused by natural organisms that can contaminate the wine. The ability of microorganisms according to the invention, the malolactic acid fermentation Starting at a pH of 3.0 is a significant benefit, further reducing the risks of wine spoilage be reduced.

Equally, there is the possibility of using the microorganisms according to the invention at lower temperatures, down to about 10.degree going down to work, another significant benefit. This temperature is a desired temperature in the winery, where it is convenient to have as many winery operations after primary fermentation as possible. Other known Microorganisms for malolactic acid fermentation must have a Have a temperature of at least 18 ° C for effective operation, which is an undesirable condition for the winery.

Occasionally it has been found that the inventive freeze-dried Microorganism preparation has difficulty in causing malolactic acid fermentation to occur spontaneously in certain types of wine to initiate. This may be due to the fact that the wine does not have the right balance of nutrients and nutrients Has acidity for this purpose. In such cases it can occur malolactic acid fermentation using the

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Preparation can be further guaranteed by adding the freeze-dried organisms to a starter broth at the beginning, which contains a GMT medium adjusted to pH 4 to 7. This * helps keep the organisms from their freeze-dried State can be traced back to a maximally active metabolic state for introduction into suboptimal wine media. To Incubation carried out in this way over a period of, for example, 8 to 72 hours with an initial amount of about

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1x10 to about 1x10 bacterial cells per milliliter of the medium All of the broth can be added to the wine and malolactic fermentation will then begin without difficulty.

The following examples illustrate the invention.

EXAMPLE 1
Selection of the tribe

Isolates have been randomly made from several natural sources. The wine samples were first passed through a sterile 0.65 m filter to remove yeast. Then, the wine passed through a sterile filter-m ^ 0.45, which was applied to a plate-Rogosa (Difco), and incubated at 30 ⁰ C, appeared to discrete colonies. In general, only one isolate was made from any sample, which was mostly the most predominant organism. Whenever possible, isolates were made during the period the wines were undergoing malolactic fermentation. This was done to ensure that the organisms isolated were in fact the predominant organisms responsible for malolactic acid fermentation.

The bacterium designated as B-44-40 was identified in this way a California Chardonnay wine.

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BE. EXAMPLE 2 Enrichment and selection of the strain

The cell mass was measured in a Klett-Summerson colorimeter. A # 66 red filter was used on wine while a # 54 green filter was used on synthetic media. 10 χ 100 mm Kimex or Pyrex test tubes with screw cap closures, each containing 5 ml of wine were vigorously agitated or agitated for the optical determination of the cell mass. Unless otherwise noted, cells were grown at 22 ° C. All of the strains selected in this way were made placed in test tubes, varying either the alcohol content or the pH value or the temperature.

EXAMPLE 3 Alcohol Fortification

Sets of six tubes (10 χ 100 mm screw top) were made filled with 5 ml of a wine sample. Ethanol of 95% purity was added to the tubes to achieve final alcohol concentrations of 13.0%, 14.0%, 14.5%, 15.0%, 15.5% and 16.0% (volume / volume) result. Each organism was placed in a set of test-tube

4 surfaces with an initial cell concentration between 5 χ 10 and Given 5 × 10 cells / ml and the growth was measured in the manner described above. The organisms were because of her high tolerance "to alcohol.

The bacterium B-44-40 grew at an alcohol concentration of 16% (volume / volume) good, and it was able to do so in all wines tested at this alcohol concentration. to carry out the malo-lactic acid fermentation.

EXAMPLE 4 pH Enrichment

The conditions for pH enrichment were the same as for alcohol selection except that in the different Tubes varied the pH instead of the alcohol content. The pH values used were 3.0, 3.3 and 3.7. The tribes were selected on the basis of growth at a low pH.

The bacterium B-44-40 grew at pH 3.0, .i and it was able to to carry out the malolactic acid fermentation at this pH value. The bacterium B-44-40 grew better at low pH than the other strains tested.

EXAMPLE 5 Temperature Tolerance

The conditions for the temperature enrichment were the same as for the alcohol selection, except that the temperature in the tubes varied instead of the alcohol content. The test temperatures were 1O ⁰ C, 2O ⁰ C and 30 ⁰ C. The strains were tested for their growth selected at low temperature.

The bacterium B-44-40 grew better at low temperature (10 ⁰ C) than any other strains tested, and it was capable to perform the Malo-lactic acid fermentation at this temperature.

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EXAMPLE 6
Selection based on malate metabolism

The conditions for malate conversion selection were the same as for the normal growing conditions in wine. After The samples were inoculated daily for malate content measured. Malate content was determined spectrometrically using Boehringer reagents and procedures Mannheim measured. Of all of the strains tested, strain B-44-40 completed malolactic acid fermentation earlier than all other tribes.

EXAMPLE 7
antibiotic resistant strains

Varying amounts of the antibiotic rifampin (rifamycin) or the antibiotic erythromycin have been added to nutrient plates such as Rogosa. The antibiotic content of the plates varied from 10 "" pLg / ml to 100 ng / ml. The rifampin and the erythromycin were of the highest purity available. Bacterial cells from B-44-40 were grown to maximum density in GMT media (as described below). 0.2 ml cells (approximately 2 × 10 cells) were spread on this plate. The plates were then ^{incubated at 30 °} C. until discrete colonies could be seen. Those colonies which appeared to grow normally on the antibiotic-containing plates (as compared to the untreated B-44-40 bacterial cells) were considered to be resistant to the respective antibiotic level. The resistant colonies were placed on separate plates for isolation. They were later retested against normal, non-antibiotic-resistant B-44-40 bacterial cells, namely the parent strain, to ensure that they were indeed resistant to the antibiotic levels described. The tribes involved were:

1. Rif-R1-50 means a rifampin resistance at 50 μg / ml, first isolate;

2. Rif-R1-100 means a rifampin resistance. Bei 100 μ9 / πι1, first isolate;

3. Ery-R1-25 means erythromycin resistance at 25 µg / ml, first isolate;

4. Ery-R2-25 means-an erythromycin resistance at 25 μg / ml, second isolate; and

5. Ery-R1-40 means erythtromycin resistance at 40 µg / ml first isolate.

EXAMPLE -8
Cryogenic protection

The high survival of bacteria during freeze-drying is based on the use of glutamic acid and the other compounds that form a hydrogen bond and two Contain acid groups. Several features of the growth and freeze-drying process are for a high survival of the bacteria is essential, namely the right growth media, the correct pH of the growth media with the cryogen, the correct percentage of cryogen used and the correct growth phase when the cells are harvested and freeze-dried.

The bacterium B-44-40 was grown in a GMT medium which was manufactured as follows:

1.0% yeast extract, 0.25% Tween 80, 0.068% KH ₂ PO ₄ , 0.057% MgSO ₄ · 7Η_0, 0.012% MnSO ₄ · · H ₂ O, 0.003% FeSO ₄ -TH ₃ O and 1% sodium citrate were used Treated with one another for 15 minutes at 121 ^{° C. in an autoclave.} After autoclaving, pre-sterilized glucose was added to a final concentration of 1% (weight / volume). The cryogenic mixture used was 1% sodium glutamate, 0.1% malate

and 0.05% cysteine. This mixture was added to the media after Autoclave treatment given as a filter-sterilized mixture. The pH of the resulting mixture was raised with glacial acetic acid 4.5 set. The bacterial cells were grown until they were

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reached a density of between 1 χ 10 and 2 χ 10 cells / ml. At this point the cells were harvested and freeze-dried. Before freeze-drying, the cells were rapidly frozen at -80 ⁰ C and placed in the frozen state as in a freeze-drying apparatus is that the frozen cells thawed.

EXAMPLE 9

Adding the freeze-dried bacteria to wine

For best results, the wine should be used during the primary Fermentation or shortly after the completion of the primary fermentation. It's not a special treatment of the wine is necessary. The freeze-dried bacterial culture is added directly to the wine, a recommended one Inoculum size is 10 cells / ml. The bacterial culture is in a special dispersant, for example diatomaceous earth, packed in order to facilitate the quick introduction of the universally usable mixture into the wine to be treated.

The bacterial B-44-40 preparation is manufactured in such a way that it can be used for wines with a pH value below 3.3 and at temperatures below 15 ^° C. Once inoculated into the wine, the malate content can be followed by simple chromatography. When all of the malate has been used, the wines can be stored on racks to remove any sediment that may have accumulated during the secondary fermentation period. The bacterium can be B-44-40 or the antibiotic-resistant

stente strain Bio Logicals 44-40. In the latter case, when the consumer wants to test the bacteria in a given wine, the strain can be for its absolute need for oleic acid
or its antibiotic label can be tested in addition to the morphological appearance.

Claims (1)

KRAUS & WEISHRT PATENT LAWYERS AND APPROVED REPRESENTATIVES BEFORE THE EUROPEAN PATENT OFFICE DR, WALTER KRAUS DIPLOMAL CHEMIST D R.-l N G. AN NEKÄTE WEISERT DIPL-ING. SPECIALIZATION CHEMISTRY IRMGARDSTRASSE15 D-SOOO MUNICH 71 TELEPHONE 08 9/79 7077-79 70 78 TELEX O5-21215 6 kpat d TELEGRAM CRAUS PATENT 2959 WK / an JACK SILVER and FRANCES McNICHOLL Santa Rosa / California Lafayette / California United States Means to promote malolactic acid fermentation PATENT CLAIMS 1 "Means to promote malolactic acid fermentation, because * characterized in that it is the microorganism strain Leuconostoc B44-40 (NRC 2477, ATCC 31688) or its counterpart t " about antibiotic resistant mutations Bio Logicals 44-40 (ATCC 31689, 31690, 31691, 31692, 31693) in biologically viable Contains form together with a biologically acceptable carrier material. 2 "Means according to claim 1, characterized in that that the biologically acceptable carrier material is residues of media used in the biological growth of the microorganism contains or consists of. 3. Composition according to claim 2, characterized in that the remainder of the media is glutamic acid and / or biological contain acceptable salts thereof. 4. Means according to claim 3, characterized in that that the microorganism and the glutamic acid and / or the salts thereof are present in lyophilized form and that the Glutamic acid and / or the salts thereof act as a cryoprotectant for the lyophilized microorganism or act around it keep in biologically active form. 5. Means according to claim 4, characterized in that that the microorganism is the antibiotic-resistant Leuconostoc strain Bio Logicals 44-40. 6. Means according to claim 5, characterized in that that it is in admixture with at least an equal amount by weight of a biologically acceptable free flowing particulate Material is present in order to increase the volume and the dispersion of the agent in an aqueous liquid support financially. 7. Agent according to claim 2, characterized in that the remnants of the media contain an acid to act as a cryoprotectant in the subsequent lyophilization of the cells, the acid from the group C-keto-glutamic acid, L-malic acid, DL-malic acid, L-aspartic acid, DL - <^ - aminopimelic acid , ^ C -methyl-L-glutamic acid, malonic acid, N-acetyl-L-glutamic acid, N-dimethyl-L-glutamic acid, N-dimethyl-L-aspartic acid, L-cysteic acid, Custeine, DL-threonine, acetylglycine, DL-5-pyrrolidone-2-carboxylic acid, D-glutamic acid, DL-glutamic acid, L-glutamic acid, aminomalonic acid, tartaronic acid and lysine. 8. Process for the production of new antibiotic-resistant lactic acid bacteria of the genus Leuconostoc or Pediococcus, the strains Bio Logicals 4 4-4 0, characterized in that the bacterial strain B 44-40 grown in a growth-promoting medium under essentially anaerobic conditions that the strain B 44-40 is mutated that the resulting mutants in the presence of an antibiotic and that the antibiotic-resistant mutant strain Bio Logicals 44-40 is grown from the mutation growth medium wins. 9. The method according to claim 8, characterized in that the cultivation of the bacterial mutants B 44-40 in a growth promoting medium containing glutamic acid and / or biologically acceptable salts thereof. 10. The method according to claim 9, characterized in that g e k e η η - ζ e i η e t · that one after the stage of breeding the mutants the biologically viable ones from the growth-promoting medium Bacterial strain B 44-40 wins together with the remaining glutamic acid and / or the glutamate salt and that the obtained Freeze Dried Medium. 11. The method according to claim 8, characterized in that one in a further subsequent Stage the freeze-dried composition in dry form with at least an equal amount by weight of a free flowing particulate biologically acceptable carrier material intimately mixed. 12. Biologically viable lactic acid bacteria of the genus Leuconostoc or Pediococcus, strains Bio Logicals 44-40, wherein the strains are resistant to at least one antibiotic and have the ability to undergo malolactic acid fermentation in wine at temperatures below 18 ^° C. and a pH value induce below 3.3. 13. Method of treating wine to be controlled therein induce rapid malolactic acid fermentation, dadurqh characterized in that an agent according to claim 1, claim 4 or claim 6 is added to the wine. J IZ44U4 -A- 14. The method according to claim 13, characterized in that about 10 to 10 viable; Organisms in the mean per milliliter of wine adds. 15. The method according to claim 13, characterized in that the addition to wine is carried out at a temperature in the range from 8 ⁰ C to 18 ° C and at a pH of less than 3.3. 16. Stabilized against malolactic acid fermentation Wine, characterized in that it contains biologically viable lactic acid bacteria of the genus Leuconostoc or Pediococcus, strains Bio Logicals 44-4 0, the strains being resistant to at least one antibiotic and are also able to induce malotaric acid fermentation in wine.