U.S. patent application number 09/895318 was filed with the patent office on 2002-02-28 for pourable feed additives containing d- pantothenic acid and/or salts thereof, and process for their preparation.
This patent application is currently assigned to DEGUSSA AG. Invention is credited to Binder, Michael, Greissinger, Dieter, Moeller, Alexander, Moll, Matthias, Pfefferle, Walter.
Application Number | 20020025563 09/895318 |
Document ID | / |
Family ID | 7647659 |
Filed Date | 2002-02-28 |
United States Patent
Application |
20020025563 |
Kind Code |
A1 |
Binder, Michael ; et
al. |
February 28, 2002 |
Pourable feed additives containing D- pantothenic acid and/or salts
thereof, and process for their preparation
Abstract
A composition including at least one of D-pantothenic acid and a
salt of D-panthothenic acid, greater than 50 percent of non-biomass
constituents of a D-pantothenic fermentation liquor, and 0 to 100%
of the biomass from D-pantothenic fermentation liquor wherein
composition is a solid, pourable composition in the form of
particles.
Inventors: |
Binder, Michael;
(Steinhagen, DE) ; Moll, Matthias; (Hanau, DE)
; Greissinger, Dieter; (Niddatal, DE) ; Moeller,
Alexander; (Gelnhausen, DE) ; Pfefferle, Walter;
(Halle, DE) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
DEGUSSA AG
Duesseldorf
DE
|
Family ID: |
7647659 |
Appl. No.: |
09/895318 |
Filed: |
July 2, 2001 |
Current U.S.
Class: |
435/106 ;
562/569 |
Current CPC
Class: |
A23K 20/174 20160501;
Y10S 426/807 20130101; A23K 40/10 20160501 |
Class at
Publication: |
435/106 ;
562/569 |
International
Class: |
C12P 013/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 4, 2000 |
DE |
100 32 349.9 |
Claims
1. A composition comprising: at least one of D-pantothenic acid and
a salt of D-panthothenic acid; greater than 50 percent of further
non-biomass constituents of a D-pantothenic fermentation liquor;
and 0 to 100% of the biomass from said D-pantothenic fermentation
liquor, wherein said composition is a solid, pourable composition
in the form of particles.
2. The composition according to claim 1, wherein the particle size
of the additive is from 20 .mu.m to 2000 .mu.m, inclusive.
3. The composition according to claim 1, wherein the particle size
of the additive is from 50 .mu.m to 800 .mu.m, inclusive.
4. The composition according to claim 1, wherein the particle size
of said additive is from 150 .mu.m to 600 .mu.m, inclusive.
5. The composition according to claim 1, further comprising
chloride containing constituents in a concentration of <3 mg/g
of additive.
6. The composition according to claim 1, further comprising
chloride containing constituents in a concentration of <2 mg/g
of additive.
7. The composition according to claim 1, further comprising
chloride containing constituents in a concentration of <1.5 mg/g
of additive.
8. The composition according to claim 1, wherein the salt is at
least one s elected from the group consisting of sodium salt of
D-pantothenic acid, potassium part of D-pantothenic acid, ammonium
salt of D-pantothenic acid, magnesium salt of D-pantothenic acid
and calcium salt of D-pantothenic acid.
9. The composition according to claim 1, wherein the feed additive
contains from 20-80 wt. % D-pantothenic acid and/or a salt of
D-pantothenic acid.
10. The composition according to claim 9, wherein the chloride
content of the feed additive is <3.0 mg/g of additive.
11. The composition according to claim 9, wherein the chloride
content of the feed additive is <2.0 mg/g of additive.
12. The composition according to claim 9, wherein the chloride
content of the feed additive is <1.5 mg/g of additive.
13. The composition according to claim 1, wherein the feed additive
contains at least one L-amino acid selected from the group
consisting of L-methionine, L-lysine, L-valine, L-alanine,
L-threonine or L-tryptophan.
14. The composition according to claim 1, wherein said composition
is an additive for animal feed.
15. A process for preparing a composition containing D-pantothenic
acid and/or at least one salt of D-pantothenic acid, said process
comprising: fermenting D-pantothenic acid and/or salts of
D-pantothenic acid in a fermentation liquor; separating a biomass
from said fermentation liquor; drying the fermentation liquor; and
granulating said dried fermentation liquor to obtain particles
having a size distribution of from 20 .mu.m to 2000 .mu.m.
16. The process according to claim 15, further comprising
concentrating the fermentation liquor prior to granulation.
17. The process according to claim 15, further comprising adding at
least one from the group consisting of a hydroxide of an alkaline
earth metal, an oxide of an alkaline earth metal, a hydroxide of an
alkali metal and an oxide of an alkali metal to the fermentation
liquor prior to drying the liquor.
18. The process according to claim 17, wherein said fermentation
liquor is concentrated prior to granulation.
19. The process according to claim 17, wherein the oxide or the
hydroxide is added in a stoichiometric ratio of from 0.8 to 1.2
based on the D-pantothenic acid.
20. The process according to claim 17, wherein the oxide or the
hydroxide is added in a stoichiometric ratio of from 0.95 to 1.1
based on the D-pantothenic acid.
21. The process according to claim 16, further comprising adding
said particles to animal feed.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention relates to pourable animal feed additives
based on fermentation liquor and containing D-pantothenic acid
and/or a salt thereof, and to a process for the preparation of such
additives.
[0003] 2. Discussion of the Background
[0004] Pantothenic acid is produced worldwide on a scale of several
thousand tons per year, and demand for the product is increasing.
Much of the pantothenic acid that is produced is used for feeding
productive livestock such as poultry and pigs.
[0005] Pantothenic acid can be produced by biotechnology or by
chemical synthesis. Biotechnology methods involve fermentation of
suitable microorganisms in appropriate nutrient solutions. In
chemical synthesis, DL-pantolactone is an important precursor. It
is prepared in a multi-step process from formaldehyde,
isobutylaldehyde and cyanide where the racemic mixture is resolved,
the D-pantolactone is condensed with .beta.-alanine, and
D-pantothenic acid is thus obtained.
[0006] The typical commercial form of pantothenic acid is the
calcium salt of D-pantothenic acid. The calcium salt of the racemic
mixture of D,L-pantothenic acid is also common.
[0007] The advantage of preparing pantothenic acid by means of
fermentation of microorganisms is that the desired stereoisomeric
form, namely the D form, which is free of L-pantothenic acid, is
formed directly.
[0008] Various types of bacteria, such as, for example, Escherichia
coli, Arthrobacter ureafaciens, Corynebacterium erythrogenes,
Brevibacterium ammoniagenes, and also yeasts, such as, for example,
Debaromyces castellii, are able to produce D-pantothenic acid under
suitable fermentation conditions, as is shown in EP-A-0 493 060,
EP-A-0 590 857 and WO 97/10340. Especially suitable microorganisms
are the Escherichia coli IF03547 derivatives described therein,
such as, for example, the strains FV5069/pFV31 or
FV5069/pFV202.
[0009] In the preparation of D-pantothenic acid by fermentation, as
is described in EP-A-0 493 060, EP-A-0 590 857 and WO 97/10340, a
microorganism capable of producing D-pantothenic acid is cultivated
in a suitable nutrient medium and the D-pantothenic acid that forms
is then isolated, purified and prepared in the form of the calcium
salt in a complicated manner.
[0010] Suitable nutrient media contain a carbon source, a nitrogen
source, a phosphorous source, salts, trace elements and vitamins,
and optionally, complex media additives, such as yeast extract.
Examples of the carbon source include glucose, starch flour
hydrolysate, sucrose and molasses. An example of the nitrogen
source is ammonium sulfate. An example of the phosphorous source is
potassium phosphate.
[0011] According to the current prior art described in WO96/33283
and EP-A-0 590857, the calcium salt of D-pantothenic acid is
obtained from the pantothenic acid-containing fermentation liquor
by means of a complex isolation and purification operation. After
first separating off the biomass by filtration or centrifugation,
the filtrate is further purified by means of activated carbon or by
column chromatography. After reaction of the resulting solutions
with calcium hydroxide, the desired Ca salt is allowed to
crystallize.
[0012] According to WO 96/33283, the filtrate is decolored with
activated carbon in the first column. A pH value of 3.0 is adjusted
using concentrated hydrochloric acid, and the liquid is then
purified continuously over two further columns packed with
activated carbon. Elution of the D-pantothenic acid takes place
with the aid of methyl alcohol. Subsequent neutralisation using
Ca(OH).sub.2 powder yields a solution from which calcium
D-pantothenate is obtained by crystallisation at 5.degree. C.
[0013] In the method described in EP-A-0 590 857, the filtrate is
first purified with the aid of cation- and anion-exchanger columns.
Elution takes place with hydrochloric acid. The eluted fraction is
then neutralised using Ca(OH).sub.2; activated carbon is added
thereto and the whole is filtered off. The filtrate that is
obtained is then extracted in a low molecular weight alcohol
(methanol, ethanol, isopropanol), and calcium D-pantothenate is
obtained by crystallisation.
[0014] The calcium D-pantothenate prepared in the described manner
is used as an additive in animal feed.
OBJECT OF THE INVENTION
[0015] According to the prior art, salts of D-pantothenic acid or
D,L-pantothenic acid are obtained by chemical synthesis or from
fermentation liquors and then added in pure form to feeds.
[0016] The object of the invention is to provide more readily
processable forms of D-pantothenic acid and its salts and processes
for the preparation thereof for feeds.
DESCRIPTION OF THE INVENTION
[0017] The present invention provides a pourable animal feed
additive based on fermentation liquor and containing D-pantothenic
acid and/or salts thereof. The feed additive is characterized in
that
[0018] a) it contains the biomass formed during the fermentation in
an amount of from .gtoreq.0 to 100%; and
[0019] b) it contains at least the predominant part of the further
constituents of the fermentation liquor; and
[0020] c) it is in solid form, in a particle size distribution of
from 20 to 2000 .mu.m, especially from 50 to 800 .mu.m, more
especially from 150 to 600 .mu.m, and is pourable.
[0021] In a preferred embodiment of the invention, the pourable
animal feed additive containing D-pantothenic acid and/or salts
thereof is characterized in that it additionally contains, in solid
form, an amount of chloride-containing constituents in a
concentration of <3 mg per g of additive, preferably <2 mg
per g of additive and especially <1.5 mg per g of additive.
[0022] The additives are generally compacted, granulated, or
fine-grained, but in any case, pourable form, according to
requirements, and contain varying amounts of biomass. The apparent
density is from 200 to 800 kg/m.sup.3, especially approximately
from 400 to 700 kg/m.sup.3.
[0023] Pourable in the context of this invention means freely
flowing non-clumped particles of a predetermined size which are
able to be dispensed from a container. The additives are readily
pourable and storage stable.
[0024] If the biomass is separated off further, inorganic solids,
for example those which have been added during the fermentation,
are generally removed. In addition, the additive according to the
invention contains at least the predominant part of the further
substances, especially organic, that have been formed or added and
are dissolved in the fermentation liquor, insofar as they have not
been separated off by suitable processes.
[0025] Such substances include organic by-products that are
produced and secreted in addition to D-pantothenic acid by the
microorganisms used in the fermentation. They include L-amino
acids, selected from the group L-methionine, L-lysine, L-valine,
L-threonine, L-alanine or L-tryptophan, especially L-valine. They
also include organic acids carrying from one to three carboxyl
groups, such as, for example, acetic acid, lactic acid, citric
acid, malic acid or fumaric acid. Finally, they also include
sugars, such as, for example, trehalose. Such compounds may be
desirable, in that they improve the nutritional value of the
additive.
[0026] In a preferred form there is prepared a fermentation liquor
containing D-pantothenic acid and/or salts thereof, wherein
[0027] a) the fermentation takes place in a substantially
chloride-free medium,
[0028] b) the resulting fermentation liquor, optionally after
separation of the biomass and concentration, is dried, compacted,
spray dried, spray granulated or granulated or applied to a carrier
or embedded in a stabilising matrix.
[0029] The fermentation liquors which are suitable for the process
according to the invention are obtained using microorganisms
suitable for the production of D-pantothenic acid and that contain
D-pantothenic acid and/or salts thereof. The salts are generally
sodium, potassium, ammonium, magnesium or calcium salt.
[0030] The microorganisms may be fungi or yeasts, such as, for
example, Debaromyces castellii, or Gram-positive bacteria, for
example of the genus Corynebacterium, or Gram-negative bacteria,
such as, for example, those of the family Enterobacteriaceae. In
the case of the family of the Enterobacteriaceae, special mention
may be made of the genus Escherichia and of the species Escherichia
coli. Within the species Escherichia coli, mention may be made of
the so-called K-12 strains, such as, for example, strains MG1655 or
W3110 (Neidhard et al.: Escherichia coli and Salmonella. Cellular
and Molecular Biology (ASM Press, Washington, D.C.)), or of the
Escherichia coli wild type strain IFO3547 (Institut fur
Fermentation, Osaka, Japan) and mutants derived therefrom both of
which are incorporated herein by reference. Of the strains produced
from IFO3547, FV5069/pFV31 (EP-A-0 590 857) and FV5069/pFV202 (WO
97/10340) are prominent. In the case of the genus Corynebacterium,
special mention may be made of the species Corynebacterium
glutamicum.
[0031] The above-described microorganisms can be cultivated for the
purposes of D-pantothenic acid production continuously or
discontinuously by the batch or fed batch or repeated fed batch
process. A summary of known cultivation methods is described in the
textbook by Chmiel (Bioprozesstechnik 1. Einfuhrung in die
Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991) or in
the textbook by Storhas (Bioreaktoren und periphere Einrichtungen
(Vieweg Verlag, Braunschweig/Wiesbaden, 1994) both of which are
incorporated herein by reference.
[0032] The culture medium to be used must meet the requirements of
the microorganisms to be used in a suitable manner. The
fermentation medium is substantially free of chloride-containing
constituents. According to the invention, the concentration of
chloride ions in the production fermenter is <300 mg/l,
preferably <200 mg/l and very especially preferably <150
mg/l. There may be used as the carbon source sugars and
carbohydrates such as glucose, saccharose, lactose, fructose,
maltose, molasses, starch and cellulose, oils and fats, such as
soybean oil, sunflower oil, groundnut oil and coconut oil, fatty
acids such as palmitic acid, stearic acid and linoleic acid,
alcohols such as glycerol and ethanol, and organic acids such as
acetic acid. Those substances may be used individually or in the
form of a mixture. There may be used as the nitrogen source organic
nitrogen-containing compounds, such as peptones, yeast extract,
meat extract, malt extract, corn steep liquor, soybean flour and
urea, or inorganic compounds, such as ammonium sulfate, ammonium
phosphate, ammonium carbonate and ammonium nitrate. The nitrogen
sources may be used individually or in the form of a mixture. There
may be used as the phosphorus source potassium dihydrogen phosphate
or dipotassium hydrogen phosphate or the corresponding
sodium-containing salts. The culture medium must also contain salts
of metals such as magnesium sulfate or iron sulfate, which are
necessary for growth. Finally, essential growth substances, such as
amino acids and vitamins, may be used in addition to the
above-mentioned substances. Precursors of D-pantothenic acid, such
as aspartate, .beta.-alanine, ketoisovalerate, ketopantoic acid or
pantoic acid, and, optionally, salts thereof, may also be added to
the culture medium. The mentioned substances may be added to the
culture in the form of a single batch, or they may be fed in a
suitable manner during the cultivation.
[0033] In order to control the pH value, ammonia or ammonia water
or other basic compounds, such as sodium hydroxide, potassium
hydroxide or calcium hydroxide are used. If acid compounds are
required to control the pH value, phosphoric acid or sulfuric acid
may expediently be used. In order to prepare the calcium salt of
pantothenic acid directly, calcium hydroxide in the form of an
aqueous suspension is used during the fermentation. In order to
control the development of foam, anti-foams such as fatty acid
polyglycol esters, may be used. In order to maintain the stability
of plasmids, suitable substances having a selective action, for
example antibiotics, are optionally added to the medium. In order
to maintain aerobic conditions, oxygen or gas mixtures containing
oxygen such as air, are introduced into the culture. The
temperature of the culture is normally from 20.degree. C. to
45.degree. C. and preferably from 25.degree. C. to 40.degree. C.
The culture is continued until the maximum amount of D-pantothenic
acid has formed, which is generally within a period of 10 hours to
160 hours.
[0034] The fermentation liquors thus obtained usually have a
content of dry matter of 7.5 to 25 wt. % and contain from 2 to 20
wt. % D-pantothenic acid. Fermentation processes in which
D-pantothenic acid is present in the dry matter in an amount of at
least 20 wt. % when the fermentation is complete are especially
advantageous. It is also advantageous for the amount of sugar in
the fermentation to be limited at least at the end of the
fermentation, but advantageously for at least 30% of the duration
of the fermentation. That means that the concentration of usable
sugar in the fermentation medium is maintained at, or reduced to
from .gtoreq.0 to 3 g/l during that time.
[0035] For the preparation of the additives according to the
invention, the fermentation liquors containing D-pantothenic acid
and/or salts thereof are preferably first freed of all or part of
the biomass by known separation methods such as centrifugation,
filtration, decantation or a combination thereof. However, it is
also possible according to the invention to leave all of the
biomass in the fermentation liquor. The suspension obtained in that
manner is then concentrated preferably to not more than 60 wt. %
dry matter and worked up to a powder, for example with the aid of a
spray drier or a lyophilising apparatus. The powder is then
converted into a coarser-grained, readily pourable, storable and
largely dust-free product by suitable compacting or granulating
processes. In the granulating or compacting operation it is
advantageous to use conventional organic or inorganic auxiliary
substances, or carriers, such as starch, gelatin, cellulose
derivatives or similar substances, as are conventionally employed
as binders, gelling agents or thickeners in the processing of
foodstuffs or feeds, or further substances such as, for example,
silicas, silicates or stearates.
[0036] Alternatively, the product may be applied to an organic or
inorganic carrier substance that is known and conventionally
employed in the processing of feeds, such as, for example, silicas,
silicates, meals, brans, flours, starches, sugars or the like, and
stabilised by means of conventional thickeners or binders. Relevant
examples and processes are described in the literature (Die
Muhle+Mischfuttertechnik 132 (1995) 49, page 817) which is
incorporated herein by reference.
[0037] The novel solid products according to the invention that
contain D-pantothenic acid and/or salts thereof and that can be
prepared by the above-described process contain from 20 to 80 wt.
%, preferably from 30 to 75 wt. %, D-pantothenic acid. They
generally contain inorganic constituents in an amount of from 2.5
to 25 wt. % and optionally organic by-products in an amount of from
>0 to 30 wt. %. The content of dry biomass is from .gtoreq.0 to
35 wt. %. The water content is preferably <5 wt. %.
[0038] The novel products according to the invention that contain
D-pantothenic acid and/or salts thereof and that are prepared by
the above-described process are distinguished by a particle size
distribution of from 20 .mu.m to 2000 .mu.m, preferably from 50
.mu.m to 800 .mu.m and especially from 150 .mu.m to 600 .mu.m. The
content of very fine dust (<10 .mu.m) is approximately from 0
wt. % to 10 wt. %, preferably approximately from 0 wt. % to 5 wt.
%. The product is used as a feed additive.
[0039] The concentration of D-pantothenic acid can be determined by
known methods (Velisek; Chromatographic Science 60, 515-560
(1992)).
[0040] The particle size distribution can be determined by methods
of laser diffraction spectrometry. Corresponding methods are
described in the textbook on "Teilchengrossenmessung in der
Laborpraxis" by R. H. Muller and R. Schuhmann, Wissenschaftliche
Verlagsgesellschaft Stuttgart (1996) or in the textbook
"Introduction to Particle Technology" by M. Rhodes, Verlag Wiley
& Sons (1998) both of which are incorporated herein by
reference.
EXAMPLES
[0041] The present invention is explained in greater detail below
by means of Examples. To that end, tests have been carried out
using the D-pantothenic acid-producing strain Escherichia coli
5069/pFV31, which has been deposited as FERM-BP 4395 in accordance
with the Budapest Treaty at the Fermentation Research Institute,
Agency of Industrial Science and Technology in 1-1-3, Higashi,
Tsukuba-shi, Ibaraki (Japan) (EP-A-0590857).
[0042] The measurements were carried out on a Cilas 920 laser
diffraction spectrometer from Quanto Chrome (Odelzhausen, Germany).
Evaluation of the measurement results was carried out as specified
in German Industrial Standard DIN 66141 for representation of the
particle size distribution.
Example 1
[0043] Preparation of the Calcium Salt of D-pantothenic acid in a
Fermentation Liquor
[0044] 1. Preparation of Inoculum (Master Cell Bank)
[0045] A sample of Escherichia coli FV5069/pFV3 1 was spread on LBG
agar to which 50 .mu.g per ml ampicillin had been added. The agar
plate culture was incubated for 17 hours at 37.degree. C. and then
stored in a refrigerator at +4.degree. C. Selected individual
colonies were then multiplied further in LBG bouillon. LBG bouillon
has the following composition: 10 g/l peptone, 5 g/l yeast extract,
5 g/l NaCl and 1 g/l glucose. LBG agar additionally contains 12 g/l
agar. Ready-made preparations can be obtained from Gibco/BRL
(Paisley, Scotland, Great Britain) as LB broth base or LB agar.
After the addition of 1 g/l glucose, the indicated media are then
obtained. 10 ml cultures, which were contained in 100 ml Erlenmeyer
flasks, were incubated for 16 hours at 37.degree. C. and 180 rpm in
an ESR incubator from Kuhner AG (Birsfelden, Switzerland). The cell
suspension was then separated by centrifuging in a J-6B centrifuge
from Beckmann (Hanover, Germany) for 15 minutes at 4000 rpm. The
cell pellet was re-suspended in 10 ml of LBG medium to which 20%
glycerol had been added; 1 ml was introduced under sterile
conditions into each of 10 aliquots and frozen at -70.degree. C.
Those cultures were used as the master cell bank.
[0046] For the preparation of a working cell bank, LBG medium to
which 50 .mu.g/ml ampicillin had been added was divided into 10 ml
portions in 100 ml Erlenmeyer flasks and then inoculated with 100
.mu.l of the above-described master cell bank. Incubation was
carried out for 16 hours at 37.degree. C. and 180 rpm in an ESR
incubator from Kuhner AG (Birsfelden, Switzerland).
[0047] After incubation, the optical density (OD) of the culture
suspension was determined at a measuring wavelength of 660 nm using
an LP2W photometer from Dr. Lange (Berlin, Germany). It was 3.5.
The cell suspension was then introduced under sterile conditions
into sterile 30 ml polyethylene tubes from Greiner (Frickenhausen,
Germany) and separated by centrifuging at 2500 rpm for 15 minutes
using a J-6B centrifuge from Beckmann (Hanover, Germany). The
biomass that had been separated off was re-suspended in 10 ml of
LBG medium to which 20% glycerol had been added. The cell
suspension was then introduced in 500 .mu.l portions, under sterile
conditions, into 1 ml sterile tubes from Nalgene (New York, U.S.A.)
and frozen at -70.degree. C. The preserved batches so prepared were
used as the working cell bank.
[0048] 2. Preparation of a Fermentation Liquor Containing Calcium
D-pantothenate
[0049] For the preparation of a fermentation liquor containing
calcium D-pantothenate, the working cell bank was first multiplied
in a shaking flask culture, and the latter was used to inoculate a
pre-fermenter. The pre-fermenter culture was used to inoculate the
production fermenter.
[0050] SKA medium (Table 1) was used for the shaking flask culture.
SKA medium was prepared as follows: 7.0 g of
(NH.sub.4).sub.2SO.sub.4, 0.5 g of KH.sub.2PO.sub.4, 1.0 g of
K.sub.2HPO.sub.4, 0.5 g of MgSO.sub.4.7H.sub.2O, 0.01 g of
MnSO.sub.4.H.sub.2O, 0.001 g of ZnSO.sub.4.7H.sub.2O, 0.005 g of
Fe.sub.2(SO.sub.4).sub.3 and 20 g of corn steep liquor, which had
previously been adjusted to pH 6.8 using 25% ammonia solution, were
weighed into a 1 liter glass beaker and then made up to 825 g with
distilled water. That salt solution containing corn steep liquor
was sterilised in an autoclave at 121.degree. C. for 20 minutes. In
addition, a solution consisting of 25 g of glucose and 0.002 g of
thiamin.HCl was made up to 125 g with distilled water and
sterilised by filtration. 10 g of CaCO.sub.3 were weighed into a
100 ml flask and sterilised in an autoclave at 123.degree. C. for
20 minutes. SKA medium was obtained by combining the
above-mentioned two components with the salt solution containing
corn steep liquor.
[0051] The SKA medium was divided into 12.5 ml portions in 100 ml
Erlenmeyer flasks and was then inoculated with 0.5 ml of a cell
suspension. The cell suspension used was a preserved batch of the
working cell culture diluted 1:100 with sterile physiological
saline. Incubation was carried out for 20 hours at 32.degree. C.
and 150 rpm on an RC-1-TK incubator from Infors AG (Bottmingen,
Switzerland). The optical density determined thereafter at a
measuring wavelength of 660 nm (OD 660) was 12.5.
[0052] For the inoculation of 20 kg of A1-102 pre-culture medium,
which was contained in a 42 liter stirred reactor fermenter from
Bioengineering (Wald, Switzerland, model LP-42), 0.5 ml of SKA
medium was diluted 1:100 and 50 ml of that suspension were added to
the fermenter. The pre-culture medium A1-102 contained the
constituents listed in Table 2. The culture was cultivated for 15.5
hours at a temperature of 37.degree. C., with volume-specific
aeration of 0.5 volume/volume/minute (vvm), at an oxygen partial
pressure of 20% of air saturation and at a pH of 6.5, until an
OD660 of 11.3 was reached.
[0053] For the inoculation of 5830 g of M1-380 main culture medium,
which was contained in 14 liter stirred reactor fermenters from B.
Braun (BBI, Germany, Melsungen, model Biostat E/ED), 423 ml of the
second pre-culture in A1-102 medium were added. M1-380 main culture
medium contained the constituents listed in Table 3. The culture
was first cultivated for 6.5 hours at a temperature of 37.degree.
C., with volume-specific aeration of 0.75 vvm, with minimum
stirring of 400 rpm and at a pH of 6.5, until an OD660 of 18.6 and
an oxygen partial pressure of 2% of air saturation had been
reached. The culture was then cultivated for a further 41 hours at
a temperature of 37.degree. C., an oxygen partial pressure of 2% of
air saturation and a pH value of 6.0, until an OD660 of 66.8 had
been reached. After a fermentation time of 13 hours, .beta.-alanine
was fed in in a concentration of 152.7 g in 570 ml of H.sub.2O over
a period of 34.5 hours. After a fermentation time of 21.5 hours, a
ten percent Ca(OH).sub.2 solution was metered in over a period of
26 hours in order to control the pH. 3.43 kg of M2-257 medium
having a glucose concentration of 650.8 g/l and a concentration of
thiamin.HCl of 35.7 g/l were fed in in the course of 41 hours.
[0054] The optical density (OD) was then determined at a measuring
wavelength of 660 nm using an LP1W type digital photometer from Dr.
Bruno Lange GmbH (Berlin, Germany), and the concentration of
D-pantothenic acid that had formed was determined by means of HPLC
(Hypersil APS 2 5 .mu.m, 250.times.5 mm, RI detection).
[0055] A calcium D-pantothenate concentration of 49.7 g/l, measured
as D-pantothenic acid, was determined in the final fermentation
sample after 70.0 hours.
[0056] The content of D-pantothenic acid was determined with the
aid of a type M321 HPLC (high-performance liquid chromatography)
apparatus from Knauer (Berlin, Germany) by means of RI (refractive
index) detection using a Hypersil APS2 amino phase of particle size
5 .mu.m.
1TABLE 1 Composition of SKA medium Concentration Component (per
litre) Glucose 25.0 g Corn steep liquor 20.0 g
(NH.sub.4).sub.2SO.sub.4 7.0 g KH.sub.2PO.sub.4 0.5 g
K.sub.2HPO.sub.4 1.0 g MgSO.sub.4 .multidot. 7H.sub.2O 0.5 g
FeSO.sub.4 .multidot. 7H.sub.2O 5 mg MnSO.sub.4.multidot. H.sub.2O
10 mg ZnSO.sub.4 .multidot. 7H.sub.2O 1 mg CaCO.sub.3 10 g Thiamin
chloride .multidot. HCl 2 mg Structol 0.7 g
[0057]
2TABLE 2 Composition of A1-102 medium Concentration Component (per
litre) Glucose 25.0 g Corn steep liquor 20.0 g
(NH.sub.4).sub.2SO.sub.4 7.0 g KH.sub.2PO.sub.4 0.5 g
K.sub.2HPO.sub.4 1.0 g MgSO.sub.4 .multidot. 7H.sub.2O 0.5 g
FeSO.sub.4 .multidot. 7H.sub.2O 10 mg MnSO.sub.4.multidot. H.sub.2O
10 mg Thiamin chloride .multidot. HCl 3 mg Structol 0.6 g
[0058]
3TABLE 3 Composition of M1-380 medium Concentration Component (per
litre) Glucose 18.0 g Corn steep liquor 40.0 g .beta.-Alanine 15.0
g (NH.sub.4).sub.2SO.sub.4 11.8 g KH.sub.2PO.sub.4 0.6 g
K.sub.2HPO.sub.4 1.2 g MgSO.sub.4 .multidot. 7H.sub.2O 0.67 g
MnSO.sub.4.multidot. H.sub.2O 10 mg Thiamin chloride .multidot. HCl
1.6 mg Structol 0.6 g
Example 2
[0059] Preparation of Finely Divided Calcium D-pantothenate From
Fermentation Liquor
[0060] The biomass was first separated from a calcium D-pantothenic
acid-containing fermentation liquor that had been prepared
according to the process of Example 1 and contained approximately
4.9 wt. % D-pantothenic acid. To that end, 90 liters of the
above-mentioned fermentation liquor were filtered off by cross-flow
filtration using a 0.22 .mu.m microfiltration membrane in a CERAFLO
MSP005756 filtration apparatus from Millipore (Bad Homburg,
Germany).
[0061] The liquor so treated was then concentrated to a liquid
content of approximately 30% dry content in a Rotavapor R-152
rotary evaporator from Buchi, Switzerland at from 40 to 80.degree.
C., in vacuo. The liquor so concentrated was then spray dried in
order to prepare the calcium salt of D-pantothenic acid. To that
end there was used a Technikum spray drier from Niro (Copenhagen,
Denmark) of the NIRO Minor type having an atomising plate (120 mm
diameter; speed of rotation 135 m/sec.) at an inlet temperature of
175.degree. C., an outlet temperature of 80.degree. C. and a drying
gas throughput of 525 m.sup.3/hour. For the purposes of better
discharge of the product, Sipernat 22 from Degussa-Huls AG
(Frankfurt am Main, Germany) was added as powder auxiliary to the
drying gas stream in a ratio of 5 wt. %, based on dry matter in the
concentrate.
[0062] The calcium D-pantothenate-containing product so prepared
had a D-pantothenic acid content of 48.5 wt. %, was pourable and
had an apparent density of 460 kg/m.sup.3 with a mean particle size
of 34 .mu.m.
4TABLE 4 Designation Diameter Content [%] Fine dust <10 .mu.m 10
Dust 10-20 .mu.m 20 Powder/dust 20-50 .mu.m 50 Powder >50 .mu.m
20
Example 3
[0063] Preparation of D-calcium Pantothenate Having a Particle Size
>100 .mu.m by Compaction in a Roll Compactor and Sieving
[0064] A calcium D-pantothenic acid-containing dust-like product,
which had been prepared according to the process of Example 2 and
contained approximately 48.5 wt. % D-pantothenic acid and had a
mean particle size of 34 .mu.m, was compacted by means of a roll
compactor having cigar-like rolls (Pharmapaktor from BEPEX of type
L200/50 P) with a compressive force of from 40 to 90 Newtons. The
speed of rotation of the rolls was 10 revolutions per minute. The
compacted product so prepared was then broken down to a particle
size distribution of from 200 to 400 .mu.m in comminution sieves.
The yield in the individual particle fractions is summarised in
Table 5.
[0065] The compacted product was distinguished by a markedly lower
content of fine dust and substantially improved flow behaviour as
compared with the pulverulent starting product.
5TABLE 5 Designation Diameter Content [%] Fine dust <10 .mu.m 5
Dust 10-50 .mu.m 5 Fines 50-200 .mu.m 20 Mean particle size 200-400
.mu.m 50 Oversize particles >400 .mu.m 20
[0066] The fraction "mean particle size" was isolated by sieving
and represents the product. The product so prepared had a content
of approximately 40.7 wt. %, measured as D-pantothenic acid, and
had an apparent density of 630 kg/m.sup.3.
Example 4
[0067] Preparation of D-calcium Pantothenate Having a Mean Particle
Size of From 200 to 400 .mu.m by Build-up Granulation in a
Fluidised-bed Granulator
[0068] 4.1 Use of Water as the Granulation Binder
[0069] Pulverulent product containing calcium D-pantothenic acid,
which had been prepared from a fermentation solution containing
calcium D-pantothenic acid by spray drying according to the process
described in Example 2, was subsequently processed further in a
fluidised-bed granulator by spraying with a particular amount of
water.
[0070] To that end, 300 g of the dust-like calcium D-pantothenic
acid-containing product prepared according to Example 2 were placed
in a laboratory fluidised-bed apparatus from Aeromatics Niro
(Copenhagen, Denmark). At a fluidised bed temperature of 50.degree.
C. and a waste gas temperature of 30.degree. C., 3 g of water per
minute were sprayed in via a metering device. The fluidised gas
temperature was from 70 to 80.degree. C. The particle size
distribution of the product so prepared is shown in Table 6.
6TABLE 6 Designation Diameter Content [%] Fine dust <10 .mu.m 1
Dust 10-50 .mu.m 4 Fines 50-200 .mu.m 20 Mean particle size 200-400
.mu.m 750 Oversize particles >400 .mu.m 0
[0071] The content, measured as D-pantothenic acid, was determined
as 38.1 wt. %. The product was almost dust-free. The apparent
density was 310 kg/m.sup.3. The product was very readily
pourable.
[0072] 4.2 Use of a Concentrate Containing Calcium Pantothenate as
the Granulation Binder
[0073] A pulverulent product containing calcium D-pantothenic acid,
which had been prepared from a calcium D-pantothenic
acid-containing fermentation solution by spray drying according to
the process described in Example 2, was processed further in
another test in a fluidised-bed granulator by spraying with a
particular amount of concentrated calcium D-pantothenate solution
having a content of dry matter of approximately 50 wt. %.
[0074] To that end, 1000 g of the dust-like calcium D-pantothenic
acid-containing product prepared according to the process described
in Example 2 were placed in a laboratory fluidised-bed apparatus,
which operated batchwise, from Glatt (Binzen, Germany). At a
fluidised bed temperature of approximately from 40 to 45.degree. C.
and an inlet air temperature of approximately 80.degree. C.,
approximately 5 g of the above-described concentrate per minute
were sprayed into the laboratory fluidised-bed apparatus. The
particle size distribution of the product so prepared is shown in
Table 7.
7TABLE 7 Designation Diameter Content [%] Fine dust <10 .mu.m 1
Dust 10-50 .mu.m 2 Fines 50-200 .mu.m 18 Mean particle size 200-400
.mu.m 79 Oversize particles >400 .mu.m 0
[0075] The content, measured as D-pantothenic acid, was determined
as 38.9 wt. %. The product was almost dust-free. The apparent
density was 400 kg/m.sup.3.
[0076] By means of the process described herein, or by means of a
different spray, fluidised-bed, stirring or mixing process, the
concentrate containing D-calcium pantothenate or D-pantothenic acid
is sprayed onto other conventional organic or inorganic carriers or
auxiliary substances, such as silicas, silicates, meals, brans,
flours, starches, sugars or the like, and granulated, optionally
with the use of binders, gelling agents or other formulation
auxiliaries.
Example 5
[0077] Preparation of D-calcium Pantothenate Having a Mean Particle
Size from 100 to 400 .mu.m by Mixing and Granulation in a Vacuum
Drier
[0078] The biomass was first separated from a calcium D-pantothenic
acid-containing fermentation liquor that had been prepared
according to the process of Example 1 and contained approximately
4.9 wt. % D-pantothenic acid. To that end, 90 liters of the
above-mentioned fermentation liquor were filtered off by cross-flow
filtration using a 0.22 .mu.m microfiltration membrane in a CERAFLO
MSP005756 filtration apparatus from Millipore (Bad Homburg,
Germany).
[0079] The liquor so treated was then concentrated to a liquid
content of approximately 50 wt. % dry content in a Rotavapor R-152
rotary evaporator from Buchi, Switzerland, at from 40 to 80.degree.
C., in vacuo. The calcium D-pantothenic acid-containing concentrate
so prepared having a D-pantothenic acid content of 28.8 wt. % was
then mixed with a silica (Sipernat 22, Degussa-Huls AG, Frankfurt
Germany) with the aid of a vacuum drier (type VT 130, Gebruder
Lodige Maschinenbau GmbH, Paderborn, Germany) to form a freely
flowable granulate. 15 kg of the silica (Sipernat 22, Degussa-Huls
AG, Germany) were first placed in the vacuum drier (type VT 130,
Gebruder Lodige Maschinenbau GmbH, Germany), and then 39.0 kg of
the calcium D-pantothenic acid-containing concentrate prepared
above were added at a rate of 2.0 kg per minute under a vacuum of
200 mbar, with the material to be mixed having a temperature of
45.degree. C., and with a stirrer performance of 120 rpm
(revolutions per minute). The mixing operation was then continued
for a further 15 minutes while increasing the vacuum to 50 mbar.
The D-calcium pantothenate-containing granulate so prepared was
then dried to a residual moisture content of less than 2 wt. % in a
vibratory fluidised-bed drier (Escher-Wyss, Linden, Germany) having
a fluidised-bed surface area of 0.3 m.sup.2 with a bed temperature
of 65.degree. C. and a drying gas throughput of 270 Nm3/hour. Table
8 shows the average particle size distribution of the product.
8TABLE 8 Designation Diameter Content [%] Fine dust <10 .mu.m 1
Dust 10-50 .mu.m 2 Fines 50-125 .mu.m 6 Mean particle size 200-400
.mu.m 89 Oversize particles >400 .mu.m 2
[0080] The content, measured as D-pantothenic acid, was determined
as 32.6 wt. %. The product was almost dust-free. The apparent
density was 650 kg/m.sup.3 after drying.
[0081] By means of the process described herein, or by means of a
different spray, fluidised-bed, stirring or mixing process, the
concentrate containing D-calcium pantothenate or D-pantothenic acid
can be sprayed onto other conventional organic or inorganic
carriers or auxiliary substances, such as silicas, silicates,
meals, brans, flours, starches, sugars, and granulated, optionally
with the use of binders, gelling agents or other formulation
auxiliaries.
[0082] Numerous modifications and variations of the present
invention are possible in light of the above teachings. It is
therefore to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described herein.
[0083] This application is based on German patent application
serial No. 100 32 349.9, filed on Jul. 4, 2000, the contents of
which are incorporated herein by reference.
* * * * *