EP1318725A2 - Tierfuttersupplement enthaltend d-pantothensäure und/oder deren salze, verbessertes verfahren zu dessen herstellung sowie dessen verwendung - Google Patents

Tierfuttersupplement enthaltend d-pantothensäure und/oder deren salze, verbessertes verfahren zu dessen herstellung sowie dessen verwendung

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Publication number
EP1318725A2
EP1318725A2 EP01985230A EP01985230A EP1318725A2 EP 1318725 A2 EP1318725 A2 EP 1318725A2 EP 01985230 A EP01985230 A EP 01985230A EP 01985230 A EP01985230 A EP 01985230A EP 1318725 A2 EP1318725 A2 EP 1318725A2
Authority
EP
European Patent Office
Prior art keywords
pantothenic acid
animal feed
feed supplement
fermentation
salts
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01985230A
Other languages
German (de)
English (en)
French (fr)
Inventor
Jürgen Müller
Knut Eichler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Original Assignee
BASF SE
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE10112207A external-priority patent/DE10112207A1/de
Application filed by BASF SE filed Critical BASF SE
Publication of EP1318725A2 publication Critical patent/EP1318725A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/174—Vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K40/00—Shaping or working-up of animal feeding-stuffs
    • A23K40/10—Shaping or working-up of animal feeding-stuffs by agglomeration; by granulation, e.g. making powders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S426/00—Food or edible material: processes, compositions, and products
    • Y10S426/807—Poultry or ruminant feed

Definitions

  • the present invention relates to an animal feed supplement containing D-pantothenic acid, an improved process for its production and its use.
  • D-pantothenate As a starting product in 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 often described for both plants and animals. The availability of D-pantothenate is therefore of great economic interest, especially in the animal feed industry.
  • GB 598,177 describes the production of pantothenic acid in the per mille range as a by-product of the production of 2,3-butylene glycol by Aerobacter aerogenes. Concentration of pantothenic acid is only possible through adsorption on charcoal and subsequent elution.
  • EP 1 006 189 a process for the preparation of pantothenate in which a content of max. 1 g / l D-pantothenic acid is reached.
  • a content of max. 1 g / l D-pantothenic acid is reached.
  • 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.
  • No. 6,013,492 describes the working up of D-pantothenic acid from the fermentation solution by filtering off insoluble constituents, such as, for. B. cell material from the culture medium, an adsorption of the filtrate on activated carbon, a subsequent elution of D-pantothenic acid with an organic solvent, preferably methanol, a neutralization with calcium hydroxide and a final crystallization of calcium D-pantothenic acid.
  • an organic solvent preferably methanol
  • a neutralization with calcium hydroxide preferably calcium hydroxide
  • DE 100 16 321 A1 discloses a process for the production of animal feed additives which are produced by fermentation of microorganisms which produce D-pantothenic acid.
  • this process requires the addition of hydroxides or oxides of the alkali or alkaline earth metals after the fermentation.
  • the object of the present invention is therefore to provide a free D-pantothenic acid and / or salts thereof
  • the invention relates to a method for producing a free animal feed supplement containing D-pantothenic acid and / or its salts, wherein
  • a D-pantothenic acid-producing organism in a culture medium containing at least one carbon and one nitrogen source is fermented without the addition of further precursors and b) the fermentation solution containing D-pantothenic acid and / or its salts is subjected to drying and / or formulation without carrying out further work-up steps.
  • the process according to the invention is further characterized in that the fermentation is carried out until a solids content of at least 6% by weight, preferably 7-25% by weight and / or a content of D-pantothenic acid of at least 2-15 % By weight, preferably 4-15% by weight, is achieved
  • the fermentation can be carried out according to known procedures in batch, fed-batch or repeated fed-batch operation or with continuous process control.
  • the solids content includes the dried fermentation solution containing, inter alia, understand dried biomass, minerals and D-pantothenate and / or their salts.
  • a sample of the fermentation solution is taken sterile and dried, for example, in a vacuum drying cabinet at 120 ° C. for 12 hours.
  • a particular advantage of the method according to the invention over the prior art is that the fermentation solution does not have to be subjected to further, complex workup, such as. B. adsorptive processes over activated carbon to deliver a product that meets the requirements for an animal feed supplement of the desired type.
  • These requirements are, for example, a relatively high content of D-. Pantothenic acid and a 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, which corresponds to the value of the chemically synthesized D-pantothenic acid.
  • the degree of purity of D-pantothenic acid as such is to be regarded as irrelevant here, since in most cases it is incorporated into feed mixtures for animal nutrition.
  • Another advantage of the method according to the invention is the saving of time-consuming processing steps of the fermentation solution for the production of a product with good biological value.
  • the process according to the invention is characterized in that the desired valuable substance is made available entirely without the use of organic solvents.
  • the amount of waste water obtained is reduced significantly. This results in further savings on complex processing and disposal systems.
  • 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.
  • 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 requirements.
  • the amount of D-pantothenic acid and / or their salts do not pre-cell internally, but ideally completely released from the organism into the culture medium. This discharge can take place actively or passively according to mechanisms known per se.
  • microorganisms are used as the organisms producing D-pantothenic acid.
  • these include fungi, yeasts and / or bacteria.
  • fungi such as, for example, Mucor or yeasts, such as. B. Saccharomyces or Debaromyces and preferably Saccharaomyces cerevisiae.
  • coryneform bacteria or Bacillaceae are advantageously used.
  • 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.
  • 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.
  • genetically modified organisms can be isolated, for example, by chemical mutagenesis and subsequent selection using a suitable “screening method”.
  • so-called production strains are also included which are used to produce the product in the sense of the present invention are suitable and have genetic changes with regard to the metabolic flow in the direction of D-pantothenic acid, which also includes changes with regard to the discharge of D-pantothenic acid and / or its salts across the cell membrane.
  • 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.
  • overexpression and / or deregulation of one or more genes individually and / or in combination, localized in the genome and / or on a vector is 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.
  • other metabolic pathways such as. B.
  • the isoleucine-valine biosynthetic pathway can advantageously be manipulated in the organisms, as described for example in EP 1 006 189, EP 1 006 192, EP 1 006 193 or EP 1 001 027.
  • branched-chain precursors of pantothenic acid biosynthesis are increasingly being made available.
  • the genes for this biosynthetic pathway ie ilvB, ilvN, ilvC and / or ilvD, are advantageously overexpressed.
  • genetic changes in the aspartate- ⁇ -decarboxylase (panD) for example by overexpression and / or deregulation, in the D-pantothenic acid-producing organism used are included according to the invention.
  • ⁇ -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 described, for example, in EP-A-0 590 857.
  • Microorganisms whose Pantothenic acid (pan) and / or isoleucine valine (ilv) biosynthesis and / or aspartic ⁇ -decarboxylase (panD) is deregulated.
  • ketopanthoat reductase panE
  • coaA gene which is required for the synthesis of coenzyme A
  • coaX the activity of this gene coaX or of the corresponding enzyme
  • coaX the activity of this gene coaX or of the corresponding enzyme
  • coaX the activity of this gene coaX or of the corresponding enzyme
  • coaX the activity of this gene coaX or of the corresponding enzyme can also be changed, preferably reduced, or even deleted, provided that coaA itself still has sufficient, albeit reduced, enzyme activity, i.e. the enzyme activity of coaA has not been completely lost.
  • 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.
  • the bacterial strains described in accordance with the appendix such as PA 668 and / or derivatives thereof.
  • the microorganism Bacillus subtilis PA 377 as described in accordance with the appendix (PCT / US application 0025993) is used in the method according to the invention. This strain Bacillus subtilis PA 377 was produced as follows:
  • the strain PY79 was generated by transduction of the Trp + marker (from the Bacillus subtilis wild type W23) classic genetic engineering methods (e.g. in Harwood, CR and Cutting, SM (editors), Molecular Biological Methods for Bacillus (1990) John Wiley & Sons, Ltd., Chichester, England) ⁇ panB and ⁇ panEI mutations were introduced.
  • the resulting strain was transformed with genomic DNA from Bacillus subtilis strain PA221 (genotype P 2 anBCD, trpC2 (Trp " )) and genomic DNA from Bacillus subtilis strain PA303 (genotype P 2 ⁇ anE1).
  • the resulting strain PA327 has the genotype P ⁇ anBCD, P 26 panE1 and is tryptophan auxotroph (Trp " ).
  • 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 ⁇ -ketoisovalerate, pantothenic acid Titer of up to 3.0 g / L (24 h) reached.
  • 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
  • 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.
  • the E. coli strain BM4062 (bir ts ) and the information that the Bacillus operon is close to the birA gene was used for cloning.
  • the panBCD operon was introduced into a plasmid replicable in E. coli.
  • 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 the native panBCD operon was replaced by the p 26 panBCD operon by homologous recombination
  • the resulting strain is called PA221 and has the genotype P 2 ⁇ anBCD, trpC2 (Trp " ).
  • Bacillus subtilis strain PA221 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 0.92 g / L (24 h ) reached.
  • the Bacillus panE sequence was analogized using the E. coli panE gene sequence. It was found that two homologs of the panE gene from E. coli exist in B. subtilis, which were designated panEl and panE2. Deletion analyzes showed that the panEl gene is responsible for 90% of pantothenic acid production, while the deletion of the panE2 gene had no significant effect on pantothenic acid production.
  • the promoter was replaced by the strong constitutive promoter P 26 and the ribosome binding site in front of the panEl gene was replaced by the artificial binding site.
  • the P ⁇ anE1 fragment was cloned into a vector that was designed so that the P 2 ⁇ anE1 fragment could integrate into the original panEl locus in the Bacillus subtilis genome.
  • the strain resulting after transformation and homologous recombination is called PA303 and has the genotype P & panE1.
  • 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 QUVBNC operon and the marker gene for spectinomycin.
  • the P 2 QUVBNC operon integrated into the amyE locus, which was verified by PCR.
  • One transformant was designated PA340 (genotype P 2 ⁇ anBCD, P 2 ⁇ anE1, P 26 ilvBNC, specR, trpC2 (Trp " )).
  • 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 24 ml Cultures with SVY medium supplemented with 5 g / L ß-alanine and 5 g / L ⁇ -ketoisovalerate achieved pantothenic acid titres of up to 4.1 g / L (24 h).
  • a deregulated // vD cassette was introduced into the strain PA340.
  • 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 QHVD gene was integrated into the original ilvD locus by homologous recombination.
  • the resulting strain PA374 has the genotype P 2 ⁇ anBCD, P 2 ⁇ anE1, P 26 ilvBNC, P Q ⁇ IVD, specR and trpC2 (Trp " ).
  • Bacillus subtilis strain PA374 was used in 10 mL cultures with SVY medium which was only available with 5 g / L ß-alanine was supplemented, pantothenic acid titer of up to 2.99 g / L (24 h) was reached.
  • 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 Bacillus subtilis panD gene was cloned from the panBCD operon into a vector carrying the tetracycline marker gene.
  • the promoter P2 6 and an artificial RBS described above were cloned in front of the panD gene.
  • a fragment containing the tetracycline marker gene and the P 2 ⁇ 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 2 ⁇ anD, tetR and trpC2 (Trp " ).
  • pantothenic acid titer of up to 0.3 g / L (24 h) was achieved in 10 mL cultures in SVY medium supplemented with 5 g / L ⁇ -ketoisovalerate.
  • pantothenic acid titers of up to 2.2 g / L (24 h) were achieved.
  • trunks can be found in the appendix of PCT / US application 0025993.
  • 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 ) 2S0 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 * 7 H 2 O and 1 ml / L a trace salt solution of the following composition: 0.15 g Na2Mo0 4 x 2 H 2 0, 2.5 g H3BO3, 0.7 g C0CI 2 x 6 H 2 O , 0.25 g CuSO 4 x 5 H 2 0.1.6 g MnCI 2 x 4 H 2 O, 0.3 g ZnSO
  • 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. That the biosynthesis of D-pantothenic acid is independent of the feeding of further precursors.
  • such precursors include substances such as to understand ⁇ -alanine and / or L-aspartate and / or L-valine and / or ⁇ -ketoisovalerate and / or their combinations.
  • the fermentation of the D-pantothenic acid-producing organism is carried out in a culture medium which contains at least one carbon and one nitrogen source as a precursor, but no ⁇ -alanine added to the medium.
  • carbon sources suitable according to the invention for use in a culture medium for fermentation of the organisms mentioned above are sugars, such as starch hydrolysates (mono-, di-, oligosaccharides), preferably glucose or sucrose, and beet or cane sugar molasses, proteins, protein hydrolyzates, soy flour, corn steep liquor, fats, free fatty acids, returned cells from already carried out fermentations or their hydrolysates and yeast extract.
  • starch hydrolysates mono-, di-, oligosaccharides
  • proteins protein hydrolyzates
  • soy flour soy flour
  • corn steep liquor fats
  • free fatty acids returned cells from already carried out fermentations or their hydrolysates and yeast extract.
  • suitable nitrogen sources such as ammonia, ammonium sulfate, urea, proteins, protein hydrolyzates or yeast extract.
  • the fermentation medium also contains mineral salts and / or trace elements such as amino acids and vitamins.
  • compositions of suitable fermentation media are widely known and accessible to the person skilled in the art.
  • the fermentation medium 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 optionally cultivated with the addition of an anti-foaming agent.
  • the fermentation is carried out according to the invention in such a way that, when it has ended, it has at least a solids content of the dried fermentation solution of at least 6% by weight and a free D-pantothenic acid content of at least 2% by weight, preferably at least 4% by weight.
  • the fermentation can be carried out in batch, fed-batch or repeated fed-batch operation with metering in of the carbon source or can be operated continuously.
  • the fermentation temperature is 10-70 ° C, preferably 20-50 ° C.
  • the fermenter is gassed with oxygen, air or mixtures with nitrogen or other inert gases.
  • the pH is adjusted to a value in the range from 4 to 8, preferably 5 to 7.5, and is regulated if necessary by adding suitable bases and / or acids.
  • 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 used up (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).
  • 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 (PO2) is reached.
  • the carbon source e.g. sugar solution
  • the fermentation solution can be subjected to drying and / or formulation without carrying out further work-up steps. That is, elaborate work-up steps for isolating the desired product containing D-pantothenic acid from the fermentation solution, such as. B. adsorptive purifications over activated carbon are not required.
  • a separation of the biomass from the fermentation solution is also not absolutely necessary, so that the protein content of the product according to the invention, i.e. of the animal feed supplement containing D-pantothenic acid can have a protein content of up to 50% by weight.
  • the drying and / or formulation of the fermentation solution takes place according to known methods such as spray drying, spray granulation, fluid 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
  • Gas outlet temperature is 50-180 ° C, preferably 60-150 ° C.
  • Fine particles can be separated and recycled to set a desired particle size distribution and the associated product properties.
  • Coarse material can also be ground in a mill and then also returned.
  • the product according to the invention has, for example, a beige to brown color.
  • FIG. 1 A schematic block flow diagram of the aforementioned method is summarized in FIG. 1.
  • the biomass may be separated from the fermentation solution before the fermentation solution is dried and / or formulated. This separation can take place almost completely or only partially. Partial separation of the biomass is preferred, as a result of which the protein content can be reduced to below 10% by weight.
  • the solids can be separated from the aqueous liquid, for example by centrifugation. Based on the dried end product, a protein content of less than 5% by weight can even be set in a further variant of the invention.
  • the separated biomass can be used in an advantageous manner to compensate for the natural fluctuations in the D-pantothenic acid content in the tolerance ranges
  • the fermentation solution can be concentrated to increase the solids content containing D-pantothenic acid and / or salts thereof before the drying and / or formulation of the fermentation solution and, if appropriate, after separation of the biomass.
  • This can be achieved, for example, by removing water by means of evaporation, which, if necessary for cost reasons, can be carried out in several stages and can also be carried out in a vacuum in addition to normal pressure to protect the product.
  • Another option is to use a membrane process. For example, methods such as nanofiltration and / or reverse osmosis can be used here.
  • the concentration can be up to a D-pantothenic acid content of 20 to 50 wt .-%.
  • the water can be returned to the fermentation process at the same time. As a result, the amount of waste water obtained is advantageously reduced, as a result of which the outlay for waste water treatment is substantially reduced. This is shown schematically in FIG. 2.
  • FIG. 3 An illustration in the block flow diagram is shown in FIG. 3.
  • the biomass or a part thereof can be used to set a constant valuable substance content in the product in the process according to the invention after the fermentation has ended.
  • B. by separation, centrifugation, ultra, micro or depth filtration or combinations.
  • the biomass obtained in this way can in turn again be further dehumidified using a decanter.
  • the clear running of the decanter is then returned to the inlet of the separator.
  • the content of D-pantothenic acid in the product can be increased or the content can be set to a constant value by mixing different fractions with one another, so that even fluctuating contents from the fermentation can be processed without problems.
  • the fermentation solution can then be further concentrated.
  • the contents based on the free D-pantothenic acid and / or its salts are 20-95% by weight, preferably 30-90% by weight.
  • the resulting product particularly preferably has a high content of free D-pantothenic acid and / or its salts of 60-80% by weight and in particular more than 80% by weight.
  • At least one of the following steps can be carried out before the fermentation solution is dried and / or formulated
  • 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 means of a temperature treatment, preferably at 80-200 ° C. and / or an acid treatment, preferably using sulfuric acid or hydrochloric acid and / or enzymatically, preferably with lysozyme.
  • a block flow diagram for clarification is shown in FIG. 4.
  • a further embodiment of the method according to the invention describes a procedure in which the fermentation solution prior to concentration and / or before drying and / or formulation further additives and / or mixtures thereof to adjust a uniform content of D-pantothenic acid and / or to improve the product properties how dust behavior, flow properties, water absorption and storage stability are added.
  • additives and / or mixtures thereof can be based on sugars e.g. B. lactose or maltodextrin, based on cereals or legumes such.
  • a calcium D-pantothenate is produced by in a late step of the method according to the invention, ie preferably before and / or during the working up of the fermentation solution, ie before and / or during the concentration and / or drying and / or formulation of the fermentation solution (see FIG. 5), calcium salts are added.
  • the calcium ion content is adjusted by adding calcium salts so that about 1 mol of calcium salt per 2 mol of D-pantothenic acid are contained in the formulated end product.
  • the calcium ion content which is already present in the fermentation solution can advantageously be taken into account.
  • calcium salts can For example, calcium oxide, calcium hydroxide, calcium hydrogen phosphate, calcium carbonate, calcium sulfate, calcium chloride and / or another calcium salt can be used.
  • the present invention thus also relates to a process in which, based on the content of D-pantothenic acid in the formulated product before and / or during the concentration, drying and / or formulation, 1 mol of calcium ions per 2 mol of D-pantothenic acid in the form of a calcium salt as an additive be added.
  • a product in another variant of the invention by the composition of the fermentation medium and here in particular the selection of the mineral salts with a special cation, a product can be produced which increasingly contains a selected salt of D-pantothenic acid.
  • a product can be produced during the fermentation that largely contains potassium D-pantothenic acid.
  • Possible salts are e.g. Calcium, potassium, magnesium, sodium or ammonium salts of D-pantothenic acid or any mixtures thereof.
  • a block flow diagram is shown in FIG. 6.
  • the present invention furthermore relates to an animal feed supplement produced by one of the processes described above on the basis of a fermentation solution obtained by fermentation of at least one D-pantothenic acid-producing organism containing at least free D-pantothenic acid and / or its salts in a concentration of at least 30-95% by weight .-%, a total Sugar content of 0.1 - 15% by weight and a protein content of less than 5 to 50% by weight based on the dry matter.
  • the animal feed supplement according to the invention is characterized in that it contains 50-95% by weight, preferably 70-95% by weight, particularly preferably 60-80% by weight and in particular more than 80% by weight of free D-pantothenic acid and / or their salts.
  • the untreated fermentation solution contains at least 10 g / l, preferably at least 20 g / l and particularly preferably at least 40 g / l of D-pantothenate and / or salts thereof as the basis for the animal feed supplement according to the invention.
  • animal feed supplement of the present invention can contain calcium, potassium, magnesium, sodium and / or ammonium salts of D-pantothenic acid and / or mixtures thereof.
  • a special variant of the animal feed supplement according to the invention is characterized by a composition of the dry matter with at least the following components: a) free D-pantothenic acid and / or its salts at least 30-95% by weight b) proteins max. 50% by weight c) total sugar max. 15% by weight d) minerals max. 20% by weight
  • the animal feed supplement can contain a protein content of at most 50% by weight as the upper limit and as the lower limit less than 10% by weight, preferably less than 7% by weight and particularly preferably less than 5% by weight.
  • the total sugar content of the animal feed supplement is a maximum of about 15% by weight and can be as lower limit less than about 0.1 wt .-% contain, all intermediate stages are conceivable.
  • the product containing D-pantothenic acid according to the invention is characterized by a residual water content of less than 5% by weight, preferably 1-3% by weight and particularly preferably 0.5-2% by weight.
  • the present invention furthermore relates to an animal feed supplement containing inanimate, living and / or reproductive fractions of organisms producing D-pantothenic acid.
  • These are preferably microorganisms, preferably fungi, yeasts and / or bacteria.
  • the animal feed supplement according to the invention particularly preferably contains inanimate, living and / or reproductive portions of fungi of the genus Mucor, yeasts of the genus Saccharomyces and / or bacteria of the Enterobacteriaceae, such as E.
  • Bacillae such as Bacillus subtilis or Bacillus cereus, coryneform bacteria, such as Corynebacterium glutamicum or Brevibacterium breve and / or Actinum mycetalis and / or mixtures thereof. Bacteria of the genus Bacillus and here of the species Bacillus subtilis are very particularly preferred. Genetically modified and / or transgenic organisms and / or production strains suitable for the production of animal feed supplements are also included according to the invention. The preceding list is not limiting for the present invention.
  • an animal feed supplement which contains further additives, preferably based on sugars and / or
  • D- Contains pantothenic acid and / or its salts and / or mixtures thereof.
  • the animal feed supplement according to the invention is further characterized by a formulation with a bulk density of 0.35 to 0.7 kg / 1, preferably 0.4 to 0.6 kg / 1. According to the invention, it has an average grain diameter in the range from 10 to 2000 ⁇ m, preferably from
  • the animal feed supplement according to the invention can be in the form of powder, granules, pellet, provided with a coating ("coated") and / or as combinations thereof.
  • the formulation of the invention
  • Animal feed supplements for example by wrapping compounds, are used, for. B. to improve the product properties, such as dust behavior, flow properties, water absorption and
  • the present invention furthermore relates to the use of the animal feed supplement with the properties described above as an additive to animal feed and / or animal feed supplements.
  • aqueous fermentation medium with the following composition was placed: Yeast extract 20 g / ⁇
  • the trace salt solution is composed as follows:
  • the trace salt solution is added via sterile filtration.
  • the initial liquid volume is 6 I.
  • the contents listed above are based on this value.
  • the pH at 7.2 was fed in by adding ammonia into the fermenter or from
  • Phosphoric acid kept. Ammonia also serves as a nitrogen source for the fermentation.
  • the speed of the agitator was controlled by keeping the dissolved oxygen content at 30% of the saturation value. After the addition of the carbon source had been stopped, the fermentation was continued until the dissolved oxygen content (PO 2 ) reached a value of 95% of the saturation value. After that the
  • Fermentation ended and the organism thermally killed.
  • the fermentation solution was kept at 100 ° C. for 1 h.
  • the killing was verified by plating.
  • the concentration of D-pantothenic acid at termination after 72 h was 28 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.
  • Potassium hydrogen phosphate Potassium dihydrogen phosphate buffer in the fermentation is adjusted so that the potassium salt of D-pantothenic acid, ie potassium D-pantothenate, is largely obtained.
  • Example 2 Cell separation and drying of D-pantothenic acid-containing fermentation solution from E. coli
  • D-pantothenic acid-containing fermentation solution is produced in accordance with Example 1 of US Pat. No. 6,013,492 with Escherichia coli IFO 814 / pFV 31.
  • the fermentation is then continued until the carbon source has been completely consumed until the dissolved oxygen content (PO 2 ) has reached over 80 % has increased.
  • the cells are then separated using a separator.
  • the D-pantothenic acid content here is 38.5 g / l.
  • the concentrate is dried in a laboratory spray dryer under the following conditions:
  • a free-flowing product with an average particle diameter of 20-300 ⁇ m is obtained.
  • Fermentation solution (1 l) from example 1 is dried in a laboratory spray dryer under the following conditions:
  • Gas inlet temperature 100 - 250 ° C
  • Gas outlet temperature 60 - 150 ° C
  • a free-flowing product with an average particle diameter of 20-300 ⁇ m is obtained.
  • Example 4 Cell separation and drying of fermentation solution containing D-pantothenic acid with lactose as additive
  • the biomass from fermentation solution (1 l) from example 1 is centrifuged off in a centrifuge.
  • the supernatant is mixed with 30 g lactose and dried in a laboratory spray dryer under the following conditions:
  • a free-flowing product with a particle diameter of 40-500 ⁇ m and a content of free D-pantothenic acid> 30% by weight is obtained.
  • Fermentation solution with chemically produced calcium D-pantothenate as an additive e.g. for setting a fixed concentration of D-pantothenic acid in the end product
  • the biomass from fermentation solution (1 l) from example 1 is centrifuged off in a centrifuge.
  • the supernatant is mixed with 100 g of chemically produced calcium D-pantothenate and dried in a laboratory spray dryer under the following conditions:
  • Gas inlet temperature 100 - 250 ° C
  • Gas outlet temperature 60 - 150 ° C
  • a free-flowing product with a particle diameter of 40-500 ⁇ m and a content of free D-pantothenic acid> 60% by weight is obtained.
  • Example 6 Adjustment of the calcium content in formulations of D-pantothenic acid from fermentation solutions
  • a fermentation solution containing D-pantothenic acid contains a solids content of 95 g / l, of which 70 g / l D-pantothenic acid and 25 g / l other solids (salts, residues of the biomass, other solid components depending on the fermentation medium, none calcium ions).
  • the resulting contents of D-pantothenic acid in the formulated product when various calcium salts are added are given below.
  • the calcium content was adjusted so that 1 mol of calcium ions are contained per 2 mol of D-pantothenic acid.
  • Drying additives are added.
  • Fig. 6 Block flow diagram of a method for producing a D-pantothenic acid salt in which the desired cation by the selection of the Fermentation medium used salts is reached.

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EP01985230A 2000-09-20 2001-09-08 Tierfuttersupplement enthaltend d-pantothensäure und/oder deren salze, verbessertes verfahren zu dessen herstellung sowie dessen verwendung Withdrawn EP1318725A2 (de)

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DE10046490 2000-09-20
DE10046490 2000-09-20
DE10112207 2001-03-14
DE10112207A DE10112207A1 (de) 2001-03-14 2001-03-14 Tierfuttersupplement enthaltend D-Pantothensäure und/oder deren Salze, verbessertes Verfahren zu dessen Herstellung sowie dessen Verwendung
PCT/EP2001/010386 WO2002024001A2 (de) 2000-09-20 2001-09-08 Tierfuttersupplement enthaltend d-pantothensäure und/oder deren salze, verbessertes verfahren zu dessen herstellung sowie dessen verwendung

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SK10422003A3 (sk) * 2001-02-21 2004-02-03 Basf Aktiengesellschaft Príprava kyseliny D-pantoténovej a/alebo jej solí ako prísad do krmív
DE10108225A1 (de) * 2001-02-21 2002-10-10 Basf Ag Verfahren zur Herstellung von D-Pantothensäure und/oder deren Salze als Zusatz zu Tierfuttermitteln
MXPA03007455A (es) * 2001-02-21 2004-07-30 Basf Ag Metodo y produccion de acido d-pantotenico y las sales de este como adyuvante en productos alimenticios para animales.
DE10108223A1 (de) 2001-02-21 2002-10-10 Basf Ag Verfahren zur Herstellung von D-Pantothensäure und/oder deren Salze als Zusatz zu Tierfuttermitteln
DE102004026152A1 (de) * 2004-05-28 2005-12-15 Basf Ag Fermentative Herstellung von Feinchemikalien
BRPI0924745B1 (pt) 2009-03-23 2021-05-11 Donaghys Limited composição e método para melhorar o crescimento ou desenvolvimento da planta
MX348659B (es) * 2010-11-19 2017-05-08 Centro De Innovacion Biotecnologica A C Alimentos funcionales con altos contenidos de harina de soya (sbm), almidon y bacillus ssp.
CN105166414A (zh) * 2015-10-10 2015-12-23 山西农业大学 肉牛过瘤胃泛酸添加剂及其制备方法
CN109161574A (zh) * 2018-09-18 2019-01-08 陕西科技大学 一种提高枯草芽孢杆菌抗菌蛋白产量的发酵方法

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JP3710497B2 (ja) * 1992-09-25 2005-10-26 ビーエーエスエフ アクチェンゲゼルシャフト Ｄ−パント酸、ｄ−パントテン酸およびそれらの塩の製造法
PT822989E (pt) * 1995-04-21 2001-07-31 Basf Ag Processo para a producao de d-pantotenato de calcio
US5932457A (en) * 1995-09-13 1999-08-03 Takeda Chemical Industries, Ltd Process for producing D-pantoic acid and D-pantothenic acid or salts thereof
DE19855312A1 (de) * 1998-12-01 2000-06-08 Degussa Verfahren zur fermentativen Herstellung von D-Pantothensäure unter Verwendung coryneformer Bakterien
US6238714B1 (en) * 1999-05-05 2001-05-29 Degussa-Huls Ag Feedstuff additive which contains D-pantothenic acid and/or its salts and a process for the preparation thereof
EP1050219B1 (de) * 1999-05-05 2002-11-20 Degussa AG D-Pantothensäure und/oder eines ihrer Salze enthaltende Futtermittel-Additive und Verfahren zu deren Herstellung
US6319528B1 (en) * 1999-05-05 2001-11-20 Degussa Aktiengesellschaft Feedstuff additive which contains D-pantothenic acid and/or its salts and a process for the preparation thereof

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NO20031261D0 (no) 2003-03-19
IL154824A0 (en) 2003-10-31
US20040050335A1 (en) 2004-03-18
JP2004508841A (ja) 2004-03-25
NO20031261L (no) 2003-03-19
HUP0301095A2 (hu) 2003-10-28
KR20030038751A (ko) 2003-05-16
BR0113399A (pt) 2003-07-15
CA2422817A1 (en) 2003-03-19
HUP0301095A3 (en) 2004-04-28
CZ2003797A3 (cs) 2003-06-18
AU2002218164A1 (en) 2002-04-02
SK3262003A3 (en) 2003-08-05
WO2002024001A2 (de) 2002-03-28
MXPA03002345A (es) 2003-06-30
CN1531398A (zh) 2004-09-22

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