U.S. patent application number 10/938273 was filed with the patent office on 2005-03-10 for high-activity phytase compositions.
Invention is credited to Andela, Carl Sidonius Maria, Barendse, Rudolf Carolus Maria, Meesters, Gabriel Marinus Henricus.
Application Number | 20050054065 10/938273 |
Document ID | / |
Family ID | 8228386 |
Filed Date | 2005-03-10 |
United States Patent
Application |
20050054065 |
Kind Code |
A1 |
Barendse, Rudolf Carolus Maria ;
et al. |
March 10, 2005 |
High-activity phytase compositions
Abstract
A process is disclosed for preparing aqueous phytase-containing
liquids involving culturing microorganisms of the genus Aspergillus
or Trichoderma in a medium containing assimilable carbon and
nitrogen sources (e.g., glucose and ammonium ions), filtering the
medium, and subjecting the resulting filtrate to ultra-filtration
to give an aqueous composition having at least 14,000 FTU/g. This
aqueous liquid optimally can be used to prepare granulates that can
be incorporated in animal feedstuffs.
Inventors: |
Barendse, Rudolf Carolus Maria;
(Delft, NL) ; Meesters, Gabriel Marinus Henricus;
(Delft, NL) ; Andela, Carl Sidonius Maria; (Delft,
NL) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
3811 VALLEY CENTRE DRIVE
SUITE 500
SAN DIEGO
CA
92130-2332
US
|
Family ID: |
8228386 |
Appl. No.: |
10/938273 |
Filed: |
September 10, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10938273 |
Sep 10, 2004 |
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09089871 |
Jun 4, 1998 |
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60048611 |
Jun 4, 1997 |
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Current U.S.
Class: |
435/169 ;
426/635; 435/41 |
Current CPC
Class: |
A23K 20/158 20160501;
C12Y 302/01008 20130101; A23K 40/10 20160501; A23K 20/163 20160501;
C12Y 301/03008 20130101; Y10S 426/805 20130101; Y10S 426/807
20130101; C12Y 302/01004 20130101; A23K 50/80 20160501; C12N 9/16
20130101; Y02A 40/818 20180101; A23K 20/189 20160501; A23K 50/10
20160501; A23K 50/40 20160501; A23K 40/20 20160501; C12N 9/98
20130101; C12N 15/80 20130101; A23K 10/14 20160501; A23K 40/25
20160501; A23K 50/75 20160501 |
Class at
Publication: |
435/169 ;
435/041; 426/635 |
International
Class: |
C12P 001/00; C12P
001/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 4, 1997 |
EP |
97201641.4 |
Claims
1. A process for the preparation of an aqueous liquid comprising a
phytase, the process comprising: (a) culturing in an aqueous medium
a microorganism of the genus Aspergillus or Trichoderma having a
heterologous phytase gene under the control of a glucoamylase (for
Aspergillus) or cellobiohydrolase (for Trichoderma) promoter, under
conditions that allow recombinant expression of the phytase, where
the medium comprises, as a feed for the microorganism an
assimilable carbon source and an assimilable nitrogen source. (b)
filtering the aqueous medium to remove the microorganisms to give
an aqueous filtrate; and (c) subjecting the filtrate from (b) to
ultrafiltration to give an aqueous liquid, having a phytase
concentration of at least about 14,000 FTU/g.
2. The process according to claim 1 where the microorganism is
Aspergillus niger, Aspergillus oryzae or Trichoderma reesei.
3. The process according to claim 1 wherein the microorganism does
not possess, or does not express, a glucoamylase (AG) gene.
4. The process according to claim 1 wherein the microorganism
possesses multiple copies of the phytase gene.
5. The process according to claim 1 wherein the aqueous liquid is
substantially free of taka-amylase.
6. The process according to claim 1 wherein substantially all of
the carbon and nitrogen sources in the medium have been consumed by
the microorganisms before filtering in (b).
7. The process according to claim 1 wherein the aqueous liquid is
free of the carbon and/or nitrogen sources.
8. The process according to claim 1 wherein the phytase is
expressed in the microorganism with a glucoamylase signal
sequence.
9. The process according to claim 1 wherein neither the aqueous
filtrate or the aqueous liquid are subjected to: (a)
crystallisation; (b) a colour-removal step; or (c) crystallization
and a colour removal step.
10. The process according to claim 1 wherein the resulting aqueous
liquid has a phytase activity of about 18,000 FTU/g or more.
11. An aqueous liquid comprising a phytase at a concentration of at
least about 14,000 FTU/g.
12. The aqueous liquid according to claim 11 which is derived from
a culture medium in which the phytase was expressed.
13-38. (Canceled)
39. The aqueous liquid according to claim 11 wherein the phytase is
a fungal phytase.
40. The aqueous liquid according to claim 40 wherein the phytase is
an Aspergillus phytase or a Trichoderma phytase.
41. The aqueous liquid according to claim 11 wherein the phytase is
at a concentration of at least about 18,000 FTU/g.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority of U.S. Provisional
Patent Application 60/048,611 filed Jun. 4, 1997, now abandoned,
and European Patent Application No. 97201641.4 filed in The
Netherlands on Jun. 4, 1997. These applications hereby are
incorporated in their entireties by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the preparation and
formulation of phytase enzymes and their use to prepare granulates
for feed-enzymes in animal feeds.
BACKGROUND OF THE INVENTION
[0003] The use of various enzymes such as phytases in animal, e.g.,
livestock, feed is becoming more common. These enzymes are included
in order to improve nutrient or mineral uptake from the feed by the
animal, and may also help digestibility. They are usually produced
by culturing microorganisms in large scale fermenters operated by
industrial enzyme producers. At the end of the fermentation the
resulting "broth" is usually subjected to a series of filtration
steps to separate the biomass (the microorganisms) from the desired
enzyme (in solution). The enzyme solution is either then sold as a
liquid (often after addition of various stabilizers) or processed
to a dry formulation.
[0004] Enzyme liquid and dry formulations are used on a commercial
scale by the animal feed industry. Liquid formulations may be added
to the feed after pelleting in order to avoid heat inactivation of
the enzyme which would occur during the pelleting process.
[0005] Dry formulations usually involve steam pelleting where the
feed is subjected to steam injection(s) prior to pelleting. In the
subsequent pelleting step the feed is forced through a matrix or
die and the resulting strips are cut into suitable pellets of
variable length. During this process temperatures may rise to
60-95.degree. C.
[0006] Phytases are enzymes that (at least partially) hydrolyse
phytate (myo-inositolhexakis phosphate) to myo-inositol and
inorganic phosphate. These enzymes are found in wheat bran, plant
seeds, animal intestines and can be produced by microorganisms.
Phytases are provided in animal feeds because, as they are able to
degrade phytate, they can increase the availability of phosphorus
and other nutritional components to the animal. Phytases can also
increase the digestibility of calcium.
[0007] Phosphorus is an essential element for the growth of
organisms. For livestock, the feed is often supplemented with
inorganic phosphorus in order to obtain good growth in monogastric
animals. There is however often no need for this in feedstuffs of
ruminants because microorganisms present in the rumen produce
enzymes that catalyse the conversion of phytate to inositol and
inorganic phosphate. The degration of phytate is often desirable
because phytic acid can be anti-nutritional as it chelates useful
minerals such as calcium, zinc, magnesium and iron, and can also
react adversely with proteins thereby decreasing their
bioavailability to the animal. The addition of phytase may also
reduce the amount of inorganic feed that needs to be added, and so
less phosphorus is excreted in the manure which is better for the
environment.
[0008] The gene for various phytase enzymes have been cloned and
expressed. EP-A-0,420,358 (Gist-Brocades) describes the expression
of microbial phytases.
[0009] In a later application EP-A-0,684,313 (Hoffmann-La Roche)
describes a DNA sequence coding for various polynucleotides having
phytase activity.
[0010] EP-A-0,758,018 (Gist-Brocades) refers to methods of
improving the stability of enzymes, especially for use as animal
feeds, and refers to phytases.
[0011] WO-A-94/03612 (Alko) describes the production of phytase
degrading enzymes in Trichoderma while WO-A-97/16076 (Novo Nordisk)
describes enzyme-containing preparations for use in the manufacture
of animal feeds comprising various hydrophobic substances.
[0012] Animal feed represents one of the largest costs incurred in
keeping livestock and other animals. Furthermore, additives such as
enzymes like phytase can add significantly to the cost of animal
feed. One aim of the present invention is to be able to provide
phytase compositions that are cheaper to produce. This can be
achieved by being able to manufacture high activity or highly
concentrated phytase compositions, as provided by the present
invention.
[0013] An additional advantage in being able to make high activity
phytase compositions as provided by the present invention is that
these compositions can show a marked increase in stability,
especially during a pelleting process in the preparation of animal
feed (pellets), and so are more likely to retain the higher phytase
activity than the compositions of the prior art over time. Further
objects and advantages of the invention will be apparent from the
description herein.
BRIEF SUMMARY OF THE INVENTION
[0014] The present invention provides high-activity phytase
compositions and methods for the preparation of same. The
compositions can be employed inter alia in the preparation of
granulates that can be incorporated into animal feeds.
DETAILED DESCRIPTION OF THE INVENTION
[0015] In a first preferred aspect of the present invention there
is provided a process for the preparation of an aqueous liquid
comprising a phytase, the process preferably comprising:
[0016] (a) culturing in an aqueous medium a microorganism of the
genus Aspergillus or Trichoderma having a heterologous (i.e.,
foreign to the host) phytase gene under the control of a
glucoamylase (for Aspergillus) or cellobiohydrolase (for
Trichoderma) promoter, under conditions that allow recombinant
expression of the phytase, where the medium comprises, as a feed
for the microorganism, an assimilable carbon source and an
assimilable nitrogen source;
[0017] (b) filtering the aqueous medium to remove the
microorganisms to give an aqueous filtrate; and
[0018] (c) subjecting the filtrate from (b) to ultrafiltration to
give an aqueous liquid having a phytase concentration of at least
about 14,000 FTU/g.
[0019] This process has been found to provide a particularly high
concentration of phytase in the resulting aqueous composition. This
has allowed the preparation of other phytase compositions, also at
high activity levels, which means that not only is the process
cheaper (per unit of enzyme activity), but also, the more
concentrated phytase-containing compositions have been found to be
much more stable than their less concentrated counterparts.
[0020] The microorganisms are preferably of the species Aspergillus
niger, Aspergillus oryzae or Trichoderma reesei. For the
Aspergillus organisms, the phytase gene is suitably under the
control of a glucoamylase (or amyloglucosidase, AG) promoter. For
Trichoderma organisms, it is preferred to use a cellobiohydrolase
promoter. Desirably the promoter is one which is capable of
commanding gene expression in these respective hosts.
[0021] The assimilable carbon source can comprise glucose and/or
maltodextrin, and/or the assimilable nitrogen source can comprise
ammonium ions. The glucose and ammonium ions can be the only
assimilable carbon or nitrogen sources in the aqueous medium. That
is to say, it is contemplated that no complex carbon or nitrogen
sources are used. The ammonium ions can be provided either as
ammonia or an ammonium salt. Preferred ammonium salts include
ammonium nitrate, ammonium sulphate and ammonium phosphate.
[0022] Preferably the carbon and/or nitrogen source is supplied to
the culture medium during the fermentation process. The rate of
supply of either source can be substantially the same as it is
consumed by the microorganisms. Thus the carbon and/or nitrogen
source can be provided in a continuous or continual manner. The
carbon and nitrogen sources can be provided separately, or in the
same supply.
[0023] The resulting aqueous liquid can have a phytase
concentration of at least about 16,000, and possibly even about
18,000 or more, FTU/g. In particular, the resulting aqueous liquid
desirably comprises a phytase concentration of from about 15,000 to
about 20,000 FTU/g, and more preferably comprises a phytase
concentration of from about 16,000 to about 18,000 FTU/g. One FTU
(phytase unit) is the amount of enzyme which liberates 1 micromole
of inorganic phosphorus per minute from 0.0051 Mol/L of
sodium-phytate at pH 5.5 and 37.degree. C. FTU can be calculated
using any standard means, and, desirably, using the method of
Engelen et al., Journal of AOAC International, 77 (3), 760-764
("Simple and Rapid Determination of Phytase Activity"), and more
particularly, as set forth in Example 2. Further, phytase activity
can be determined according to the procedure "ISL-method 61696
(manual vanadate assay) which is obtainable on request from
Gist-brocades, Food Specialties, Agri Ingredients Group,
Wateringseweg 1, P.O. Box 1, 2600 MA, Delft, The Netherlands.
[0024] By using these particular organisms under these conditions
the filtrate will be relatively concentrated. This allows it to be
subjected to ultra-filtration. In some prior art methods, the
resulting filtrate contains too much debris and other substances to
allow ultra-filtration (the filter clogs). However, in the process
of the present invention, the filtrate is relatively "clean" which
allows the filtrate to be subjected to ultra-filtration, without
any further processing, and by ultra-filtration a particularly high
concentrated aqueous composition can therefore be obtained.
[0025] Prior art methods have discussed the possibility of
subjecting either the filtrate or the aqueous composition to either
crystallization and/or colour removal steps, for example charcoal
filtration. However, both of these additional steps (which would
add to the cost of producing the phytase) can be dispensed with in
the present invention.
[0026] Preferably the microorganisms do not possess, or at least do
not express, a glucoamylase gene. This means that the microorganism
can devote more energy to the production of the phytase.
[0027] The microorganism can possess multiple copies of the phytase
gene. This has been found to increase the production levels of the
phytase because there are more phytase genes to be expressed.
[0028] The aqueous composition can be substantially free of
taka-amylase (i.e., a well-known .alpha.-amylase such as described
in Toda et al., Proc. Japan Acad., 58 Ser. B 208-212 (1982)).
[0029] In the process of the invention it is preferred that
(substantially) all of the carbon and/or nitrogen sources have been
consumed by the microorganisms before the filtering in (b) takes
place. This can be achieved by allowing fermentation to continue
for some time after the last supply of carbon and/or nitrogen
sources have taken place. Alternatively, one can allow the
fermentation to continue beyond the stage when all of the carbon
and/or nitrogen sources have been added. The advantage of this, as
will be apparent, is that the aqueous composition can then be
(substantially) free of the carbon and/or nitrogen sources (e.g.,
the glucose and/or ammonium ions). Once again, this can make for a
cleaner aqueous liquid, which can contain fewer by-products. By
reducing the number of by-products one can minimise the number of
processing steps required to be able to either use the aqueous
liquid, or to be able to obtain a desired high phytase
activity.
[0030] The most preferred organism is Aspergillus niger. Also
preferred is that the phytase is expressed in a microorganism with
a glucoamylase signal sequence.
[0031] The resulting aqueous phytase-containing liquid can then be
used for a variety of purposes, although its application in animal
feeds is specifically contemplated here. A second aspect of the
invention relates to this aqueous liquid, such as preparable by a
process of the first aspect, comprising phytase at a concentration
of at least about 14,000 FTU/g, desirably from about 15,000 to
about 20,000 FTU/g, and even more preferably from about 16,000 to
about 18,000 FTU/g.
[0032] In the specification a "phytase" means not only naturally
occurring phytase enzymes, but any enzyme (i.e., produced by
recombinant, synthetic and any other means) that possesses phytase
activity, for example, the ability to catalyse the reaction
involving the removal or liberation of inorganic phosphorous
(phosphate) from myo-inositol phosphates. Preferably the phytase
will belong to the class EC 3.1.3.8. The phytase itself is
preferably a fungal phytase, such as derived from an Aspergillus or
Trichoderma species.
[0033] The invention can also provide processes for the preparation
of phytase formulations in the form of granulates that use an
edible carbohydrate polymer as a carrier. The carrier may be in
particulate or powder form. The phytase-containing aqueous liquid,
such as a solution or a slurry, can be mixed with the solid carrier
and allowed to absorb onto the carrier. During or after the mixing,
the phytase-containing liquid and the carrier are processed into a
granulate, which can then subsequently be dried. The use of the
carbohydrate carrier may allow the absorption of large amounts of
the composition (and therefore phytase). The mixture can be used to
form a plastic paste or non-elastic dough that can readily be
processed into granules, for example it is extrudable. Suitably the
carrier is non-fibrous which allows for easier granulation: fibrous
materials can prevent granulation by extrusion.
[0034] A number of prior art documents refer to pellets containing
various enzymes, but these find use as detergents, often in washing
compositions. In contrast, the present application finds use in
animal feeds and for that reason the granulates of the invention
are edible (by animals) and preferably also digestible. It
therefore is a surprising and unexpected aspect of the present
invention that the granulates, granules, and compositions of the
invention are free of soap, detergents and bleach or bleaching
compounds, zeolites, binders, fillers (TiO.sub.2, kaolin,
silicates, talc, etc.) to name but a few.
[0035] The edible carbohydrate polymer should be chosen so that it
is edible by the animal for whom the feed is intended, and
preferably digestible as well. The polymer preferably comprises
glucose (e.g., a glucose-containing polymer), or
(C.sub.6H.sub.10O.sub.5).sub.n, units. Preferably the carbohydrate
polymer comprises .alpha.-D-glucopyranose units, amylose (a linear
(1.fwdarw.4) .alpha.-D-glucan polymer) and/or amylopectin (a
branched D-glucan with .alpha.-D-(1.fwdarw.4) and
.alpha.-D-(1.fwdarw.6) linkages). Starch is the preferred
carbohydrate polymer. Other suitable glucose-containing polymers
that can be used instead of, or in addition to starch, include
.alpha.-glucans, .beta.-glucans, pectin (such as proto-pectin), and
glycogen. Derivatives of these carbohydrate polymers, such as
ethers and/or esters, thereof are also contemplated, although
gelatinised starch is often avoided. Suitably the carbohydrate
polymer is water-insoluble.
[0036] In the examples described herein corn-, potato- and
rice-starch is used. However, starch obtained from other (e.g.,
plant, such as vegetable or crop) sources such as tapioca, cassava,
wheat, maize, sago, rye, oat, barley, yam, sorghum, or arrowroot is
equally applicable. Similarly both native or modified (e.g.,
dextrin) types of starch can be used in the invention. Preferably
the carbohydrate (e.g., starch) contains little or no protein,
e.g., less than about 5% (w/w), such as less than about 2% (w/w)
preferably less than about 1% (w/w). Even more desirably the
carbohydrate contains from about 0.005% (w/w) to about 1%
(w/w).
[0037] At least about 15% (w/w) of the solid carrier can comprise
the carbohydrate polymer (such as starch). Preferably, however, at
least about 30% (w/w) of the solid carrier comprises the
carbohydrate, optimally at least about 40% (w/w). Advantageously
the major component of the solid carrier is the carbohydrate (e.g.,
starch), for example more than about 50% (w/w), preferably at least
about 60% (w/w), suitably at least about 70% (w/w), and optimally
at least about 80% (w/w). Thus, desirably the carbohydrate
comprises from about 40% to about 100% (w/w), particularly from
about 70% to about 90% (w/w) of the solid carrier. These weight
percentages are based on the total weight of the non-enzymatic
components in the final dry granulate.
[0038] The amount of phytase-containing liquid that can be absorbed
onto the carrier is usually limited by the amount of water that can
be absorbed. For natural, granular, starch this can vary between
from about 25% (w/w) to about 30% (w/w), without using elevated
temperatures (that cause the starch to swell). In practice the
percentage of enzyme liquid to be added to the carbohydrate will
often be much larger than this because the enzyme containing liquid
will usually contain a significant amount of solids. The phytase
solution can contain about 25% (w/w) solids (optimally from about
15% to about 40% (w/w), as a result of which the carbohydrate
(e.g., starch) and phytase solution can be mixed at a ratio of
carbohydrate:phytase solution of from about 0.5:1 to about 2:1,
e.g., from about 1.2:1 to about 1.6:1, such as at a ratio of about
60% (w/w):40% (w/w), respectively. Preferably the amount of liquid
added to the solid carrier is such that (substantially) all the
water in the (aqueous) liquid is absorbed by the carbohydrate
present in the solid carrier.
[0039] At elevated temperatures starch and other carbohydrate
polymers can absorb much larger amounts of water under swelling.
For this reason the carbohydrate polymer is desirably able to
absorb water (or enzyme-containing aqueous liquids). For example,
corn starch can absorb up to three times its weight of water at
60.degree. C. and up to ten times at 70.degree. C. The use of
higher temperatures in order to absorb a greater amount
enzyme-containing liquid is thus contemplated by the present
invention, and indeed is preferable especially when dealing with
thermostable phytase enzymes. For these enzymes therefore the
mixing of the solid carrier and liquid can be conducted at elevated
temperatures (e.g., above ambient temperature), such as above about
30.degree. C., preferably above about 40.degree. C., and optimally
above about 50.degree. C. In particular, depending on the
thermostability of the enzymes employed, desirably mixing can be
done at temperatures from about 30.degree. C. to about 80.degree.
C. Alternatively, or in addition, the liquid may be provided at
this temperature.
[0040] However, in general, non-swelling conditions at lower (e.g.,
ambient) temperatures are preferred to minimise activity loss
arising from instability of (heat sensitive) phytases at higher
temperatures. Suitably the temperature during the mixing of the
enzyme and the liquid is from about 20.degree. C. to about
35.degree. C., and more preferably from about 20.degree. C. to
about 25.degree. C.
[0041] The mechanical processing used in the present invention for
making the mixture of the phytase-containing liquid and the solid
carrier into granules (in other words granulating) can employ known
techniques frequently used in food, feed and enzyme formulation
processes. This can comprise expansion, extrusion, spheronisation,
pelleting, high shear granulation, drum granulation, fluid bed
agglomeration or a combination thereof. These processes are usually
characterised by an input of mechanical energy, such as the drive
of a screw, the rotation of a mixing mechanism, the pressure of a
rolling mechanism of a pelleting apparatus, the movement of
particles by a rotating bottom plate of a fluid bed agglomerator or
the movement of the particles by a gas stream, or a combination
thereof. These processes allow the solid carrier (e.g., in the form
of a powder), to be mixed with the phytase-containing liquid (an
aqueous solution or slurry), and so subsequently granulated.
[0042] In yet a further preferred embodiment of the invention the
granulate (e.g., an agglomerate) is formed by spraying or coating
the phytase-containing liquid onto the carrier, such as in a fluid
bed agglomerator. Here the resulting granules can include an
agglomerate as can be produced in a fluid bed agglomerator.
[0043] Preferably the mixing of the phytase-containing liquid and
the solid carrier additionally comprises kneading of the mixture.
This can improve the plasticity of the mixture in order to
facilitate granulation (e.g., extrusion).
[0044] If the granulate is formed by extrusion this is preferably
performed at low pressure. This can offer the advantage that the
temperature of the mixture being extruded will not increase, or
increases only slightly. Low-pressure extrusion includes extrusion
for example in a Fuji Paudal basket- or dome-extruder. Preferably
extrusion does not result in the temperature of the material being
extruded to rise above about 40.degree. C. The extrusion can
naturally produce granules (the granules can break off after
passage through a die) or a cutter can be employed.
[0045] Suitably the granules will have a water content of from
about 30% to about 40%, such as from about 33% to about 37%. The
enzyme content is preferably from about 3% to about 10%, e.g., from
about 5% to about 9%.
[0046] The granules obtained desirably can be subjected to rounding
off (e.g., spheronisation) such as in a spheromiser, e.g., a
MARUMERISER.TM. machine and/or compaction. The granules preferably
can be spheronised prior to drying since this can reduce dust
formation in the final granulate and/or can facilitate any coating
of the granulate.
[0047] The granules preferably can then be dried, such as in a
fluid bed drier or, in case of the fluid bed agglomeration,
desirably can be immediately dried (in the agglomerator) to obtain
(solid dry) granulates. Other known methods for drying granules in
the food, feed or enzyme industry can be used by the skilled
person. Suitably the granulate is flowable.
[0048] The drying preferably takes place at a temperature of from
about 25.degree. C. to about 60.degree. C., such as from about
30.degree. C. to about 50.degree. C. Here the drying may last from
about 10 minutes to about several hours, such as from about 15
minutes to about 10 hours, preferably from about 15 minutes to
about 3 hours, or desirably from about 15 minutes to about 30
minutes. The length of time required will of course depend on the
amount of granules to be dried, but as a guide this is from about 1
to about 2 seconds per kg of granules.
[0049] After drying the granules, the resulting granulate
preferably has a water content of from about 3% to about 10%, such
as from about 5% to about 9%.
[0050] A coating preferably can be applied to the granulate to give
additional (e.g., flavoured) characteristics or properties, like
low dust content, colour, protection of the enzyme from the
surrounding environment, different enzyme activities in one
granulate, or a combination thereof. The granules can be coated
with a fat, wax, polymer, salt, unguent and/or ointment or a
coating (e.g., liquid) containing a (second) enzyme, or a
combination thereof. It will be apparent that if desired several
layers of (different) coatings can be applied. To apply the
coating(s) onto the granulates a number of known methods are
available which include the use of a fluidised bed, a high shear
granulator, a mixer granulator, or a Nauta-mixer.
[0051] In other embodiments additional ingredients can be
incorporated into the granulate where desirable, e.g., as
processing aids, for further improvement of the pelleting stability
and/or the storage stability of the granulate. A number of such
preferred additives are discussed below.
[0052] Salts preferably can be included in the granulate (e.g.,
with the solid carrier or liquid). Preferably (as suggested in
EP-A-0,758,018) inorganic salt(s) can be added, which can improve
the processing and storage stability of the dry phytase
preparation. Preferred inorganic salts comprise a divalent cation,
such as zinc, magnesium, and calcium. Sulphate is the most favoured
anion.
[0053] Preferably (as suggested in EP-A-0,758,018) inorganic
salt(s) can be added, which can improve the processing and storage
stability of the dry enzyme preparation. Preferred inorganic salts
are water soluble. They can comprise a divalent cation, such as
zinc (in particular), magnesium, and calcium. Sulphate is the most
favoured anion although other anions resulting in water solubility
can be used. The salts can be added (e.g., to the mixture) in solid
form. However, the salt(s) can be dissolved in the water or
enzyme-containing liquid prior to mixing with the solid carrier.
Suitably the salt is provided at an amount that is at least about
15% (w/w based on the enzyme), such as at least about 30%. However,
it can be as high as at least about 60%, or even about 70%, for a
maximum range of from about 15% to about 70% (again, w/w based on
the enzyme). These amounts can apply either to the granules or to
the granulate. The granulate therefore can comprise less than about
12% (w/w) of the salt, for example from about 2.5% to about 7.5%
(w/w), e.g., from about 4% to about 6% (w/w).
[0054] If the salt is provided in the water then it optimally can
be in an amount of from about 5% to about 30% (w/w), such as from
about 15% to about 25% (w/w).
[0055] Further improvement of the pelleting stability can be
obtained where desirable by the incorporation of hydrophobic,
gel-forming or slow dissolving (e.g., in water) compounds. These
can be provided at from about 1% to about 10%, such as from about
2% to about 8%, and preferably from about 4% to about 6% by weight
(based on the weight of water and solid carrier ingredients).
Suitable substances include but are not limited to derivatised
celluloses, such as HPMC (hydroxy-propyl-methyl-ce- llulose), CMC
(carboxy-methyl-cellulose), HEC (hydroxy-ethyl-cellulose);
polyvinyl alcohols (PVA); and/or edible oils. Edible oils, such as
soy oil or canola oil or other appropriate oil, can be added (e.g.,
to the mixture to be granulated) as a processing aid, although as a
rule hydrophobic substances (e.g., palm oil) are preferably
absent.
[0056] Preferably the granules have a relatively narrow size
distribution (e.g., they are monodisperse). This can facilitate a
homogeneous distribution of the phytase in the granules and/or the
enzyme granulate in the animal feed. The process of the invention
tends to produce granulates with a narrow size distribution.
However, if necessary, an additional step can be included in the
process to further narrow the size distribution of the granules,
such as screening. The size distribution of the granulate is
suitably between about 100 .mu.m and about 2000 .mu.m, preferably
between about 200 .mu.m and about 1800 .mu.m, and optimally between
about 300 .mu.m and about 1600 .mu.m. The granules can be of
irregular (but preferably are regular) shape, for example,
approximately spherical.
[0057] Other suitable enzyme(s) can be included in the animal feed
which includes pet food. The function of these enzymes is often to
improve the feed conversion rate, e.g., by reducing the viscosity
or by reducing the anti-nutritional effect of certain feed
compounds. Feed enzymes can also be used where desirable, such as
to reduce the amount of compounds which are harmful to the
environment in the manure. Preferred enzymes for these purposes
include but are not limited to: carbohydrases, such as amylolytic
enzymes and plant cell wall degrading enzymes which include
cellulases such as .beta.-glucanases, hemicelluloses such as
xylanases, or galactanases; peptidases, galactosidases, pectinases,
esterases; proteases, preferably with a neutral and/or acidic pH
optimum; and lipases, preferably phospholipases such as the
mammalian pancreatic phospholipases A2. Preferably, the enzyme does
not include starch degrading enzymes (for example amylases). In
some embodiments proteases desirably can be excluded as these may
cause harm if ingested. If the enzyme is a plant cell wall
degrading enzyme, for example a cellulase, and in particular a
hemicellulose such as xylanase, then the final granulate desirably
can have an activity of the enzyme ranging from about 3,000 to
about 100,000, preferably from about 5,000 to about 80,000, and
optimally from about 8,000 to about 70,000, EXU/g. If the enzyme is
a cellulase, such as .beta.-gluconase, then the final granulate
preferably can have an enzyme activity of from about 500 to about
15,000, preferably from about 1,000 to about 10,000, and optimally
from about 1,500 to about 7,000, BGU/g.
[0058] One EXU (endoxylanase unit) is the amount of enzyme which
liberates 1 micromole of reducing sugars (measured as xylose
equivalents) per minute from a 1% xylan solution at pH 3.5 and
40.degree. C. One BGU (Betaglucanase unit) is the amount of enzyme
which liberates 0.278 micromole reducing sugars (measured as
glucose equivalents) per minute at pH 3.5 and 40.degree. C. at a
substrate concentration of 0.5% betaglucan from barley. EXU and BGU
can be calculated using any standard means, and desirably, using
the method of Engelen et al., Journal of AOAC International 79 (5),
"Viscosimetric determination of .beta.-glucanase and endoxylanase
activity in feed" (1996). Furthermore, EXU and BGU can be
calculated, respectively, according to the procedures "ISL-method
62170" (manual viscosimetric assay) and "ISL-method 62169" (manual
viscosimetric assay), both protocols of which are obtainable on
request for Gist-brocades (Delft, The Netherlands).
[0059] The granules desirably can comprise from about 5% to about
20%, e.g., from about 7% to about 15% of the enzyme(s). The enzyme
can be naturally occurring or recombinant (i.e., including
synthetic).
[0060] A preferred process according to the invention therefore
comprises:
[0061] a. mixing the aqueous phytase-containing liquid and solid
carrier comprising at least about 15% (w/w) or an edible
carbohydrate polymer, for example mixing the solid carrier with an
aqueous enzyme-containing liquid;
[0062] b. optionally kneading the resulting mixture;
[0063] c. granulating, for example by mechanical processing, the
mixture in order to obtain enzyme-containing granules, for example
by using a granulator or by extrusion;
[0064] d. optionally spheronising the granules;
[0065] e. drying the resultant granules to obtain an
enzyme-containing granulate.
[0066] During the entire process one will aim to keep the maximum
temperature to which the enzyme(s) are exposed to below about
80.degree. C. In particular, desirably mixing can be done at
temperatures of from about 30.degree. C. to about 80.degree. C.,
and drying can be done at temperatures of from about 25.degree. C.
to about 60.degree. C.
[0067] The granulates of the invention are suitable for use in the
preparation of an animal feed. At perhaps its broadest, this aspect
of the invention covers a granulate comprising a phytase and an
edible carbohydrate polymer, the granulate having an activity of at
least about 6,000 FTU/g. In particular, preferably the final
granulate has an activity of from about 5,000 to about 10,000
FTU/g, such as from about 6,000 to about 8,000 FTU/g. In such
processes, the granulates desirably are mixed with feed substances,
either as such, or as part of a premix. The characteristics of the
granulates according to the invention allows their use as a
component of a mixture which is well suited as an animal feed,
especially if the mixture is steam treated and subsequently
pelleted. The dried granules desirably can be visible or
distinguishable in such pellets.
[0068] Thus a third preferred aspect of the present invention
relates to a process for the preparation of animal feed, or a
premix or precursor to an animal feed, the process preferably
comprising mixing a composition of the second aspect with one or
more animal feed substances (e.g., seeds) or ingredients. This
desirably can then be sterilised, e.g., subjected to heat
treatment. The resulting composition is then suitably processed
into pellets.
[0069] A fourth preferred aspect of the invention relates to a
composition comprising a granulate of the second preferred aspect,
which is preferably an edible feed composition such as an animal
feed. This composition is preferably in the form of pellets (there
may be from about 1 to about 5, e.g., from about 2 to about 4,
dried granules per pellet).
[0070] Suitably the composition comprises from about 0.05 to about
2.0, such as from about 0.3 to about 1.0, optimally from about 0.4
to about 0.6 FTU/g of the phytase. A xylanase can be present at
from about 0.5 to about 50 EXU/g, e.g., from about 1 to about 40
EXU/g. Alternatively, or in addition, a cellulase can be present at
from about 0.1 to about 1.0, e.g., from about 0.2 to about 0.4
BGU/g.
[0071] The composition desirably can have a water content of from
about 10% to about 20%, e.g., from about 12% to about 15%. The
amount of enzyme(s) is suitably from about 0.0005% to about
0.0012%, such as at least about 5 ppm.
[0072] A fifth preferred aspect relates to a process for promoting
the growth of an animal, the process comprising feeding an animal
with a diet that comprises a composition of the second aspect or a
composition of the fourth aspect. Here, the animal diet can include
either the granulate itself, or the granulate present in a
feed.
[0073] A sixth preferred aspect of the present invention relates to
the use of compositions in, or as a component of, an animal feed or
for use in an animal diet.
[0074] A seventh preferred aspect of the present invention also
relates to the use of a composition comprising at least about 15%
(w/w) of an edible carbohydrate polymer as a carrier for a phytase
to improve the pelleting stability of the phytase.
[0075] Suitable animals include farm animals (e.g., pigs, poultry,
livestock and the like), non-ruminants or monogastric animals
(e.g., pigs, fowl, poultry, marine animals such as fish and the
like), ruminants (e.g., bovine or ovine, such as cows, sheep,
goats, deer, calves, lambs and the like). Poultry includes but is
not limited to chickens, hens and turkeys.
[0076] Preferred features and characteristics of one aspect of the
invention are equally applicable to another mutatis mutandis.
[0077] The following Examples are presented merely to illustrate
the invention, and are not intended, or to be construed as, being
limiting.
EXAMPLES
EXAMPLE 1
[0078] Fermentation of A. niger CBS 513.88
[0079] Aspergillus niger fungal spore preparations were made
following standard techniques.
[0080] Spores and subsequently cells were transferred through a
series of batch fermentations in Erlenmeyer flasks to a 101
fermenter. After growth in batch culture, the contents of this
fermenter were used as inoculum for a final 500 litre batch
fermentation.
[0081] The media used contains: 91 g/l corn starch (BDH chemicals);
ammonium 38 g/l glucose.H.sub.2O; 0.6 g/l MgSO.sub.4. 7H.sub.2O;
0.6 g/l KCl; 0.2 g/l FeSO.sub.4.7H.sub.2O and 12 g/l KNO.sub.3. The
pH was maintained at 4.6.+-.0.3 by automatic titration with either
4N NaOH or 4N H.sub.2SO.sub.4.
[0082] Cells were grown at 28.degree. C. at an automatically
controlled dissolved oxygen concentration at 25% air saturation.
Phytase production reached a maximum level of 5-10 U/ml after 10
days of fermentation.
[0083] The fermentation was repeated using ammonium sulphate in
place of corn starch (to give an equivalent assimilable nitrogen
content).
EXAMPLE 2
[0084] Purification and Characterization of Phytase: Phytase
Activity Assay
[0085] 100 .mu.l of broth filtrate (diluted when necessary) or
supernatant or 100 .mu.l of demiwater as reference are added to an
incubation mixture having the following composition:
[0086] 0.25M sodium acetate buffer pH 5.5, or
[0087] glycine HCl-buffer; pH 2.5
[0088] 1 mM phytic acid, sodium salt
[0089] demiwater up to 900 .mu.l
[0090] The resulting mixture was incubated for 30 minutes at
37.degree. C. The reaction was stopped by the addition of 1 ml of
10% TCA (trichloroacetic acid). After the reaction had terminated,
2 ml of reagent (3.66 g of FeSO.sub.4.7H.sub.2O in 50 ml of
ammonium molybdate solution (2.5 g
(NH.sub.4).sub.6Mo.sub.7O.sub.24.4H.sub.2O and 8 ml of
H.sub.2SO.sub.4, diluted up to 250 ml with demiwater) was
added.
[0091] The intensity of the blue colour was measured
spectro-photometrically at 750 nm. The measurements are 15
indicative of the quantity of phosphate released in relation to a
calibration curve of phosphate in the range of 0-1 mMol/l.
EXAMPLE 3
[0092] A. Phytase Expression in A. niger CBS 513.86 Transformed
with Expression Vectors Containing the A. Ficuum Phytase Gene Fused
to the Promoter and/or Signal Sequences of the A. niger
Amyloglucosidase (AG) Gene
[0093] To obtain overexpression of phytase in A. niger an
expression cassette was derived in which the A. ficuum phytase gene
was under control of the A. niger amyloglucosidase (AG) promoter in
combination a signal sequence. For the longer leader sequence the
AG promoter sequence was fused to the phytase encoding sequence
including the phytase leader sequence which was fused to the
phytase gene fragment encoding the mature protein (see for
reference Example 10 of EP-A-0,420,358).
[0094] B. Expression of the Phytase Gene Under the Control of the
AG Promoter in A. niger
[0095] The A. Niger strain CBS 513.88 (deposited 10 Oct. 1988 with
Centraal bureau Voor Schimmelcultures (CBS), Oosterstraat 1,
Postbus 273, NL-3740 AG Baam,--Netherlands) was transformed with 10
.mu.g DNA fragment by known procedures (e.g., see Example 9 of
EP-A-0,420,358). Single A. niger transformants from each expression
cassette were isolated, and spores were streaked on selective
acetamide-agar plates. Spores of each transformant were collected
from cells grown for 3 days at 37.degree. C. on 0.4%
potato-dextrose (Oxoid, England) agar plates. Phytase production
was tested in shake flasks under the following conditions:
[0096] Approximately 1.times.10.sup.8 spores were inoculated in 100
ml pre-culture medium containing (per litre): 1 g KH.sub.2PO.sub.4;
30 g maltose; 5 g yeast extract; 10 g casein-hydrolysate; 0.5 g
MgSO.sub.4.7H.sub.2O and 3 g Tween 80. The pH was adjusted to
5.5.
[0097] After growing overnight at 34.degree. C. in a rotary shaker,
1 ml of the growing culture was inoculated in a 100 ml main-culture
containing (per litre): 2 g KH.sub.2PO.sub.4; 70 g maltodextrin
(maldex MDO.sub.3, Amylum); 12.5 g yeast extract; 25 g
casein-hydrolysate; 2 g K.sub.2SO.sub.4; 5 g MgSO.sub.4.7H.sub.2O;
0.03 g ZnCl.sub.2; 0.02 g CaCl.sub.2; 0.05 MnSO.sub.4.4H.sub.2O and
FeSO.sub.4. The pH was adjusted to 5.6.
[0098] The mycelium was grown for at least 140 hours. Phytase
production was measured as described in Example 2. The fermentation
was repeated using equivalent amounts of glucose and ammonium
sulphate as the carbon and nitrogen sources. The broth was filtered
to give a filtrate which was separated from the biomass. Using the
expression cassette PFYT3 (AG-promoter/phytase leader) a maximum
phytase activity of 280 U/ml was obtained.
EXAMPLE 4
[0099] Purification of Phytase from Filtrate
[0100] The purification to obtain highly purified phytase was as
follows:
[0101] 1. Cation exchange chromatography at pH 4.9
[0102] 2. Cation exchange chromatography at pH 3.8
[0103] 3. Anion exchange chromatography at pH 6.3
[0104] 4. Ultrafiltration
[0105] 1. The phytase filtrate was diluted 20 times with water and
the pH was set at 4.9. This material was passed through a S
Sepharose Fast Flow column equilibrated with a 20 mM citric
acid/NaOH pH 4.9 buffer. The unbound material, with phytase, was
collected and used for the next step.
[0106] 2. The pH 4.9 material was brought to pH 3.8 and the phytase
was bound on a S Sepharose Fast Flow column equilibrated with 2-mM
citric acid/NaOH pH 3.8 buffer. The phytase was eluted from the
column with a 20 mM NaPO.sub.4, 50 mM NaCl pH 7.6 buffer.
[0107] 3. The pooled phytase fractions from the second cation
exchange step were pH adjusted to 6.3 and the phytase was bound
onto a Q Sepharose Fast Flow column equilibrated with a 10 mM
KPO.sub.4 pH 6.3 buffer. The phytase was eluted using a gradient to
1M NaCl in the same buffer.
1TABLE 1 Summary Purification Results Sample Purification Factor
Starting Filtrate 1 After Cation exchanger pH 4.9 1.07 After Cation
exchanger pH 3.8 1.2 After Anion exchanger 1.46
[0108] The final (anion exchanged) product containing 10 mg
protein/ml was concentrated ten-fold by ultrafiltration using an
Amicon Stirred Cell (2 L module) with a Kalle E35 membrane at 3
bar.
[0109] The final concentration for purified phytase reached 280-300
g/l (28-30%). With a specific activity of 100 FTU/mg protein, this
results in a phytase activity of 28,000-30,000 FTU/g.
EXAMPLE 5
[0110] High Activity Phytase Stability Tests
[0111] To demonstrate that a higher enzyme concentration (in
granules made using the high activity phytase liquid) gives a
higher pelleting stability, granulates with an increasing enzyme
concentration were made and the pelleting stability of these
samples were tested.
[0112] Comparative Sample A: Preparation of a Corn Starch-Based Low
Active Enzyme Granulate by Mixing, Kneading, Extrusion,
Spheronisation and Drying.
[0113] A mixture was prepared by mixing and kneading 73% (w/w) corn
starch and low concentration 4% (w/w) phytase Ultra Filtrate and
23% (w/w) water. This mixture was extruded using a Nica E-220
basket extruder to obtain a wet extrudate which was spheronised in
a Fuji Paudal Marumeriser.TM. for 2 minutes to obtain round
particles of an average diameter of 600 .mu.m. These particles were
subsequently dried in a Glatt GPCG 1.1 fluid bed dryer. The final
activity of the granulate was 610 FTU/g.
[0114] Comparative Sample B: Preparation of a Corn Starch-Based
Middle Active Enzyme Granulate by Mixing, Kneading, Extrusion,
Spheronisation and Drying.
[0115] A mixture was prepared by mixing and kneading 70% (w/w) corn
starch and 17% (w/w) phytase Ultra Filtrate and 13% (w/w) water.
This mixture was extruded using a Nica E-220 basket extruder to
obtain a wet extrudate which was spheronised in a Fuji Paudal
Marumeriser for 2 minutes to obtain round particles of an average
diameter of 600 .mu.m. These particles were subsequently dried in a
Glatt GPCG 1.1 fluid bed dryer. The final activity of the granulate
was 4170 FTU/g.
[0116] Sample C
[0117] Preparation of a Corn Starch-Based High Active Enzyme
Granulate by Mixing, Kneading, Extrusion, Spheronisation and
Drying.
[0118] A mixture was prepared by mixing and kneading 67% (w/w) corn
starch and 30% (w/w) of the phytase Ultra Filtrate prepared in
Example 4 (but diluted to 18,400 FTU/g) and 3% (w/w) water. This
mixture was extruded using a Nica E-220 basket extruder to obtain a
wet extrudate which was spheronised in a Fuji Paudal Marumeriser
for 2 minutes to obtain round particles of an average diameter of
600 .mu.m. These particles were subsequently dried in a Glatt GPCG
1.1 fluid bed dryer. The final activity of the granulate was 6830
FTU/g.
[0119] Comparison of the Pelleting Stabilities
[0120] The different enzyme granulates were subsequently placed in
a pelleting trial and their pelleting stability compared. The
pelleting trial consists of mixing the enzyme granulates with a
feed premix at respectively 1500, 320 and 200 ppm. These mixtures
were pre-treated by steam injection to give a temperature rise to
75.degree. C., after which the mixtures were pelleted in a
pelleting machine to obtain the feed pellets at a temperature of
82.degree. C., which were subsequently dried. This type of process
is typical for the feed industry to obtain feed pellets.
[0121] Table 2 summarises the results of the pelleting trials. It
is apparent that the two granules with the highest enzyme
concentration had much higher pelleting stability.
2TABLE 2 Results of the pelleting tests Granulate Temp. Temp.
Enzyme yield Sample activity of meal Pellets after pelleting number
in FTU/g (.degree. C.) (.degree. C.) (%) Comp. (A) 610 75 82 <17
Comp. (B) 4,170 75 82 37 (C) 6,830 75 82 48
EXAMPLE 6
[0122] Preparation of a Potato Starch-Based Enzyme Granulate
Containing Soy Oil and MgSO.sub.4 Additions by Mixing, Kneading,
Pelleting and Drying
[0123] In a mixer/kneader 30 kg of potato starch was added and 2.5
kg of Soy oil was mixed in. Subsequently a phytase ultra-filtrate
derived from Aspergillus (16,840 FTU/g) was added containing
MgSO.sub.4.7H.sub.2O (3.5 kg of MgSO.sub.4.7H.sub.2O was dissolved
in 14 kg of ultra-filtrate). The product was mixed thoroughly in
the kneader, then extruded and dried in a fluid bed drier as in
Example 1. This resulted in a product of 5870 FTU/g.
EXAMPLE 7
[0124] Preparation of a Rice Starch-Based Enzyme Granulate by
Mixing, Kneading, Extrusion, Spheronisation and Drying
[0125] A mixture was prepared by mixing and kneading 62% (w/w) rice
starch and 38% (w/w) of the same phytase ultra-filtrate used in
Example 6. This mixture was extruded using the Fuji Paudal basket
extruder to obtain a wet extrudate which was then spheronised in
the MARUMERISER.TM. for one minute to obtain round particles of an
average diameter of 785 .mu.m. These particles were subsequently
dried in a fluid bed drier as in Example 1. The final activity of
the granulate was 7280 FTU/g.
EXAMPLE 8
[0126] Preparation of a Corn Starch-Based Enzyme Granulate
Containing an HPMC Addition by Mixing, Kneading, Extrusion,
Spheronisation and Drying
[0127] An enzyme preparation was obtained by kneading a mixture of
54% (w/w) of corn starch, 5% of HPMC
(hydroxy-propyl-methyl-cellulose) and 41% (w/w) of the phytase
ultra-filtrate used in Example 6. This mixture was extruded using
the Fuji Paudal basket extruder to obtain a wet extrudate which was
spheronised in the MARUMERISER.TM. for one minute to obtain round
particles of an average diameter of 780 .mu.m. These were
subsequently dried in a fluid bed drier for 20 minutes at
40.degree. C. bed temperature, and 75.degree. C. inlet temperature.
The thus obtained dry enzyme granulate had an activity of 8470
FTU/g.
EXAMPLE 9
[0128] Preparation of a Corn Starch-Based Enzyme Granulate
Containing an HEC Addition by Mixing, Kneading, Extrusion,
Spheronisation and Drying
[0129] An enzyme preparation was obtained by mixing and kneading
54% (w/w) of corn starch, 5% (w/w) of HEC (hydroxy-ethyl-cellulose)
with 41% (w/w) of the same phytase ultra-filtrate used in Example
6. This mixture was extruded using the Fuji Paudal basket extruder
to obtain a wet extrudate which was spheronised in the
MARUMERISER.TM. for one minute to obtain round particles of an
average diameter of 780 .mu.m. These were subsequently dried in a
fluid bed drier for 20 minutes at 40.degree. C. bed temperature,
and 75.degree. C. inlet temperature. The thus obtained dry enzyme
granulates had an activity of 8410 FTU/g.
EXAMPLE 10
[0130] An ultrafiltrate of 18,000 FTU/g was employed, derived from
the ultrafiltrate from Example 4, and diluted.
[0131] Samples
[0132] The activity of the 3 samples prepared were 610 (A,
Comparative); 4170 (B, Comparative) and 6830 (C) FTU/g. This gave
three feeds of activity 1.153, 1.685 and 1.745 FTU/g feed,
respectively.
[0133] The first sample, 150 g was mixed with 20 kg feed as
described below. After this the premix was mixed with 80 kg feed
and divided in two parts to become feed for two trials at two
different temperatures. The second sample was 153.6 g in 20 kg
feed. This 20,153.6 g sample was divided in two equal portions of
10.076 kg. Each portion was then mixed with 230 kg feed to get the
meal for the tests.
[0134] For the third trial 96 g of granulate was mixed with 20 kg
feed and divided in two portions of 10.048 g. Each portion was then
mixed with 230 kg feed to get the meal for the tests. The pelleting
speed was 600 kg/h. The feed mixture is set out in Table 3.
3TABLE 3 Feed Mixture Corn 20.00% Wheat 30.00% Soybeans (heated)
10.00% Soy (coarse meal 46.7/3.7) 18.20% Tapioca (65% starch) 6.97%
Animal meal (56.5/10.9) 4.00% Fish meal (70.6% re) 1.00% Feather
meal, hydr. 1.00% Soy oil/maize oil 1.30% Animal fat 4.00%
Vit./Min. premix (maize) 1.00% Calcium carbonate 0.85% Mono-calcium
phosphate 1.05% Salt 0.26% L-Lysine HCl 0.16% DL-Methionine
0.21%
[0135] The three mixtures were then pelleted. The feed was fed into
a conditioner where direct steam was added to the meal. The
temperature rose to 75.degree. C. Subsequently the meal exited the
pelletiser where it was pushed through a die plate with 5 mm holes
and 65 mm thick. The temperature of the feed at this point rose
another 4.degree. C. to 79.degree. C.
[0136] The activity of the three feeds was 10.11 (A); 10.04(B) and
9.81 (C).
[0137] The results of this test for residual activity were: 63(A);
66(B) and 72%(C) respectively for the original 610; 4170; 6830 and
FTU/g samples. This shows that even with similar activities (B and
C) the highest activity formulation (C; 6830 FTU/g, of the
invention) gave a much higher pelleting stability. This was 6%
higher than for (comparative) Sample B, remarkable as only a 3%
increase was observed (from A to B) with a very large increase in
activity (610 to 4170 FTU/g).
[0138] All the references cited herein, including patents, patent
applications, and publications, are hereby incorporated in their
entireties by reference.
* * * * *