U.S. patent application number 11/374661 was filed with the patent office on 2007-04-12 for l-lysine-containing feed additives.
Invention is credited to Ulrich Becker, Frank Dubner, Lawrence Edward Fosdick, Friederike Kaeppke, Ralf Kelle, Hermann Lotter, Joachim Pohlisch, Cory M. Sander.
Application Number | 20070082031 11/374661 |
Document ID | / |
Family ID | 37911271 |
Filed Date | 2007-04-12 |
United States Patent
Application |
20070082031 |
Kind Code |
A1 |
Lotter; Hermann ; et
al. |
April 12, 2007 |
L-lysine-containing feed additives
Abstract
The invention relates to relatively light and thermally stable
granulated, fermentation-broth-based animal feed additives having a
high content of L-lysine and to low-loss methods for their
production from broths obtained by fermentation.
Inventors: |
Lotter; Hermann;
(Altenstadt, DE) ; Becker; Ulrich;
(Freigericht-Horbach, DE) ; Dubner; Frank;
(Friedberg, DE) ; Kaeppke; Friederike;
(Gelnhausen, DE) ; Pohlisch; Joachim; (Gelnhausen,
DE) ; Kelle; Ralf; (Gutersloh, DE) ; Sander;
Cory M.; (Omaha, NE) ; Fosdick; Lawrence Edward;
(Oskaloosa, IA) |
Correspondence
Address: |
SMITH, GAMBRELL & RUSSELL
SUITE 3100, PROMENADE II
1230 PEACHTREE STREET, N.E.
ATLANTA
GA
30307-3592
US
|
Family ID: |
37911271 |
Appl. No.: |
11/374661 |
Filed: |
March 13, 2006 |
Current U.S.
Class: |
424/442 ;
426/635; 514/566 |
Current CPC
Class: |
C12P 13/08 20130101;
A23K 40/30 20160501; C12P 13/008 20130101; A23K 20/147 20160501;
A23K 10/12 20160501; A23K 40/10 20160501; A23K 20/142 20160501;
A61K 31/198 20130101 |
Class at
Publication: |
424/442 ;
514/566; 426/635 |
International
Class: |
A61K 31/198 20060101
A61K031/198; A23K 1/165 20060101 A23K001/165 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 8, 2005 |
DE |
102005048315.1 |
Claims
1. Granulated fermentation-broth-based feed additive having a) an
L-lysine content of 10 to 70% by weight (calculated as base, based
on the total weight), in particular 30 to 60% by weight, b) a water
content of 0.1 to 5% by weight (based on total weight), and c) a
sulphate/L-lysine ratio of 0.85 to 1.2, this ratio being calculated
according to the formula
2.times.[SO.sub.4.sup.2-]/[L-lysine]=ratio.
2. Feed additive according to claim 1 having a pH of 3.5 to 5.1,
measured in a 10% strength by weight aqueous suspension.
3. Feed additive according to claim 1, up to >97% of which has a
mean particle size of 0.1 to 1.8 mm.
4. Feed additive according to claim 1, the surface of which is
coated with an oil in an amount of 0.02 to 2% by weight, based on
the total amount of the feed additive.
5. Feed additive according to claim 1, the colour values of which
are in the following ranges: (measurement of diffuse reflection of
the sample at an angle of 8.degree.) L*65-80 (black-white range)
a*4-8 (red-green range) b*20-30 (yellow-blue range).
6. Method for producing a granulated L-lysine-containing feed
additive by fermentation of an L-lysine-producing microorganism in
an aqueous medium under aerobic conditions, wherein, after
completion of the fermentation a) the sulphate/L-lysine ratio in
the fermentation broth is optionally determined, b) subsequently
ammonium sulphate is optionally added and c) the pH is lowered by
adding sulphuric acid to 4.9 to 5.2, a sulphate/L-lysine ratio of
0.85 to 1.2 being set in the broth by addition of the sulphate
containing compounds, and d) the resultant mixture is optionally
concentrated, granulated and a product is obtained which has an
L-lysine content of 10 to 70% by weight (determined as lysine base,
based on the total amount).
7. Method according to claim 6, in which the broth is concentrated
by removal of water between steps c) and d).
8. Method according to claim 6, in which the sequence of the
additions of ammonium sulphate and sulphuric acid is turned the
other way round.
9. Method according to claim 6, in which, before the concentration,
ammonium-, earthalkali- or an alkali metal hydrogensulphite or
mixtures thereof are added in an amount of 0.01 to 0.5% by weight,
based on fermentation broth.
10. Method according to claim 6, in which, after completion of the
fermentation, 0 to 100% of the biomass formed during the
fermentation is removed and steps a) to d) follow.
11. Method according to claim 6, in which the surface of the
granules is coated with an oil in an amount of 0.02 to 2% by
weight, based on the total amount of the feed additive.
12. Method according to claim 6, in which coryneform microorganisms
are used.
13. Method according to claim 6, in which corynebacteria are used.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of German priority
Application No. 10 2005 048 315.1 filed Oct. 8, 2005, which is
relied on and incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The invention relates to relatively light and thermally
stable granulated, fermentation-broth-based animal feed additives
having a high content of L-lysine and to low-loss methods for their
production from broths obtained by fermentation.
PRIOR ART
[0003] Animal feeds are supplemented with individual amino acids in
accordance with the requirements of the animals. For
supplementation of animal feeds, e.g. with L-lysine, to date use is
very predominantly made of L-lysine monohydrochloride having an
L-lysine content of 78%. Since the L-lysine is produced by
fermentation, it must, to produce the monohydrochloride, first be
separated off from all other constituents of the crude fermentation
broth in complex process steps, then converted into the
monohydrochloride and the latter is crystallized. In the process a
large number of byproducts and the reagents required for workup
arise as waste. Since a high purity of the animal feed supplement
is not always necessary, and in addition, in the fermentation
byproducts, frequently nutritionally active valuable materials are
still present, there has therefore been no lack of attempts in the
past to avoid the complex production of feed amino acids, in
particular pure L-lysine monohydrochloride, and to convert the
crude fermentation broth more inexpensively into a solid animal
feed.
[0004] The complex composition of such media has proved a serious
disadvantage, since they can generally only be dried with
difficulty, and are then hygroscopic, virtually not free-flowing,
at risk of lump formation and unsuitable for the technically
demanding processing in mixed feed works. This applies especially
to L-lysine-containing fermentation products. Simple dewatering of
the crude fermentation broth by spray drying led to a dusty, very
hygroscopic concentrate which becomes lumpy after a short storage
time, and in this form cannot be used as animal feed.
[0005] EP 0 533 039 relates to methods for producing an amino acid
fermentation-broth-based animal feed supplement, the supplement
being obtainable directly from the fermentation broth by spray
drying. For this, in a variant, a part of the biomass is separated
off upstream of the spray drying. By means of a very clean
fermentation procedure, i.e. obtaining a fermentation broth which
is low in residues of organic substances, the broth can be dried
even without the biomass and without additional carrier to give
handlable granules.
[0006] Solid concentrates containing approximately 20% by weight of
L-lysine are disclosed by GB 1 439 121, in which
L-lysine-containing fermentation broths having a pH of 4.5 and a
bisulphite content are also described.
[0007] EP 0 615 693 discloses a method for producing a
fermentation-broth-based animal feed additive in which the
fermentation broth, if appropriate after removing part of the
constituents, is spray-dried to produce a fine grain, at least 70%
by weight of which has a maximum particle size of 100 .mu.m, and
this fine grain, in a second stage, is built up to produce granules
containing the fine grain at least 30% by weight.
[0008] According to GB 1 439 728, an L-lysine-containing
concentrate is produced from a fermentation broth which, upstream
of the concentration, is acidified by HCl to a pH of approximately
6.4 and to which bisulphite is added for stabilization.
[0009] After concentration by evaporation, it is further acidified
to a pH of 4.0, and the desired product obtained by spray
drying.
[0010] EP-A 1 331 220 (.apprxeq.US 2003/152633) relates to
granulated feed additives which contain L-lysine as main
component.
[0011] There, it was found that the amount of the counterions for
the lysine, such as, for example, that of the sulphate ions, can be
decreased by using as counterion carbon dioxide formed in the
fermentation. Overall, an anion/lysine ratio of 0.68 to 0.95 is
claimed.
[0012] The reduction of the counterions, such as, e.g. sulphate, in
the L-lysine-containing product is said to lead to an improvement
of the hygroscopic properties and caking tendency.
OBJECT OF THE INVENTION
[0013] It is an object of the invention to provide an
L-lysine-containing feed additive having improved properties which
is produced using the broths arising in the fermentation, and a
method having lower lysine loss during workup of the broths than
known from the state of art.
DESCRIPTION OF THE INVENTION
[0014] The invention relates to a granulated
fermentation-broth-based feed additive having
[0015] a) an L-lysine content of 10 to 70% by weight (calculated as
L-lysine base), in particular 30 to 60% by weight,
[0016] b) a water content of 0.1 to 5% by weight of water, and
[0017] a sulphate/L-lysine ratio of 0.85 to 1.2, preferably 0.9 to
1.0, in particular >0.9 to <0.95, this ratio being calculated
according to the formula V=2.times.[SO.sub.4.sup.2-]/[L-lysine]
[0018] this ratio being calculated from the formula
V=2.times.[SO.sub.4.sup.2-]/[L-lysine].
[0019] The difference from 100% by weight is made up by the further
constituents of the fermentation broth and if appropriate the
biomass.
[0020] Preferably, the granules have a pH of 3.5 to 5.1, in
particular 4.0 to 5.0, preferably 4.2 to 4.8, measured in a 10%
strength by weight suspension in deionized water at 25.degree. C.
using a pH electrode. The measured value establishes itself at a
constant value after approximately 1 min.
[0021] The molar ratio of sulphate to L-lysine is set subsequently
to the fermentation, an SO.sub.4.sup.2--containing compounds, in
particular ammonium sulphate and sulphuric acid, being added in a
suitable dilution.
[0022] "Fermentation-broth-based" means that an L-lysine-containing
fermentation broth is worked up which contains the biomass formed
during the fermentation at 0 to 100%.
[0023] The invention likewise relates to a method for producing a
granulated L-lysine-containing feed additive by fermentation of an
L-lysine-producing microorganism in an aqueous medium using
ammonium sulphate and under aerobic conditions and producing a
granulated product by intrinsically known methods, wherein, after
completion of the fermentation
[0024] optionally the ratio sulphate/L-lysine in the fermentation
broth is measured.
[0025] The sulfate concentration in the fermentation broth is
known. This is in general also true for the produced amounts of
L-lysine. Said amounts are usually measured in the case of
uncertainty.
BRIEF DESCRIPTION OF DRAWING
[0026] The accompanying drawing is a graph of hygroscopicity
characteristics of product of the invention E compared to products
A to D of the prior art.
DETAILED DESCRIPTION OF INVENTION
[0027] The process comprises the following steps:
[0028] a) Optionally subsequently ammonium sulphate is added
and
[0029] b) the pH is lowered by adding sulphuric acid to 4.0 to 5.2,
in particular 4.9 to 5.1, a sulphate/L-lysine ratio of 0.85 to 1.2,
in particular 0.9 to 1.0, being set in the broth by the addition of
the sulfate containing compound(s), and
[0030] c) the resultant mixture is preferably heated and
concentrated by dehydration, granulated and a product is obtained
which has an L-lysine content of 10 to 70% by weight (calculated as
lysine base, based on the total amount) and a sulphate/L-lysine
ratio of 0.85 to 1.2, in particular 0.9 to 1.0, in particular
>0.9 to <0.95, the ratio being calculated from the formula
2.times.[SO.sub.4.sup.2-]/[L-lysine]=V.
[0031] A ratio V=1 means, for example, that a stoichiometrically
composed (SO.sub.4)Lys.sub.2 is present, whereas at a ratio of 0.9,
a 10% sulphate deficit is found.
[0032] Step b) can also be carried out before step a).
[0033] Preferably the product has a L-Lysine content of 20 to 65,
most preferably 30 to 65% by weight.
[0034] It is possible to carry out the fermentation in the presence
of an amount of ammonium sulphate such that after completion of the
fermentation a sulphate/lysine ratio is already present which is in
the range claimed by the invention.
[0035] Further addition of ammonium sulphate may then no longer be
necessary.
[0036] If acid is added beyond the inventive pH reduction, because
of the buffering action of the compounds present in the broth,
increased amounts of acid are necessary, which can then lead to
unwanted denaturation and disintegration of the cells.
[0037] Even without addition of an acid, the pH of the broth during
the concentration by evaporation falls to a pH of approximately
5.4.
[0038] The granules produced according to the invention have a pH
of 3.5 to 5.1 (measured in the suspension, see above). The addition
of hydrochloric acid is preferably excluded. Its portion is in
general at max. 1%, preferably 0.01 to 0.1% by weight, related to
the amount of sulphuric acid.
[0039] The granules, despite the increased sulphate content, have a
markedly higher degree of whiteness, a lower hygroscopicity and a
better stability under thermal stress than granules having the same
L-lysine content, as are known from the prior art (see examples)
which, in suspension, exhibit a pH of 5.3 to 5.7, and a
sulphate/L-lysine ratio in the range of, for example, from 0.75 to
0.87.
[0040] The said properties may be further improved by adding
sulphites in an amount of 0.01 to 0.5%, preferably 0.1 to 0.3, by
weight, in particular 0.1 to 0.2% by weight, based on the
fermentation broth.
[0041] Said sulphites, especially ammonium-, earthalkali or
alkalisalts of sulphurous acid or mixtures thereof, in particular
sodium hydrogensulphite, are preferably added as solution to the
fermentation broth before concentration. The amount used is
preferably taken into consideration during setting the
sulfate/L-lysine ratio.
[0042] The granules may be produced, for example, by the methods
according to EP-B 0 615 693 or EP-B 0 809 940, U.S. Pat. No.
5,840,358 or WO 2005/006875 or WO 2004/054381. Generally, >97%
have a mean particle size of >0.1 to 1.8 mm, and a bulk density
of 600 to 950 kg/m.sup.3, in particular 650 to 900 kg/m.sup.3.
[0043] The colour values of the granules are preferably in the
ranges:
[0044] without hydrogensulphite addition: L*65-70, a*6-8,
b*20-25
[0045] with hydrogensulphite addition: L*>70-80, a*4-<6,
b*>25-30.
[0046] However, the inventive method does not only lead to products
showing advantageous properties.
[0047] It is found, at the same time, that the lysine loss which
generally occurs in the workup of fermentation broth can be lowered
by approximately 50% by the acidification according to the
invention and setting the sulphate/lysine ratio before
concentration.
[0048] Fermentation of the coryneform bacteria preferably used
according to the invention, in particular of the species
Corynebacterium glutamicum can be carried out continuously, for
example as described in PCT/EP 2004/008882, or discontinuously in
the batch method, or in the fed batch method or repeated fed batch
method for the purpose of producing L-lysine. A general summary on
known cultivation methods is available in the textbook by Chmiel
(Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik
[Bioprocess technology 1. Introduction to bioengineering
technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the
textbook by Storhas (Bioreaktoren und periphere Einrichtungen
[Bioreactors and peripheral devices] (Vieweg Verlag,
Brunswick/Wiesbaden, 1994)).
[0049] The culture medium and fermentation medium to be used must
appropriately satisfy the requirements of the respective strains.
Descriptions of culture media of various microorganisms are present
in the handbook "Manual of Methods for General Bacteriology" of the
American Society for Bacteriology (Washington D.C., USA, 1981). The
terms culture medium and fermentation medium or medium are mutually
exchangeable.
[0050] As carbon source, use can be made of sugars and
carbohydrates, for example glucose, sucrose, lactose, fructose,
maltose, molasses, sucrose-containing solutions from sugar beet or
sugar cane production, starch, starch hydrolysate and cellulose,
oils and fats, for example soya oil, sunflower oil, peanut oil and
coconut fat, fatty acids, for example palmitic acid, stearic acid
and linoleic acid, alcohols, for example glycerol, methanol and
ethanol, and organic acids, for example acetic acid. These
substances can be used individually or as a mixture.
[0051] As nitrogen source, use can be made of organic nitrogenous
compounds such as peptones, yeast extract, meat extract, malt
extract, maize steep liquor, soya bean meal and urea, or inorganic
compounds such as ammonium sulphate, ammonium phosphate, ammonium
carbonate and ammonium nitrate, preferably ammonium sulphate. The
nitrogen sources can be used individually or as a mixture.
[0052] As phosphorus source, use can be made of phosphoric acid,
potassium dihydrogenphosphate or dipotassium hydrogenphosphate or
the corresponding sodium salts.
[0053] The culture medium must in addition contain salts which are
necessary for growth, for example in the form of sulphates of
metals, for example sodium, potassium, magnesium, calcium and iron,
for example magnesium sulphate or iron sulphate. Finally, use can
be made of essential growth substances such as amino acids, for
example homoserine and vitamins, for example thiamin, biotin or
pantothenic acid, in addition to the abovementioned substances. In
addition, suitable precursors of the respective amino acid can be
added to the culture medium.
[0054] The said feed materials can be added to the culture in the
form of a single batch, or fed in a suitable manner during the
culturing.
[0055] For pH control of the culture, use is made of basic
compounds, such as sodium hydroxide, potassium hydroxide, ammonia
or ammonia water, or acidic compounds such as phosphoric acid or
sulphuric acid. The pH is generally set to 6.0 to 9.0, preferably
6.5 to 8. To control foam development, use can be made of
antifoams, for example polyglycol esters of fatty acids. To
maintain the stability of plasmids, suitable selectively active
substances, for example antibiotics, can be added to the medium. To
maintain aerobic conditions, oxygen or oxygen-containing gas
mixtures, for example air, are introduced into the culture. The use
of liquids which are enriched with hydrogen peroxide is likewise
possible. If appropriate the fermentation is run at
superatmospheric pressure, for example at a pressure of 0.03 to 0.2
MPa. The temperature of the culture is usually 20.degree. C. to
45.degree. C., and preferably 25.degree. C. to 40.degree. C. In the
case of batch methods, culturing is continued until a maximum of
the desired amino acid has formed. This target is usually achieved
within 10 hours to 160 hours. In the case of continuous methods,
longer culturing times are possible.
[0056] Examples of suitable fermentation media are found, inter
alia, in the patent documents U.S. Pat. No. 5,770,409, U.S. Pat.
No. 5,840,551 and U.S. Pat. No. 5,990,350, or U.S. Pat. No.
5,275,940.
[0057] Methods for determining L-amino acids are known from the
prior art. The analysis can, for example, be performed as described
in Spackman et al. (Analytical Chemistry, 30, (1958), 1190) by
anion-exchange chromatography with subsequent ninhydrin
derivatization, or it can be performed by reverse phase HPLC, as
described in Lindroth et al. (Analytical Chemistry (1979) 51:
1167-1174).
[0058] The invention accordingly relates to a method for producing
an L-amino acid in which
[0059] a) a coryneform bacterium is fermented in a suitable medium
and
[0060] b) the L-amino acid is enriched in the fermentation broth or
in the cells of the isolated coryneform bacterium.
[0061] The fermentation broth produced in this manner is
subsequently further processed to form a solid or liquid
product.
[0062] A fermentation broth is taken to mean a fermentation medium
in which a microorganism was cultured for a certain time and at a
certain temperature. The fermentation medium and the medium used
during the fermentation contains/contain all of the substances and
components which ensure multiplication of the microorganism and
formation of the desired amino acid.
[0063] At completion of the fermentation, the resultant
fermentation broth accordingly contains a) the biomass of the
microorganism resulting from multiplication of the microorganism
cells, b) the L-lysine formed in the course of fermentation, c) the
organic byproducts formed in the course of fermentation and d) the
constituents of the fermentation medium/fermentation media used
which were not consumed by the fermentation and of the feed
materials, for example vitamins such as biotin, amino acids such as
homoserine, or salts such as magnesium sulphate.
[0064] The organic byproducts include substances which may be
produced in addition to L-lysine, and excreted, by the
microorganisms used in the fermentation. These include L-amino
acids which make up less than 30%, 20% or 10%, relative to the
desired amino acid. In addition, these include organic acids which
bear one to three carboxyl groups, for example acetic acid, lactic
acid, citric acid, malic acid or fumaric acid. Finally, these also
include sugars, for example trehalose.
[0065] Typical fermentation broths suitable for industrial purposes
have an L-lysine content of 40 g/kg to 180 g/kg, or 50 g/kg to 150
g/kg. The biomass content (as dried biomass) is generally 20 to 50
g/kg.
[0066] In the case of methods for producing lysine, those methods
are preferred in which products are obtained which contain
constituents of the fermentation broth. These are used in
particular as animal feed additives.
[0067] Depending on requirements, the biomass can be entirely or in
part removed from the fermentation broth by separation methods, for
example centrifugation, filtration, decantation or a combination
thereof, or left completely in it. If appropriate, the biomass or
the biomass-containing fermentation broth is inactivated during a
suitable process step.
[0068] In one processing method, the biomass is completely or
virtually completely removed, so that none (0%) or at most 30%, at
most 20%, at most 10%, at most 5%, at most 1%, or at most 0.1%, of
biomass remains in the product produced. In a preferred processing
method, the biomass is not removed or removed only in minor
amounts, so that all (100%) or more than 70%, 80%, 90%, 95%, 99% or
99.9% of biomass remains in the product produced.
[0069] Fermentation broths from which the biomass has been in part
or entirely removed can be used for standardizing the product.
Obviously, this also applies to the pure compounds L-lysine base
and lysine sulphate.
[0070] According to the invention, before the concentration, the
fermentation broth obtained after the fermentation is acidified by
sulphuric acid and if appropriate admixed with ammonium sulphate.
Finally, the broth can also be stabilized and brightened by adding
preferably sodium hydrogensulphite or another salt, for example
ammonium salt, alkali metal salt or alkaline earth metal salt of
sulphurous acid.
[0071] If biomass is separated off, this is preferably performed
before the inventive lowering of the pH and the addition of
ammonium sulphate and sulphite salt.
[0072] If the biomass is separated off, if appropriate, organic or
inorganic solids present in the fermentation broth are in part or
entirely removed. The organic byproducts dissolved in the
fermentation broth and the dissolved non-consumed constituents of
the fermentation medium (feed materials) remain at least in part in
the product (>0%), preferably at least 25%, particularly
preferably at least 50%, and very particularly preferably at least
75%. If appropriate, these also remain entirely (100%) in the
product, or virtually entirely, that is >95%, or >98%. In
this sense, the term "fermentation broth base" means that a product
contains at least a part of the constituents of the fermentation
broth.
[0073] Subsequently, water is removed from the broth using known
methods, for example using a rotary evaporator, thin-film
evaporator, falling-film evaporator, by reverse osmosis or by
nanofiltration, or the broth is thickened or concentrated. This
concentrated fermentation broth can subsequently be worked up to
form free-flowing products, in particular granules, by methods of
freeze drying, spray drying, spray granulation or by other methods,
for example in the circulating fluidized bed according to PCT/EP
2004/006655. If appropriate, from the resultant granules, a product
having the desired particle size is isolated by sieving or dust
separation.
[0074] It is likewise possible to obtain a finely divided powder
from the fermentation broth directly, i.e. without previous
concentration by spray drying or spray granulation.
[0075] The particle size determination can be carried out using
methods of laser diffraction spectrometry. The corresponding
methods are described in the textbook on
"Teilchengro.beta.enmessung in der Laborpraxis" [Particle size
measurement in laboratory practice] by R. H. Muller and R.
Schuhmann, Wissenschaftliche Verlagsgesellschaft Stuttgart (1996)
or in the textbook "Introduction to Particle Technology" by M.
Rhodes, Wiley & Sons (1998).
[0076] The free-flowing, finely divided powder can be converted in
turn into a coarse-grained, free-flowing, storable and
substantially dust-free product by suitable compacting or
granulation methods.
[0077] "Free-flowing" is taken to mean powders which, of a series
of glass outlet vessels having different-sized outlet orifices,
flow unimpeded at least out of the vessel having an orifice size of
5 mm (millimetres) (Klein: Seifen, Ole, Fette, Wachse 94, 12
(1968)).
[0078] "Finely divided" means a powder having a majority (>50%)
of a particle size of 20 to 200 .mu.m in diameter.
[0079] "Coarse-grained" means a product having a majority (>50%)
of a particle size of 200 to 2000 .mu.m in diameter.
[0080] The term "dust-free" means that the product only contains
small fractions (<5%) of particle sizes <100 .mu.m in
diameter.
[0081] In the granulation or compacting, it is advantageous to use
customary organic or inorganic aids, or carriers, such as starch,
gelatin, cellulose derivatives or similar substances as are
customarily used in food or feed processing as binders, gelation
agents or thickeners, or of further substances, for example silicic
acids, silicates (EP-A 0 743 016) stearates.
[0082] In addition, it is advantageous to provide the surface of
the resultant granules with oils, as described in WO 04/054381. As
oils, use can be made of mineral oils, vegetable oils or mixtures
of vegetable oils. Examples of such oils are soya oil, olive oil,
soya oil/lecithin mixtures. Similarly, silicone oils, polyethylene
glycols or hydroxyethylcellulose are also suitable. Treating the
surfaces with the said oils achieves an increased abrasion
resistance of the product and a reduction of the dust fraction. The
oil content in the product is 0.02 to 2.0% by weight, preferably
0.02 to 1.0% by weight, and very particularly preferably 0.2 to
1.0% by weight, based on the total amount of the feed additive.
[0083] Preference is given to products having a fraction of
.gtoreq.97% by weight of a particle size of .gtoreq.100 to 1800
.mu.m, or a fraction of .gtoreq.95% by weight of a particle size of
.gtoreq.300 to 1800 .mu.m in diameter. The fraction of dust, that
is particles having a particle size <100 .mu.m, is preferably
>0 to 1% by weight, particularly preferably at most 0.5% by
weight.
[0084] Alternatively, the product can also be taken up on an
organic or inorganic carrier known and customary in feed
processing, for example silicic acids, silicates, meals, brans,
flours, starches, sugars or others and/or mixed and stabilized
using customary thickeners or binders. Application examples and
methods for these are described in the literature (Die
Muhle+Mischfuttertechnik 132 (1995) 49, page 817).
[0085] Finally, the product can also, by means of coating methods
using film-forming agents, for example metal carbonates, silicic
acids, silicates, alginates, stearates, starches, gums and
cellulose ethers, as described in DE-C 41 00 920, be brought into a
state in which it is stable against digestion by animal stomachs,
in particular the stomach of ruminants.
[0086] To set a desired amino acid concentration in the product,
depending on requirements, the corresponding amino acid can be
added during the method in liquid or solid form in the form of a
concentrate or if appropriate a substantially pure substance or
salt thereof. These can be added individually or as mixtures to the
resultant or concentrated fermentation broth, or else during the
drying or granulation process.
[0087] In the case of lysine, the solid product produced in this
manner has, on a fermentation broth basis, a lysine content
(calculated as lysine base) of 10% by weight to 70% by weight,
preferably 30% by weight to 60% by weight, and very particularly
preferably 40% by weight to 60% by weight, based on the total
amount of the product.
[0088] In studies it has been found that setting the pH in the
fermentation broth to .ltoreq.pH 5.2, increasing the
sulphate/lysine ratio and optionally sulphite addition of, 0.01 to
0.5% by weight in the fermentation broth after the fermentation
significantly reduces the lysine loss during the workup of the
fermentation broth.
[0089] Combination of the various measures before the workup leads
here to a synergistic effect compared with the sum of the
individual effects.
[0090] In untreated fermentation broths (no addition of one of the
additives), during the concentration to form the concentrate, an
average lysine loss of approximately 3.5% results (without
granulation step). Increasing the sulphate ratio by adding ammonium
sulphate leads at the end to an average lysine loss of
approximately 3.2%, setting the pH alone reduces the average lysine
loss to approximately 1.4%.
[0091] The combination of pH setting and increasing the sulphate
ratio shows a higher protective action for the lysine and results
in an average lysine loss of approximately 0.9%. The combination of
pH setting and sodium hydrogensulphite addition, together with the
combination of all three additives, gives an average lysine loss of
only approximately 0.6% or approximately 0.7%, respectively.
Therefore in general, lysine loss has not be taken into account
during calculating the sulfate/L-lysine ratio.
[0092] Thus it has been clearly shown that the pretreatment of
lysine-containing fermentation broth by lowering the pH, increasing
the sulphate balance and adding sulphite has a protective effect on
the lysine present. In addition, the light colour of the product
and stability under thermal stress is improved.
[0093] 1. Experimental Procedures
[0094] 1.1 Fermentation
[0095] Fermentations were carried out according to EP 0 533 039.
The granules were produced therefrom according to the method
described in EP-B 0 809 940. The granules A to D thus produced were
compared with the granules E and F produced according to the
invention. The L-lysine content of the samples was standardized and
set to approximately 51 to 52%.
[0096] 1.2 Colour Measurement
[0097] The L*a*b* colour measurement was carried out as
follows:
[0098] Principle
[0099] The 3-range calorimeter for measurement of colour and
reflectance operates by the principle described in DIN 5033,
according to which the diffuse reflection of the sample is measured
at an angle of 8.degree.. The reflected light is transmitted into
the instrument via a light guide for splitting on the exactly
defined standard colour filter. Measurement is performed against a
calibration standard.
[0100] Equipment:
[0101] Colorimeter Micro Color II LMC (manufacturer Dr. Lange)
[0102] Micro Color II Laboratory Station LDC 20
[0103] White standard LZM 076
[0104] Positioning cap
[0105] Light-protection cap O 50 mm
[0106] Powder cuvette O 34 mm
[0107] Procedure [0108] calibrate Micro Color II (in accordance
with operating instructions) [0109] select analysis programme
(.fwdarw.L*a*b*) [0110] after calibration attach positioning cap
[0111] charge product into the clean cuvette up to two thirds full
loosely [0112] carefully shake product to achieve uniform filling
[0113] clean cuvette base using a soft cloth [0114] place the
cuvette on the measurement orifice and cover using the
light-protection cap [0115] measure
[0116] Note
[0117] In the measurement of pulverulent substances, care must be
taken to ensure a uniform particle size (as small as possible).
[0118] With coarse-grained substances, duplicate measurement is
carried out.
[0119] Explanations of the L*a*b* system:
[0120] L*: black-white range
[0121] a*: red-green range
[0122] b*: yellow-blue range
[0123] 1.2.1 Colour measurement of products from comparative
experiments and products according to the invention. TABLE-US-00001
TABLE 1 Lysine pH [%] [%] [%] Sulphate/ [%] (10% in water) L* a* b*
lysine ratio A 51.4 5.6 60 9 16 0.83 B 52.3 5.7 62 8 17 0.82 C 51.3
5.6 60 8 19 0.84 D 52.5 5.3 61 9 21 0.83 E 51.6 4.6 67 8 25 0.9 F
52.5 4.5 76.0 5.0 26.0 0.95
[0124] Table 1 lists the results of the colour determination for
the comparative experiments A to D. Even without acidifying the
fermentation broth, products are obtained having an acidic pH, the
colour values of which, however, do not achieve those of the
inventive products.
[0125] Samples E and F correspond to the inventive products which
were obtained after acidifying the broth to pH 5.1, 0.2% by weight
of sodium bisulphite being additionally added to the broth used to
produce sample F.
[0126] It is found that samples E and F are markedly lighter than
the products of the prior art. The sulphate/lysine ratio is
determined from the formula
2.times.[SO.sub.4.sup.2-]/[L-lysine]=ratio.
[0127] 1.3 Product Stability after Thermal Stress
[0128] Table 2 shows the superiority of the inventive products E
and F under thermal stress in relation to lower L-lysine loss.
[0129] 1.4 Water Uptake (Hygroscopicity)
[0130] 1.4.1 Measurement of Water Uptake (Hygroscopicity-Test)
[0131] Principle: The water uptake of powdered or granulated
substances is determined by exposing them for a certain period of
time to standardized climatic conditions of 40.degree. C. and 75%
re. humidity (according to ICH). The water uptake is determined
gravimetricly. [0132] Equipment: Climatic chamber standard climate
40.degree. C./75% rel. humidity, flat weighing bottles with glass
lid (5 cm in diameter), analytical balance (readability 0.0001 g)
[0133] Prodedure: Determine the tare of the weighing bottle with
lid [0134] accurately weigh of 5 g of the homogeneous mixed
substance into weighing bottle [0135] the open weighing bottle is
stored in the climatic chamber under the following conditions:
[0136] Temperature=4.degree. C. [0137] Humdidity, rel.=75% [0138]
Time=1 h, 4 h [0139] weigh closed weighing bottle after 1 h and 4 h
calculate Calculation ##EQU1## Water .times. .times. uptake .times.
.times. ( ( A - T ) - E ) .times. 100 E .function. [ % ] ##EQU1.2##
[0140] A=weight after 1 h, 4 h in g [0141] T=tare of the weighing
bottle with lid in g [0142] E=weight substance in g [0143] Note: To
determine the course of the water uptake weigh every hour during
the first 6 hours of the test and after 24 h. [0144] Diagram:
[0145] x-axis: time in h [0146] y-axis: water uptake
[0147] 1.4.2 Water Absorption of Products from Comparative
Experiments and Products According to the Invention
[0148] FIG. 1 shows that the inventive product E absorbs lower
amounts of water/time than the products A to D according to the
prior art.
[0149] A lower hydroscopicity is of great importance for
processability. TABLE-US-00002 TABLE 2 Stability under thermal
stress (85.degree. C.). A B D E F Lys (%) Lys (%) Lys (%) Lys (%)
Lys (%) 85.degree. C. Initial 51.4 52.3 52.5 51.6 52.2 1 week 48.4
49.4 49.1 50.8 52.0 2 weeks 48.5 48.1 47.1 50.3 51.2 3 weeks 48.2
46.8 47.3 49.8 51.1 diff. % abs -3.2 -5.5 -5.2 -1.8 -1.4
[0150] 1. Production Experiments
[0151] The fermentations were carried out according to EP-B 0 533
039.
[0152] Granulation was carried out according to EP-B 0 809 940
(U.S. Pat. No. 5,840,358).
[0153] 2.1 Comparative Experiment, Prior Art
[0154] Fermentation was carried out as described in EP 0 533 039
and no biomass was separated off. The following values are obtained
(L-lysine content is calculated as content of lysine base in the
dry mass): TABLE-US-00003 TABLE 3 L-Lysine content (% by
Sulphate/lysine weight) (without water) pH ratio 57.2 7.8 0.82
[0155] 100 kg of the fermentation broth were heated to 65.degree.
C., transferred to an evaporator and concentrated there at
82.degree. C. and -0.5 bar vacuum.
[0156] The concentrated broth was granulated according to EP-B 0
809 940.
[0157] This gave an L-lysine loss of 5.1%. TABLE-US-00004 TABLE 4
L-Lysine content (% by weight) (without water) Water (%) 52.1 2
[0158] 2.2 Addition of Ammonium Sulphate and Sulphuric Acid
[0159] The specification of the fermentation broth was as follows:
TABLE-US-00005 TABLE 5 L-Lysine content (% by Sulphate/lysine
weight) (without water) pH ratio 57.7 7.5 0.9
[0160] 1.35 kg of ammonium sulphate solution (37% solids fraction)
were added to 100 kg of the fermentation broth so that the
sulphate/lysine ratio was increased to 0.93.
[0161] The pH was lowered to pH 5.2 by adding 0.54 kg of sulphuric
acid (approximately 93% strength), so that the initial L-lysine
content decreases from 57.7% to 55.7%. TABLE-US-00006 TABLE 6
L-Lysine content (% by Sulphate/lysine weight) (without water) pH
ratio 55.7 5.2 0.93
[0162] The resultant fermentation broth was heated to 55.degree. C.
and then concentrated and granulated as in example 2.1.
[0163] This gave a loss of 3.3% and thus an improvement of
approximately 35% compared with the comparative experiment.
TABLE-US-00007 TABLE 7 L-Lysine content (% by weight) (without
water) Water (%) 52.4 2.55
[0164] 2.3 Addition of Ammonium Sulphate, Sulphuric Acid and Sodium
Hydrogensulphite
[0165] The specification of the fermentation broth used was as
follows: TABLE-US-00008 TABLE 8 L-Lysine content (% by
Sulphate/lysine weight) (without water) pH ratio 57.3 7.8 0.95
[0166] The sulphate/L-lysine ratio after fermentation amounted to
0.95, so that no further ammonium sulphate was added.
[0167] 0.105 kg of sodium hydrogensulphite was added to 100 kg of
the fermentation broth, stirred for 30 min and subsequently 0.61 kg
of sulphuric acid was added, a pH of 5.2 being established.
[0168] Adding the hydrogensulphite lowers the L-lysine content to
57.0%, and the acid addition lowers it further to 55.1% owing to
the dilution effect, the amount of dry mass increases.
[0169] The specification of the fermentation broth was as follows:
TABLE-US-00009 TABLE 9 L-Lysine content (% by Sulphate/lysine
weight) (without water) pH ratio 55.1 5.2 0.96
[0170] The resultant fermentation broth was heated to 55.degree. C.
and then concentrated and granulated as in example 2.1.
TABLE-US-00010 TABLE 10 L-Lysine content (% by weight) (without
water) Water (%) 53.0 1.9
[0171] This gave an L-lysine loss of 2.1% and thus an improvement
by virtually 60% compared with the comparative experiment.
* * * * *