SK6452000A3 - Feed additive containing d-pantothenic acid and/or its salts and method for their preparation - Google Patents

Abstract

Animal feed additive based on fermentation liquor containing D-pantothenic acid (I) and/or its salts, contains (a) (I) and/o its salts, (b) 0-100% biomass from microorganisms producing (I) during fermentation and (c) mainly other contents of the fermentatio liquor and (d) is in solid, finely-divided or granulated, free-running form. Independent claims are also included for methods of producing animal feed additives containing (I) and/or its salts.

Description

Feed additives containing D-pantothenic acid and / or salts thereof and a process for their preparation

Technical field

The present invention relates to an animal feed additive based on fermentation mash 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 tons per year. Much of the pantothenic acid produced is used to feed livestock such as poultry and pigs. Its consumption is increasing.

Pantothenic acid can be produced by chemical synthesis or biotechnological 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 calcium salt of Dpantothenic acid. The calcium salt of the racemic mixture of D, Lpantothenic 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.

D-pantothenic acid may be produced under various fermentation conditions by a variety of bacteria such as Escherichia coli, Arthrobacter ureafaciens, Corynebacterium erythrogenes, Brevibacterium ammoniagenes and also yeasts such as Debaromyces castellii, as shown in EP-A-0 493 060, A-0 590 857 and WO 97/10340. Particularly suitable are 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 D-pantothenic acid formed. is subsequently costly isolated, purified and recovered as a calcium salt.

Suitable nutrient media include a carbon source such as glucose or starch flour 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 value with appropriate aeration and stirring, excreting D-pantothenic acid.

According to the state of the art which is described in U.S. Pat

WO96 / 33283 and EP-A-0 590857, the calcium acid salt

D-pantothenic acid is obtained by costly isolation and purification from a fermentation mash containing pantothenic acid. After the first separation of the biomass by filtration or centrifugation, further processing of the filtrate by activated charcoal purification or column chromatography is carried out. 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 decolorized with activated carbon in the first column. The pH is adjusted to 3.0 with concentrated hydrochloric acid, and then the liquid is purified continuously through two additional charcoal columns. The elution of D-pantothenic acid is carried out 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, treated with activated carbon and filtered off. 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.

The object of the invention is 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 current known methods.

SUMMARY OF THE INVENTION

The subject of the invention is a feed additive for animals based on fermentation mash, 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 lyophilized, fine-grained flowable powders, but also in granular form, which may contain different proportions of biomass. The bulk density is in particular about 500 kg / m ³ . The ingredients are storage stable.

If biomass is separated, other, for example, inorganic solids are naturally removed. In addition, the additive according to the invention contains at least a 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, L-lysine, L-valine, L-threonine, L-alanine and L-tryptophan, especially L-valine. They also include organic acids having one to three carboxylic groups, such as acetic acid, lactic acid, citric acid, malic acid or fumaric acid. Finally, these include poorly usable sugars such as trehalose. These compounds are optionally desired when they improve the value of the additive.

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

A further object of the invention is a process for the preparation of a feed additive comprising D-pantothenic acid and / or a salt thereof, characterized in that

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

(b) where appropriate, all or part of the biomass is separated;

(c) the solution or mash thus obtained is mixed 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),

(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 according to the invention

(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, the genus Escherichia with the species Escherichia coli should be mentioned in particular. Within Escherichia coli, so-called K-12 strains, such as strains MG1655 or W3110 (Neidhard et al .: Escherichia coli and Salmonella. Cellular and Molecular Biology) are to be mentioned.

AS (ASM Press, Washington, D. C.)) or wild-type Escherichia coli strain IFO3547 (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.

The above-described microorganisms can be cultivated continuously or batchwise for batch production or batch fed or batch fed batch or batch fed batch production to produce D-pantothenic acid. A summary of known cultivation methods is described in a textbook from Hop (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in a textbook from Storhasa (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 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 substances can 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 phosphorus sources, potassium dihydrogen phosphate or potassium hydrogen phosphate or the corresponding sodium-containing salts can be used. The culture medium must further comprise metal salts such as magnesium sulfate or iron sulfate that are required for growth. Finally, essential growth agents such as amino acids and vitamins may be used in addition to the above-mentioned substances. In addition, suitable precursors of D-pantothenic acid, 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. Antifoams, such as polyglycol esters of fatty acids, can be used to control foaming. Optionally, suitable selective agents, for example antibiotics, may be added to the medium to maintain plasmid stability. In order to maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, for example 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 the maximum 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 from 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 in a sugar-limiting manner at least at the end, but preferably at least 30% of the duration of the fermentation. That is, during this time, the concentration of usable sugar in the fermentation broth is maintained at a value greater than or equal to 0 to 3 g / l, or decreases to this value.

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 a combination thereof. According to the invention, however, the biomass can be left completely in the fermentation mash. Subsequently, this fermentation mash is generally mixed with 0.8 to 1.2, in particular 0.95 to 1.1, equivalent of an alkali metal or alkaline earth metal oxide or hydroxide, in particular NaOH, KOH, Ca (OH) 2 or MgO, relative to D-pantothenic acid. At low concentrations of D-pantothenic acid, it may be advantageous to use significantly larger amounts of oxides or hydroxides, for example in the range of 1.2 to 4 equivalents. The suspension obtained in this way 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 in a conventional drier or, for example, by means of a film-flow evaporator or a thin-film evaporator or spray dryer or spray granulator or lyophilizer as a free-flowing, free-flowing, fine-grained powder or granulate. The granulation can also be carried out after drying, for example in the form of 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, the fermentation broth containing D-pantothenic acid, produced using the appropriate hydroxyl compounds or, if appropriate, by first known separation methods, such as centrifugation, filtration, decantation or a combination thereof, is totally or partially freed of biomass. According to the invention, however, it is also possible to completely leave the biomass in the fermentation mash. Subsequently, the pretreated mash, if necessary, is concentrated or dried by known methods, for example by means of a rotary evaporator or a thin-film evaporator or a flow-through evaporator. The optionally concentrated mash is then processed by spray drying, spray granulation or other methods to give a free-flowing, free-flowing, fine-grained powder or 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 salts thereof, based on the total amount. In addition, the inorganic components additionally generally comprise from 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 obtained by adding the corresponding 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)).

The present invention is explained in more detail below with reference to exemplary embodiments. For this purpose, experiments were carried out with D-pantothenic acid-producing strain 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 1-1-3, Higashi, Tsukubashi, Ibaraki (Japan).

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Preparation of fermentation broth containing D-pantothenic acid

1. Inoculum production

A sample of Escherichia coli FV5069 / pFV31 was plated on LBG agar supplemented with 50 gg of 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. broth

LBG has the following composition: 10 g / l peptone, 5 g / l yeast extract, 5 g / l NaCl and 1 g / l glucose. LBG agar contains • an additional 12 g / l of 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. 10 ml cultures in 100-ml Erlenmeyer flasks were incubated for 16 hours at 37 ° C and 180 rpm in an ESR incubator from Kínerner AG (Birsfelden, Switzerland). Then, the cell suspension was centrifuged in a centrifuge from Beckmann (Hannover, Germany) for 15 minutes at 4000 rpm. The cell pellet was resuspended in 10 ml of LBG medium supplemented with 20% glycerol 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 an LP2W photometer from Dr. Henderson. 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. Thereafter, 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 Dpantothenic acid

To prepare a fermentation broth containing Dpantothenic acid, the working cell bank 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.

SKA medium was used for shake flask culture. SKA medium was prepared as follows. In a 1 L beaker, 7.0 g (NH 4 SO 4), 0.5 g KH 2 PO 4, 1.0 g K 2 HPO 4, 0.5 g MgSO ₄ .7H ₂ O, 0.01 g MnSO ₄ .H ₂ O were weighed. 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 were added. This soaking salt solution was autoclaved at 121 [deg.] C. for 20 minutes, then a solution containing 125 g of distilled water, 28.7 g of glucose and 0.002 g of thiamine.HCl was sterilized by filtration, weighing 10 g of CaCO3 in a 100-ml flask and sterilizing in an autoclave at 123 [deg.] C. for 20 minutes by combining both of the above ingredients with a solution containing dipping water to obtain a SKA medium.

This SKA medium was divided into 12.5 ml portions 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 measuring wavelength of 660 nm, consequently, was 12.5.

0.5 ml of this culture was diluted in a shake flask

4.5 ml of physiological saline solution and 0.7 ml of it were used to inoculate 1300 ml of culture medium which was 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 0.014 g of Fe ₂ (SO ₄ ) ₃ 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 which contained 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 growth medium for main fermentation in a 21 Biostat ® MD fermenter.

Growth medium was prepared as follows. Solution consisting of 14,14 g (ΝΗ ₄ ) ₂ 3Ο 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 βalaninu, 0.74 Struktolu J647 and 49.72 g of maize steep liquor in 1144 ml of tap water was adjusted to pH 6.5 with 25% strength ammonia solution and sterilized 20 minutes in an autoclave at 121 ° C. To this steeping water containing the salt solution was added, under sterile conditions, a separately sterile filtered solution containing in 100 ml of distilled water 35.92 g of glucose and 0.002 g of thiamine.HCl.

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 contained 465.29 g of glucose and 0.0261 d of thiamine HCl in 584 ml of tap water, which were sterile filtered. Production medium 2 contains 37.5 g of βalanine in 140 ml of tap water, which was autoclaved at 121 ° C for 20 minutes. After a fermentation time 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 in the fermentation broth was 6.1%.

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

Example 2

In a fermentation experiment carried out under the same conditions as described in Example 1, a pantothenic acid concentration of 5.4% by weight could be detected in the fermentation broth after 43 hours of culture. The L-valine concentration was 8 g / L.

Example 3

Preparation of calcium D-pantothenate

The biomass was first separated from the pantothenic acid-containing fermentation broth produced by the methods of Examples 1 and 2 and containing approximately 6.1% by weight of D-pantothenic acid. For this purpose, 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 centrifuged supernatant was then further purified by cross-flow ultrafiltration with 30kD polymer membrane in UF equipment from ICT GmbH (Bad Homburg, Germany).

10.1 g of solid Ca (OH) ₂ (96%; MERCK, Darmstadt, Germany) were then added with stirring. 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) under vacuum at 60 ° C to a liquid fraction of approximately 50% of the dry fraction. The concentrated mash was then spray dried to obtain the calcium salt of D-pantothenic acid. 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-containing 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

The biomass was first separated from the pantothenic acid-containing fermentation broth prepared by the methods 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 3.

10.6 g NaOH (99%; MERCK) was then added with stirring. The pH was then approximately 10. The treated mash was then concentrated under vacuum at 50-60 ° C in a Rotavapor RE-120 rotary evaporator from Buchi-Labortechnik GmbH (Konstanz, Germany) to a liquid content of approximately 50% of the dry portion. The thickened mash was then lyophilized to obtain the sodium salt of D-pantothenic acid 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 content of Dpantothenic acid was 63.0% by weight.

Example 5

Preparation of magnesium D-pantothenate

The biomass was first separated from the fermentation broth containing pantothenic acid, 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 5.4 g of solid MgO (97%; MERCK) were added under stirring with breaks. The pH was then about 9-10. The treated mash was then concentrated in a Rotavapor RE-120 rotary evaporator from Buchi-Labortechnik GmbH under vacuum at 50-60 ° C to a liquid fraction of approximately 50% of the dry fraction. The thickened mash was then lyophilized to obtain the magnesium salt of Dpantothenic acid in a LYOVAC GT 2 type lyophilizer from Leybold.

The magnesium D-pantothenate-containing 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 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. For this purpose, 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 under stirring with breaks. The pH was then about 10 to 10

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

The ammonium D-pantothenate-containing 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 a fermentation broth containing biomass

The pantothenic acid-containing fermentation mash prepared by the methods of Examples 1 and 2 and containing approximately 6.1% by weight of D-pantothenic acid was first concentrated in a Rotavapor RE-120 rotary evaporator from Buchi under vacuum at 60 ° C. -Labortechnik GmbH (Konstanz, Germany) volume 1.0 L per liquid fraction approximately 30% of the dry fraction. 10.1 g of solid Ca (OH) ₂ (96%; MERCK, Darmstadt, Germany) were then added with stirring. The pH was then approximately

10. The thus treated and concentrated biomass-containing mash was then spray-dried to obtain the calcium salt of D-pantothenic acid. 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-containing 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 Lvalin-producing strain of Brevibacterium lactofermentum FERM BP-1763. 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 an ATCC13032 / pND-DBC2 strain as DSM 12437. Transformation of the FERM BP-1763 strain with plasmid pND-DBC2 gave the FERM BP1763 / pND-DBC2 producing strain.

2. Preparation of a fermentation broth containing pantothenic acid

A sample of Brevibacterium lactofermentum FERM ΒΡ-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. The selected individual colonies were then further propagated on the same medium. Using a loop, the cell material of one clone was removed from HHK agar and transferred to a 100 ml HHK broth, which was contained in a shake flask with a total volume of 1000 ml.

The HHK broth consists of brain-heart medium obtained from Merck KgaA (Darmstadt, Germany) and supplemented with glucose and kanamycin. The HHK broth composition 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 the SK-71 medium were submitted directly corresponding to the working concentrations in the fermenter and sterilized in situ.

Table 8c: Medium SK-1

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 Tiamín.HC1 0.0002 g D - (+) - Biotin 0.0003 g Struktol 0.60 g

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

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

The batch was cultivated for the entire duration of the fermentation at 30 ° C, 0.75 vvm volume specific aeration, stirring at 800 to 1,700 rpm depending on oxygen consumption and pH 7.0 and partial oxygen pressure of 20% air saturation . The culture was cultivated for 49 hours under the above conditions until an optical density of 26.2 was reached at 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 type LP1W from the company Dr. Bruno Lange GmbH (Berlin, Germany) at a wavelength of 660 nm and the concentration of D-pantothenic acid formed by HPLC (Hypersil APS 25 µm, 250x5 mm, RI detection).

A concentration of D-pantothenic acid of about 0.2 g / l was measured after 49 hours in the final fermentation sample.

‘3. Preparation of 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 under vacuum at 60 ° C in a Rotavapor RE-120 rotary evaporator from BuchiLabortechnik GmbH (Konstanz, Germany) to a mash with a dry content of approximately 15%.

Then 27.1 g of solid Ca (OH) 2 (96%; MERCK, Darmstadt, Germany) were added with stirring. The pH was then about 10.0. The thus processed and concentrated biomass-containing mash was then mixed with 37.7 g of calcium D-pantothenate (> 98%, Euro OTC Pharma GmbH, Stone, Germany). The following spray drying was carried out using a Buchi-190 laboratory spray dryer from Bííchi-Labortechnik GmbH (Konstanz, Germany) 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-containing 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 reading grid YHR063c of Saccharomyces cerevisiae (accession number U00061 National Center for Biotechnology, Bethesda, MD, USA), the following PCR primers were synthesized (MWG-Biotech, Ebersberg, Germany). The beginning or end of the reading grid is indicated by a period (.):

• OJD539 (5 'EcoRI-NotI START):

5'- GCG CGA ATT CAG ATC GCC GGC CG AAA GAG GAG AAA TTA ACT.ATG ACT GCA CCA CAC AGA AG -3 '• OJD540 (3' Spel-PstI STOP):

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

The genomic DNA of S. cerevisiae JD242, which was isolated according to a method from C. Guthrie and GR Fink, served as template (Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, Academic Press, San Diego, 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 (leu2A1 allele) and uracil (ura352 allele). 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:

For expression of the reading grid YHR063c in S. cerevisiae, a PCR amplification was inserted into the E. coli pJDCEX2 shuttle vector.

S. cerevisiae (Figure 2 and Dohmen at al., 1995, Journal of

Biological Chemistry 270, 18099-18109).

The PCR product was first restricted with EcoRI and Spel (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 µg / 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 screened by restriction with Note and PstI and subsequent separation in a 0.8% agarose gel. The plasmid with the desired structure was named pJD-YHR063c (Figure 3).

Preparation of JD242 / pJD-YHR063c strain:

The 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 leucine-free minimal SD medium with 1.8% agar (see Tables 9a and 9b).

Table 9a: Minimum SD media

compound Quantity per liter (NH ₄ ) ₂ SO ₄ 5 g kh ₂ Mon ₄ 1 g MgSO ₄ .7H ₂ O 0.5 g NaCl 0.1 g CaCl ₂ 0.1 g H3BO3 500 gg CuSO ₄ 40 gg KI 100 gg FeCl ₃ .6H ₂ O 200 gg MnSO ₄ . H ₂ 0 400 gg Na ₂ Mo0 ₄ .2H ₂ O 400 gg ZnSO ₄ 7H ₂ O 200 gg biotin 2 gg Folic acid 2 gg inositol 2 mg niacin 400 gg P-Aminobenzoic acid 200 gg pyridoxine 400 gg riboflavin 200 gg thiamin 400 gg

Table 9b: Minimum SD media

ingredients Quantity per liter glucose 20 g uracil 40 mg CuSO ₄ 24 mg -Leu DO supplement 650 mg ketopanthoate 100 mg β-Alanine 100 mg

2. Preparation of a fermentation broth containing pantothenic acid

To prepare a 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 shaking flask, cells were subsequently rinsed with 5 ml of minimal SD medium. A shake flask (total volume 500 ml) was then seeded with 2.5 ml cell suspension 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 1000-ml flask with baffles 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 this end, a round-bottomed flask was seeded with 90 ml of preculture 2 and subsequently incubated at 30 ° C and 60 rpm for 30 hours.

Optical Density (OD) was measured with a LP1W type digital camera 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 D-pantothenic 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 under vacuum at 60 ° C in a Rotavapor RE-151 rotary evaporator from Buchi-Labortechnik GmbH (Konstanz, Germany) to a melt with approximately 16% dry fraction. 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 (> 98%, Euro OTC Pharma GmbH, Stone, Germany). The mash was then lyophilized in a LYOVAC GT 2 type lyophilizer from Leybold (Cologne, Germany).

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.

BRIEF DESCRIPTION OF THE DRAWINGS

The following images are attached:

Figure 1: Map of plasmid pND-DBC2

Figure 2: Map of plasmid pJDCEX2

Figure 3: Map of plasmid pJD-YHR063c

The abbreviations used in the figures have the following meanings:

To the picture 1: rrnBTlT2: Transcript terminator of rrnB gene Birds: the tac promoter pang: the coding region of the panB gene panC: the coding region of the panC gene Panda: the panD gene coding region C. G-rep .: DNA region for replication in C. glutamicum oriV-E.C.. : origin for vegetative transfer in E. Coli kan: kanamycin resistance gene BglII: the BglII restriction enzyme splice site Clal: instead of the restriction enzyme cut Clal

ncol: the place shear restriction enzyme ncol NruI: the place shear restriction enzyme NruI PvuI: the place shear restriction enzyme PvuI suction: the place shear restriction enzyme sucking Sali the place shear restriction enzyme Sali Scala the place shear restriction enzyme scal SphI: the place shear restriction enzyme SphI XhoI: the place shear restriction enzyme XhoI

To Figures 2 and 3:

LEU2: beta-isopropylmalate dehydrogenase gene

Sa echa romyces cerevisiae

2μ: endogenous plasmid sequence 2μ Saccharomyces cerevisiae Ap ^R : beta-lactamase gene P-CUP1: CUP1 gene promoter Saccharomyces cerevisiae (metallothionein) T-CYC1: the CYC1 (cytochrome C) gene terminator of Saccharomyces cerevisiae SD: Shine-Dalgarno sequence

3¼

EcoRI: the place shear restriction enzyme EcoRI Noti: the place shear restriction enzyme Noti Spel: the place shear restriction enzyme Spel XbaI: the place shear restriction enzyme XbaI

Claims (14)

PATENT CLAIMS Animal feed additives containing Dpantothenic 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 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 the group consisting of sodium, potassium, ammonium, magnesium or calcium D-pantothenic acid. Animal feed additives according to claim 1 or 2, characterized in that they contain D-pantothenic acid and / 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 the form of spray-dried or spray-granulated. 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, Llysine, L-valine, L-threonine, L-alanine and L-tryptophan. Method for producing feed additives containing D-pantothenic acid and / or salts thereof according to claims 1 to 5 from fermentation mash, characterized in that (a) where appropriate, completely or partially separate biomass and / or part of the other constituents from the fermentation broth containing Dpantothenic acid and / or its salts, (b) the solution or mash thus obtained is mixed with an alkali earth or alkali metal hydroxide or oxide; and c) the mixture thus obtained is spray dried or spray-dried, spray-granulated or granulated. Method according to claim 6, characterized in that an oxide or a 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. Process for the preparation of feed additives containing D-pantothenic acid and / or its sodium, potassium, ammonium, magnesium or calcium salt according to claims 1 to 5, characterized in that (a) if appropriate, completely or partially separate biomass and / or part of the constituents from the fermentation broth obtained using the desired hydroxy compounds containing Dpantothenate; (b) the mixture thus obtained is concentrated in particular by removing water; and (c) the feed additive containing the pantothenate in question is obtained in solid form by drying, spray drying or spray granulation. A process for 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 broth, characterized by the following 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 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, and 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 metals or alkaline earth metals before or after the preferably carried out concentration. Process according to claims 6 to 9, characterized in that fungi or yeast are used in the fermentation. The method of claim 9, wherein one or more L-amino acids selected from L-lysine, L-valine, L-alanine, L-threonine, and L-tryptophan are added to the optionally concentrated fermentation broth. Process according to claims 6 to 11, characterized in that bacteria of the genus Corynebacterium are used in the fermentation. Process according to claims 6 to 11, characterized in that bacteria of the family Enterobacteriaceae are used in the fermentation.