CZ20001546A3 - Feed ingredients containing D-pantothenic acid and/or salts thereof and process of its preparation - Google Patents

Abstract

Fermentation-based feed additives are obtained fermentation of D-pantothenic-forming microorganisms acid. They contain D-pantothenic acid and / or its the sodium, potassium, ammonium, magnesium and calcium salts thereof preferably to fermentation mashes. Preferably, the feed the additive enriches L-amino acids.

Description

Technical field

The present invention relates to a fermentation mash based animal feed additive comprising D-pantothenic acid and / or one of its salts and a process for the preparation of the additive.

BACKGROUND OF THE INVENTION

Pantothenic acid is produced worldwide in the order of several thousand pools per year. Much of the pantothenic acid produced is used to feed livestock such as poultry and pigs. Its consumption increases.

Pantothenic acid can be produced by chemical synthesis or biotechnology by fermentation of suitable microorganisms in suitable nutrient solutions. D, L-pantolactone is an important precursor in chemical synthesis. It is produced in a multi-step process from formaldehyde, isobutylaldehyde and cyanide, in the next process steps the racemic mixture is separated, D-pantolactone is condensed with β-alanine to give D-pantothenic acid.

A typical commercial form is the D-pantothenic acid calcium salt. The calcium salt of the racemic mixture of D, L-pantothenic acid is also conventional.

The advantage of fermentation by microorganisms lies in the direct formation of the desired stereoisomeric form, namely the D-form, which does not contain L-pantothenic acid.

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D-Pantotenic acid can be produced under suitable fermentation conditions by a variety of bacteria such as Escherichia coli, Arthrobacter ureafaciens, Corynebacterium erythrogenes, Brevibacterium ammoniagenes, as well as yeasts such as Debaromyces castellii, as shown in EP-A-0 493 060, A-0 590 857 and WO 97/10340. Particularly suitable are the derivatives of Escherichia coli IFO3547 described herein, such as strains FV5069 / pFV31 or FV5069 / pFV202.

In the fermentative production of D-pantothenic acid as described in EP-A-0 493 060, EP-A-0 590 857 and WO 97/10340, the microorganism capable of producing D-pantothenic acid is cultured in a suitable nutrient medium and the formed D- pantothenic acid is subsequently costly isolated, purified and recovered as a calcium salt.

Suitable nutrient media include a carbon source such as glucose or starch meal hydrolyzate or sucrose or molasses, precursors such as β-alanine, D, L-pantoic acid or D, L-pantolactone, a nitrogen source such as ammonium sulfate, a phosphorus source such as potassium phosphate and other salts, trace elements and vitamins, and optionally complex media additives such as yeast extract. The microorganisms are then incubated in this medium at a suitable pH with appropriate aeration and agitation to secrete D-pantothenic acid.

According to the prior art described in WO96 / 33283 and EP-A-0 590857, D-pantothenic acid calcium salt is obtained by costly isolation and purification from a fermentation broth containing pantothenic acid. After the first separation of the biomass by filtration or centrifugation, the filtrate is further processed by treatment with activated charcoal or column chromatography. After the solutions thus obtained are reacted with calcium hydroxide, the desired calcium salt can be crystallized.

According to WO 96/33283, the filtrate is decolourised with activated carbon in the first column. The pH was adjusted to 3.0 with concentrated hydrochloric acid, and then the liquid was purified continuously over two additional charcoal columns. The elution of D-pantothenic acid is accomplished with methanol. Subsequent neutralization with Ca (OH) ₂ powder gives a solution from which calcium D-pantothenate is obtained by crystallization at 5 ° C.

In the method described in EP-A-0 590 857, the filtrate is first purified by means of cation exchange and anion exchange columns. Elution is carried out with hydrochloric acid. The eluted fraction is then neutralized with Ca (OH) ₂ , mixed with activated carbon and filtered. The filtrate obtained is then extracted in a low molecular weight alcohol (methanol, ethanol, isopropyl alcohol) and calcium D-pantothenate is obtained by crystallization.

The calcium D-pantothenate produced as described above is used as an additive in animal feed.

According to the prior art, salts of D-pantothenic acid and D, L-pantothenic acid are prepared by reacting acids produced by chemical synthesis or fermentation with the desired salt solutions.

SUMMARY OF THE INVENTION It is an object of the present invention to provide novel forms of D-pantothenic acid formulations and salts thereof as feed additives.

It is a further object of the present invention to provide a production method that is more economical and efficient than the currently known methods.

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SUMMARY OF THE INVENTION

The subject of the invention is a fermentation mash based animal feed additive characterized in that:

(a) it contains D-pantothenic acid and / or its salts, in particular alkali or alkaline earth metal salts;

(b) contains between 0 and 100% of biomass produced during fermentation; and

(c) it contains at least the bulk of the other dissolved constituents of the fermentation broth; and

(d) exists in solid form, in particular fine-grained or granulated and in bulk.

Additives generally exist as desired as spray-dried or freeze-dried, fine-grained particulate powders, but also in granular form, which may contain different proportions of biomass. In particular, the bulk density is approximately 500 kg / m ³ . The ingredients are storage stable.

If biomass is separated, other, for example, inorganic solids are naturally also removed. In addition, the additive according to the invention contains at least the major part of the substances which have been dissolved in the fermentation broth, further formed or added, unless they have been appropriately separated.

These substances may include organic by-products which, in addition to D-pantothenic acid, form and secrete microorganisms used in fermentation. These include L-amino acids selected from the group consisting of L-methionine, Llysine, L-valine, L-threonine, L-alanine and L-tryptophan, especially L-valine. Also included are organic acids having one to three carboxylic groups, such as acetic acid, tt tt tt tt tt tt tt tt tt tt tt Lactic acid, citric acid, malic acid or fumaric acid. Finally, these also include poorly utilizable sugars such as trehalose. These compounds are optionally desired when improving the additive value.

These substances further include residues of the useful sugars used, for example glucose or sucrose.

Another object of the invention is a process for the manufacture of a feed additive comprising D-pantothenic acid and / or salts thereof, characterized in that

(a) the fermentation is produced by a mash containing generally the sodium, potassium, ammonium, magnesium or calcium salts of the D-pantothenic acid contained;

(b) where appropriate, the biomass is wholly or partly separated,

c) mixing the solution or mash thus obtained with an alkali earth or alkali metal hydroxide or oxide, in particular in stoichiometric amounts with respect to D-pantothenic acid; and

d) the mixture thus obtained is dried, spray-dried, spray-granulated or granulated.

A further object of the invention is a method of producing animal feed additives containing D-pantothenic acid and / or its sodium, potassium, ammonium, magnesium or calcium salt in the range of about 20 to 80% by weight (dry matter) of fermentation mash, characterized by these steps

a) preferably by removing water from the fermentation broth (concentration), optionally removing biomass generated during fermentation in an amount of 0 to 100%,

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c) adding one or more of said compounds to the fermentation broths obtained according to a) and b), wherein the amount of added compounds is measured such that their total concentration in the animal feed additive is in the range of about 20 to 80% by weight, in particular 50 to 80% by weight and

d) drying the fermentation broth obtained according to c) to obtain animal feed additive in the desired powder or granulate form.

Fermentation mashes obtained by using microorganisms suitable for the production of D-pantothenic acid and containing D-pantothenic acid and / or salts thereof are suitable for the process of the invention.

The microorganisms may be fungi or yeasts, for example Debaromyces castellii or gram-positive bacteria, for example of the genus Corynebacterium, or gram-negative bacteria, for example of the family Enterobacteriaceae. In the case of the Enterobacteriaceae family, mention should be made in particular of the genus Escherichia with Escherichia coli. In the case of Escherichia coli, so-called K-12 strains such as MG1655 or W3110 strains (Neidhard et al. Escherichia coli and Salmonella. Cellular and Molecular Biology (ASM Press, Washington DC)) or wild type IFO3547 Escherichia coli strain (Institute for fermentation, Osaka, Japan) and mutants derived therefrom. Among the strains produced from IFO3547, FV5069 / pFV31 (EP-A-0 590 857) and FV5069 / pFV202 (WO 97/10340) stand out. In the case of the genus Corynebacterium, the species Corynebacterium glutamicum should be mentioned.

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The above-described microorganisms can be cultivated continuously or discontinuously for batch production or batch fed or fed batch or repeated fed batch. A summary of known cultivation methods is described in a textbook from Chmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in a textbook from Storhas (Bioreactor and Periphere Einrichtungen (Viewegh Verlag, Braunschweig / Wiesbaden, 1994)).

The culture medium to be used must suitably meet the requirements of the microorganisms in question. Descriptions of the culture media of various microorganisms can be found in the Manual of Methods for General Bacteriology of the American Society for Bacteriology (Washington, D.C., USA, 1981). As carbon sources, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose can be used; oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as palmitic acid, stearic acid, linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These may be used as individual components or as a mixture. Nitrogen-containing organic compounds such as peptones, yeast extract, meat extract, malt extract, soaking water, soy flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used singly or as a mixture. As the phosphorus source, potassium dihydrogen phosphate or potassium hydrogen phosphate or the corresponding sodium-containing salts can be used. The culture medium must furthermore have a culture medium of at least one of the following: musí «médium musí musí musí musí musí musí musí musí musí musí dále dále dále dále dále dále dále dále dále dále dále dále dále dále dále dále Contain metal salts, such as magnesium sulfate or iron sulfate, that are needed for growth. Finally, essential growth substances such as amino acids and vitamins may be used in addition to the above-mentioned substances. In addition, suitable D-pantothenic acid precursors such as aspartate, β-alanine, ketoisovalerate, ketopantoic acid or pantoic acid and optionally salts thereof may be added to the culture medium. Said components may be added to the culture in the form of a single batch or may be added in a suitable manner during the cultivation.

Preferably, ammonia or ammonia water is used to control the pH of the culture. Other basic compounds such as sodium hydroxide or potassium hydroxide are also suitable. If acidic compounds are required, phosphoric acid or sulfuric acid is suitably used. Anti-foaming agents such as polyglycol esters of fatty acids may be used to control foaming. To maintain the stability of the plasmids, suitable selectively acting substances, for example antibiotics, may optionally be added to the medium. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as air, are introduced into the culture. The temperature of the culture is usually about 20 ° C to 45 ° C, and especially about 25 ° C to 40 ° C. The culture is maintained until a maximum of D-pantothenic acid is formed. This goal is usually achieved in 10 hours to 160 hours.

The fermentation broths obtained in this way usually have 7.5 to 25% by weight of dry matter and contain D-pantothenic acid in a concentration higher than 0 to 20% by weight. Particularly preferred are fermentation processes in which 2 to 20% by weight of D-pantothenic acid is present in the dry matter after the fermentation has ended. In addition, it is also advantageous if the fermentation is carried out at least at the end, but preferably at least during the fermentation.

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00 00 00% of the duration of the fermentation, leads in a sugar-limiting manner. That is, during this time, the concentration of usable sugar in the fermentation broth is maintained at or greater than or equal to 0 to 3 g / l.

In a variant of the production of additives according to the invention comprising ammonium ions, the fermentation broths containing D-pantothenic acid are optionally first or totally freed of biomass by known separation methods, such as centrifugation, filtration, decantation or combinations thereof.

According to the invention, however, the biomass can be left completely in the fermentation broth. Subsequently, this fermentation mash is generally mixed with 0.8 to 1.2, in particular 0.95 to 1.1, equivalent of an alkali or alkaline earth metal oxide or hydroxide, in particular NaOH, KOH, Ca (OH) ₂ or MgO, relative to Dpantothenic acid. At low concentrations of D-pantothenic acid, it may also be advantageous to use significantly larger amounts of oxides or hydroxides, for example in the range of 1.2 to 4 equivalents. The suspension thus obtained is generally concentrated to a maximum of 60% by weight of dry matter before drying. Similarly, the fermentation broth may be first concentrated and then oxides or hydroxides may be added. The concentrate obtained is then obtained as a free-flowing, fine-grained powder or free-flowing granulate in a conventional drier or, for example, by means of a film-flowing film evaporator or a thin-film evaporator or spray dryer or spray granulator or lyophilizer. The granulation can also be carried out after drying, for example in the form of a superstructure granulation.

The inventors have further found a novel method for producing powders containing ammonium, potassium, sodium, magnesium or calcium salts of D-pantothenic acid or the formulations containing them in a rapid and inexpensive manner. For this purpose, a fermentation broth containing D-pantothenic acid is used.

ft ftftft * ftft ftft ftft ftft ftftft ftftftft ftftft ftftftft ftftftftftftftftftftftftftftftftftftftftftft ftft ftft ftft ftft produced using appropriate hydroxy compounds, or by first known separation methods such as centrifugation, filtration, decantation or a combination thereof completely or partially frees biomass. However, it is also possible according to the invention to completely leave the biomass in the fermentation broth. Subsequently, the optionally pretreated mash is concentrated or dried by known methods, for example by means of a rotary evaporator or a thin-film evaporator or a liquid-bed evaporator. The optionally concentrated mash is then processed by spray drying, spray granulation or other methods to form a free-flowing, fine-grained powder or granular granulate.

The novel animal feed additives according to the invention generally contain 20 to 80% by weight, in particular 30 to 75% by weight, of D-pantothenic acid and / or its salts, based on the total amount. In addition, the inorganic components additionally generally comprise 2.5 to 25% by weight and optionally organic by-products in an amount of greater than 0 to 30% by weight. The proportion of dry biomass is 0 to 35% by weight. The water content is preferably less than or equal to 5% by weight. The desired content of D-pantothenic acid and / or one or more of said salts is optionally achieved by adding the appropriate compounds to the products produced by fermentation. The desired compounds are preferably added to the mixture before drying or spray drying, especially after concentration, in the form of solutions or dry substances. The product thus obtained is used as a feed additive.

The concentration of D-pantothenic acid can be determined in a known manner (Velisek; Chromatographic Science 60, 515560 (1992)).

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The present invention is explained in more detail below with reference to exemplary embodiments. To this end, experiments were carried out with a D-pantothenic acid-producing strain of Escherichia coli 5069 / pFV31, deposited as FERM-BP 4395 under the Budapest Treaty at the Fermentation Research Institute, Agency of Industrial Science and Technology at 11-3, Higashi, Tsukubashi, Ibaraki (Japan).

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Preparation of fermentation mash containing D-pantothenic acid

1. Inoculum production

A sample of Escherichia coli FV5069 / pFV31 was plated on LBG agar supplemented with 50 µg ampicillin per ml. This culture on an agar plate was incubated for 17 hours at 37 ° C and then stored in a refrigerator at +4 ° C. Selected individual colonies were subsequently propagated in LBG broth. The LBG broth has the following composition: 10 g / l peptone, 5 g / l yeast extract, 5 g / l NaCl and 1 g / l glucose. LBG agar additionally contains 12 g / l agar. Pre-formulations can be obtained from Gibco / BRL (Paisley, Scotland, UK) as LB Broth Base or LB-agar. After adding 1 g / l of glucose, the above media are then obtained. The 10 ml cultures in 100 ml Erlenmeyer flasks were incubated for 16 hours at 37 ° C and 180 rpm in an ESR incubator from Kühner AG (Birsfelden, Switzerland). Then the cell suspension was centrifuged in a Beckmann centrifuge (Hannover, Germany) for 15 minutes at 4000 rpm. The cell pellet was resuspended in 10 ml of LBG medium supplemented with 20% glycine. * Ftft. * Ftft. * Ftft. Ftft roll and filled in 10 aliquots per ml under sterile conditions and frozen at -70 ° C. These cultures were used as a master cell bank.

To form a working cell bank, LBG medium supplemented with 50 µg / ml ampicillin was divided into 10 ml portions into 100 ml Erlenmeyer flasks and then inoculated with 100 μΐ of the above described master cell bank. Incubation was carried out for 16 hours at 37 ° C and 180 rpm in an ESR incubator from Kuhner AG (Brisfelden, Switzerland). After incubation, the optical density (OD) of the culture suspension was determined using a LP2W photometer from Dr. R &amp; Lange (Berlin, Germany) at a measuring wavelength of 660 nm. It was 3.5. Subsequently, the cell suspension was filled into sterile 30 ml polyethylene tubes from Greiner (Frickenhausen, Germany) under sterile conditions and centrifuged at 2500 rpm for 15 minutes in a J-6B centrifuge from Beckmann (Hannover,

Germany). The separated biomass was resuspended in 10 ml of LBG medium supplemented with 20% glycerol. Then, the cell suspension was filled in 500 µl portions into 1 ml sterile from Nalgene (New York, USA) under sterile conditions and frozen at -70 ° C. The cans produced in this way were used as a working cell bank.

2. Preparation of the fermentation broth containing D-pantothenic acid

To prepare a fermentation broth containing D-pantothenic acid, a working flask of cells was first propagated in a shake flask culture and used to inoculate a pre-fermenter. The pre-fermenter culture was used to inoculate the production fermenter.

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SKA medium was used for shake flask culture. SKA medium was prepared as follows. In a 1 liter beaker, 7.0 g (NH ₄ ) ₂ SO ₄ , 0.5 g KH ₂ PO ₄ , 1.0 g K ₂ HPO ₄ , 0.5 g MgSO ₄ .7H ₂ O, 0 were weighed. 0.01 g of MnSO ₄ .H ₂ O, 0.01 g of ZnSO ₄ .7H ₂ O, 0.005 g of Fe ₂ (SO ₄ ) ₃ and 20 g of soaked water previously adjusted to pH 6.8 with 25% ammonia solution , and then 875 ml of distilled water was added. This soaking salt solution was sterilized by autoclaving at 121 ° C for 20 minutes. Next, a solution containing 125 g of distilled water, 28.7 g of glucose and 0.002 g of thiamine.HCl was sterilized by filtration. 10 g CaCO ₃ was weighed in a 100 ml flask and autoclaved at 123 ° C for 20 minutes. Combining both of the above components with a soaking water-containing solution resulted in SKA medium.

This SKA medium was divided into 12.5 ml aliquots into 100 ml Erlenmeyer flasks and then seeded with 0.5 ml cell suspension. A cell suspension of a working cell culture diluted with 1: 100 sterile saline was used as the cell suspension. Incubation was performed for 20 hours at 32 ° C and 150 rpm in an RC-1-TK incubator from Infors (Bottmingen, Switzerland). The optical density determined at a measurement wavelength of 660 nm was 12.5.

0.5 ml of this shake flask culture was diluted with 4.5 ml of physiological saline and 0.7 ml was used to inoculate 1300 ml of culture medium contained in a 21 Biostat® MD laboratory fermenter from Braun Diessel Biotech GmbH (Melsungen, Germany).

The culture medium was prepared as follows. Solution consisting of 9.81 g (NH ₄ ) ₂ SO ₄ , 0.7 g KH ₂ PO ₄ , 1.402 g K ₂ HPO ₄ , 0.70 g MgSO ₄ .7H ₂ O, 0.014 g MnSO ₄ . H ₂ 0, 0.014 g Fe ₂ (SO ₄ ) ₃ · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 28 ΦΦ č φφ and 28.04 g of soaking water in 1300 ml of tap water were adjusted to pH 6.5 with 25% ammonia solution and autoclaved at 121 ° C for 20 minutes. To this steeping water containing the salt solution was added, under sterile conditions, a separately sterile filtered solution containing 40.62 g of glucose and 0.0042 g of thiamine.HCl in 100 g of distilled water.

Fermentation was carried out for 16 hours at 37 ° C and with aeration of 1 vvm. The dissolved oxygen was maintained at 20% and at pH 6.5. A 25% ammonia solution was used as the pH correction agent. The optical density was 13.1. 90 ml of this culture was used to inoculate 1144 ml of main fermentation growth medium in a 21 laboratory Biostat® MD fermenter.

Growth medium was prepared as follows. Solution consisting of 14.14 g (NH ₄ ) ₂ SO ₄ , 0.744 g KH ₂ PO ₄ , 1.0 g K ₂ HPO ₄ , 0.83 g MgSO ₄ .7H ₂ O, 0.0124 g MnSO ₄ . H ₂ O, 18.87 g βalanine, 0.74 Structure J647 and 49.72 g steeping water in 1144 ml tap water were adjusted to pH 6.5 with 25% ammonia solution and autoclaved at 121 ° for 20 minutes C.

To this steeping water containing the salt solution was added, under sterile conditions, a separately sterile filtered solution which contained in 100 ml of distilled water 35.92 g of glucose and 0.002 g of thiamine.HCl.

The fermentation was carried out for 40 hours at 37 ° C. In the growth phase, the pH was 6.5 and the aeration was 1 vvm. In the production phase the pH was 6.0 and the aeration was 1.5 vvm. The dissolved oxygen was kept below 2% in both phases. A 25% ammonia solution was used as the pH correction agent. During fermentation, the production medium 1 and the production medium 2 were fed in stages. Dipping water was added once during the cultivation. The production medium contains 465.29 g of glucose in 584 ml of tap water and «· · φ · · φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ

0.0261 g of thiamine.HCl, which were sterile filtered. Production medium 2 contains 37.5 g β-alanine in 140 ml tap water, which was autoclaved at 121 ° C for 20 minutes. After a fermentation period of 7.5 hours until the end of the cultivation, the production medium 1 was fed in stages. After 10.5 hours of culture, an additional 49.5 g of steeping water was added under sterile conditions, which was dissolved in 100 ml of tap water and sterilized for 20 minutes at 121 ° C in an autoclave. After a fermentation time of 12.5 hours until the end of the cultivation, the production medium 2 was fed at a feed rate of 3.5 g / h.

After 41 hours of cultivation, the concentration of pantothenic acid was 6.1% by weight in the fermentation broth.

The D-pantothenic acid content was determined by HPLC (high performance liquid chromatography) type M321 from Knauer (Berlin, Germany) by RI (refractive index) detection using Hypersil APS2 Aminophase with a grain size of 5 gm.

Example 2

In a fermentation experiment carried out under the same conditions as described in Example 1, the concentration of pantothenic acid in the fermentation broth after 5.4 hours was 5.4%. The L-valine concentration was 8 g / L.

Example 3

Preparation of calcium D-pantothenate

From the fermentation broth containing pantothenic acid produced by the methods of Examples 1 and 2 and which contained about 6 ftftft ftft ftft ftft ftft ftftftft ftftftft ftftftft ftftftft ftft ftftft ftft ftftftftft ftft ftft ftft ftft ftft 1% by weight of D-pantothenic acid, the biomass was first separated. To this end, 1 L of the above fermentation broth was centrifuged for 20 minutes at 4000 rpm in a Biofuge-Stratos laboratory centrifuge from Heraeus (Dusseldorf, Germany) and the centrifugation supernatant was then further purified by cross-flow ultrafiltration with a polymer membrane. 30kD in UF from ICT GmbH (Bad Homburg, Germany).

Then 10.1 g of solid Ca (OH) ₂ (96%; MERCK, Darmstadt, Germany) were added with stirring with breaks. The pH was then about 10.3. The treated mash was then concentrated in a Rotavapor RE-120 rotary evaporator from Buchi-Labortechnik GmbH (Konstanz, Germany) to a liquid fraction of approximately 50% of the dry fraction under vacuum at 60 ° C. The concentrated mash was then spray dried to obtain D-pantothenic calcium salt. For this purpose, a Büchi190 laboratory spray dryer from Buchi-Labortechnik GmbH (Konstanz, Germany) was used at an inlet temperature of 107 ° C, an outlet temperature of 85 ° C, a pressure difference of -4000 Pa and an air flow rate of 600 l / h.

The calcium D-pantothenate product thus produced had a D-pantothenic acid content of 68.5% by weight, was free-flowing and had a bulk density of 460 mg / ml. After a storage period of 5 months, the D-pantothenic acid content was 67.6% by weight.

Example 4

Preparation of sodium D-pantothenate

From a fermentation broth containing pantothenic acid, prepared according to the methods of Examples 1 and 2, and which: 0 00 0 0 0 0 0

0,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000-000-000-000-000-000-000-000-7- To this end, 1 L of the above fermentation broth was centrifuged and ultrafiltered as described in Example 3.

10.6 g of NaOH (99%; MERCK) were then added with stirring. The pH was then about 10.

The treated mash was then concentrated in a Rotavapor RE-120 rotary evaporator from Buchi-Labortechnik GmbH (Konstanz, Germany) to a liquid fraction of approximately 50% of the dry fraction under vacuum at 50-60 ° C. The concentrated mash was then lyophilized to obtain Dpantotenic acid sodium salt in a LYOVAC GT 2 type lyophilizer from Leybold (Cologne, Germany).

The sodium D-pantothenate product thus formed had a D-pantothenic acid content of 63.8% by weight and was free-flowing. After a storage period of 5 months, the D-pantothenic acid content was 63.0% by weight.

Example 5

Preparation of magnesium D-pantothenate

The biomass was first separated from the pantothenic acid-containing fermentation broth prepared by the method of Examples 1 and 2 and containing approximately 6.1% by weight of D-pantothenic acid. To this end, 1 L of the above fermentation broth was centrifuged and ultrafiltered as described in Example 2.

5.4 g of solid MgO (97%; MERCK) were then added with stirring. The pH was then about 9-10. The mash was then treated under vacuum.

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9 9 9 4 4 9 9 9 9 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 50-60 ° C in a Rotavapor RE-120 rotary evaporator from Buchi-Labortechnik GmbH to a liquid fraction of approximately 50% of the dry fraction. The concentrated mash was then lyophilized to obtain the magnesium salt of D-pantothenic acid in a LYOVAC GT 2 type lyophilizer from Leybold.

The magnesium D-pantothenate product thus formed had a D-pantothenic acid content of 64.7% by weight and was free-flowing. After a storage period of 5 months, the D-pantothenic acid content was 64.4% by weight.

Example 6

Preparation of potassium D-pantothenate

The biomass was separated from the pantothenic acid-containing fermentation broth prepared by the method of Examples 1 and 2 and containing approximately 6.1% by weight of D-pantothenic acid. To this end, 1 L of the above fermentation broth was centrifuged and ultrafiltered as described in Example 2.

Then 17.4 g of KOH (85%; MERCK) were added with stirring with breaks. The pH was then about 10 to 11. The treated mash was then concentrated in a Rotavapor RE-120 rotary evaporator from Buchi-Labortechnik GmbH under vacuum at 60 ° C to a liquid fraction of approximately 50% of the dry fraction. The concentrated mash was then lyophilized to obtain the D-pantothenic acid potassium salt in a LYOVAC GT 2 type lyophilizer from Leybold.

The potassium D-pantothenate-containing product thus formed had a D-pantothenic acid content of 63.5% by weight, and this is the following: It was loose. 9 It was loose. After a storage period of 5 months, the D-pantothenic acid content was 62.9% by weight.

Example 7

Preparation of ammonium D-pantothenate

The biomass was first separated from the pantothenic acid-containing fermentation broth prepared by the method of Examples 1 and 2 and containing approximately 6.1% by weight of D-pantothenic acid. To this end, 1 L of the above fermentation broth was centrifuged and ultrafiltered as described in Example 2.

The treated mash was then concentrated in a Rotavapor RE-120 rotary evaporator from Büchi-Labortechnik GmbH under vacuum at 60 ° C to a liquid fraction of approximately 50% of the dry fraction. The concentrated mash was then lyophilized to obtain the D-pantothenic acid ammonium salt in a LYOVAC GT 2 type lyophilizer from Leybold.

The ammonium D-pantothenate product thus formed had a D-pantothenic acid content of 66.8% by weight and was free-flowing.

Example 8

Preparation of calcium D-pantothenate from fermentation broth containing biomass

The fermentation broth containing pantothenic acid, prepared according to the methods of Examples 1 and 2, and containing about 6.1% by weight of D-pantothenic acid, was first concentrated in a rotary vacuum at 60 ° C in a rotary phase. * ΦΦφφ · v v «« «« «•φ • φφφφφφφφφφφφφφφφφφφφ v v v

Rotavapor RE-120 evaporator from Buchi-Labortechnik GmbH (Konstanz, Germany) with a volume of 1.0 L per liquid fraction of approximately 30% of the dry fraction. Then 10.1 g of solid Ca (OH) ₂ was added with stirring. (96%; MERCK, Darmstadt, Germany). The pH was then about 10. The treated and concentrated biomass containing mash was then spray dried to obtain D-pantothenic acid calcium salt. A laboratory spray dryer of the Buchi-190 type from Buchi-Labortechnik GmbH (Konstanz, Germany) was used at an inlet temperature of 107 ° C, an outlet temperature of 85 ° C, a pressure difference of -4000 Pa and an air flow rate of 600 l / h.

The calcium D-pantothenate product thus formed had a D-pantothenic acid content of 49.8% by weight, was free-flowing and had a bulk density of 480 mg / ml. The biomass content was approximately 30% by weight.

Example 9

Preparation of a product containing calcium D-pantothenate and Corynebacterium glutamicum biomass

1. Preparation of a pantothenic acid producing strain of Corynebacterium glutamicum

US-A-5,188,948 discloses a strain of Brevibacterium lactofermentum FERM BP-1763 producing Lvalin. DE 19855313.7 discloses the plasmid pND-DBC2 (FIG

1) which carries the panB, panC and pnaD genes of Corynebacterium glutamicum. The plasmid is deposited in the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as ATCC13032 / pND-DBC2 strain as DSM 12437. Transformation of tttt ·· tttt ·· • tt tttt strain FERM BP-1763 with plasmid pND-DBC2 gave strain FERM BP1763 / pND-DBC2 produces 2 / pantothenic acid.

2. Preparation of the fermentation broth containing pantothenic acid

A sample of Brevibacterium lactofermentum FERM BP-1763 / pNDDBC2 is spread on HHK agar.

HHK Agar consists of brain-heart agar, which was obtained from Merck KgaA (Darmstadt, Germany) and supplemented with kanamycin. The composition of HHK agar is shown in Table 8a.

This agar plate culture was incubated at 37 ° C for 17 hours and then stored in a refrigerator at +4 ° C. Selected individual colonies were subsequently propagated on the same medium. Using the loop, the cell material of one clone was removed from HHK agar and transferred to 100 ml of HHK broth contained in a 1000 ml shake flask.

The HHK broth consists of a brain-heart medium, which was obtained from Merck KgaA (Darmstadt, Germany) and supplemented with glucose and kanamycin. The composition of the HHK broth is shown in Table 8b.

Table 8a: HHK Agar

Substance Quantity per liter Brain-heart agar 52.0 g Kanamycin 25 mg

Table 8b: HHK broth

Substance Quantity per liter Brain-heart medium 37,0 g Kanamycin 25 mg Glucose 20.0 g

The batch was incubated at 30 ° C and 150 rpm for 22 hours. After cultivation, an optical density of 6.1 was measured with a photometer at 660 nm (OD 660). This culture of the FERM BP-1763 / pND-DBC2 strain was used to inoculate a production fermenter.

The SK-71 medium shown in Table 8c was used for fermentation. All components of SK-71 medium were submitted directly to the working concentrations in the fermenter and sterilized in situ.

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9 9 9 9 9 9 9 9 · · · · ···········

Table 8c: SK-1 Medium

Compound Quantity per liter Glucose hydrate 110,0000 g Cornsteep Liquor 5,0000 g β-Alanine 5,0000 g Nicotinic acid 0.0050 g L-Isoleucine 0.1500 g Homoserine 0.1500 g (NH ₄ ) ₂ SO ₄ 25,0000 g kh ₂ Mon ₄ 0.1000 g MgSO ₄ .7H ₂ O 1,0000 g FeSO ₄ .7H ₂ O 0.0100 g MnSO ₄ .H ₂ O 0.0050 g CaCl ₂ 2H ₂ O 0.0100 g Thiamin.HCI 0.0002 g D - (+) - Biotin 0.0003 g Struktol 0.60 g

10 L reactors from B. Braun (BBI, Germany, Melsungen, Modell Biostat E / ED) were used as fermenters.

To inoculate 1,950 g of SK-71 fermentation medium, 100 ml of the shake flask preculture described above was used in the HHK broth.

The batch was cultured for the entire duration of the fermentation at 30 ° C, 0.75 vvm volume specific aeration, stirring at 800 to 1700 rpm depending on oxygen consumption and pH 7.0 and partial oxygen pressure of 20% air saturation. The culture was cultivated for a total of 49 hours under the above conditions until an optical density of 26.2 was reached at 26.2 at an optical density of 26.2. · · · · · ·

660 nm (OD660 nm). Aqueous ammonia solution (25% w / v) was used as a pH correction agent.

Subsequently, the optical density (OD) was determined using a digital photometer of the type LP1W from the company Dr. Bruno Lange GmbH (Berlin, Germany) at a measuring wavelength of 660 nm and the concentration of D-pantothenic acid formed by HPLC (Hypersil APS 25 µm, 250x5 mm, detection R1).

The concentration of D-pantothenic acid was approximately 0.2 g / L in the final fermentation sample after 49 hours.

3. Preparation of a pantothenic acid product

According to the method of Example 9.2, a fermentation broth containing D-pantothenic acid containing about 0.02% by weight of D-pantothenic acid was prepared. 1.4 L of this fermentation mash was first concentrated in vacuo at 60 ° C in a Rotavapor RE-120 rotary evaporator from Buchi-Labortechnik GmbH (Konstanz, Germany) to a mash with a dry content of approximately 15%.

27.1 g of solid Ca (OH) 2 (96%; MERCK, Darmstadt, Germany) were then added with stirring. The pH was then about 10.0. The thus treated and concentrated biomass-containing mash was then mixed with 37.7 g of calcium D-pantothenate (&gt; 98%, Euro OTC Pharma GmbH, Kamen, Germany). For subsequent spray drying, a Büchi-190 laboratory spray dryer from Buchi-Labortechnik GmbH (Konstanz, Germany) was used at an inlet temperature of 107 ° C, an outlet temperature of 85 ° C, a pressure difference of -4000 Pa and an air flow rate of 600 l / h. .

· · * Λ λ * λ λ λ λ λ λ λ λ λ λ λ λ λ λ λ λ λ λ λ λ λ λ λ ·· · * ·· · · ·

The calcium D-pantothenate product thus formed had a D-pantothenic acid content of approximately 35% by weight, was free-flowing and had a bulk density of 600 mg / ml. The biomass fraction of C. glutamicum was approximately 3.5% by weight.

Example 10

Preparation of a product containing calcium D-pantothenate and Saccharomyces cerevisiae biomass

1. Preparation of a pantothenic acid producing strain of Saccharomyces cerevisiae

Reading grid amplification YHR063c:

Starting from the nucleotide sequence of the YHR063c reading screen of Saccharomyces cerevisiae (accession number U00061, National Center for Biotechnology, Bethesda, MD), the following PCR primers were synthesized (MWG-Biotech, Ebersberg, Germany). The beginning or end of the reading grid is indicated by a dot (.):

• OJD539 (5 'EcoRI-Notl START):

5'- GCG CGA ATT CAG ATC GCC GGC CG AAA GAG GAG AAA TTA ACT.ATG ACT GCA CCA CAC AGA AG -3 '

5'- CGC GAC TAG TCT GCA GTC CTC TTC TCC AGT CAC-3 '

Genomic DNA of S. cerevisiae JD242 strain, which was isolated from C. Guthrie and G. according to the method, served as template.

R. Finka (Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, Academic Press, San Diego) 9 9 9 9 * · · · · ·

CA, 1991). This strain is a haploid segregant of the diploid strain SC288C (Winston et al., Yeast 11, 53 et seq., (1995)), whose genome was sequenced (Goffeau et al., Science 274, p. 546, (1996)). Tetradene analysis was performed according to the method of C. Guthrie and G. R. Fink (Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, Academic Press, San Diego, CA, 1991). The JD242 strain is auxotrophic for leucine (allele leu2A1) and uracil (allele ura352). A DNA fragment of approximately 1.2 kB could be amplified using the High Fidelity Expand Polymerase kit from Roche (Mannheim) for 28 cycles of PCR under the conditions specified by the manufacturer. Size was determined by electrophoretic separation in a 0.8% agarose gel.

2. Construction of pJD-YHR063c:

To express the YHR063c reading screen in S. cerevisiae, a PCR amplification was inserted into the E. coli -S. Cerevisiae shuttle vector pJDCEX2 (Figure 2 and Dohmen at al., 1995, Journal of Biological Chemistry 270, 18099-18109).

The PCR product was first restricted with EcoRI and SpeI (AGS, Heidelberg, Germany). Subsequently, it was mixed with pJDCEX2DNA, which had been treated with EcoRI and XbaI (AGS, Heidelberg, Germany), and ligated with T4 DNA ligase (Roche, Mannheim, Germany). The ligation batch was transformed into XL1-Blue E. coli strain (Bullock et al., 1987, Biotechniques 5, 376). The transformant was obtained by selection on LB agar containing 150 gg / ml ampicillin (Sigma, Diesenhofen, Germany). Plasmid DNA from ampicillin resistant clones was prepared by alkaline lysis (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press).

1989). The isolated plasmid DNA was then examined by Notl and PstI restriction and subsequent splitting in 0.8% agarose <RTIgt;% </RTI> ftftftft. • ft · · · ft ft ftft · ft ·· ftft «· ftft ftft ftft gel. The plasmid with the desired structure was named pJD-YHR063c (Figure 3).

Preparation of JD242 / pJD-YHR063c strain:

S. cerevisiae strain JD242 was transformed with plasmid pJDYHR063c according to the method of Dohmen et al. (Dohmen et al., Yeast 7: 691 (1991)). Transformant selection was performed on minimal leucine-free SD medium with 1.8% agar (see Tables 9a and 9b).

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9 9 9 9 9

9 9 9 9 9 · 9 · 99

Table 9a: Minimum SD media

Compound Quantity per liter (NH ₄ ) ₂ SO ₄ 5 g kh ₂ Mon ₄ i g MgSO ₄ .7H ₂ O 0.5 g NaCl 0.1 g CaCl ₂ 0.1 g h ₃ or ₃ 500 μg CuSO ₄ 4 0 μg KI 100 μg FeCl ₃ .6H ₂ O 200 μρ MnSO ₄ .H ₂ O 400 μg Na ₂ MoO ₄ .2H ₂ O 400 μg ZnSO ₄ .7H ₂ O 200 μg Biotin 2 μρ Folic acid 2 μg Inositol 2 mg Niacin 400 μg p-Aminobenzoic acid 200 μg Pyridoxine hydrochloride 400 μg Riboflavin 200 μg Thiamine hydrochloride 400 μg

• 9 9 9 9 9 9 9 9 9 9 9 9 9 9

9

Table 9b: Minimum SD media

Ingredients Quantity per liter Glucose 20 g Uráčil 40 mg CuSO ₄ 24 mg -Leu DO supplement 650 mg Ketopantoate 100 mg β-Alanine 100 mg

2. Preparation of the fermentation broth containing pantothenic acid

To prepare the fermentation broth containing D-pantothenate, a single colony of S. cerevisiae strain JD242 / pJD-YHR063c was first plated on an agar plate with minimal SD medium and incubated for 3 days at 30 ° C. To prepare this first preculture in a shake flask, cells were subsequently rinsed with 5 ml of minimal SD medium. Using a 2.5 ml cell suspension, a shake flask (total volume 500 ml) was seeded with 50 ml minimal SD medium (Tables 9a and 9b) and cultured at 30 ° C and 130 rpm in an RC-1-TK incubator from from Infors AG (Bottmingen, Switzerland) for 6 hours until the optical density of 1.9 at 660 nm (OD660) could be measured. A second preculture was placed in a 100 ml obstruction flask in 150 ml of minimal SD medium (Tables 9a and 9b) and seeded with 50 ml of the above-described preculture. Incubation was performed at 30 ° C and 80 rpm for 20 hours until the optical density reached approximately 3.8 at a wavelength of 660 nm (OD 660). The main fermentation for pantothenic acid production was performed in a round bottom flask with a total volume of 6000 ml in 1500 ml of minimal SD medium (Tables 9a and 9b). To do this, the flask is: · ňka ňka φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ Round-bottom inoculated 90 ml of preculture 2 and incubated at 30 ° C and 60 rpm for 30 hours.

Optical Density (OD) was measured with a digital photometer type LP1W from Dr. Bruno Lange GmbH (Berlin, Germany) at a measurement wavelength of 660 nm. The concentration of D-pantothenic acid formed was determined using the Lactobacillus plantarum ATCC® 8014 strain according to the DIFCO MANUAL of DIFCO (Michigan, USA; 10 ^th Edition, 1100-1102 (1984)).

The optical density (OD660) was approximately 4 and the Dpantotenic acid content was approximately 1 mg / l.

3. Preparation of a pantothenic acid product

A fermentation broth containing pantothenic acid containing approximately 1 mg / ml D-pantothenic acid was prepared by the method of Example 10.2. The 6.0 L volume was first concentrated in a rotavaporator of type Rotavapor RE-151 from Büchi-Labortechnik GmbH (Konstanz, Germany) to a melt with approximately 16% dry fraction under vacuum at 60 ° C. 15.9 g of solid Ca (OH) 2 (96%; MERCK, Darmstadt, Germany) were then added with stirring. The pH was then about 9.2. The thus processed and concentrated biomass-containing mash was then mixed with 28.4 g of calcium D-pantothenate (&gt; 98%, Euro OTC Pharma GmbH, Kamen, Germany). The mash was then lyophilized in a LYOVAC GT 2 type lyophilizer from Leybold (Cologne, Germany).

················ 4 9 4 9 9 4 4 9

449 4 44444 444

9 9 4 4 9 4 4 4 5

444 4 9 9 4 4 4 4 4 44

The calcium D-pantothenate product thus prepared had a D-pantothenic acid content of 26.5% by weight, was free-flowing and had a bulk density of 450 mg / ml. The biomass fraction of S. cerevisiae was approximately 6.8% by weight.

Ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft

BRIEF DESCRIPTION OF THE DRAWINGS

The following pictures are attached:

Figure 1: Map of plasmids pND-DBC2

Figure 2: Map of plasmids pJDCEX2

Figure 3: Map of plasmids pJD-YHR063c

The abbreviations used in the figures have the following meanings:

To Figure 1:

rrnBT1T2: rrnB gene transcriptional terminator

Bird: tac-promoter

panB: coding region gene panB panC: coding region gene panC panD: coding region gene panD

rep-C.g .: DNA region for replication in C.glutamicum oriV-E.c .: origin for vegetative transfer in E. Coli kan: kanamycin resistance gene

BglII: place cut restriction enzyme BglII Clal: place cut restriction enzyme Clal Ncol: place cut restriction enzyme Ncol

···· • tt · · · · tt · tttt tt

33 • ♦ • · · • · »Tt Nrul: place cut restriction enzyme Nrul Pvul: place cut restriction enzyme Pvul Suction: place cut restriction enzyme Suction Halls: place cut restriction enzyme Sáli Seal: place cut restriction enzyme Seal Sphl: place cut restriction enzyme Sphl Xhol: place cut restriction enzyme Xhol

To Figures 2 and 3:

LEU2: beta-isopropylmalate dehydrogenase gene

Saccharomyces cerevisiae

2μ: endogenous plasmid sequence 2μ

Saccharomyces cerevisiae

Ap ^R : beta-lactamase gene

P-CUP1: Saccharomyces cerevisiae CUP1 promoter (metallothionein)

T-CYC1: CYC1 (cytochrome C) terminator of Saccharomyces cerevisiae

SD: Shine-Dalgarno Sequence ·······

4 4

4 9

4 4

4 9

EcoRI:

Notl:

Spěl:

Xbal:

restriction enzyme cut EcoRI restriction enzyme cut Notl restriction enzyme cut Spěl restriction enzyme cut Xbal

Claims (14)

PATENT CLAIMS Animal feed additives comprising D-pantothenic acid and / or its salts based on fermentation mash, characterized in that: (a) contain D-pantothenic acid and / or its salts; (b) contain 0 to 100% of biomass produced during fermentation of D-pantothenic acid producing micro-organisms; and (c) contain at least the bulk of the other dissolved constituents of the fermentation broth; and (d) they exist in solid, fine-grained or granular bulk form. Animal feed additives according to claim 1, characterized in that they contain one or more salts selected from sodium, potassium, ammonium, magnesium or calcium salts of D-pantothenic acid. Animal feed additives according to claim 1 or 2, characterized in that they contain D-pantothequine or one of its salts in an amount of greater than 0, in particular 20 to 80% by weight (dry matter). Animal feed additives according to claim 1, characterized in that they exist in spray-dried or spray-granulated form. Animal feed additives according to one or more of the preceding claims, characterized in that they additionally comprise one or more L-amino acids selected from the group consisting of L-methionine, L-lysine, Lvaline, L-threonine, L-alanine and L-alanine. -tryptophan. · · Tt t t t t t t t t t tt tt tt t tt Method for producing feed additives containing Dpantotenic acid and / or its salts according to claims 1 to 5 from fermentation mash, characterized in that (a) completely or partially separate biomass and / or part of the other constituents from the fermentation broth containing D-pantothenic acid and / or its salts, b) the solution or mash thus obtained is mixed with an alkaline earth or alkali metal hydroxide or oxide; and c) the mixture thus obtained is spray dried or spray-dried, spray-granulated or granulated. The method according to claim 6, characterized in that an oxide or hydroxide selected from sodium, potassium, ammonium, magnesium and calcium compounds is added in a stoichiometric ratio of 0.8 to 1.2, in particular 0.95 to 1, 1, with respect to D-pantothenic acid. A process for producing feed additives comprising D-pantothenic acid and / or its sodium, potassium, ammonium, magnesium or calcium salt according to claims 1 to 5, characterized in that (b) c) from the fermentation broth obtained by using the desired hydroxy compounds containing D-pantothenate optionally completely or partially separating the biomass and / or part of the ingredients, the mixture thus obtained is concentrated in particular by removing water and the feed additive containing the pantothenate in solid form by drying, spray drying or granulation spraying. ···· A process for producing animal feed additives comprising D-pantothenic acid and / or its sodium, potassium, ammonium, magnesium or calcium salt in the range of about 20 to 80% by weight (dry matter) of fermentation mash, characterized by. these steps a) preferably by removing water from the fermentation broth (concentration), b) optionally removing 0 to 100% of the biomass generated during fermentation, c) adding one or more of said compounds to the fermentation broths obtained in accordance with a) and b), wherein the amount of added compounds is measured such that their total concentration in the animal feed additive is in the range of about 20 to 80% by weight; d) drying the fermentation broth obtained according to c) to obtain animal feed additive in the desired powder or granulate form. Process according to claim 9, characterized in that the fermentation mash is mixed with the oxide or hydroxide of said alkali or alkaline earth metals before or after the preferential concentration. Process according to Claims 6 to 9, characterized in that fungi or yeast are used in the fermentation. A method according to claim 9, characterized in that one or more L-amino acids selected from the group consisting of: Φ · φφ φφ φφφφ · Zahrnφ zahrn zahrn L zahrn zahrn L L L L L L L L L L L L Lujícího L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L zahrn L zahrn L L zahrn L zahrn L zahrn L zahrn L zahrn L zahrn L zahrn L zahrn L zahrn L zahrn L zahrn L zahrn zahrn zahrn L zahrn zahrn zahrn zahrn zahrn zahrn zahrn zahrn zahrn zahrn zahrn zahrn zahrn zahrn zahrn zahrn zahrn zahrn zahrn zahrn L zahrn L zahrn L zahrn L zahrn L zahrn L zahrn L Lφ zahrn L zahrn zahrn Process according to Claims 6 to 11, characterized in that bacteria of the genus Corynebacterium are used in the fermentation. 14. The method according to claims 6 to 11, characterized in that bacteria of the family Enterobacteriaceae are used in the fermentation.