EP1362117A2

EP1362117A2 - Method for producing d-pantothenic acid and/or the salt thereof as an additive for other metallic surface (2) are provided.animal food

Info

Publication number: EP1362117A2
Application number: EP02719872A
Authority: EP
Inventors: Kai-Uwe Baldenius; Christine Beck; Hans-Peter Harz; Markus Lohscheidt; Daniela Klein
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 2001-02-21
Filing date: 2002-02-20
Publication date: 2003-11-19
Also published as: WO2002066663A2; AU2002250976A1; US20040048344A1; MXPA03007385A; BR0207475A; NO20033704D0; PL367284A1; CZ20032236A3; US7781192B2; CN1294272C; RU2003128529A; EE200300404A; DE10108225A1; IN2003CH01298A; WO2002066663A3; KR20030075203A; NO20033704L; IL157472A0; JP2004529626A; HUP0303216A3

Abstract

The invention relates to an improved method for producing D-pantothenic acid and/or the salts thereof and the use of said substance as an additive for animal food.

Description

Process for the preparation of D-pantothenic acid and / or its salts as an additive to animal feed

The present invention relates to an improved process for the preparation of D-pantothenic acid and / or its salts and the use as an additive to animal feed.

As a starting product for the biosynthesis of coenzyme A, D-pantothenate is widely used in plants and animals. In contrast to humans, who ingest sufficient amounts of pantothenic acid through food, deficiency symptoms for D-pantothenate are frequently described for both plants and animals. The availability of D-pantothenate is therefore of great economic interest, especially in the animal feed industry.

Conventionally, the production of D-pantothenate by chemical synthesis from D-pantolactone and calcium-.beta.-alaninate (Ullmann's Encyclopedia of Industrial Chemistry, 6 ^th edition, 1999, electronic release, chapter "Vitamins"). In order to provide D-pantolactone The complex sales product resulting from chemical synthesis is usually the calcium salt of D-pantothenic acid, calcium D-pantothenate.

Compared to chemical synthesis, the advantage of biotechnological manufacturing processes with microorganisms lies in the selective (enantiomerically pure) provision of the D-form of pantothenic acid that can be used by the higher organism. A complex resolution of racemates, as is required in chemical synthesis, is thus eliminated.

Fermentative processes for the production of D-pantothenic acid with microorganisms are well known, e.g. from EP 0 590 857, WO 96/33283, US 6,013,492, WO 97/10340, DE 198 46499, EP 1 001 027, EP 1 006 189, EP 1 006 192 and EP 1 006 193.

For example, EP 1 006 189 and EP 1 001 027 describe processes for the production of Pantothenate in which a content of at most 1 g / l D-pantothenic acid is reached in the fermentation solution. Such low pantothenic acid contents in the fermentation solution, ie less than 10% by weight based on the solids content, are unsuitable for the economical production of animal feed supplements containing D-pantothenic acid. Another disadvantage of the processes described so far is that the isolation of the product from the fermentation medium requires numerous complex work-up steps. An economical manufacturing process for the industrial scale is not disclosed.

The published patent application DE 100 16 321 describes a fermentation process for the production of an animal feed supplement containing D-pantothenic acid. A major disadvantage of this process, however, as with the fermentative processes for producing D-pantothenic acid mentioned above, is that the pantothenic acid precursor β-alanine must be added to the microorganism via the fermentation medium in order to obtain economic yields of the desired product.

Furthermore, US Pat. No. 6,013,492 and WO 96/332839 describe working up the D-pantothenic acid from the fermentation solution by filtering off insoluble constituents (for example cell material) from the culture medium, adsorbing the filtrate on activated carbon, then eluting the D-pantothenic acid with an organic one Solvent, preferably methanol, neutralization with calcium hydroxide and a final crystallization of calcium D-pantothenate. Significant disadvantages are the loss of valuable product that occurs during crystallization and the use of an organic solvent that is difficult to remove from the product and that requires expensive solvent recovery.

EP 0 590 857 describes a fermentation process for the production of D-pantothenic acid, in which the cultivation of a microorganism absolutely requires the addition of β-alanine. The fermentation solution is filtered to separate the biomass, then passed through a cation exchanger and then through an anion exchanger, followed by calcium hydroxide neutralized, evaporated, mixed with activated carbon, filtered again and crystallized with the addition of methanol and calcium chloride. The resulting calcium pantothenate-containing product contains, in addition to D-pantothenic acid in the form of the potassium salt, calcium chloride in a molar ratio of 1: 1. Electrodialysis followed by spray drying is required to reduce the calcium chloride content. The disadvantage of this process is that it is neither economical nor ecological because of the large number of complex process steps and the use of organic solvents.

The object of the present invention is to provide an animal feed supplement containing D-pantothenic acid and / or its salts, and the production thereof by an improved process for producing D-pantothenic acid and / or its salts which does not have the disadvantages mentioned above. For economic reasons, a process is desirable in which the addition of β-alanine is drastically reduced or is not required at all. Furthermore, the production of D-pantothenic acid in the form of its divalent salts, and especially the alkaline earth metal salts, is desirable since the divalent salts have less hygroscopic properties than monovalent salts of pantothenic acid and for further use, e.g. B. as an animal feed supplement, thus less prone to clumping.

This object is achieved in an advantageous manner by the present invention.

The present invention relates to a process for the preparation of D-pantothenic acid and / or its salts, characterized in that a) at least one organism producing D-pantothenic acid is used, the pantothenic acid (pan) and / or isoleucine / valine ( ilv) - Biosynthesis is deregulated and forms at least 2 g / l of salts of D-pantothenic acid by fermentation in a culture medium, 0-20 g / l of free ß-alanine and / or ß-alanine salt being added to the culture medium, b) the fermentation solution containing D-pantothenate is passed over a cation exchanger, free D-pantothenic acid being formed from the salts of D-pantothenic acid, c) the solution containing free D-pantothenic acid is adjusted to a pH of 3-10 by the addition of calcium and / or magnesium base, a solution is obtained which contains calcium and / or magnesium pantothenic acid and d) the solution containing calcium and / or magnesium pantothenate

Drying and / or formulation is subjected.

In a variant of the process according to the invention, a suspension which contains calcium and / or magnesium chloride can also be obtained in step c). In the following step d), this suspension is then subjected to drying and / or formulation.

The fermentation in step a) of the process according to the invention is carried out according to procedures known per se in batch, fed-batch or repeated fed-batch operation or with continuous process control. To neutralize the resulting pantothenic acid ^' usual buffer systems, such as. B. phosphate buffer with NaOH, KOH or ammonia.

In further variants of the process according to the invention, in step a) at least 10 g / l, preferably at least 20 g / l, particularly preferably at least 40 g / l, most particularly preferably at least 60 g / l and in particular at least 70 g / l of salts of D-pantothenic acid formed by fermentation in the culture medium.

Here, the phrase “produce” means according to the invention that the organism can synthesize larger amounts of D-pantothenic acid and / or its salts than are required for its own metabolic needs. In a variant which is advantageous according to the invention, the amount of D-pantothenic acid and / or their salts do not pre-exist inside the cell, but are ideally completely released from the organism into the culture medium, which can be actively or passively discharged according to mechanisms known per se.

According to the invention, microorganisms are used as the organisms producing D-pantothenic acid. According to the invention, these include mushrooms, yeasts and / or Bacteria. According to the invention, fungi such as, for example, Mucor or yeasts, such as. B. Saccharomyces or Debaromyces and preferably Saccharaomyces cerevisiae. According to the invention, coryneform bacteria or Bacillaceae are advantageously used. According to the invention are preferably z. B. bacteria of the genera Corynebacterium, Escherichia, Bacillus, Arthrobacter, Bevibacterium, Pseudomonas, Salmonella, Klebsiella, Proteus, Acinetobacter or Rhizobium. Corynebacterium glutamicum, Brevibacterium breve or Bacillus subtilis, B. licheniformis, B. amyloliquefaciens, B. cereus, B. lentimorbus, B. lentus, B. firmus, B.pantothenticus, B. circulans, B. coagulans, are particularly preferred here, for example B. megaterium, B. pumilus, B. thuringiensis, B. brevis, B. stearothermophilus and other group 1 Bacillus species characterized by their 16sRNA or Actinum mycetalis. This list is for the purpose of explanation and is in no way limitative of the present invention.

In addition, the present invention also includes the use of genetically modified organisms for the production according to the invention of an animal feed supplement containing free D-pantothenic acid and / or its salts. Such genetically modified organisms can be isolated, for example, by chemical mutagenesis and subsequent selection by means of a suitable “screening method”. According to the invention, so-called “production strains” are also included which are suitable for the production of the product in the sense of the present invention and have genetic changes with regard to the metabolic flow in the direction D-pantothenic acid, which also includes changes in the discharge of D-pantothenic acid and / or its salts across the cell membrane. This can e.g. B. by changes in key positions in relevant metabolic biosynthetic pathways of the organism used.

It is also conceivable to use transgenic organisms which result from the transfer of homologous and / or heterologous nucleotide sequences which are necessary or can be beneficial for the synthesis of the desired product. Here, the overexpression and / or deregulation is one or more genes individually and / or in combination, localized in the genome and / or on a vector, conceivable.

Such transgenic organisms can advantageously contain additional copies and / or genetically modified genes selected from the group of panB, panC, panD, panE and / or their combinations and / or even organizational units, such as the panBCD operon. Further metabolic pathways, e.g. the isoleucine-valine biosynthetic pathway in the organisms can advantageously be manipulated, as described, for example, in EP 1 006 189, EP 1 006 192, EP 1 006 193 or EP 1 001 027. As a result, branched-chain precursors of pantothenic acid biosynthesis are increasingly being made available. The genes for this biosynthetic pathway may be advantageous, i.e. ilvB, ilvN, ilvC and / or ilvD overexpressed.

In addition, genetic changes in aspartate α-decarboxylase (panD), e.g. by overexpression and / or deregulation, in the D-pantothenic acid-producing organism used according to the invention.

According to the invention, the term "deregulation" means the following: Change or modification of at least one gene which codes for an enzyme in a biosynthetic pathway, so that the activity of the enzyme in the microorganism is changed or modified. It is preferred that at least one gene which codes for an enzyme of a biosynthetic pathway is modified in such a way that the gene product is produced more or has an increased activity. The term "deregulated pathway" also includes a biosynthetic pathway in which more than one gene is responsible for more encoded as an enzyme is modified or modified so that the activities of more than one enzyme are changed or modified.

Changes or modifications may include, but are not limited to: removing the endogenous promoter or regulatory elements; Inserting strong promoters, inducible promoters, or multiple promoters simultaneously; Removing regulatory sequences so that the expression of the gene product is changed; Change in the chromosomal location of the gene; Alteration of the DNA sequence in the vicinity of the gene or within the gene such as e.g. B. the ribosomal binding site (RBS); Increasing the copy number of the gene in the genome or by introducing plasmids of different copy numbers; Modification of proteins (eg regulatory proteins, suppressors, enhancers, transcriptional activators, and the like) that play a role in the transcription of the gene and / or in the translation to the gene product. This also includes all other possibilities for deregulation of the expression of genes which are state of the art, such as, for. B. the use of antisense oligonucleotides, or the blocking of repressor proteins.

Deregulation can also involve changes in the coding region of genes, e.g. B. lead to cancellation of feedback regulation in the gene product or to a greater or lesser specific activity of the gene product.

In addition, genetic engineering changes to enzymes are advantageous according to the invention which influence the outflow of precursors of pantothenic acid and / or the flow of pantothenic acid to coenzyme A. Examples of genes encoding such enzymes are: alsD, avtA, ilvE, ansB, coaA, coaX and others. This list is for the purpose of explanation and is in no way limitative of the present invention.

Furthermore, genetic engineering changes are advantageous which ensure the cellular provision of cofactors (e.g. of methylene tetrahydrofolate, redox equivalents and the like) in an optimal amount for pantothenic acid production.

Advantageously, ß-alanine is already present in the cells in increased concentrations compared to correspondingly non-genetically modified organisms and thus does not have to be added to the culture medium as a precursor, as is the case, for. B. in EP-A-0 590 857 is required. Microorganisms whose pantothenic acid (pan) and / or isoleucine valine (ilv) biosynthesis and / or aspartic decarboxylase (panD) is deregulated are advantageous. An additional overexpression of ketopanthoat reductase (panE) in the microorganisms is also advantageous.

It is also advantageous according to the invention if the activity of the coaA gene which is required for the synthesis of coenzyme A is reduced or if it is completely switched off (for example in Bacillus species). In addition to coaA, Bacillus contains another gene for this enzymatic function (= coaX). The activity of this gene coaX or of the corresponding enzyme can also be changed, are preferably reduced, or even deleted, provided that coaA itself still has sufficient, albeit reduced, enzyme activity, ie the enzyme activity of coaA has not been completely lost. In addition to the overexpression of the various genes, genetic manipulation of the promoter regions of these genes is advantageous in such a way that this manipulation leads to overexpression of the gene products.

In one embodiment variant of the present invention, the bacterial strains described in accordance with the appendix (PCT / US application 0025993), such as Bacillus subtilis PA 824 and / or derivatives thereof. In a further embodiment variant, the microorganism Bacillus subtilis PA 668, as described in accordance with the appendix (US Serial No. 60 / 262,995), is used in the method according to the invention. These strains Bacillus subtilis PA 824 and PA 668 were produced as follows:

Starting from the strain Bacillus subtilis 168 (Marburg strain ATCC 6051), which has the genotype trpC2 (Trp ^" ), the strain PY79 was generated by transduction of the Trp ⁺ marker (from the Bacillus subtilis wild type W23) classical genetic engineering methods (as described, for example, in Harwood, CR and Cutting, SM (editors), Molecular Biological Methods for Bacillus (1990) John Wiley & Sons, Ltd., Chichester, England) introduced ΔpanB and ΔpanEI mutations.

The resulting strain was transformed with genomic DNA from Bacillus subtilis strain PA221 (genotype P ₂ "panBCD, trpC2 (Trp ^" )) and genomic DNA from Bacillus subtilis strain PA303 (genotype P ₂₆ panE7). The resulting strain PA327 has genotype P ₂ anBCD, P ₂ βpanE1 and is tryptophan auxotroph (Trp ^" ). The Bacillus subtilis strain PA327 was made up to 740 in 10 mL cultures with SVY medium (25 g / L Difco Veal Infusion Broth, 5 g / L Difco Yeast Extract, 5 g / L Na glutamate, 2.7 g / L ammonium sulfate Fill up with water, autoclave, then add 200 mL 1 M potassium phosphate, pH 7.0 and 60 mL 50% sterile glucose solution), which was supplemented with 5 g / L ß-alanine and 5 g / L α-ketoisovalerate, pantothenic acid Titer of up to 3.0 g / L (24 h) reached. The preparation of the Bacillus subtilis strain PA221 (genotype P _2G panBCD, trpC2 (Trp ^" )) is described in the following section:

The panBCD operon from. Was derived from a Bacillus subtilis GP275 plasmid library using classical genetic engineering methods with the aid of the sequence information of the panBCD operon from E. coli (see Merkel et al., FEMS Microbiol. Lett., 143, 1996: 247-252) Bacillus cloned. For cloning, the E. coli strain BM4062 (bir ^te ) and the information that the Bacillus operon is close to the birA gene was used. The panBCD operon was introduced into a plasmid replicable in E. coli. To improve the expression of the panBCD operon, strong, constitutive promoters of Bacillus subtilis phage SP01 (P ₂ β) were used and the ribosome binding site (= RBS) in front of the panß gene was replaced by an artificial RBS. A DNA fragment immediately upstream of the native panB gene in Bacillus was ligated in front of the P ₂ anBCD cassette on the plasmid. This plasmid was transformed into Bacillus subtilis strain RL-1 (derivative of Bacillus subtilis 168 (Marburg strain ATCC 6051), genotype trpC2 (Trp) obtained by classic mutagenesis) and by homologous recombination the native panBCD operon was replaced by the p ₂₆ panBCD operon replaced. The resulting strain is called PA221 and has the genotype P ₂₆ panßCD, trpC2 (Trp ^" ). The Bacillus subtilis strain PA221 was in 10 mL cultures with SVY medium containing 5 g / L ß-alanine and 5 g / L α Ketoisovalerate was supplemented, pantothenic acid titer of up to 0.92 g / L (24 h) was reached.

The preparation of the Bacillus subtilis strain PA303 (genotype P _panE1) is described in the following section: The Bacillus panE sequence was cloned analogously using the E. coli panE gene sequence. It was found that two homologs of the E. coli panE gene exist in B. subtilis and were designated panE1 and panE2. Deletion analyzes showed that the panEY gene is responsible for 90% of pantothenic acid production, while the deletion of the panE2 gene had no significant effect on pantothenic acid production. Here too, analogous to the cloning of the panBCD operon, the promoter was replaced by the strong constitutive promoter ₆ and the ribosome binding site in front of the panE1 gene was replaced by the artificial binding site. The P _2e panE1 fragment was cloned into a vector that was designed so that the P ₂ ψanE1 fragment was in the original panE1 locus in the genome from Bacillus subtilis. The strain resulting after transformation and homologous recombination is called PA303 and has the genotype P ₂ βpanE1. Bacillus subtilis strain PA303 was used in 10 mL cultures with SVY medium supplemented with 5 g / L ß-alanine and 5 g / L ketoisovalerate to produce pantothenic acid titers of up to 1.66 g / L (24 h ) reached.

The further stem construction was carried out by transforming PA327 with a plasmid which contained the P ₂ _iivBNC operon and the marker gene for spectinomycin. The P ₂₆ // ß / VC operon integrated into the amyE locus, which was verified by PCR. One transformant was designated PA340 (genotype P epanBCD, P ₂ anE1, P ₂ _ilvBNC, specR, trpC2 (Trp)).

With the Bacillus subtilis strain PA340, pantothenic acid titer of up to 3.6 g / L (24 h) was achieved in 10 ml cultures with SVY medium supplemented only with 5 g / L ß-alanine, in 10 ml Cultures with SVY medium supplemented with 5 g / L ß-alanine and 5 g / L α-ketoisovalerate achieved pantothenic acid titers of up to 4.1 g / L (24 h).

A deregulated / Λ / D cassette was also introduced into the strain PA340. For this purpose, a plasmid which contains the ilvD gene under the control of the P ₂₆ promoter with the artificial RBS2 was transformed into PA340. The P_eilvD gene was integrated into the original ilvD locus by homologous recombination.

The resulting strain PA374 has the genotype P ₂ _panBCD, P ₂₆ panE1, P ₂ _ilvBNC,

P ₂ , ilvD, specR and trpC2 (Trp ^" ).

With the Bacillus subtilis strain PA374, pantothenic acid titer of up to 2.99 g / L (24 h) was achieved in 10 mL cultures with SVY medium which was supplemented only with 5 g / L ß-alanine.

In order to produce feed-free pantothenic acid with the strain PA374 ß-alanine, additional copies of the gene panD coding for the aspartate α-decarboxylase were introduced into the strain PA374. For this, chromosomal DNA of the strain PA401 was transformed into PA374. The strain PA377 was obtained by selection for tetracycline.

The resulting strain PA377 has the genotype P ₂₆ panBCD, P _2e panE1, P ₂₆ ilvBNC, P _2G ilvD, specR, tetR and trpC2 (Trp ^" ). With the Bacillus subtilis strain PA377, pantothenic acid titers of up to 1.31 g / L (24 h) were achieved in 10 mL cultures with SVY medium without precursor feeds.

The preparation of the Bacillus subtilis strain PA401 (genotype P ₂ φanD) is described in the following section:

The Bacillus subtilis panD gene was cloned from the panBCD operon into a vector that carries the tetracycline marker gene. The promoter P ₂₆ and an artificial RBS described above were cloned in front of the panD gene. A fragment containing the tetracycline marker gene and the P ₂₆ panD gene was produced by restriction digestion. This fragment was religated and transformed into the strain PA221 described above. The fragment integrated into the genome of the PA211 strain. The resulting strain PA401 has the genotype P ₂₆ panBCD, P ₂₆ panD, tetR and trpC2 (Trp ^" ). The Bacillus subtilis strain PA401 was in 10 mL cultures in SVY medium, which was supplemented with 5 g / L α-ketoisovalerate , Pantothenic acid titer of up to 0.3 g / L (24 h) was reached, in 10 mL cultures with SVY medium supplemented with 5 g / L D-pantoic acid and 10 g / L L-aspartate, pantothenic acid Titer of up to 2.2 g / L (24 h) reached.

Starting from strain PA377, a tryptophan prototrophic strain was generated by transformation with chromosomal DNA from strain PY79. This strain PA824 has the genotype P ₂₆ panBCD, P ₂₆ panE1, P ₂₆ ilvBNC, P ₂₆ // VD, specR, tetR and Trp ⁺ . Bacillus subtilis strain PA824 was used in 10 mL cultures in SVY medium to pre-feed-free pantothenic acid titer of up to 4.9 g / L (48 h) (comparison PA377: up to 3.6 g / L in 48 h) reached.

The preparation of PA668 is described in the following section: The Bacillus panB gene was cloned from the wild-type panBCD operon and inserted into a vector which, in addition to a chloramphenicol resistance gene, also ß. contains subtilis sequences of the vpr locus.

The strong constitutive promoter P ₂₆ was introduced before the 5 'end of the panB gene. A fragment containing the P ₂₆ panß gene, the marker gene for chloramphenicol resistance and Bacillus subtilis vpr sequences obtained by restriction digestion. The isolated fragment was religated and the strain PA824 was transformed with it. The strain obtained was designated PA668. The genotype of PA668 is: P ₂ φanBCD, P ₂ φanE1, P ₂₆ ilvBNC, P ₂₆ ilvD, P ₂ epanB, specR, tetR, CmR and Trp ⁺ . Two colonies of PA668 were isolated and designated PA668-2A, the other PA668-24.

With B. subtilis strain PA668-2A_, pantothenic acid titers of 1.5 g / L are achieved in 48 h in 10 ml cultures in SVY medium without addition of precursors. In 10 mL cultures supplemented with 10g / L aspartate, titres up to 5 g / L are achieved.

With ß. subtilis strain PA668-24 are achieved in 10 ml cultures in SVY medium without addition of precursors pantothenic acid titer of 1.8 g / L in 48 h. In 10 mL cultures supplemented with 10g / L L-aspartate, titres up to 4.9 g / L are achieved.

The exact construction of the strains is according to the appendices of PCT / US application 0025993 and US serial no. 60 / 262,995.

The above-described strain PA377 is used for glucose-limited fermentation in SVY medium (25 g / L Difco Veal Infusion Broth, 5 g / L Difco Yeast Extract, 5 g / L tryptophan, 5 g / L Na glutamate, 2 g / L (NH ₄ ) ₂ SO ₄ , 10 g / L KH ₂ PO ₄ , 20 g / LK ₂ HPO ₄ , 0.1 g / L CaCl ₂ , 1 g / L MgSO ₄ , 1 g / L sodium citrate, 0 , 01 g / L FeSO ₄ x7 H ₂ O and 1 ml / L of a trace salt solution of the following composition: 0.15 g Na ₂ MoO x 2 H ₂ O, 2.5 g H ₃ BO ₃ , 0.7 g CoCI ₂ x 6 H ₂ O, 0.25 g CuSO ₄ x 5 H ₂ O, 1.6 g MnCl ₂ x 4 H ₂ O, 0.3 g ZnSO ₄ x 7 H ₂ O, made up to 1 I with water)) im 10 L scale with continuous feeding of a glucose solution in 36 h (48 h) pantothenic acid concentrations in the fermentation broth of 18-19 g / L (22-25 g / L) reached.

When glucose-limited fermentation of PA824, the tryptophan-prototrophic derivative of PA377, in yeast extract medium (10 g / L Difco Yeast Extract, 5 g / L Na-Glutamate, 8 g / L (NH ₄ ) ₂ SO ₄₎ 10 g / L KH ₂ PO ₄ , 20 g / LK ₂ HPO _4j 0.1 g / L CaCI ₂ , 1 g / L MgSO ₄ , 1 g / L sodium citrate, 0.01 g / L FeSO ₄ x7 H ₂ O and 1 ml / L of the trace salt solution described above) on a 10 L scale at continuous Feeding a glucose solution in 36 h, 48 h and 72 h reached the following pantothenic acid concentrations in fermentation broths: 20 g / L, 28 g / L and 36 g / L. Through further media optimization, strain PA824 is used for glucose-limited fermentation in a medium consisting of 10 g / L Difco Yeast Extract, 10 g / L NZ Amine A (Quest International GmbH, Erftstadt), 10 g / L Na-Glutamate, 4 g / L (NH ₄ ) ₂ SO ₄ , 10 g / L KH ₂ PO ₄ , 20 g / LK ₂ HPO ₄ , 0.1 g / L CaCI ₂₎ 1 g / L MgSO ₄ , 1 g / L sodium citrate, 0 , 01 g / L FeSO ₄ x7 H ₂ O and 1 ml / L of the trace salt solution described above on a 10 L scale with continuous addition of a glucose solution in 36 h (48 h) pantothenic acid concentrations of 37 g / L (48 g / L) reached in fermentation broths.

Further increases in the pantothenic acid concentration in the fermentation broth are conceivable by further media optimization, by increasing the fermentation time, by improving the process and strain, and by combinations of the individual steps. The pantothenic acid concentrations described above can also be achieved by fermentation of strains which are derivatives of the PA824 described above. Derivatives can be produced through classic strain development as well as through further genetic engineering manipulations. Through media, strain and fermentation process development, the pantothenic acid titers in the fermentation broths can be increased to over 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, and> 90 g / L.

An important advantage of the process according to the invention is that the fermentation is carried out in a culture medium which, apart from at least one carbon and nitrogen source, contains no further precursors as starting compounds. This means that the biosynthesis of D-pantothenic acid is independent of the feeding of further precursors. According to the invention, such precursors are understood to mean substances such as, for example, β-alanine and / or L-aspartate and / or L-valine and / or α-ketoisovalerate and / or combinations thereof. In a preferred variant of the process according to the invention, the fermentation of the D-pantothenic acid-producing organism is carried out in a culture medium which contains a carbon and a nitrogen source, but to which no free ß-alanine and / or ß-alanine salts have been added or during the course is fed to the fermentation. That is, for the production of D-pantothenic acid in ranges of at least 10 g / l culture medium, preferably of at least 20 g / l, particularly preferably of at least 40 g / l, most particularly preferably of at least 60 g / l and in particular of at least 70 g / l, no additional β-alanine and / or β-alanine salts need to be added according to the invention. The independence from the feeding of precursors represents in particular a significant economic advantage of the method according to the invention over known methods, since a large number of precursors are very expensive.

However, the addition of ß-alanine and / or ß-alanine salts is not excluded according to the invention, so that consequently the yield of D-pantothenic acid can be further improved by the addition of ß-alanine and / or ß-alanine salts. If, for example, it is assumed that all necessary precursors of pantothenic acid are present in sufficient quantity, only the activity of the panD gene limits a further increase in pantothenic acid production, the yield of pantothenic acid can e.g. can be increased by a further 50% by adding free ß-alanine and / or ß-alanine salts.

In an advantageous variant of the present invention, up to 20 g / l of free .beta.-alanine and / or .beta.-alanine salts can be added to the culture medium to further increase the pantothenic acid yield by more than 50%. It is preferred to add about 15 g / l of free β-alanine and / or β-alanine salts to the culture medium.

Examples of carbon sources suitable according to the invention for use in a culture medium for fermentation of the aforementioned organisms are sugars, such as starch hydrolysates (mono-, di-, oligosaccharides), preferably glucose or sucrose, and beet or cane sugar molasses, proteins, protein hydrolysates, soybean meal, corn steep liquor, fats, free fatty acids, recycled cells from fermentations already carried out or their hydrolyzates as well as yeast extract. These lists are not limiting for the present invention.

Furthermore, the present method is advantageously characterized in that the total sugar content is reduced to a minimum until the end of the fermentation, since this would otherwise make subsequent drying and / or formulation of the fermentation solution more difficult by sticking together. This can be achieved according to the invention in that the fermentation is continued for some time, after the carbon source has been consumed (in cultivation in batch mode) or after the carbon supply (in process control in fed-batch or repeated fed-batch mode) has been interrupted and / or regulated in such a way that the concentration of the carbon source is almost zero is (with fed-batch, repeated-fed-batch or continuous process control).

This is done according to the invention in that after the metering in of the carbon source (for example sugar solution) has been interrupted, the fermentation is continued in the fermentation solution to at least 80%, preferably 90% and particularly preferably 95% of the saturation value until the dissolved oxygen concentration (pO ₂ ) is reached ,

Examples of nitrogen sources suitable according to the invention are ammonia, ammonium sulfate, urea, proteins, protein hydrolyzates or yeast extract. This list is also not limiting for the present invention.

The fermentation medium also contains mineral salts and / or trace elements such as amino acids and vitamins. The exact compositions of suitable fermentation media are widely known and accessible to the person skilled in the art. After the fermentation medium has been inoculated with a suitable D-pantothenic acid-producing organism (with cell densities known to the person skilled in the art), the organism is cultivated, if appropriate with the addition of an anti-foaming agent. If necessary, the pH of the medium can be regulated using various inorganic or organic alkalis or acids, e.g. NaOH, KOH, ammonia, phosphoric acid, sulfuric acid, hydrochloric acid, formic acid, succinic acid, citric acid etc.

Due to the buffer systems used during the fermentation, which can be e.g. NaOH, KOH, ammonia, phosphoric acid, sulfuric acid, hydrochloric acid, formic acid, succinic acid, citric acid or the like, the D-pantothenic acid formed is in the fermentation solution in the form depending on the buffer system used of the respective salt (s). Since the salts of D-pantothenic acid in the form of their monovalent cations are particularly disadvantageous, the fermentation solution is prepared according to the invention using a cation exchanger. The present invention encompasses all commercially available cation exchangers. According to the invention suitable cation exchanger for double decomposition of pantothenate salts for pantothenic acid are in the H ⁺ form present, acidic resins based on acrylate with carboxyl functionalities and / or polystyrene resins having sulfonate groups, for example. Lewatit CNP 80, CNP LF, S 100, S1468 and SP 112 Monoplus (Bayer, Leverkusen, Germany), Diaion PK 216 (Mitsubishi Chemical Corp, Tokyo, Japan), Amberlite 200 and Amberlite 252 (Rohm and Haas, Philadelphia, USA). Preferred cation exchangers are strongly acidic ion exchangers in the H ⁺ form based on sulfonated polystyrene. Monodisperse cation exchangers, such as, for. B. Lewatit S1468. The preceding list of cation exchangers is exemplary and is not limiting for the present invention. As is familiar to the specialist, the ion exchangers can be regenerated into the H ⁺ form after exhaustion with mineral acids and can thus be used many times.

By using the cation exchanger, the monovalent cations, e.g. Ammonium, potassium or sodium are advantageously almost completely removed from the fermentation solution, so that free D-pantothenic acid is formed in the solution from the D-pantothenic acid salts. According to the invention, the content of monovalent cations, preferably ammonium, potassium and / or sodium ions, is reduced to a concentration of <1 g / kg of solution.

The solution of free pantothenic acid thus obtained is adjusted according to the invention to a pH of 3-10 by adding calcium and / or magnesium base. A pH value of 5-10 is advantageous. The pH is preferably adjusted to 5-9, particularly preferably 6-9 and very particularly preferably 6-8. In this way, a solution or suspension of calcium and / or magnesium pantothenate is obtained. Calcium hydroxide, calcium carbonate, calcium oxide, magnesium hydroxide and / or basic magnesium carbonate is preferably added to the solution for neutralization in the form of a solid and / or as an aqueous suspension.

It is preferred according to the invention if the neutralization of the free solution containing D-pantothenic acid with the aid of a calcium and / or Magnesium base takes the form of an aqueous suspension. By using an aqueous suspension, the neutralization takes place faster and without greater pH fluctuations than is the case when using an appropriate solid.

According to the invention, the process is characterized in that an aqueous suspension containing 2-55% by weight, preferably 10-50% by weight and particularly preferably 20-40% by weight of calcium hydroxide is added to the solution in step c). According to the invention, a method is also included in which an aqueous suspension containing 2-65% by weight, preferably 10-50% by weight and particularly preferably 20-40% by weight of calcium carbonate is added to the solution in step c). In a further embodiment variant of the present invention, an aqueous suspension containing 2-60% by weight, preferably 10-50% by weight and particularly preferably 20-40% by weight of magnesium hydroxide is added to the solution in step c) of the method according to the invention , Also included in the invention is a method in which the solution in step c) is added with an aqueous suspension containing 2-25% by weight, preferably 10-20% by weight, of basic magnesium carbonate.

In a further variant, the free solution containing D-pantothenic acid can be obtained

Step b) in a subsequent step c) an acidic inorganic and / or organic calcium and / or magnesium salt is added, one

Solution or suspension is obtained, the calcium and / or magnesium

Contains pantothenate.

Are advantageous according to the invention under acidic inorganic calcium and / or

Magnesium salts to understand the corresponding calcium and / or magnesium halides. According to the invention, these are preferably CaCl ₂ and / or MgCl ₂ . The acidic organic calcium and / or magnesium salts are understood to mean, for example, readily water-soluble salts of organic anions. These are preferably z.

B. calcium and / or magnesium formate, acetate, propionate, glycinate and / or

Lactate. In the subsequent step d), the solution or suspension of the

Calcium and / or magnesium pantothenate undergoes drying and / or formulation. The calcium and / or magnesium D-pantothenate-containing solution or suspension obtained is dried and / or formulated by methods known per se, such as spray drying, spray granulation, fluidized bed drying, fluidized bed granulation, drum drying or spin-flash drying (Ullmann's Encyclopedia of Industrial Chemistry, 6 ^th edition, 1999, electronic release, chapter "Drying of Solid Materials"). The gas inlet temperature for convection drying is in the range of 100-280 ° C, preferably 120-210 ° C. The gas outlet temperature is 50-180 ° C., preferably at 60-150 ° C. Fine particles can be separated and returned to set a desired particle size distribution and the associated product properties, and coarse material can also be ground in a mill and then also returned.

According to the invention, it is advantageous in the method described above to reduce complicated work-up steps, in particular to dispense with the use of organic solvents, while at the same time providing a desired product with good biological value. Furthermore, the amount of waste water obtained is substantially reduced according to the invention. This results in further savings on complex processing and disposal systems. Thus, the method according to the invention is advantageously characterized in that it is simpler, less prone to failure, less time-consuming, significantly less expensive and therefore more economical than conventional methods.

However, this does not rule out that the method according to the invention can be varied. The method steps a) to d) according to the invention mentioned at the beginning can be supplemented by one or more of the following method steps which are familiar to the person skilled in the art. All conceivable combinations of the additional (optional) process steps with the (essential) process steps a) to d) are encompassed according to the invention.

So the solutions or suspensions resulting from process steps a) - c) z. B. can be sterilized by heating (sterilization) or other methods such as pasteurization or sterile filtration. In further variants of the method according to the invention, at least one and / or combinations of the following steps can be carried out before the drying and / or formulation of the solution or suspension, including lysis and / or killing of the biomass and / or separation of the biomass from the fermentation solution and / or Adding further additives and / or concentrating the fermentation solution, preferably by removing water.

The present invention thus also relates to a method in which the lysis and / or killing of the biomass is still carried out in the fermentation solution or only after the biomass has been separated from the fermentation solution. This can be done, for example, by a temperature treatment, preferably at 80-200 ° C. and / or an acid treatment, preferably with sulfuric acid or hydrochloric acid and / or enzymatically, preferably with lysozyme.

In a further embodiment of the present invention, the cells of the fermented microorganisms can be removed from the solutions or suspensions of steps a), b) or c) of the method according to the invention by filtration, separation (e.g. centrifugation) and / or decanting. It is also conceivable that the solutions or suspensions of steps a), b) or c) are passed directly over a cation exchanger without removing the organisms contained. If the processing step by means of cation exchangers (step b)) of the method according to the invention is not preceded by any separation of the biomass, the fermentation solution containing biomass can advantageously also be passed through the ion exchanger bed from bottom to top, ie counter to gravity. This procedure is generally advantageous if suspended solids are present in the solution to be cleaned.

The solution or suspension resulting from the work-up on the cation exchanger can, after neutralization, be used on a suitable evaporator, e.g. B. falling film evaporator, thin film evaporator or rotary evaporator. In a further variant, the actual drying and / or formulation of the solution or suspension in step d) of the aforementioned method according to the invention Go ahead with concentration. For this purpose, the solution or suspension from step c) z. B. concentrated in a falling film evaporator and / or a thin film evaporator. Such evaporators are e.g. B. by the companies GIG (4800 Attnang Puchheim, Austria), GEA Canzler (52303 Düren, Germany), Diessel (31103 Hildesheim, Germany) and Pitton (35274 Kirchhain, Germany).

In order to improve the color properties of the end product, an additional filtration step can be carried out, in which some activated carbon is added to the solutions or suspensions obtained during the process and the suspension containing activated carbon is then filtered. Or the solutions obtained during fermentation can be passed through a small bed of activated carbon. The amounts of activated carbon required for this are in the range of a few% by weight of the fermentation solution and are within the knowledge and discretion of the person skilled in the art. These filtrations can be made easier by adding a commercially available flocculant (e.g. Sedipur CF 902 or Sedipur CL 930 from BASF AG, Ludwigshafen) to the respective solution before filtration.

In an advantageous embodiment of the present invention, the fermentation discharge (fermentation broth) is sterilized by heating and then freed from the cell mass by centrifuging, filtering or decanting. After adding 50-1000 mg / kg, preferably 100-200 mg / kg, of a commercially available flocculant based on the fermentation output, the mixture is filtered through a short bed of activated carbon and sand in order to obtain a biomass-free solution with a high D-pantothenic acid content , This prepared solution is then conveyed through the ion exchange bed (in H ⁺ form).

If the processing step according to the invention is not preceded by a separation of the biomass via a cation exchanger, the fermentation solution containing biomass can advantageously also be passed through the ion exchanger bed from bottom to top, that is to say against the force of gravity.

The subsequent drying of the solution or suspension containing calcium and / or magnesium pantothenate can, for example, by spray drying respectively. This can be done in cocurrent, countercurrent or mixed flow. All known atomizers can be used for atomization, in particular centrifugal atomizers (atomizer disks), single-substance nozzles or two-substance nozzles. Preferred drying temperature conditions are 150 - 250 ° C tower inlet temperature and 70 - 130 ° C tower outlet temperature. However, it can also be dried at a higher or lower temperature level. In order to achieve a very low residual moisture, a further drying step can be carried out in a fluidized bed.

Spray drying can also be carried out in an FSD or SBD dryer (FSD: Fluidized Spray Dryer; SBD: Spray Bed Dryer), as built by the companies Niro (Copenhagen, Denmark) and APV-Anhydro (Copenhagen, Denmark), perform, which are a combination of spray dryer and fluidized bed. A flow aid can be added during spray drying. As a result, the deposits on the dryer wall can be reduced and the flow behavior, especially with fine-grained powders, improved. Silicates, stearates, phosphates and corn starch are particularly suitable as flow aids. In principle, drying can also take place in a spray fluidized bed, which can be operated both continuously and batchwise. The solution or suspension can be sprayed in from above (top spray), from below (bottom spray) and from the side (sidespray).

The present invention furthermore relates to a composition for use as an animal feed additive and / or animal feed supplement, it being producible by a) using at least one organism producing D-pantothenic acid, the pantothenic acid (pan) and / or isoleucine / valine / valine (ilv) biosynthesis is deregulated and forms at least 2 g / l of salts of D-pantothenic acid by fermentation in a culture medium, the culture medium being 0-20 g / l, preferably 0 g / l, of free β-alanine and / or β Alanine salt is added, b) the fermentation solution containing D-pantothenate is passed over a cation exchanger, free salts of D-pantothenic acid resulting in free D-pantothenic acid, c) the free D-pantothenic acid solution by adding a calcium and / or magnesium base to a pH A value of 3-10 is set, a solution is obtained which contains calcium and / or magnesium pantothenate and d) the solution containing calcium and / or magnesium pantothenate is subjected to drying and / or formulation.

In an advantageous variant, a suspension which contains calcium and / or magnesium pantothenate is obtained or presented in step c) or step d).

In a further variant, an acidic inorganic and / or organic calcium and / or magnesium salt can be added to the free D-pantothenic acid-containing solution from step b) in a subsequent step c), a solution or suspension being obtained, which contains calcium and / or magnesium pantothenate.

According to the invention, acidic inorganic calcium and / or magnesium salts are advantageously to be understood as meaning the corresponding calcium and / or magnesium halides. According to the invention, these are preferably CaCl ₂ and / or MgCl ₂ . The acidic organic calcium and / or magnesium salts are understood to mean, for example, readily water-soluble salts of organic anions. These are preferably z. B. calcium and / or magnesium formate, acetate, propionate, glycinate and / or lactate.

In the subsequent step d), the solution or suspension of the calcium and / or magnesium pantothenate is then subjected to drying and / or formulation.

Furthermore, the composition according to the invention is characterized in that it contains salts of D-pantothenic acid in a concentration of at least 1-100% by weight, preferably at least 20-100% by weight and particularly preferably at least 50% by weight. The present invention relates to a composition which comprises salts of D-pantothenic acid in the form of divalent cations, preferably calcium and / or contains magnesium D-pantothenate. According to the invention, a composition is preferred which is characterized in that the content of salts of D-pantothenic acid in the form of monovalent cations is 1 1 g / kg.

According to the invention, a calcium or magnesium D-pantothenate is obtained by the process described above, which meets the requirements for a feed additive. These requirements are, for example, a relatively high content of D-pantothenate and good tolerance for the target organism as well as a biological value in the sense of the "vitamin effect" of the product according to the invention.

The present invention is explained in more detail by the following examples, which, however, are not limiting for the invention:

Example 1:

In a laboratory fermenter with stirrer and gassing device with 14 l content, aqueous fermentation medium with the following composition is placed:

The trace salt solution is composed as follows:

0.15 g Na ₂ MoO ₄ x 2 H ₂ O, 2.5 g H ₃ BO ₃ , 0J g CoCI ₂ x 6 H ₂ O, 0.25 g CuSO ₄ x 5 H ₂ O, 1.6 g MnCI ₂ x 4 H ₂ O, 0.3 g ZnSO ₄ x 7 H ₂ O are made up to 1 I with water. The trace salt solution is added via sterile filtration. The Initial liquid volume is 6 I. The above-mentioned contents are based on this value.

60 ml of Bacillus subtilis PA824 seed culture (OD = 10) is added to this solution and fermented at 43 ° C. with vigorous stirring at a gassing rate of 12 l / min. This strain is described in the Appendix in PCT / US application 0025993.

6.5 l of a sterile aqueous solution are added within 47 h, the composition of which is as follows:

The fermentation is carried out glucose-limited. During the fermentation, the pH value is adjusted to 7.2 by adding 25% ammonia solution or 20% phosphoric acid. Ammonia also serves as a nitrogen source for the fermentation. The speed of the agitator is regulated by keeping the dissolved oxygen content at 30% of the saturation value. After the addition of the carbon source has been terminated, the fermentation is continued until the dissolved oxygen content (pO ₂ ) has reached a value of 95% of the saturation value. The fermentation is then ended and the organism thermally killed. For this, the fermentation solution is sterilized for 45 minutes. Successful killing is demonstrated by plating.

The cells are then separated by centrifugation. After cell separation, the concentration of D-pantothenate is 22.8 g / l after 48 h.

Fermentation broths with ß-alanine feed-free pantothenic acid titers of over 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, and> can also be produced in an analogous manner Have 90 g / L. A fermentation discharge which has been sterilized by heating as described above and largely freed of the biomass by centrifuging is mixed with additional D-pantothenic acid and the pH is adjusted to a value of about 7 with 25% ammonium hydroxide solution (dispersion) , The solution or suspension thus obtained is filtered over a small bed of sand / activated carbon. The content of D-pantothenic acid in the filtrate is 70 g / l, the D-pantothenic acid being mainly in the form of the ammonium salt.

Using hydrostatic pressure, 1000 ml of this filtrate is passed through a 250 ml glass column containing approx. 100 ml of Lewatit S100 G1 ion exchanger. The flow is regulated to approx. 20 ml / min.

Fractions of 100 ml were collected from the flow and analyzed for their pH, the concentration of D-pantothenate and the ions Ca ²⁺ , Mg ²⁺ , NH ₄ ⁺ , K ⁺ and Na ⁺ . Fractions 1-3 are given as examples in the table below.

75 ml of fraction 2, which contained 4.8 g of pantothenate, were brought to a pH of 7 with stirring by adding solid calcium hydroxide. The calcium D-pantothenate solution or suspension thus obtained was then dried by evaporating off the water on a rotary evaporator and 8.98 g of a slightly brownish calcium D-pantothenate powder were obtained which contained of 57% calcium D-pantothenate. This powder is not prone to sticking and has good product properties.

Example 2:

The trace salt solution is composed as follows:

0.15 g Na ₂ MoO ₄ x 2 H ₂ O, 2.5 g H ₃ BO ₃ , 0.7 g CoCI ₂ x 6 H ₂ O, 0.25 g CuSO ₄ x 5 H ₂ O, 1, 6 g MnCI ₂ x 4 H ₂ O, 0.3 g ZnSO ₄ x 7 H ₂ O are made up to 1 I with water. The trace salt solution is added via sterile filtration. The Initial liquid volume is 5.5 I. The above-mentioned contents are based on this value.

55 ml of Bacillus subtilis PA824 seed culture (OD = 10) are added to this solution and fermented at 43 ° C. with vigorous stirring at a gassing rate of 12 l / min. This strain is described in the Appendix in PCT / US application 0025993.

6 l of a sterile aqueous solution are metered in within 48 h, the composition of which is as follows:

The fermentation is carried out glucose-limited. During the fermentation, the pH was regulated to a value of approximately 7.2 by metering in 25% ammonia solution or 20% phosphoric acid. Ammonia also serves as a nitrogen source for the fermentation. The speed of the agitator is regulated by keeping the dissolved oxygen content at 30% of the saturation value. After the addition of the carbon source has been terminated, the fermentation is continued until the dissolved oxygen content (pO ₂ ) has reached a value of 95% of the saturation value. The fermentation is then ended and the organism thermally killed. For this purpose, the fermentation solution is sterilized for 30 minutes. Successful killing is demonstrated by plating.

The cells are then separated by centrifugation. After cell separation, the concentration of D-pantothenate when terminated after 48 h is 24.1 g / l.

Fermentation broths with ß-alanine feed-free pantothenic acid titers of over 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, and> can also be produced in an analogous manner Have 90 g / L. 2817 ml of the fermenter discharge are mixed with 1183 ml of a pantothenic acid solution with a concentration of 178.9 g / l and neutralized with 25% ammonia solution. It is then filtered through sand / activated carbon. The content of D-pantothenic acid in the filtrate is 67 g / l, the D-pantothenic acid being mainly in the form of the ammonium salt.

1300 ml of the filtrate were conveyed through a bed (volume about 1 liter) of the Lewatit S100 G1 ion exchanger (in the H ⁺ form) and rinsed with water. The flow is regulated to approx. 20 ml / min.

After a preliminary run of 200 ml, 1810 g of the eluate were collected. The D-pantothenic acid content was 45 g / l. The following ion concentrations are present: NH ⁺ : 66 mg / kg eluate and K ⁺ : <0.001 g / 100 g eluate. The phosphorus content is 0.072 g / 100 g eluate.

With stirring, the free pantothenic acid is neutralized with solid calcium hydroxide, i.e. adjusted to a pH of about 7.

1840 g of an aqueous calcium D-pantothenate solution or suspension with a content of D-calcium pantothenate of 49 g / l were obtained.

This aqueous calcium D-pantothenate solution or suspension is dried in a laboratory spray dryer from Niro, type Minor. The inlet temperature is about 200 ° C, the outlet temperature 85 - 90 ° C. The atomization takes place using a two-fluid nozzle at a pressure of 4 bar. The water content was carried out according to the Karl Fischer method. The powdery product has the following specification (data in% by weight): water content: 2% calcium D-pantothenate: 56.3% ammonium ions: 0.049% potassium ions: <0.01% sodium ions: <0 , 01%. Example 3:

A water fermentation medium with the following composition is placed in a laboratory fermenter with stirrer and gassing device with a capacity of 300 l:

After sterilization, the following sterile media components are also added:

The trace salt solution is composed as follows:

0.15 g Na ₂ MoO ₄ x 2 H ₂ O, 2.5 g H ₃ BO ₃ , 0J g CoCI ₂ x 6 H ₂ O, 0.25 g CuSO ₄ x 5 H ₂ O, 1.6 g MnCI ₂ x 4 H ₂ O, 0.3 g ZnSO ₄ x 7 H ₂ O are made up to 1 I with water. The trace salt solution is added via sterile filtration. The initial liquid volume is 100 I. The contents listed above are based on this value. 4 l of seed culture (OD = 120) of Bacillus subtilis PA824 is added to this solution and fermented at 43 ° C. with vigorous stirring at a gassing rate of 1.8 m ³ / h. This strain is described in the Appendix in PCT / US application 0025993.

1 13 l of a sterile aqueous solution are metered in within 43 h, the composition of which is as follows:

The fermentation is carried out glucose-limited. During the fermentation, the pH at 7.2 is regulated by adding 25% ammonia solution or 20% phosphoric acid. Ammonia also serves as a nitrogen source for the fermentation. The speed of the agitator is regulated by keeping the dissolved oxygen content at 30% of the saturation value. After the addition of the carbon source has been terminated, the fermentation is continued until the dissolved oxygen content (pO ₂ ) has reached a value of 95% of the saturation value. The fermentation is ended after 43 h. The cells are separated by separation in a plate centrifuge. The cell-free fermentation solution is then sterilized for 60 minutes. Successful killing is demonstrated by plating.

The concentration of D-pantothenate after cell separation and sterilization is 21 g / l. Fermentation broths with ß-alanine feed-free pantothenic acid titers of over 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, and> can also be produced in an analogous manner Have 90 g / L.

Then 3270 ml of the fermentation discharge obtained are topped up with 1750 ml of sodium pantothenate solution at a concentration of 170 g / l. Then about 7 g of powdered activated carbon are stirred into this solution. To improve the filterability of the suspension, one half of it is mixed with Sedipur CF 902 (48 g of a 1% solution) and the other half with Sedipur CL 930 (30 g of a 1% solution). After stirring briefly, both halves are filtered through a small bed of Celite and then combined again. 1800 ml of the filtrate thus obtained, containing 65.4 g / l of D-pantothenic acid, are conveyed through a bed (volume 1 liter) of the Lewatit S100 G1 ion exchanger (in the H ⁺ form) and rinsed with water. The flow is approx. 20 ml / min.

After a flow of 250 ml, which is discarded, 2.2 l of the eluate are collected and the pH is adjusted to a value of approximately 7 by adding an aqueous 20% strength calcium hydroxide dispersion (lime milk). It is filtered again through a paper filter and 2174 g of an aqueous calcium D-pantothenate solution or suspension containing 58 g / l calcium D-pantothenate is obtained as the filtrate after sampling.

The subsequent spray drying takes place in a laboratory spray dryer from Niro, type Minor. The inlet temperature is approximately 185 ° C, the outlet temperature is approximately 95 ° C. The atomization takes place using a two-fluid nozzle at a pressure of 2 bar. The water content was carried out according to the Karl Fischer method. The powdery product obtained has the following specification (data in% by weight): water content: 1.4% calcium D-pantothenate: 63.2% ammonium ions: 0.043% potassium ions: <0.01% sodium ions : <0.01%.

Example 4: In a fermenter with stirrer and gassing device with 20L content, 200g soy flour, 50g 50% yeast extract, 25g Na glutamate, 40g ammonium sulfate and 5mL anti-foaming agent Tego KS911 were mixed with 3.9L demineralized water and sterilized for 30 minutes at 121 ° C.

Then solutions 1, 2, 3 and 4 were added.

Solution 1 was prepared as follows: 12.5 g glucose, 0.5 g CaCl ₂ x 2 H ₂ O and 5 g MgCl ₂ x 6 H ₂ O were dissolved in 100 ml demineralized water and sterile filtered.

Solution 2 was prepared as follows: 25 ml of citrate-iron solution (200 g / l sodium citrate, 2 g / L FeSO x 7 H2O, sterile filtered) were mixed with 5 ml of trace salt solution (0.15 g Na ₂ MoO ₄ x 2 H ₂ O, 2.5 g H ₃ BO ₃ , OJg CoCI ₂ x 6 H ₂ O, 0.25g CuSO ₄ x 5 H ₂ O, 1.6g MnCI ₂ x 4 H ₂ O, 0.3g ZnSO x 7 H ₂ O were made up to 1L with water , sterile filtered) added.

Solution 3 was prepared as follows: 87.5 g of glucose were made up to 500 ml with water and sterilized.

Solution 4 was prepared as follows: 25 g KH PO, 50 g K ₂ HPO, 25 g NaH ₂ PO and 50 g Na ₂ HPO were made up to 500 ml with water and sterilized.

The mixed medium (volume after addition of all solutions: 5L) was added with 100 ml of seed culture (OD = 9.5 in SVY medium (SVY medium: Difco Veal Infusion broth 25 g, Difco Yeast extract 5 g, sodium glutamate 5 g, (NH ₄ ) ₂ SO ₄ 2.7 g in 740 ml H ₂ O, sterilize; add 200mL sterile 1M K ₂ HPO ₄ (pH 7) and 60mL sterile 50% glucose solution (final volume 1 L))) from Bacillus subtilis PA668-2A and fermented at 43 ° C with vigorous stirring at a gassing rate of 12L / min. This strain is listed in the appendix in U.S. application serial no. 60 / 262,995.

About 3.5L of a sterile aqueous glucose solution were metered in within 48 h. The solution was prepared as follows: 5kg glucose x H ₂ O and 3.6g CaCl ₂ x 2 H ₂ O were mixed with 1.2 kg demineralized water and sterilized for 30 minutes. Then 9 ml of trace salt solution (composition see above) and 37.5 ml of citrate iron solution (composition see above) were added. Then 60 ml of sterile-filtered 375 g / L sodium glutamate solution were added. The fermentation was carried out glucose-limited. During the fermentation, the pH was kept at 7.2 by adding 25% ammonia solution or 20% phosphoric acid. Ammonia also served as a nitrogen source for the fermentation. The speed of the stirrer was controlled by keeping the dissolved oxygen content at 20% of the saturation value. The foaming was controlled by occasionally adding the anti-foaming agent Tego KS 911. After the addition of the carbon source had been discontinued, the fermentation was continued until the dissolved oxygen content (pO ₂ ) had reached a value of 95% of the saturation value. The fermentation was then ended. The cells were separated by centrifugation. The remaining cells in the supernatant were thermally killed by sterilization. The killing was verified by plating. After separation and sterilization, the concentration of D-pantothenate was 35.8 g / L. Fermentation broths with a ß-alanine-free feed pantothenic acid titer of more than 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 can also be produced in an analogous manner , and have more than 90 g / L.

Example 5:

1000 ml of the centrifugate from Example 4 were made up to 70 g / l with synthetic sodium pantothenate and pumped from top to bottom through a 1000 ml glass column containing approximately 1000 ml of Lewatit S100 G1 ion exchanger. The flow was regulated to approx. 20 ml / min.

Fractions of 250 ml were collected from the flow and analyzed for their pH value, the concentration of D-pantothenate and the ions Ca ²⁺ , Mg ²⁺ , NH ⁺ , K ⁺ and Na ⁺ . Fractions 1-3 are listed as examples in the table below.

250 ml of fraction 2 was brought to pH 7 with stirring by adding solid calcium hydroxide. A white precipitate precipitated out, which was filtered off after addition of Sedipur CF902. The filtrate was then dried by evaporating the water on a rotary evaporator and 25 g of a slightly brownish calcium D-pantothenate powder was obtained, which had a content of 55% pantothenate. This powder is not prone to sticking and has good product properties.

Example 6:

1000 ml of the spiked centrifugate from Example 5 were pumped from bottom to top through a bed (volume about 1 liter) of the Lewatit S100 G1 ion exchanger (in the H ⁺ form). Then it was rinsed with water. The flow was regulated to approx. 20 ml / min.

After a preliminary run of 500 ml, 1750 g of the eluate were collected. The D-pantothenate content was 35 g / l. The following ion concentrations were present in the eluate: NH ₄ ⁺ : <0.01%, Na ⁺ : 0.0064%, K ⁺ : 0.006%, PO ₄ ^{3 "} : 0.29%, SO ₄ ^2" : 0.1% and Cl ^" : 0.043%.

With stirring, the free pantothenic acid was neutralized with solid calcium hydroxide, ie a pH of about 7 was set. Approx. 1800 g of an aqueous calcium D-pantothenate suspension with a pantothenate content of approx. 35 g / l were obtained.

This aqueous calcium D-pantothenate suspension was dried in a laboratory spray dryer from Niro, type Minor. The inlet temperature was about 200 ° C, the outlet temperature 85 - 90 ° C. The atomization was carried out using a two-component nozzle at a pressure of 4 bar. The water content was carried out according to the Karl Fischer method. The powdery product had the following specification (data in% by weight):

Water content: 2% D-pantothenate: 56.3% ammonium ions: 0.03% potassium ions: <0.015% sodium ions: <0.019% calcium ions: 9.5%

Example 7:

3270 ml of the fermentation discharge obtained in Example 4 were replenished with 1750 ml of sodium pantothenate solution at a concentration of 125.6 g / l pantothenate. Then about 7 g of powdered activated carbon were stirred into this solution. To improve the filterability of the suspension, half of it was mixed with Sedipur CF 902 (48 g of a 1% solution). After brief stirring, both halves were filtered through a small bed of Celite and then combined again.

Of the filtrate obtained in this way, containing 66.5 g / l of D-pantothenic acid, 1000 ml were conveyed through a bed (volume 1 liter) of the Lewatit S100 G1 ion exchanger (in the H ⁺ form). It was rinsed with water. The flow rate was approx. 20 ml / min.

After the forerun of 500 ml, which was discarded, 2 l of the eluate were collected and the pH was adjusted to a value of approximately 7 by adding an aqueous 20% strength calcium hydroxide dispersion (milk of lime). It was filtered again through a paper filter and the filtrate was about 1960 ml of an aqueous Calcium D-pantothenate solution or suspension containing 16 g / l D-pantothenate obtained after sampling.

The subsequent spray drying was carried out in a laboratory spray dryer from Niro, type Minor. The inlet temperature was about 185 ° C, the outlet temperature was about 95 ° C. The atomization was carried out using a two-component nozzle at a pressure of 2 bar. The water content was carried out according to the Karl Fischer method. The powdery product obtained had the following specification (data in% by weight):

Water content: 1.4% D-pantothenate: 51% ammonium ions: 0.01% potassium ions: 0.012% sodium ions: 0.01% calcium ions: 7.7%

Example 8:

Analogously to Example 4, a fermentation broth with a pantothenic acid concentration of 22 g / L was obtained in a further fermentation with strain PA668-2A.

1000 ml of the centrifugate were made up to 70 g / l with sodium pantothenate and conveyed through a bed (volume about 1 liter) of the ion exchanger Amberlite 200 (in the H ⁺ form) and rinsed with water.

After a preliminary run of 500 ml, 2000 ml of the eluate were collected. The content of D-pantothenic acid was 32.6 g / l. The following ion concentrations were present in the eluate: NH ⁺ : 0.01%, Na ⁺ : 0.0062%, K ⁺ : 0.00045%, PO ₄ ^{3 "} : 0.06%, SO ₄ ^2" : 0.06% and CI ^" : 0.02%. For regeneration, the ion exchanger column was first rinsed with 1000 ml of 1N sodium hydroxide solution and 2500 ml of water. The ion exchanger was then regenerated with 1000 ml of 10% hydrochloric acid and the column was then rinsed neutral with 2500 ml of water.

With stirring, the free pantothenic acid was neutralized with solid calcium hydroxide, ie a pH of about 7 was set. About 2000 g of an aqueous calcium D-pantothenate suspension with a pantothenate content of 27.35 g / l were obtained.

Water content: 1.7% D-pantothenate: 64.6% ammonium ions: 0.08% potassium ions: 0.006% sodium ions: 0.086% calcium ions: 6.5%

Example 9:

Analogously to Example 4, a fermentation broth was obtained in a 40-hour fermentation with strain PA668-2A. This was freed from biomass by centrifugation and had a pantothenic acid concentration of 42.9 g / L. 1000 ml of this unsterilized fermenter broth were conveyed through a bed (volume about 1.0 liter) of the ion exchanger S1468 (in the H ⁺ form) and rinsed with water. The flow was regulated to approx. 20 ml / min.

After a preliminary run of 500 ml, 2100 ml of the eluate were collected. The content of D-pantothenic acid was 19.2 g / l. For regeneration, the ion exchanger column was first rinsed with 1000 ml of 1N sodium hydroxide solution and 2500 ml of water. The ion exchanger was then regenerated with 1000 ml of 10% hydrochloric acid and the column was then rinsed neutral with 2500 ml of water.

With stirring, the free pantothenic acid was neutralized with solid calcium hydroxide, ie a pH of about 7 was set. About 2000 g of an aqueous calcium D-pantothenate suspension were obtained, which was sterilized for 60 min. Successful killing was demonstrated by plating. This aqueous calcium D-pantothenate suspension was dried in a laboratory spray dryer from Niro, type Minor. The inlet temperature was about 200 ° C, the outlet temperature 85 - 90 ° C. The atomization was carried out using a two-component nozzle at a pressure of 4 bar. The water content was carried out according to the Karl Fischer method. The powdery product had the following specification (data in% by weight):

Water content: 2.6% D-pantothenate: 8.5% ammonium ions: 0.01% potassium ions: 0.003% sodium ions: 0.016% calcium ions: 0.61%

Example 10:

895 ml of sodium pantothenate solution containing 158.6 g / l pantothenate were added to 2305 ml of the centrifugate obtained from Example 4 and having a D-pantothenic acid content of 35.8 g / l. 1000 ml of this broth were conveyed through a bed (volume 1 liter) of the ion exchanger Lewatit S100 G1 (in the H ⁺ form) and rinsed with water. The flow rate was approx. 20 ml / min.

After the forerun of 500 ml, which was discarded, 2 l of the eluate were collected. The D-pantothenate content of the eluate was 25.3 g / l. 1 equivalent of calcium ions in the form of calcium chloride based on pantothenic acid were added.

Water content: 2.1% D-pantothenate: 50% ammonium ions: 0.01% potassium ions: 0.015% sodium ions: 0.01% calcium ions: 8.0%

Claims

Expectations:

1. A process for the preparation of D-pantothenic acid and / or its salts, characterized in that a) at least one organism producing D-pantothenic acid is used, the pantothenic acid (pan) and / or isoleucine / valine (ilv) biosynthesis deregulated and which forms at least 2 g / l of salts of D-pantothenic acid by fermentation in a culture medium, 0-20 g / l of free β-alanine and / or β-alanine salt being added to the culture medium, b) the D -Pantothenate-containing fermentation solution is passed over a cation exchanger, free D-pantothenic acid resulting from the salts of D-pantothenic acid, c) the solution containing free D-pantothenic acid by adding a calcium and / or magnesium base to a pH of 3 -10 is set, whereby a solution is obtained which contains calcium and / or magnesium pantothenate and d) the solution containing a calcium and / or magnesium pantothenate of a drying and / or formulation below is brought up.

2. The method according to claim 1, characterized in that no free ß-alanine and / or ß-alanine salt is fed to the culture medium.

3. The method according to any one of claims 1 or 2, characterized in that a bacterium, a yeast or a fungus is used as the organism producing D-pantothenic acid.

4. The method according to any one of claims 1 to 3, characterized in that a bacterium from the Bacillaceae family is used as the microorganism.

5. The method according to any one of claims 1 to 4, characterized in that a bacterium of the genus Bacillus and preferably of the type B. subtils, B. licheniformis or B. amyloliquefaciens is used.

6. The method according to any one of claims 1 to 5, characterized in that in step a) a content of D-pantothenic acid and / or its salts of at least 10 g / l culture medium, preferably at least 20 g / l, particularly preferably at least 40 g / l and most preferably at least 60 g / l culture medium.

7. The method according to any one of claims 1 to 6, characterized in that in step b) the content of monovalent cations, preferably ammonium, potassium and / or sodium ions, is reduced to a concentration of <1g / kg solution.

8. The method according to any one of claims 1 to 7, characterized in that in step c) the pH of the solution is adjusted to a value of 5-10.

9. The method according to any one of claims 1 to 8, characterized in that in step c) the pH of the solution is adjusted to a value of 5-9, preferably 6-9 and particularly preferably 6-8.

10. The method according to any one of claims 1 to 9, characterized in that the solution in step c) for neutralization calcium hydroxide, calcium carbonate,

Calcium oxide, magnesium hydroxide and / or basic magnesium carbonate is added in the form of a solid and / or as an aqueous suspension.

11. The method according to any one of claims 1 to 10, characterized in that the solution in step c) comprises an aqueous suspension containing 2-55% by weight, preferably 10-50% by weight and particularly preferably 20-40% by weight. % of calcium hydroxide is added.

12. The method according to any one of claims 1 to 11, characterized in that the solution in step c) an aqueous suspension containing 2-65 wt .-%, preferably 10-50 wt .-% and particularly preferably 20-40 wt .-% % of calcium carbonate is added.

13. The method according to any one of claims 1 to 12, characterized in that the solution in step c) an aqueous suspension containing 2-60 wt .-%, preferably 10-50 wt .-% and particularly preferably 20-40 wt .-% % of magnesium hydroxide is added.

14. The method according to any one of claims 1 to 13, characterized in that the solution in step c) is added an aqueous suspension containing 2-25 wt .-%, preferably 10-20 wt .-% of basic magnesium carbonate.

15. The method according to any one of claims 1 to 14, characterized in that in step c) an acidic inorganic and / or organic calcium and / or magnesium salt is added to the solution containing free D-pantothenic acid.

16. The method according to any one of claims 1 to 15, characterized in that in step c) a corresponding halide is used as the acidic inorganic calcium and / or magnesium salt.

17. The method according to any one of claims 1 to 16, characterized in that in step c) CaCl ₂ and / or MgCl _{2 is} used as the acidic inorganic calcium and / or magnesium salt.

18. The method according to any one of claims 1 to 17, characterized in that in step c) as the acidic organic calcium and / or magnesium salt calcium and / or magnesium formate, acetate, propionate, glycinate and / or -Lactate is used.

19. The method according to any one of claims 1 to 18, characterized in that in step c) or d) a suspension is obtained or presented which contains calcium and / or magnesium pantothenate.

20. Composition for use as an animal feed additive and / or animal feed supplement, characterized in that it can be produced by a) at least one organism producing D-pantothenic acid is used, the pantothenic acid (pan) and / or isoleucintvaline (ilv) biosynthesis is deregulated and which forms at least 2 g / l of salts of D-pantothenic acid by fermentation in a culture medium , wherein the culture medium 0-20 g / l, preferably 0 g / l free ß-alanine and / or ß-

Alanine salt is supplied, b) the fermentation solution containing D-pantothenate is passed over a cation exchanger, free D-pantothenic acid being formed from the salts of D-pantothenic acid, c) the solution containing free D-pantothenic acid by the addition of a

Calcium and / or magnesium base is adjusted to a pH of 3-10, whereby a solution is obtained which contains calcium and / or magnesium pantothenate and d) the drying solution containing calcium and / or magnesium pantothenate and / or formulation is subjected.

2 I .Composition according to claim 20, characterized in that in step c) an acidic inorganic and / or organic calcium and / or

Magnesium salt is added, whereby a solution is obtained which is a polyvalent salt of pantothenic acid, preferably calcium and / or

Magnesium pantothenate, contains.

22. Composition according to one of claims 20 or 21, characterized in that in step c) a calcium and / or halide is added as the acidic inorganic calcium and / or magnesium salt.

23. Composition according to one of claims 20 to 22, characterized in that in step c) CaCl ₂ and / or MgCl _{2 is} used as the acidic inorganic calcium and / or magnesium salt.

24. The composition according to any one of claims 20 to 23, characterized in that in step c) as acidic organic calcium and / or magnesium salt calcium and / or magnesium formate, acetate, propionate, glycinate and / or -Lactate is used.

25. Composition according to one of claims 20 to 24, characterized in that a suspension is obtained or is present in step c) or d) which contains calcium and / or magnesium pantothenate.

26. The composition according to any one of claims 20 to 25, characterized in that it contains salts of D-pantothenic acid in the form of divalent cations, preferably calcium and / or magnesium D-pantothenate.

27. The composition according to any one of claims 20 to 26, characterized in that it contains salts of D-pantothenic acid in a concentration of at least 1-100% by weight, preferably at least 20-100% by weight and particularly preferably at least 50% by weight. -% contains.

28. Composition according to one of claims 20 to 27, characterized in that the content of salts of D-pantothenic acid in the form of monovalent cations is <1 g / kg.