U.S. patent application number 16/149708 was filed with the patent office on 2019-04-25 for feed additive composition.
The applicant listed for this patent is DUPONT NUTRITION BIOSCIENCES APS. Invention is credited to MARION BERNARDEAU, MARI ELLEN DAVIS, SOFIA FORSSTEN, ELIZABETH ANN GALBRAITH, MAI FAURSCHOU ISAKSEN, ELIJAH G. KIARIE, SUSAN ARENT LUND, Paivi NURMINEN, DANIEL PETRI, LUIS FERNANDO ROMERO MLLAN.
Application Number | 20190117706 16/149708 |
Document ID | / |
Family ID | 48914311 |
Filed Date | 2019-04-25 |
United States Patent
Application |
20190117706 |
Kind Code |
A1 |
ISAKSEN; MAI FAURSCHOU ; et
al. |
April 25, 2019 |
FEED ADDITIVE COMPOSITION
Abstract
A feed additive composition comprising a direct fed microbial
(DFM), in combination with a xylanase (e.g.
endo-1,4-.beta.-d-xylanase) and a .beta.-glucanase (and optionally
a further fibre degrading enzyme), wherein the DFM is selected from
the group consisting of an enzyme producing strain; a C5
sugar-fermenting strain; a short-chain fatty acid-producing strain;
a fibrolytic, endogenous microflora-promoting strain; or
combinations thereof. The DFM may be selected from the group
consisting of: Bacillus subtilis AGTP BS3BP5, Bacillus subtilis
AGTP BS442, B. subtilis AGTP BS521, B. subtilis AGTP BS918,
Bacillus subtilis AGTP BS1013, B. subtilis AGTP BS1069, B. subtilis
AGTP 944, B. pumilus AGTP BS 1068 or B. pumilus KX11-1,
Enterococcus faecium ID7, Propionibacterium acidipropionici P169,
Lactobacillus rhamnosus CNCM-I-3698, Lactobacillus farciminis
CNCM-I-3699, a strain having all the characteristics thereof, any
derivative or variant thereof, and combinations thereof and the
further fibre degrading enzyme may be selected from the group
consisting of a cellobiohydrolase (E.C. 3.2.1.176 and E.C.
3.2.1.91), a .beta.-glucosidase (E.C. 3.2.1.21), a
.beta.-xylosidase (E.C. 3.2.1.37), a feruloyl esterase (E.C.
3.1.1.73), an .alpha.-arabinofuranosidase (E.C. 3.2.1.55), a
pectinase (e.g. an endopolygalacturonase (E.C. 3.2.1.15), an
exopolygalacturonase (E.C. 3.2.1.67) or a pectate lyase (E.C.
4.2.2.2)), or combinations thereof.
Inventors: |
ISAKSEN; MAI FAURSCHOU;
(Hojbjerg, DK) ; BERNARDEAU; MARION; (Caen,
FR) ; ROMERO MLLAN; LUIS FERNANDO; (Wilshire, GB)
; KIARIE; ELIJAH G.; (Guelph, CA) ; LUND; SUSAN
ARENT; (Brabrand, DK) ; NURMINEN; Paivi;
(Siuntio, FI) ; FORSSTEN; SOFIA; (Kantvik, FI)
; PETRI; DANIEL; (Waukesha, WI) ; GALBRAITH;
ELIZABETH ANN; (Wauwatosa, WI) ; DAVIS; MARI
ELLEN; (Waukesha, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DUPONT NUTRITION BIOSCIENCES APS |
Copenhagen |
|
DK |
|
|
Family ID: |
48914311 |
Appl. No.: |
16/149708 |
Filed: |
October 2, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14609911 |
Jan 30, 2015 |
|
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|
PCT/EP2013/066254 |
Aug 2, 2013 |
|
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16149708 |
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61679084 |
Aug 3, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Y 302/01008 20130101;
C12Y 302/01021 20130101; A23K 50/30 20160501; A23K 50/60 20160501;
A61K 35/742 20130101; A61K 38/47 20130101; A23K 10/18 20160501;
A23Y 2220/00 20130101; A61K 35/747 20130101; A23K 10/16 20160501;
A23K 50/75 20160501; A23K 20/189 20160501 |
International
Class: |
A61K 35/742 20060101
A61K035/742; A23K 50/60 20060101 A23K050/60; A23K 50/30 20060101
A23K050/30; A23K 50/75 20060101 A23K050/75; A23K 20/189 20060101
A23K020/189; A23K 10/18 20060101 A23K010/18; A23K 10/16 20060101
A23K010/16; A61K 35/747 20060101 A61K035/747; A61K 38/47 20060101
A61K038/47 |
Claims
1. (canceled)
2. A method for improving the performance of a subject or for
improving digestibility of a raw material in a feed, or for
improving nitrogen retention, or for improving feed conversion
ratio (FCR), or for improving weight gain in a subject, or for
improving feed efficiency in a subject, or for shifting the
fermentation process in the subject's gastrointestinal tract
towards the production of butyric acid and/or propionic acid, the
method comprising administering a direct fed microbial (DFM) to the
subject, in combination with a xylanase and a .beta.-glucanase,
wherein the DFM is selected from the group consisting of an enzyme
producing strain; a C5 sugar-fermenting strain; a short-chain fatty
acid-producing strain; a fibrolytic, endogenous
microflora-promoting strain; and combinations thereof.
3. The method of claim 2, wherein the DFM is an enzyme producing
strain.
4. The method of claim 2, wherein the DFM is a C5 sugar-fermenting
strain.
5. The method of claim 2, wherein the DFM is a short-chain fatty
acid-producing strain.
6. The method of claim 2, wherein the DFM is a fibrolytic,
endogenous microflora-promoting strain.
7. The method of claim 2, wherein the DFM is at least one strain of
bacterium selected from the group consisting of the following
genera: Bacillus, Enterococcus, Lactobacillus, Propionibacterium
and combinations thereof.
8. The method of claim 2, wherein the DFM is at least one strain
selected from the Bacillus genus.
9. The method of claim 2, wherein the DFM is selected from the
group consisting of: Bacillus subtilis AGTP BS3BP5, Bacillus
subtilis AGTP BS442, Bacillus subtilis AGTP BS521, Bacillus
subtilis AGTP BS918, Bacillus subtilis AGTP BS1013, Bacillus
subtilis AGTP BS1069, Bacillus subtilis AGTP 944, B. pumilus AGTP
BS 1068 or B. pumilus KX11-1, Enterococcus faecium ID7,
Propionibacterium acidipropionici P169, Lactobacillus rhamnosus
CNCM-I-3698, Lactobacillus farciminis CNCM-I-3699, and combinations
thereof.
10. The method of claim 2, wherein the DFM is a viable
bacterium.
11. The method of claim 2, wherein the direct fed microbial is in
the form of an endospore.
12. The method of claim 2, wherein the xylanase is an
endo-1,4-.beta.-d-xylanase.
13. (canceled)
14. The method of claim 2, wherein the method comprises
administering to a subject a direct fed microbial (DFM), in
combination with a xylanase and a .beta.-glucanase and a further
fibre degrading enzyme.
15. The method of claim 14, wherein the further fibre degrading
enzyme is selected from the group consisting of a cellobiohydrolase
(E.C. 3.2.1.176 and E.C. 3.2.1.91), a .beta.-glucosidase (E.C.
3.2.1.21), a .beta.-xylosidase (E.C. 3.2.1.37), a feruloyl esterase
(E.C. 3.1.1.73), an .alpha.-arabinofuranosidase (E.C. 3.2.1.55), a
pectinase (e.g. an endopolygalacturonase (E.C. 3.2.1.15), an
exopolygalacturonase (E.C. 3.2.1.67) or a pectate lyase (E.C.
4.2.2.2)), or combinations thereof.
16. The method of claim 15, wherein the further fibre degrading
enzyme is selected from the group consisting of a cellobiohydrolase
(E.C. 3.2.1.176 and E.C. 3.2.1.91), a .beta.-glucosidase (E.C.
3.2.1.21) or combinations thereof.
17. The method of claim 2, further comprising administering at
least one vitamin and/or at least one mineral to the subject.
18-25. (canceled)
26. The method of claim 2, wherein the DFM is selected from the
group consisting of Bacillus subtilis, B. licheniformis, B.
amyloliquefaciens, and B. pumilus.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. patent
application Ser. No. 14/609,911, filed Jan. 30, 2015, which is a
371 of International Patent Application No. PCT/EP2013/066254,
filed Aug. 2, 2013, which claims priority to U.S. Provisional
Patent Application No. 61/679,084, which was filed on Aug. 3, 2012,
and all of which are herein incorporated by reference.
FIELD OF INVENTION
[0002] The present invention relates to methods for improving feed
compositions using a specific direct fed microbial in combination
with a xylanase and a .beta.-glucanase, and to a feed additive
composition comprising a direct fed microbial in combination with a
xylanase and a .beta.-glucanase. The present invention further
relates to uses and kits.
BACKGROUND OF THE INVENTION
[0003] Supplemental enzymes are used as additives to animal feed,
particularly poultry and swine feeds, as a means to improve
nutrient utilization and production performance characteristics.
Enzyme blends are available to improve the nutritional value of
diets containing cereal grains, soybean meal, animal protein meals,
or high fibre food and industrial by-products.
[0004] The concept of direct fed microbials (DFM) involves the
feeding of live beneficial microbes to animals like chickens or
pigs, such that when administered in adequate amounts confer a
health benefit on the host. Probiotics is another term for this
category of feed additives. Probiotics or DFM have been shown to
improve animal performance in controlled studies. DFM includes
direct fed bacteria and or yeast-based products.
[0005] Although combinations of DFMs with some enzymes have been
contemplated, the interaction between DFMs and enzymes has never
been fully understood. The present invention relates to novel
specific combinations which surprisingly significantly improve
production performance characteristics of animals.
[0006] Continued pressure on global feed grain markets has resulted
in trends for the swine and poultry industries to seek alternative
cost-effective ingredient options such as co-products (by-products)
from the biofuel and milling industries. However, a characteristic
of alternative ingredients is the high content of non-starch
polysaccharides (NSP; fibre) which for the non-ruminants, are of
low nutritive value as they are indigestible, limit the nutrient
intake of an animal and negatively influence energy and nutrient
utilization. It follows that successful application of alternative
fibrous ingredients in monogastric diets will be dependent on the
availability of technologies for efficiently utilizing the energy
contained in the dietary fibre, mitigating risks associated with
their anti-nutritional properties and potential economic benefits
when formulated correctly into diets.
SUMMARY OF INVENTION
[0007] A seminal finding of the present invention is that the
degradation of dietary material derived from plant cell wall
particles which is high in non-starch polysaccharides (NSP) by
xylanases can be optimized for improved animal performance when
combining xylanase and a .beta.-glucanase with one or more specific
direct fed-microbials (DFMs) selected for their capacity to digest
plant cell wall structural carbohydrates and/or their capacity of
producing Short Chain Fatty Acids (SCFA) from pentoses (e.g.
arabinoxylans) contained in the NSP fraction of ingredients in
anaerobic conditions.
[0008] The reason why this combination improves performance is that
the solubilisation of fibre, specifically hemicellulose, from the
diet is maximized in the gastro intestinal tract (GIT) of the
animals. This solubilisation of hemicellulose would not always be
sufficient to increase performance because C5-sugars released are
not an efficient source of energy for animals when they are
absorbed (Savory C., J. Br. J. Nut. 1992, 67: 103-114), but they
are a more efficient source of energy when converted into short
chain fatty acids (SCFA) either by microorganisms in the GIT or by
DFMs.
[0009] Therefore the energy value from plant products (e.g. wheat,
corn, oats, barley and cereals co-products (by-products) or mixed
grain diet readily accessible for monogastrics) can be optimized by
combining xylanase and a .beta.-glucanase and specific DFMs that
can either produce SCFAs from NSP fraction pentoses in anaerobic
conditions or that can modulate the microbial populations in the
GIT to increase SCFA production from the sugars released. The DFMs
may adapt their metabolism to synergistically increase the fibre
hydrolysis in combination with xylanase and .beta.-glucanase. Using
DFMs with fibrolytic enzymes can provide additional benefits and
maximize the benefits of the carbohydrases.
[0010] Specific DFMs selected for their enzymatic activities can be
considered as a glycan-driven bacterial food chain. The
specifically selected DFMs taught herein may preferentially utilize
dietary fibres, a trait that allows them to carry out the initial
glycan digestion steps to liberate shorter, more soluble
polysaccharides for other bacteria, e.g. other endogenous GIT
microflora. The specific DFMs have been selected for their
metabolism which adjusts according to the glycans released by
enzymes (e.g. xylanase and .beta.-glucanase) to improve the
efficacy of the enzymes taught herein and the DFM(s) combination
compared to use of a combination of enzymes alone or the use of
DFM(s) alone.
[0011] Without wishing to be bound by theory, in the present
invention dietary material derived from plant cell wall particles
which is rich in source-specific glycans, such as cellulose,
hemicellulose and pectin (plant material) or glycosaminoglycans
enter the distal gut in particulate forms that are attacked by the
specific DFMs glycan degraders which are capable of directly
binding to these insoluble particles and digesting their glycan
components. After this initial degradation of glycan-containing
particles, more-soluble glycan fragments can be digested by
secondary glycan degraders present in the caecum, which contribute
to the liberated pool of short-chain fatty acid (SCFA) fermentation
products that is derived from both types of degraders. As SCFAs
arise from carbohydrate fermentation and/or protein fermentation
and deamination by the indigenous anaerobic microflora in the GIT,
SCFA concentration can be an index of the anaerobic-organism
population. SCFA may actually provide a number of benefits to the
host animal, acting as metabolic fuel for intestine, muscle,
kidney, heart, liver and brain tissue, and also affording
bacteriostatic and bacteriocidal properties against organisms such
as Salmonella and E. coli.
[0012] The nutritional value of fibre in non-ruminants can mainly
be derived through short chain fatty acids (SCFA) production via
fermentation of solubilized or degraded fibres by effective fibre
degrading enzymes (e.g. a xylanase and a .beta.-glucanase, suitably
in combination with a further fibre degrading enzyme). Feed
xylanase alone is not enough to use fibrous ingredients in animal
(especially non-ruminant) diets. A large array of chemical
characteristics exists among plant-based feed ingredients. An
enzyme application depends on the characteristics of the plant
(feed) material. By way of example only, in wheat grain
arabinoxylans predominates, however in wheat middlings (a
co-product or by-product of wheat milling), the content of
.beta.-glucan increases from 8 g.sup.-1 DM (in grain) to an excess
of 26 g kg.sup.-1 DM.
[0013] SCFAs have different energy values and some can serve as
precursors of glucose and some can contribute to the maintenance of
intestinal integrity and health. The inventors have found that the
specific combinations taught herein preferentially move the
fermentation process in an animal's GIT towards the production of
more valuable/useful SCFA's such as butyric acid and/or propionic
acids.
[0014] In one aspect, the present invention provides a feed
additive composition comprising a direct fed microbial (DFM), in
combination with a xylanase and a .beta.-glucanase, wherein the DFM
is selected from the group consisting of an enzyme producing
strain; a C5 sugar-fermenting strain; a short-chain fatty
acid-producing strain; a fibrolytic, endogenous
microflora-promoting strain; or combinations thereof.
[0015] The present invention further provides a method for: [0016]
i) improving the performance of a subject, or [0017] ii) for
improving digestibility of a raw material in a feed (e.g. nutrient
digestibility, such as amino acid digestibility), or [0018] iii)
for improving nitrogen retention, or [0019] iv) for improving feed
conversion ratio (FCR), or [0020] v) for improving weight gain in a
subject, or [0021] vi) for improving feed efficiency in a subject,
or [0022] vii) for shifting the fermentation process in the
subject's gastrointestinal tract towards the production of butyric
acid and/or propionic acid, which method comprising administering
to a subject a direct fed microbial (DFM), in combination with a
xylanase and a .beta.-glucanase, wherein the DFM is selected from
the group consisting of an enzyme producing strain; a C5
sugar-fermenting strain; a short-chain fatty acid-producing strain;
a fibrolytic, endogenous microflora-promoting strain; or
combinations thereof.
[0023] The present invention yet further provides a premix
comprising a feed additive composition according to the present
invention or a direct fed microbial (DFM), a xylanase and a
.beta.-glucanase, wherein the DFM is selected from the group
consisting of an enzyme producing strain; a C5 sugar-fermenting
strain; a short-chain fatty acid-producing strain; a fibrolytic,
endogenous microflora-promoting strain; or combinations thereof,
and at least one vitamin and/or at least one mineral.
[0024] In a yet further aspect, the present invention provides a
feed comprising a feed additive composition according to the
present invention or a premix according to the present
invention.
[0025] The present invention yet further provides a feed comprising
a direct fed microbial (DFM), in combination with a xylanase and a
.beta.-glucanase, wherein the DFM is selected from the group
consisting of an enzyme producing strain; a C5 sugar-fermenting
strain; a short-chain fatty acid-producing strain; a fibrolytic,
endogenous microflora-promoting strain; or combinations
thereof.
[0026] In another aspect, there is provided a method of preparing a
feedstuff comprising admixing a feed component with a feed additive
composition according to the present invention or a premix
according to the present invention.
[0027] A further aspect of the present invention is a method of
preparing a feedstuff comprising admixing a feed component with a
direct fed microbial (DFM), in combination with a xylanase and a
.beta.-glucanase, wherein the DFM is selected from the group
consisting of an enzyme producing strain; a C5 sugar-fermenting
strain; a short-chain fatty acid-producing strain; a fibrolytic,
endogenous microflora-promoting strain; or combinations
thereof.
[0028] The present invention yet further provides use of a direct
fed microbial (DFM), in combination with a xylanase and a
.beta.-glucanase, wherein the DFM is selected from the group
consisting of an enzyme producing strain; a C5 sugar-fermenting
strain; a short-chain fatty acid-producing strain; a fibrolytic,
endogenous microflora-promoting strain; or combinations thereof:
[0029] i) for improving the performance of a subject, or [0030] ii)
for improving digestibility of a raw material in a feed (e.g.
nutrient digestibility, such as amino acid digestibility), or
[0031] iii) for improving nitrogen retention), or [0032] iv) for
improving feed conversion ratio (FCR), or [0033] v) for improving
weight gain in a subject, or [0034] vi) for improving feed
efficiency in a subject, or [0035] vii) for shifting the
fermentation process in the subject's gastrointestinal tract
towards the production of butyric acid and/or propionic acid.
[0036] A further aspect relates to a kit comprising a direct fed
microbial (DFM), a xylanase and a .beta.-glucanase, wherein the DFM
is selected from the group consisting of an enzyme producing
strain; a C5 sugar-fermenting strain; a short-chain fatty
acid-producing strain; a fibrolytic, endogenous
microflora-promoting strain; or combinations thereof (and
optionally at least one vitamin and/or optionally at least one
mineral) and instructions for administration.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 shows the effects of xylanase and .beta.-glucanase
without or with Bacillus direct fed microbial (DFM) on fecal
lactobacillus and E. coli counts (log transformed colony forming
unit/gram of feces, Log 10 cfu/g).
DETAILED DESCRIPTION OF THE INVENTION
[0038] Preferably the enzyme(s) used in the present invention are
exogenous to the DFM. In other words the enzyme(s) are preferably
added to or admixed with the DFM.
[0039] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR
BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale
& Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper
Perennial, N.Y. (1991) provide one of skill with a general
dictionary of many of the terms used in this disclosure.
[0040] This disclosure is not limited by the exemplary methods and
materials disclosed herein, and any methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of embodiments of this disclosure. Numeric ranges are
inclusive of the numbers defining the range. Unless otherwise
indicated, any nucleic acid sequences are written left to right in
5' to 3' orientation; amino acid sequences are written left to
right in amino to carboxy orientation, respectively.
[0041] The headings provided herein are not limitations of the
various aspects or embodiments of this disclosure which can be had
by reference to the specification as a whole. Accordingly, the
terms defined immediately below are more fully defined by reference
to the specification as a whole.
[0042] Amino acids are referred to herein using the name of the
amino acid, the three letter abbreviation or the single letter
abbreviation.
[0043] The term "protein", as used herein, includes proteins,
polypeptides, and peptides.
[0044] As used herein, the term "amino acid sequence" is synonymous
with the term "polypeptide" and/or the term "protein". In some
instances, the term "amino acid sequence" is synonymous with the
term "peptide". In some instances, the term "amino acid sequence"
is synonymous with the term "enzyme".
[0045] The terms "protein" and "polypeptide" are used
interchangeably herein. In the present disclosure and claims, the
conventional one-letter and three-letter codes for amino acid
residues may be used. The 3-letter code for amino acids as defined
in conformity with the IUPACIUB Joint Commission on Biochemical
Nomenclature (JCBN). It is also understood that a polypeptide may
be coded for by more than one nucleotide sequence due to the
degeneracy of the genetic code.
[0046] All E.C. enzyme classifications referred to herein relate to
the classifications provided in Enzyme
Nomenclature--Recommendations (1992) of the nomenclature committee
of the International Union of Biochemistry and Molecular
Biology--ISBN 0-12-226164-3.
[0047] Other definitions of terms may appear throughout the
specification. Before the exemplary embodiments are described in
more detail, it is to understand that this disclosure is not
limited to particular embodiments described, as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting, since the scope of the
present disclosure will be limited only by the appended claims.
[0048] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within this disclosure. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within this disclosure, subject to any specifically excluded limit
in the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in this disclosure.
[0049] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "an enzyme" includes a plurality of such
candidate agents and reference to "the feed" includes reference to
one or more feeds and equivalents thereof known to those skilled in
the art, and so forth.
[0050] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
such publications constitute prior art to the claims appended
hereto.
[0051] The enzymes for use in the present invention can be produced
either by solid or submerged culture, including batch, fed-batch
and continuous-flow processes. Culturing is accomplished in a
growth medium comprising an aqueous mineral salts medium, organic
growth factors, the carbon and energy source material, molecular
oxygen, and, of course, a starting inoculum of one or more
particular microorganism species to be employed.
[0052] The DFM for use in the present invention may be an enzyme
producing strain.
[0053] The DFM for use in the present invention may be a C5
sugar-fermenting strain.
[0054] The DFM for use in the present invention may be a
short-chain fatty acid-producing strain.
[0055] The DFM for use in the present invention may be a
fibrolytic, endogenous microflora-promoting strain.
[0056] The enzyme producing strain and/or the C-5 sugar-fermenting
strain and/or the short-chain fatty acid-producing strains and/or
the fibrolytic, endogenous microflora-promoting strain according to
the present invention may be selected from the group consisting of
the following genera: Bacillus, Enterococcus, Lactobacillus,
Propionibacterium and combinations thereof.
[0057] The enzyme producing strain and/or the C-5 sugar-fermenting
strain and/or the short-chain fatty acid-producing strains and/or
the fibrolytic, endogenous microflora-promoting strain according to
the present invention may be at least one strain selected from the
Bacillus genus, particularly Bacillus subtilis, B. licheniformis,
B. amyloliquefaciens or B. pumilus.
[0058] The enzyme producing strain and/or the C-5 sugar-fermenting
strain and/or the short-chain fatty acid-producing strains and/or
the fibrolytic, endogenous microflora-promoting strain according to
the present invention may be at least one strain selected from the
Enterococcus genus, particularly Enterococcus faecium.
[0059] The enzyme producing strain and/or the C-5 sugar-fermenting
strain and/or the short-chain fatty acid-producing strains and/or
the fibrolytic, endogenous microflora-promoting strain according to
the present invention may be selected from the group consisting of:
Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP BS442, B.
subtilis AGTP BS521, B. subtilis AGTP BS918, Bacillus subtilis AGTP
BS1013, B. subtilis AGTP BS1069, B. subtilis AGTP 944, Bacillus
subtilis BS 2084 (NRRL B-50013), Bacillus subtilis LSSAO1 (NRRL
B-50104), Bacillus subtilis 3A-P4 (PTA-6506), Bacillus subtilis
22C-P1 (PTA-6508), Bacillus licheniformis BL21 (NRRL B-50134),
Bacillus subtilis BS-27 (NRRL B-50105), Bacillus subtilis BS18
(NRRL B-50633), Bacillus subtilis 15A-P4 (PTA-6507), Bacillus
subtilis BS278 (NRRL B-50634), Bacillus licheniformis BL842 (NRRL
B-50516), B. pumilus AGTP BS 1068, B. pumilus KX11-1, Enterococcus
faecium ID7, Propionibacterium acidipropionici P169, Lactobacillus
rhamnosus CNCM-I-3698, Lactobacillus farciminis CNCM-I-3699, or a
strain having all the characteristics thereof, any derivative or
variant thereof, and combinations thereof.
[0060] The enzyme producing strain and/or the C-5 sugar-fermenting
strain and/or the short-chain fatty acid-producing strains and/or
the fibrolytic, endogenous microflora-promoting strain for use in
the present invention is preferably a viable bacterium.
[0061] The enzyme producing strain and/or the C-5 sugar-fermenting
strain and/or the short-chain fatty acid-producing strains and/or
the fibrolytic, endogenous microflora-promoting strain for use in
the present invention may be in the form of an endospore.
[0062] The xylanase for use in the present invention is preferably
an endo-1,4-.beta.-d-xylanase (E.C. 3.2.1.8).
[0063] In some embodiments preferably the xylanase and the
.beta.-glucanase are used in combination with at least one further
fibre degrading enzyme. The (further) fibre degrading enzyme may be
selected from the group consisting of a cellobiohydrolase (E.C.
3.2.1.176 and E.C. 3.2.1.91), a .beta.-glucosidase (E.C. 3.2.1.21),
a .beta.-xylosidase (E.C. 3.2.1.37), a feruloyl esterase (E.C.
3.1.1.73), an .alpha.-arabinofuranosidase (E.C. 3.2.1.55), a
pectinase (e.g. an endopolygalacturonase (E.C. 3.2.1.15), an
exopolygalacturonase (E.C. 3.2.1.67) or a pectate lyase (E.C.
4.2.2.2)), or combinations thereof.
[0064] Suitably there may be more than one further fibre degrading
enzyme, suitably more than two, suitably more than three, suitably
more than four, suitably more than five.
[0065] Suitably the feed additive composition according to the
present invention or the composition comprising a DFM in
combination with a xylanase, a .beta.-glucanase and at least one
further degrading enzyme move the fermentation process in the
subject's gastrointestinal tract towards the production of butyric
acid and/or propionic acid.
[0066] Direct Fed Microbial (DFM)
[0067] The term "microbial" herein is used interchangeably with
"microorganism".
[0068] The DFM for use in the present invention may be any suitable
DFM which is an "enzyme producing strain"--such as an enzyme
producing Bacillus strain. To determine if a DFM is an "enzyme
producing strain" the DFM assay defined herein as "enzyme producing
DFM assay" may be used. A DFM is considered to be an enzyme
producing DFM if it is classed as an enzyme producing DFM using the
"enzyme producing DFM assay" taught herein.
[0069] The DFM for use in the present invention may be any suitable
DFM which is a "C5 sugar-fermenting strain". To determine if a DFM
is a "C5 sugar-fermenting strain" the DFM assay defined herein as
"C5 sugar-fermenting DFM assay" may be used. A DFM is considered to
be a C5 sugar-fermenting DFM if it is classed as C5 sugar
fermenting using the "C5 sugar-fermenting DFM assay" taught
herein.
[0070] The DFM for use in the present invention may be any suitable
DFM which is a "short chain fatty acid (SCFA)-producing strain". To
determine if a DFM is a "SCFA-producing strain" the DFM assay
defined herein as "SCFA-producing DFM assay" may be used. A DFM is
considered to be a SCFA-producing DFM if it is classed as SCFA
producing using the "SCFA-producing DFM assay" taught herein.
[0071] The DFM for use in the in present invention may be any
suitable DFM which is a "fibrolytic, endogenous
microflora-promoting strain". To determine if a DFM is a
"fibrolytic, endogenous microflora-promoting strain" the DFM assay
defined herein as "fibrolytic, endogenous microflora-promoting DFM
assay" may be used. A DFM is considered to be a fibrolytic,
endogenous microflora-promoting DFM if it promotes or stimulates
endogenous fibrolytic microflora using the assay taught herein.
[0072] The DFM for use in the present invention may be any suitable
DFM which is an "enzyme producing strain", a "C5 sugar-fermenting
strain", a "SCFA-producing strain", a "fibrolytic, endogenous
microflora-promoting strain" or combinations thereof.
[0073] Suitably the DFM for use in the present invention may be a
DFM which is a strain that would be classified as being an "enzyme
producing strain" and/or a "C5 sugar-fermenting strain" and/or a
"SCFA-producing strain" and/or a "fibrolytic, endogenous
microflora-promoting strain". Suitably the DFM may be a strain that
is classified as having more than one type of activity, e.g. at
least 2, suitably at least 3, suitably all 4 activities, e.g.
enzyme producing activity, C5 sugar-fermenting activity,
SCFA-producing activity and/or fibrolytic, endogenous
microflora-promoting activity.
[0074] The DFMs according to the present invention provide benefits
to animals fed high levels of high-fibre plant by-products, such as
dried distillers grains with solubles (DDGS).
[0075] Enzyme Producing DFM Assay:
[0076] High-throughput screening of these test strains was
performed by replicate spot plating of 2 microliters liquid culture
onto 15.0 ml of various substrate media types of interest in
100.times.100.times.15 mm grid plates. Cellulase, .alpha.-amylase,
zeinase, soy protease, esterase, lipase and xylanase activities
were determined based on specific substrate utilization by the
individual strains. Media components used to assay the substrate
utilization properties from enzymatic activity of the
environmentally derived strains are described in Table 1. Assay
plates were left to dry for 30 minutes following culture
application, and then incubated at 32.degree. C. for 24 hours.
Enzymatic activities for each strain were determined by measuring
the zone of substrate degradation in millimeters, as indicated by
clearing of the surrounding edge of colony growth. Mean values from
replicate plates were recorded.
TABLE-US-00001 TABLE 1 Media components used to assay the enzymatic
activities illustrated by substrate utilization properties of
environmentally derived Bacillus. Plate Extra Visualization Assay
Media Composition Requirements .alpha.-Amylase Nutrient Agar, 2%
Corn Starch .05% Iodine Stain Solution Soy Nutrient agar, 2%
Purified Soy Protein None; Measure Zone of Protease Clearing in
opaque media Cellulase 0.1% Ammonium Sulfate, 0.1% Potassium 30
minute 0.05% Congo Phosphate Dibasic, 0.1% Yeast Extract, 1.0% Red
Dye stain, followed by Polypeptone, 1.5% Agar, 0.75% 1M NaCl rinse.
Carboxymethyl Cellulose (CMC) Esterase/Lipase 1.0% Polypeptone,
1.5% Agar, 0.5% Yeast None; Measure Zone of Extract, 1.5% Tween 80,
1.5% Tributyrin, Clearing in opaque media 0.01% Victoria Blue B Dye
(filtered). Zeinase Nutrient Agar, 2% Purified Zein, solubilized in
None; Measure Zone of 70% methanol Clearing in opaque media
Xylanase Nutrient Agar, 2% Xylan None; Measure Zone of Clearing in
opaque media
[0077] In one embodiment the enzyme producing strain produces one
or more the following enzyme activities: cellulase activity,
.alpha.-amylase activity, xylanase activity, esterase activity,
lipase activity, .beta.-mannanase activity, protease activity (e.g.
zeinase or soy protease activity) and combinations thereof.
[0078] In one embodiment preferably the enzyme producing strain
produced one or more of the following enzyme activities: cellulose
activity, xylanase activity .beta.-mannanase activity, or
combinations thereof.
[0079] In one embodiment the enzyme producing DFM is a strain
selected from the group consisting of the species Bacillus
subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus
amyloliquefaciens or mixtures thereof.
[0080] In one embodiment preferably the enzyme producing DFM strain
is selected from the group consisting of:
[0081] Bacillus subtilis AGTP BS3BP5 (NRRL B-50510),
[0082] Bacillus subtilis AGTP BS442 (NRRL B-50542),
[0083] Bacillus subtilis AGTP BS521 (NRRL B-50545),
[0084] Bacillus subtilis AGTP BS918 (NRRL B-50508),
[0085] Bacillus subtilis AGTP BS1013 (NRRL B-50509),
[0086] Bacillus pumilus AGTP BS 1068 (NRRL B-50543),
[0087] Bacillus subtilis AGTP BS1069 (NRRL B-50544),
[0088] Bacillus subtilis AGTP 944 (NRRL B-50548),
[0089] Bacillus pumilus AGTP KXII-1 (NRRL B-50546),
[0090] Bacillus subtilis 15A-P4 (PTA-6507),
[0091] Bacillus subtilis BS 2084 (NRRL B-50013),
[0092] Bacillus subtilis LSSAO1 (NRRL B-50104),
[0093] Bacillus subtilis 3A-P4 (PTA-6506),
[0094] Bacillus subtilis 22C-P1 (PTA-6508),
[0095] Bacillus licheniformis BL21 (NRRL B-50134),
[0096] Bacillus subtilis BS-27 (NRRL B-50105),
[0097] Bacillus subtilis BS18 (NRRL B-50633),
[0098] Bacillus subtilis BS278 (NRRL B-50634),
[0099] Bacillus licheniformis BL842 (NRRL B-50516).
[0100] or any derivative or variant thereof,
[0101] and combinations thereof.
[0102] The enzyme producing strain of DFM may be one or more of the
strains taught in U.S. 61/527,371 and U.S. 61/526,881, both of
which are incorporated herein by reference.
[0103] C5 Sugar Fermenting DFM Assay:
[0104] Bacillus strains are grown overnight on plates of Tryptic
soy agar (Difco) at 32.degree. C., and lactic acid bacteria are
grown overnight on MRS agar (Difco) under anaerobic conditions at
37.degree. C. API 50 CHB and API 50 CHL media (bioMerieux, Marcy
l'Etoile, France) are inoculated with pure culture DFM (either
Bacillus or lactic acid bacteria respectively) and applied to API
50CH.RTM. strips as per manufacturer's instructions. Strips are
incubated at 32.degree. C. (Bacillus) or 37.degree. C. under
anaerobic conditions (lactic acid bacteria) and monitored at 24 and
48 hours for colorimetric changes.
[0105] There term "C5 sugar" as used herein means any sugar having
5 carbons. C5 sugars may be referred to herein as pentoses.
[0106] The C5 sugars include D-arabinose, L-arabinose, D-ribose,
D-xylose and L-xylose.
[0107] In one embodiment the C5 sugar-fermenting strain of DFM is
selected from the group consisting of:
[0108] Bacillus subtilis 15A-P4 (PTA-6507)
[0109] Bacillus subtilis AGTP BS918 (NRRL B-50508)
[0110] Bacillus subtilis BS 2084 (NRRL B-50013)
[0111] Bacillus subtilis LSSAO1 (NRRL B-50104)
[0112] Enterococcus faecium ID7
[0113] Lactobacillus lactis DJ6 (PTA 6102)
[0114] Lactococcus lactis ID7 (PTA 6103),
[0115] or combinations thereof.
[0116] Short Chain Fatty Acid (SCFA)-Producing DFM Assay:
[0117] A 1% vol/vol inoculum of a 48 hr culture of a DFM is used to
inoculate 10 ml tubes of modified Sodium Lactate Broth (NLB) (1%
sodium lactate; Sigma-Aldrich, St Louis, Mo.; 1% tryptone; Oxoid
Ltd., Hampshire, England, 0.5% yeast extract; Oxoid Ltd. and 0.5%
KH.sub.2PO.sub.4) devoid of sodium lactate and supplemented with a
commensurate amount (1% wt/vol) of one of nine different
carbohydrates (lactate, glucose, galactose, arabinose, sucrose,
starch, xylose, cellobiose, fructose; Sigma-Aldrich, St. Louis,
Mo.). Cultures are grown under anaerobic conditions at 32.degree.
C., and after 0, 24, 48, and 72 hours of incubation, duplicate
tubes are centrifuged at 5000.times.g for 10 min and spent broth
collected from each culture. Production of short chain fatty acids
in the spent broth was measured via high performance liquid
chromatography (HPLC). Duplicate 1 ml samples of spent culture
broth are removed from each sampling tube and mixed with 10 ml
0.005M H.sub.2SO.sub.4. Three mis of each diluted sample are
filtered through a 0.2 micron filter into HPLC vials and capped.
Samples are analysed for acetate, lactate, propionic acid, and
butyric acid with a Waters 2695 separation module (Milford, Ma)
using a 300.times.7.8 mm Bio-Rad (Hercules, Calif.) Aminex HPX-87H
column. All analytes are detected with a Waters 2410 RI
detector.
[0118] In one embodiment the short chain fatty acid
(SCFA)-producing strain may be Propionibacterium acidipropionici
P169.
[0119] In another embodiment the short chain fatty acid
(SCFA)-producing strain may be Enterococcus faecium ID7.
[0120] The term "short chain fatty acid" as used herein includes
volatile fatty acids as well as lactic acid.
[0121] In one embodiment the SCFA may be selected from the group
consisting of: acetic acid, propionic acid, butyric acid,
isobutyric acid, valeric acid, isovaleric acid, 2-methylbutyric
acids and lactic acid.
[0122] In one embodiment the SCFA may be butyric acid.
[0123] Fibrolytic, Endogenous Microflora-Promoting DFM Assay:
[0124] A pen trial is conducted to determine the effects of a DFM
on broiler chickens compared to a control without DFM. Samples are
collected on days 11 and 42 of the trial. At each sampling date one
bird is collected from each pen for a total of eight birds per
treatment. Birds are euthanized and the total gastrointestinal
tract (GIT) from below the gizzard to the ileal-cecal junction is
collected from each bird. Cecal samples from each bird are sliced
open and digesta and cecal tissue are collected in a whirl-pak bag
and masticated in 99 ml of 0.1% peptone at 7.0 strokes/s for 60
seconds to release mucosa-associated bacterial cells from the cecal
tissue. Aliquots of the masticated solution containing bacteria
from the cecal mucosa and digesta are flash-frozen in liquid
nitrogen and stored at -20.degree. C. until further analysis.
Genomic DNA is isolated from 250 .mu.l of each sample by phenol
chloroform extraction and purified using Roche Applied Science High
Pure PCR Template Purification Kit (Roche Diagnostics Corp.,
Indianapolis, Ind.). DNA from two birds per treatment is pooled in
equal amounts and submitted for pyrosequencing as a single sample,
resulting in four samples per treatment from each age. Bacterial
tag-encoded FLX amplicon pyrosequencing is performed as described
previously (Dowd, et al BMC Microbiol. 2008 Jul. 24; 8:125). The
V1-V3 region of the 16S rRNA gene is amplified in each pooled
sample using the primers 28 F (5'-GAGTTTGATCNTGGCTCAG) and 519R
(5'-GTNTTACNGCGGCKGCTG). Pyrosequencing data is processed and
analysed using the Qiime v.1.4.0. software pipeline. Briefly, raw
sequence data is screened and trimmed based on quality. All
sequences are trimmed to 350 bp. Sequences are binned by individual
samples based on barcode sequences. Barcode tags and primers are
removed from the sequences and non-bacterial ribosomal sequences
are removed. Sequences are clustered into operational taxonomic
units (OTUs) at 97% similarity using uclust. Representative
sequences from each OTU are then aligned using PyNAST and taxonomy
is assigned by sequence comparison to known bacterial 16S rRNA gene
sequences in the SILVA database using the RDP classifier. A random
subsampling of sequences is performed to normalize each sample so
that the same number of sequences are analyzed. Analysis of
Variance (ANOVA) analysis is used to determine if any fibrolytic
microflora (taxa) are significantly affected by treatment.
[0125] The term "fibrolytic microflora" as used herein means a
group of microorganisms that are able to process complex plant
polysaccharides due to their ability to synthesize cellulolytic and
hemicellulolytic enzymes.
[0126] The term "endogenous" as used herein means present in (or
originating in) the GIT of a subject (e.g. an animal). In other
words the fibrolytic, endogenous microflora is not a DFM.
[0127] The fibrolytic, endogenous microflora is not added to the
subject's feed.
[0128] Preferably the enzyme producing strain and/or the C-5
sugar-fermenting strain and/or the short-chain fatty acid-producing
strains and/or the fibrolytic, endogenous microflora-promoting
strain for use in the present invention comprises a viable
microorganism.
[0129] Preferably the enzyme producing strain and/or the C-5
sugar-fermenting strain and/or the short-chain fatty acid-producing
strains and/or the fibrolytic, endogenous microflora-promoting
strain comprises a viable bacterium or a viable yeast or a viable
fungi.
[0130] In one preferred embodiment the enzyme producing strain
and/or the C-5 sugar-fermenting strain and/or the short-chain fatty
acid-producing strains and/or the fibrolytic, endogenous
microflora-promoting strain comprises a viable bacterium.
[0131] The term "viable microorganism" means a microorganism which
is metabolically active or able to differentiate.
[0132] In one embodiment the enzyme producing strain and/or the C-5
sugar-fermenting strain and/or the short-chain fatty acid-producing
strains and/or the fibrolytic, endogenous microflora-promoting
strain may be a spore forming bacterium and hence the term DFM may
be comprised of or contain spores, e.g. bacterial spores. Therefore
in one embodiment the term "viable microorganism" as used herein
may include microbial spores, such as endospores or conidia.
[0133] In another embodiment the enzyme producing strain and/or the
C-5 sugar-fermenting strain and/or the short-chain fatty
acid-producing strains and/or the fibrolytic, endogenous
microflora-promoting strain in the feed additive composition
according to the present invention is not comprised of or does not
contain microbial spores, e.g. endospores or conidia.
[0134] The microorganism may be a naturally occurring microorganism
or it may be a transformed microorganism. The microorganism may
also be a combination of suitable microorganisms.
[0135] In some aspects, the enzyme producing strain and/or the C-5
sugar-fermenting strain and/or the short-chain fatty acid-producing
strains and/or the fibrolytic, endogenous microflora-promoting
strain according to the present invention may be one or more of the
following: a bacterium, a yeast or a fungi.
[0136] Preferably the enzyme producing strain and/or the C-5
sugar-fermenting strain and/or the short-chain fatty acid-producing
strains and/or the fibrolytic, endogenous microflora-promoting
strain according to the present invention is a probiotic
microorganism.
[0137] In the present invention, the term direct fed microbial
(DFM) encompasses direct fed bacteria, direct fed yeast, direct fed
fungi and combinations thereof.
[0138] Preferably the enzyme producing strain and/or the C-5
sugar-fermenting strain and/or the short-chain fatty acid-producing
strains and/or the fibrolytic, endogenous microflora-promoting
strain is a direct fed bacterium.
[0139] Suitably the enzyme producing strain and/or the C-5
sugar-fermenting strain and/or the short-chain fatty acid-producing
strains and/or the fibrolytic, endogenous microflora-promoting
strain may comprise a bacterium from one or more of the following
genera: Bacillus, Lactobacillus, Propionibacterium and combinations
thereof.
[0140] In one embodiment the enzyme producing strain and/or the C-5
sugar-fermenting strain and/or the short-chain fatty acid-producing
strains and/or the fibrolytic, endogenous microflora-promoting
strain may be a strain selected from the Bacillus genus.
[0141] In one embodiment the enzyme producing strain and/or the C-5
sugar-fermenting strain and/or the short-chain fatty acid-producing
strains and/or the fibrolytic, endogenous microflora-promoting
strain may be selected from the following Bacillus spp: Bacillus
subtilis, Bacillus cereus, Bacillus licheniformis, B. pumilus, B.
coagulans, B. amyloliquefaciens, B. stearothermophilus, B. brevis,
B. alkalophilus, B. clausii, B. halodurans, B. megaterium, B.
circulans, B. lautus, B. thuringiensis and B. lentus strains.
[0142] In at least some embodiments the B. subtilis strain(s) is
(are) Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP BS442,
B. subtilis AGTP BS521, B. subtilis AGTP BS918, Bacillus subtilis
AGTP BS1013, B. subtilis AGTP BS1069, B. subtilis AGTP 944.
[0143] In at least some embodiments the B. subtilis strain(s) is
(are) Bacillus subtilis 15A-P4 (PTA-6507), LSSAO1 (NRRL
B-50104).
[0144] In at least some embodiments the B. pumilus strain is B.
pumilus AGTP BS 1068 or B. pumilus KX11-1.
[0145] Strains 3A-P4 (PTA-6506), 15A-P4 (PTA-6507) and 22C-P1
(PTA-6508) are publically available from American Type Culture
Collection (ATCC). Strains 2084 (NRRL B-500130); LSSA01
(NRRL-B-50104); BS27 (NRRL B-50105) are publically available from
the Agricultural Research Service Culture Collection (NRRL). Strain
Bacillus subtilis LSSA01 is sometimes referred to as B. subtilis 8.
These strains are taught in U.S. Pat. No. 7,754,469 B2.
[0146] Danisco USA, Inc. of Waukesha, Wis., USA deposited under the
Budapest Treaty the following biological deposits with the
Agricultural Research Service Culture Collection (NRRL) with the
dates of the original deposits and accession numbers detailed
below:
TABLE-US-00002 Deposit Accession Number Deposit date Bacillus
subtilis AGTP NRRL B-50510 13 May 2011 BS3BP5 Bacillus subtilis
AGTP NRRL B-50542 4 Aug. 2011 BS442 Bacillus subtilis AGTP NRRL
B-50545 4 Aug. 2011 BS521 Bacillus subtilis AGTP NRRL B-50508 13
May 2011 BS918 Bacillus subtilis AGTP NRRL B-50509 13 May 2011
BS1013 Bacillus subtilis AGTP NRRL B-50544 4 Aug. 2011 BS1069
Bacillus subtilis AGTP 944 NRRL B-50548 11 Aug. 2011 Bacillus
pumilus AGTP NRRL B-50543 4 Aug. 2011 BS1068 Bacillus pumilus AGTP
NRRL B-50546 5 Aug. 2011 KXII-1 Bacillus subtilis BS18 NRRL B-50633
9 Jan. 2012 Bacillus subtilis BS278 NRRL B-50634 9 Jan. 2012
Bacillus licheniformis NRRL B-50516 20 May 2011 BL842
[0147] Danisco USA, Inc. of Waukesha, Wis., USA has authorised
DuPont Nutrition Biosciences ApS of Langebrogade 1, PO Box 17,
DK-1001, Copenhagen K, Denmark to refer to these deposited
biological materials in this patent application and has given
unreserved and irrevocable consent to the deposited material being
made available to the public.
[0148] AgTech Products, Inc. of W227 N752 Westmound Drive,
Waukesha, Wis. 53186, USA deposited under the Budapest Treaty the
following biological deposit with the Agricultural Research Service
Culture Collection (NRRL) with the date of the original deposit and
accession number detailed below:
TABLE-US-00003 Bacillus licheniformis BL21 NRRL B-50134 15 Apr.
2008
[0149] AgTech Products, Inc has authorised DuPont Nutrition
Biosciences ApS of Langebrogade 1, PO Box 17, DK-1001, Copenhagen
K, Denmark to refer to this deposited biological material in this
patent application and has given unreserved and irrevocable consent
to the deposited material being made available to the public.
[0150] The table below summarises the enzyme producing capabilities
of the selected strains using the "Enzyme producing DFM assay"
above:
[0151] Summary of direct fed microbial candidate strains enzymatic
activity..sup.a
TABLE-US-00004 TABLE 2 Cellulase, xylanase, and .beta.-mannanase
activities of Bacillus strains. Isolate CMCase .beta.- Name
(Cellulase) Xylanase Mannanase.sup.1 BS27 0.0 4.0 3.0 BL21 3.0 0.0
2.5 BL842 1.0 0.0 2.5 BS18 3.0 3.0 3.5 15AP4 4.0 2.0 2.5 22CP1 3.0
5.0 2.0 3AP4 4.0 2.5 1.5 BS278 4.0 3.0 1.0 LSSAO1 3.5 4.0 3.3
BS2084 4.0 3.0 1.0 BS3BP5 3.3 3.0 N/A BS442 1.8 2.5 2.0 BS521 6.0
4.0 2.0 BS918 4.0 5.5 3.3 BS1013 6.5 4.0 2.5 BP1068 3.0 6.0 4.5
BS1069 4.0 4.0 2.5 944 6.5 3.5 1.0 KXII-1 2.5 5.0 N/A
.sup.1Mannanase (e.g. .beta.-mannanase) is the name given to a
class of enzymes which can hydrolyze 1,4-.beta.-D-glycosidic bonds
of .beta.-mannan, galactomannan and glucomannan into mannan
oligosaccharides and mannose, thus breaking down mannan containing
hemicellulose, one of the major components of plant cell walls.
.beta.-mannanase is endo-1,4-.beta.-D-mannanase (E.C.
3.2.1.78).
[0152] Suitably the enzyme producing strain and/or the C-5
sugar-fermenting strain and/or the short-chain fatty acid-producing
strains and/or the fibrolytic, endogenous microflora-promoting
strain for use in the present invention may be a strain selected
from the Propionibacterium genus. In one embodiment the DFM for use
in the present invention may be selected from the species
Propionibacterium acidipropionici.
[0153] In one embodiment the DFM for use in the present invention
is Propionibacterium acidipropionici P169.
[0154] Agtech Products, Inc. of W227 N752 Westmound Dr. Waukesha,
Wis. 53186, USA deposited on 28 Jul. 2003 under the Budapest Treaty
Propionibacterium acidipropionici P169 with the American Type
Culture Collection (ATCC), Manassas, Va. 20110-2209, USA as
Accession no. PTA-5271. Propionibacterium acidipropionici P169 was
referenced in granted patent U.S. Pat. No. 6,951,643B2 and is
publically available from ATCC.
[0155] In one embodiment the enzyme producing strain and/or the C-5
sugar-fermenting strain and/or the short-chain fatty acid-producing
strains and/or the fibrolytic, endogenous microflora-promoting
strain for use in the present invention may be a strain from the
Enterococcus genus.
[0156] In one embodiment the DFM for use in the present invention
may be selected from the species Enterococcus faecium.
[0157] In one embodiment the DFM for use in the present invention
may be Enterococcus faecium ID7.
[0158] Lactococcus lactis ID7 (which was later reclassified as
Enterococcus faecium ID7) was deposited on 22 Jun. 2004 under the
Budapest Treaty as Lactococcus lactis ID7 with the American Type
Culture Collection (ATCC), Manassas, Va. 20110-2209, USA as
Accession no. PTA-6103. Lactococcus lactis ID7 (which was later
reclassified as Enterococcus faecium ID7) was referenced in granted
patent U.S. Pat. No. 7,384,628 and is publically available from
ATCC. When "Enterococcus faecium ID7" is used herein it will be
understood that this organism's name is interchangeable with
"Lactococcus lactis ID7" which was deposited as Accession no.
PTA-6103. Enterococcus faecium ID7 is also publically available
from Danisco Animal Nutrition, Denmark.
[0159] In one embodiment the enzyme producing strain and/or the C-5
sugar-fermenting strain and/or the short-chain fatty acid-producing
strains and/or the fibrolytic, endogenous microflora-promoting
strain for use in the present invention may be a strain from
Lactobacillus genus.
[0160] In one embodiment the enzyme producing strain and/or the C-5
sugar-fermenting strain and/or the short-chain fatty acid-producing
strains and/or the fibrolytic, endogenous microflora-promoting
strain may be selected from the following Lactobacillus spp:
Lactobacillus buchneri, Lactobacillus acidophilus, Lactobacillus
casei, Lactobacillus kefiri, Lactobacillus bifidus, Lactobacillus
brevis, Lactobacillus helveticus, Lactobacillus paracasei,
Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus
curvatus, Lactobacillus bulgaricus, Lactobacillus sakei,
Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus
farciminis, Lactobacillus lactis, Lactobacillus delbreuckii,
Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus
farciminis, Lactobacillus rhamnosus, Lactobacillus crispatus,
Lactobacillus gasseri, Lactobacillus johnsonii and Lactobacillus
jensenii, and combinations of any thereof.
[0161] In one embodiment the DFM may be selected from one or more
of the following strains: Lactobacillus rhamnosus CNCM-I-3698 and
Lactobacillus farciminis CNCM-I-3699. These strains were deposited
at the Collection Nationale de Cultures de Microorganims (CNCM) 25,
Rue due Docteur Roux, F75724 Paris Cedex 15, France on 8 Dec. 2006
by Sorbial, Route de Spay 72700 Allonnes, France and all right,
title and interest in the deposits were subsequently transferred to
Danisco France SAS of 20, Rue de Brunel, 75017 Paris, France.
[0162] Danisco France SAS has authorised DuPont Nutrition
Biosciences ApS of Langebrogade 1, PO Box 17, DK-1001, Copenhagen
K, Denmark to refer to these deposited biological materials in this
patent application and have given unreserved and irrevocable
consent to the deposited material being made available to the
public.
[0163] In at least some embodiments the DFM may be selected from
Lactobacillus lactis DJ6 (PTA 6102) and/or Lactococcus lactis ID7
(PTA 6103).
[0164] AgTech Products, Inc. of W227 N752 Westmound Drive,
Waukesha, Wis. 53186, USA deposited under the Budapest Treaty the
following biological deposits with the American Type Culture
Collection (ATCC), Manassas, Va. 20110-2209, USA with the dates of
the original deposits and accession numbers detailed below:
TABLE-US-00005 Lactobacillus lactis DJ6 PTA 6102 22 Jun. 2004
Lactococcus lactis ID7 PTA 6103 22 Jun. 2004
[0165] AgTech Products, Inc. has authorised DuPont Nutrition
Biosciences ApS of Langebrogade 1, PO Box 17, DK-1001, Copenhagen
K, Denmark to refer to these deposited biological materials in this
patent application and has given unreserved and irrevocable consent
to the deposited material being made available to the public.
[0166] In at least one embodiment, more than one of the strain(s)
described herein is (are) combined.
[0167] Therefore the enzyme producing strain and/or the C-5
sugar-fermenting strain and/or the short-chain fatty acid-producing
strain and/or the fibrolytic, endogenous microflora-promoting
strain used in the present invention may be a combination of at
least two, suitably at least three, suitably at least four DFM
strains described herein, e.g. DFM strains selected from the group
consisting of Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP
BS442, B. subtilis AGTP BS521, B. subtilis AGTP BS918, Bacillus
subtilis AGTP BS1013, B. subtilis AGTP BS1069, B. subtilis AGTP
944, B. pumilus AGTP BS 1068, B. pumilus KX11-1, Propionibacterium
P169, Lactobacillus rhamnosus CNCM-I-3698 or Lactobacillus
farciminis CNCM-I-3699.
[0168] In one embodiment preferably the DFM may be one or more of
the group consisting of Bacillus subtilis AGTP BS3BP5, Bacillus
subtilis AGTP BS442, B. subtilis AGTP BS521, B. subtilis AGTP
BS918, Bacillus subtilis AGTP BS1013, B. subtilis AGTP BS1069, B.
subtilis AGTP 944, B. pumilus AGTP BS 1068, B. pumilus KX11-1 and a
combination thereof.
[0169] Any Bacillus, Lactobacillus or Propionibacterium derivative
or variant is also included and is useful in the methods described
and claimed herein.
[0170] In some embodiments, Bacillus variant strains having all the
characteristics of Bacillus subtilis AGTP BS3BP5, Bacillus subtilis
AGTP BS442, B. subtilis AGTP BS521, B. subtilis AGTP BS918,
Bacillus subtilis AGTP BS1013, B. subtilis AGTP BS1069, B. subtilis
AGTP 944, B. pumilus AGTP BS 1068 or B. pumilus KX11-1 are also
included and are useful in the methods described and claimed
herein.
[0171] As used herein, a "variant" has at least 80% identity of
genetic sequences with the disclosed strains using random amplified
polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. The
degree of identity of genetic sequences can vary. In some
embodiments, the variant has at least 85%, 90%, 95%, 96%, 97%, 98%
or 99% identity of genetic sequences with the disclosed strains
using RAPD-PCR analysis.
[0172] Six primers that can be used for RAPD-PCR analysis include
the following: Primer 1 (5'-GGTGCGGGAA-3'), Primer 2
(5'-GTTTCGCTCC-3'), Primer 3 (5'-GTAGACCCGT-3'), Primer 4
(5'-AAGAGCCCGT-3'), Primer 5 (5'-AACGCGCAAC-3'), Primer 6
(5'-CCCGTCAGCA-3'). RAPD analysis can be performed using
Ready-to-Go.TM. RAPD Analysis Beads (Amersham Biosciences, Sweden),
which are designed as pre-mixed, pre-dispensed reactions for
performing RAPD analysis.
[0173] The direct fed bacterium used in the present invention may
be of the same type (genus, species and strain) or may comprise a
mixture of genera, species and/or strains.
[0174] Preferably the DFM to be used in accordance with the present
invention is a microorganism which is generally recognised as safe
and, which is preferably GRAS approved.
[0175] A skilled person will readily be aware of specific species
and or strains of microorganisms from within the genera described
herein which are used in the food and/or agricultural industries
and which are generally considered suitable for animal
consumption.
[0176] Preferably, the DFM used in accordance with the present
invention is one which is suitable for animal consumption.
[0177] Advantageously, where the product is a feed or feed additive
composition, the viable DFM should remain effective through the
normal "sell-by" or "expiration" date of the product during which
the feed or feed additive composition is offered for sale by the
retailer. The desired lengths of time and normal shelf life will
vary from feedstuff to feedstuff and those of ordinary skill in the
art will recognise that shelf-life times will vary upon the type of
feedstuff, the size of the feedstuff, storage temperatures,
processing conditions, packaging material and packaging
equipment.
[0178] In some embodiments it is important that the DFM is tolerant
to heat, i.e. is thermotolerant.
[0179] This is particularly the case where the feed is pelleted.
Therefore in one embodiment the DFM may be a thermotolerant
microorganism, such as a thermotolerant bacteria, including for
example Bacillus spp.
[0180] In some embodiments it may be preferable that the DFM is a
spore producing bacteria, such as Bacilli, e.g. Bacillus spp.
Bacilli are able to from stable endospores when conditions for
growth are unfavorable and are very resistant to heat, pH, moisture
and disinfectants.
[0181] Suitably the DFM is not an inactivated microorganism.
[0182] In one embodiment the DFM may be a viable or inviable
microorganism which is used in isolated or semi-isolated form. The
DFM may be used in combination with or without the growth medium in
which it was cultured.
[0183] In one embodiment, the DFM is capable of producing colony
forming units when grown on an appropriate media. The appropriate
media may comprise (or consist of) a feed or a feed
constituent.
[0184] In one embodiment, the DFM is incapable of producing colony
forming units when grown on an appropriate media. The appropriate
media may comprise (or consist of) a feed or a feed
constituent.
[0185] Irrespective of whether the DFM is capable or incapable of
producing colony forming units when grown on an appropriate
media--the cells may be still metabolically active (e.g. even if
they are unable to divide).
[0186] In one embodiment the DFM may be administered as inviable
cells.
[0187] In one embodiment the DFM may be administered as a viable
microorganism.
[0188] The DFM may be dosed appropriately.
[0189] Suitably dosages of DFM in the feed may be between about
1.times.10.sup.3 CFU/g feed to about 1.times.10.sup.9 CFU/g feed,
suitably between about 1.times.10.sup.4 CFU/g feed to about
1.times.10.sup.8 CFU/g feed, suitably between about
7.5.times.10.sup.4 CFU/g feed to about 1.times.10.sup.7 CFU/g
feed.
[0190] In one embodiment the DFM is dosed in the feedstuff at more
than about 1.times.10.sup.3 CFU/g feed, suitably more than about
1.times.10.sup.4 CFU/g feed, suitably more than about
7.5.times.10.sup.4 CFU/g feed.
[0191] Suitably dosages of DFM in the feed additive composition may
be between about 1.times.10.sup.5 CFU/g composition to about
1.times.10.sup.13 CFU/g composition, suitably between about
1.times.10.sup.6 CFU/g composition to about 1.times.10.sup.12 CFU/g
composition, suitably between about 3.75.times.10.sup.7 CFU/g
composition to about 1.times.10.sup.11 CFU/g composition.
[0192] In one embodiment the DFM is dosed in the feed additive
composition at more than about 1.times.10.sup.5 CFU/g composition,
suitably more than about 1.times.10.sup.6 CFU/g composition,
suitably more than about 3.75.times.10.sup.7 CFU/g composition.
[0193] In a preferred embodiment the DFM may be dosed in the feed
additive composition at between about 5.times.10.sup.7 to about
1.times.10.sup.9 CFU/g, suitably at between about 1.times.10.sup.8
to about 5.times.10.sup.8 CFU/g composition.
[0194] In another preferred embodiment the DFM may be dosed in the
feed additive composition at between about 5.times.10.sup.3 to
about 5.times.10.sup.5 U/g, suitably at between about
1.times.10.sup.4 to about 1.times.10.sup.5 CFU/g composition.
[0195] Fibre Degrading Enzymes
[0196] The DFM as taught herein may be used in combination with at
least one xylanase and at least one .beta.-glucanase (and
optionally at least one further fibre degrading enzyme).
[0197] .beta.-glucanase or endo-glucanase is the name given to a
class of enzymes which can hydrolyze (1,3)-.beta.-D-glycosidic
and/or (1,4)-.beta.-D-glycosidic bonds of (1,4)-.beta.-glucan,
(1,3;1,4)-.beta.-glucan and cellulose into glucose oligosaccharides
and glucose, thus breaking down cellulose and hemicellulose, the
major components of plant cell walls.
[0198] The .beta.-glucanase for use in the present invention may be
any commercially available .beta.-glucanase.
[0199] In one embodiment the .beta.-glucanase is an endoglucanase,
e.g. an endo-1,4-.beta.-D-glucanase (classified as E.C.
3.2.1.4).
[0200] Suitably, the .beta.-glucanase for use in the present
invention may be a .beta.-glucanase from Bacillus, Trichoderma,
Aspergillus, Thermomyces, Fusarium and Penicillium.
[0201] In one embodiment the fibre degrading enzyme may be a
.beta.-glucanase produced from one or more of the expression hosts
selected from the group consisting of: Bacillus lentus, Aspergillus
niger, Trichoderma reesei, Penicillium funiculosum, Trichoderma
longibrachiatum, Humicola insolens, Bacillus amyloliquefaciens,
Aspergillus aculeatus, Aspergillus aculeatus.
[0202] In one embodiment the fibre degrading enzyme may be one or
more of the following commercial products which comprises at least
a .beta.-glucanase fibre degrading enzyme:
[0203] Econase.RTM. GT or Econase.RTM. BG (available from AB
Vista), Rovabio Excel.RTM. (available from Adisseo), Endofeed.RTM.
DC and Amylofeed.RTM. (available from Andres Pintaluba S.A.),
AveMix.RTM. XG10 (from Aveve), Natugrain.RTM., Natugrain.RTM. TS,
or Natugrain.RTM. TS/L (available from BASF), Avizyme.RTM. 1210,
Avizyme.RTM. SX, Grindazym.RTM. GP, Grindazym.RTM. GV, Porzyme.RTM.
8100, Porzyme.RTM. 9102, Porzyme.RTM. tp100, AXTRA.RTM. XB,
Avizyme.RTM. 1100, Avizyme.RTM. 1110, Avizyme.RTM. 1202,
Porzyme.RTM. sf or Porzyme.RTM. SP (available from Danisco Animal
Nutrition), Bio-Feed Plus.RTM., Ronozyme A.RTM., Ronozyme VP.RTM.
or Roxazyme G2.RTM. (available from DSM), Hostazym C.RTM.
(available from Huvepharma), Kemzyme W dry or Kemzyme W liquid
(available from Kemin), Biogalactosidase BL (available from Kerry
Ingredients), Safizyme G (available from Le Saffre), or Feedlyve
AGL (available from Lyven).
[0204] In one embodiment the .beta.-glucanase may be obtained from
Axtra.RTM. XB.
[0205] .beta.-glucanase may be dosed in any suitable amount.
[0206] In one embodiment the .beta.-glucanase for use in the
present invention may be present in the feedstuff in a range of
about 50 BGU/kg feed to about 50000 BGU/kg feed, suitably about 100
BGU/kg feed to about 1000 BGU/kg feed.
[0207] The .beta.-glucanase for use in the present invention may be
present in the feedstuff in a range of about 75 BGU/kg feed to
about 400 BGU/kg feed, suitably about 150 BGU/kg feed to about 200
BGU/kg feed.
[0208] In one embodiment the .beta.-glucanase is present in the
feedstuff at less than 1000 BGU/kg feed, suitably less than about
500 BGU/kg feed, suitably less than 250 BGU/kg feed.
[0209] In one embodiment the .beta.-glucanase is present in the
feedstuff at more than 75 BGU/kg feed, suitably more than 100
BGU/kg feed.
[0210] Suitably, the .beta.-glucanase is present in the feed
additive composition in the range of about 150 BGU/g composition to
about 3000 BGU/g composition, suitably in the range of about 300
BGU/g composition to about 1500 BGU/g composition.
[0211] In one embodiment the .beta.-glucanase is present in the
feed additive composition at less than 5000 BGU/g composition,
suitably at less than 4000 BGU/g composition, suitably at less than
3000 BGU/g composition, suitably at less than 2000 BGU/g
composition.
[0212] In one embodiment the .beta.-glucanase is present in the
feed additive composition at more than 50 BGU/g composition,
suitably at more than 100 BGU/g composition, suitably at more than
125 BGU/g composition.
[0213] In some embodiments the activity of .beta.-glucanase can be
calculated using the ".beta.-glucanase Activity Assay (BGU)" as
taught herein.
[0214] In one embodiment the .beta.-glucanase for use in the
present invention may have .beta.-glucanase activity as determined
using the ".beta.-glucanase Activity Assay (CMC U/g)" taught
herein.
[0215] The term "fibre degrading enzyme" as used herein may include
one or more of the following fibre degrading enzymes: a xylanase
(e.g. an endo-1,4-.beta.-D-xylanase (E.C. 3.2.1.8) or a 1,4
.beta.-xylosidase (E.C. 3.2.1.37)), a .beta.-glucanase (E.C.
3.2.1.4), a cellobiohydrolase (E.C. 3.2.1.176 and E.C. 3.2.1.91), a
.beta.-glucosidase (E.C. 3.2.1.21), a feruloyl esterase (E.C.
3.1.1.73), an .alpha.-arabinofuranosidase (E.C. 3.2.1.55), a
pectinase (e.g. an endopolygalacturonase (E.C. 3.2.1.15), an
exopolygalacturonase (E.C. 3.2.1.67) or a pectate lyase (E.C.
4.2.2.2)), or combinations thereof.
[0216] The term "further fibre degrading enzyme" as used herein may
include one or more of the following fibre degrading enzymes: a
cellobiohydrolase (E.C. 3.2.1.176 and E.C. 3.2.1.91), a
.beta.-glucosidase (E.C. 3.2.1.21), a .beta.-xylosidase (E.C.
3.2.1.37), a feruloyl esterase (E.C. 3.1.1.73), an
.alpha.-arabinofuranosidase (E.C. 3.2.1.55), a pectinase (e.g. an
endopolygalacturonase (E.C. 3.2.1.15), an exopolygalacturonase
(E.C. 3.2.1.67) or a pectate lyase (E.C. 4.2.2.2)), or combinations
thereof.
[0217] It will also be understood by a person skilled in the art
that "a further fibre degrading enzyme" may encompass multiple
further fibre degrading enzymes.
[0218] In one embodiment the DFM as taught herein may be used in
combination with at least one xylanase, at least one
.beta.-glucanase and at least one further fibre degrading
enzyme.
[0219] In another embodiment the DFM as taught herein may be used
in combination with at least one xylanase, at least one
.beta.-glucanase and two (or at least two) further fibre degrading
enzymes.
[0220] In another embodiment the DFM as taught herein may be used
in combination with at least one xylanase, at least one
.beta.-glucanase and three (or at least three) further fibre
degrading enzymes.
[0221] In another embodiment the DFM as taught herein may be used
in combination with at least one xylanase, at least one
.beta.-glucanase and four (or at least four) further fibre
degrading enzymes.
[0222] In one embodiment the DFM as taught herein may be used in
combination with a broth or a solid-state fermentation product
containing measurable enzyme activity or activities of the present
invention.
[0223] In one embodiment the DFM as taught herein may be used in
combination with the enzymes of the present invention, which
enzymes are in isolated or purified form.
[0224] In one embodiment the DFM as taught herein may be used in
combination with the enzymes of the present invention, which
enzymes are exogenous to the DFM in the composition (e.g. if the
DFM is an enzyme producing strain).
[0225] Preferably, the fibre degrading enzyme(s) is present in the
feedstuff in the range of about 0.05 to 5 g of enzyme protein per
metric ton (MT) of feed (or mg/kg).
[0226] Suitably, each fibre degrading enzyme may be present in the
feedstuff in the range of about 0.05 to 5 g of enzyme protein per
metric ton (MT) of feed (or mg/kg).
[0227] Suitably, the fibre degrading enzymes in total are present
in the feedstuff in the range of about 0.05 to 5 g of enzyme
protein per metric ton (MT) of feed (or mg/kg).
[0228] Preferably, the fibre degrading enzyme(s) is present in the
feed additive composition (or premix) in the range of about 0.05 to
100 mg protein/g of composition (e.g. at a total inclusion in the
diet of 50 to 1000 g/MT).
[0229] Suitably, each fibre degrading enzyme is present in the feed
additive composition (or premix) in the range of about 0.05 to 100
mg protein/g of composition (e.g. at a total inclusion in the diet
of 50 to 1000 g/MT).
[0230] Suitably, the fibre degrading enzymes in total is present in
the feed additive composition (or premix) in the range of about
0.05 to 100 mg protein/g of composition (e.g. at a total inclusion
in the diet of 50 to 1000 g/MT).
[0231] In a preferred embodiment the fibre degrading enzyme (e.g.
each fibre degrading enzyme or the fibre degrading enzymes in
total) may be in the feed additive composition (or premix) in the
range of about 50 to about 700 g/MT of feed. Suitably the fibre
degrading enzyme (e.g. each fibre degrading enzyme or the fibre
degrading enzymes in total) may be in the feed additive composition
(or premix) at about 100 to about 500 g/MT of feed.
[0232] In one embodiment the further fibre degrading enzyme(s) for
use in the present invention may comprise (or consist essentially
of, or consist of) a cellobiohydrolase (E.C. 3.2.1.176 and E.C.
3.2.1.91).
[0233] In another embodiment the further fibre degrading enzyme(s)
for use in the present invention may comprise (or consist
essentially of, or consist of) a .beta.-glucosidase (E.C.
3.2.1.21).
[0234] Suitably the further fibre degrading enzyme may comprise (or
consist essentially of, or consist of) a cellobiohydrolase (E.C.
3.2.1.176 and E.C. 3.2.1.91), a .beta.-glucosidase (E.C. 3.2.1.21)
or combinations thereof.
[0235] In another one embodiment the further fibre degrading
enzyme(s) for use in the present invention may comprise (or consist
essentially of, or consist of) a .beta.-xylosidase (E.C.
3.2.1.37).
[0236] In one embodiment the fibre degrading enzyme(s) for use in
the present invention may comprise (or consist essentially of, or
consist of) a feruloyl esterase (E.C. 3.1.1.73).
[0237] In another embodiment the further fibre degrading enzyme for
use in the present invention may comprise (or consist essentially
of, or consist of) an .alpha.-arabinofuranosidase (E.C.
3.2.1.55).
[0238] In a yet further embodiment the further fibre degrading
enzyme(s) for use in the present invention may comprise (or consist
essentially of, or consist of) a pectinase (e.g. an
endopolygalacturonase (E.C. 3.2.1.15), an exopolygalacturonase
(E.C. 3.2.1.67) or a pectate lyase (E.C. 4.2.2.2)).
[0239] In a preferred embodiment the further fibre degrading
enzyme(s) for use in the present invention may comprise (or consist
essentially of, or consist of) one or more (suitably two or two or
more, suitably three) pectinase(s) selected from the group
consisting of: an endopolygalacturonase (E.C. 3.2.1.15), an
exopolygalacturonase (E.C. 3.2.1.67) and a pectate lyase (E.C.
4.2.2.2).
[0240] In one embodiment the further fibre degrading enzyme(s) for
use in the present invention may comprise (or consist essentially
of, or consist of) a cellobiohydrolase (E.C. 3.2.1.176 and E.C.
3.2.1.91), a .beta.-glucosidase (E.C. 3.2.1.21), a
.beta.-xylosidase (E.C. 3.2.1.37), a feruloyl esterase (E.C.
3.1.1.73), an .alpha.-arabinofuranosidase (E.C. 3.2.1.55), and/or a
pectinase (e.g. an endopolygalacturonase (E.C. 3.2.1.15), an
exopolygalacturonase (E.C. 3.2.1.67) or a pectate lyase (E.C.
4.2.2.2).
[0241] The present invention relates to the combination of at least
one xylanase, with at least one .beta.-glucanase and at least one
specific DFM as taught herein.
[0242] In a preferred embodiment, the at least one xylanase, the at
least one .beta.-glucanase and the at least one specific DFM as
taught herein may be combined with a further fibre degrading enzyme
as taught herein.
[0243] The present invention further relates to the combination of
at least one xylanase and at least one .beta.-glucanase, with at
least two, such as at least three or at least four or at least
five, further fibre degrading enzymes and at least one specific DFM
as taught herein.
[0244] Xylanase is the name given to a class of enzymes which
degrade the linear polysaccharide beta-1,4-xylan into xylose, thus
breaking down hemicellulose, one of the major components of plant
cell walls.
[0245] The xylanase for use in the present invention may be any
commercially available xylanase.
[0246] Suitably the xylanase may be an endo-1,4-.beta.-d-xylanase
(classified as E.C. 3.2.1.8).
[0247] In one embodiment preferably the xylanase is an
endoxylanase, e.g. an endo-1,4-.beta.-d-xylanase. The
classification for an endo-1,4-.beta.-d-xylanase is E.C.
3.2.1.8.
[0248] In one embodiment the present invention relates to a DFM in
combination with an endoxylanase, e.g. an
endo-1,4-.beta.-d-xylanase, and another enzyme.
[0249] All E.C. enzyme classifications referred to here relate to
the classifications provided in Enzyme
Nomenclature--Recommendations (1992) of the nomenclature committee
of the International Union of Biochemistry and Molecular
Biology--ISBN 0-12-226164-3.
[0250] Suitably, the xylanase for use in the present invention may
be a xylanase from Bacillus or Trichoderma.
[0251] In one embodiment the xylanase may be a xylanase comprising
(or consisting of) an amino acid sequence shown herein as SEQ ID
No. 1, a xylanase comprising (or consisting of) an amino acid
sequence shown herein as SEQ ID No. 2 or a xylanase comprising (or
consisting of) an amino acid sequence shown herein as SEQ ID No. 3
(FveXyn4), a xylanase from Trichoderma reesei, Econase XT.TM. or
Rovabio Excel.TM..
[0252] In one embodiment the xylanase may be the xylanase in Axtra
XAP.RTM. or Avizyme 1502.RTM. or AxtraXB.TM., both commercially
available products from Danisco A/S.
[0253] In one preferred embodiment the xylanase for use in the
present invention may be one or more of the xylanases in one or
more of the commercial products below:
TABLE-US-00006 Commercial Name .RTM. Company Xylanase type Xylanase
source Allzyme PT Alltech endo-1,4-.beta.-xylanase Aspergillus
Niger Amylofeed Andres Pintaluba S.A endo-1,4-.beta.-xylanase
Aspergillus Niger (phoenicis) Avemix 02 CS Aveve
endo-1,4-.beta.-xylanase Trichoderma reesei AveMix XG 10 Aveve, NL
endo-1,4-.beta.-xylanase Trichoderma reesei Avizyme 1100 Danisco
endo-1,4-.beta.-xylanase Trichoderma longibrachiatum Avizyme 1110
Danisco endo-1,4-.beta.-xylanase Trichoderma longibrachiatum
Avizyme 1202 Danisco endo-1,4-.beta.-xylanase Trichoderma
longibrachiatum Avizyme 1210 Danisco endo-1,4-.beta.-xylanase
Trichoderma longibrachiatum Avizyme 1302 Danisco
endo-1,4-.beta.-xylanase Trichoderma longibrachiatum Avizyme 1500
Danisco endo-1,4-.beta.-xylanase Trichoderma longibrachiatum
Avizyme 1502 Danisco endo-1,4-.beta.-xylanase Trichoderma
longibrachiatum Avizyme 1505 Danisco endo-1,4-.beta.-xylanase
Trichoderma longibrachiatum Avizyme SX Danisco
endo-1,4-.beta.-xylanase Trichoderma longibrachiatum Axtra XAP
Danisco endo-1,4-.beta.-xylanase Trichoderma longibrachiatum Axtra
XB Danisco endo-1,4-.beta.-xylanase Trichoderma longibrachiatum
Belfeed Beldem endo-1,4-.beta.-xylanase Bacillus subtilis MP100
Biofeed Combi Novozymes A/S endo-1,4-.beta.-xylanase Produced in
Aspergillus oryzae carrying a gene from Thermomyces lanuginosis and
Aspergillus aculeatus Biofeed Plus DSM endo-1,4-.beta.-xylanase
Humicola insolens Biofeed Wheat Novozymes A/S
endo-1,4-.beta.-xylanase Produced in Aspergillus oryzae carrying a
gene from Thermomyces lanuginosis Danisco Danisco Animal
endo-1,4-.beta.-xylanase Trichoderma reesei Glycosidase Nutrition
(TPT/L) Danisco Danisco endo-1,4-.beta.-xylanase Trichoderma reesei
Xylanase Econase ABenzymes/ABVista endo-1,4-.beta.-xylanase
Trichoderma reesei Wheat Plus Econase XT ABVista
endo-1,4-.beta.-xylanase Trichoderma reesei Endofeed .RTM. DC
Andres Pintaluba S.A. endo-1,4-.beta.-xylanase Aspergillus Niger
Feedlyve AXC Lyven endo-1,4-.beta.-xylanase Trichoderma koningii
Feedlyve AXL Lyven endo-1,4-.beta.-xylanase Trichoderma
longibrachiatum Grindazym GP Danisco endo-1,4-.beta.-xylanase
Aspergillus Niger Grindazym GV Danisco endo-1,4-.beta.-xylanase
Aspergillus Niger Hostazym X Huvepharma endo-1,4-.beta.-xylanase
Trichoderma longibrachiatum Kemzyme Plus Kemin
endo-1,4-.beta.-xylanase Trichoderma viride Dry Kemzyme Plus Kemin
endo-1,4-.beta.-xylanase Trichoderma viride Liquid Kemzyme W Kemin
endo-1,4-.beta.-xylanase Trichoderma viride dry Kemzyme W Kemin
endo-1,4-.beta.-xylanase Trichoderma viride liquid Natugrain BASF
endo-1,4-.beta.-xylanase Trichoderma longibrachiatum Natugrain TS
BASF endo-1,4-.beta.-xylanase Aspergillus Niger Plus Natugrain BASF
endo-1,4-.beta.-xylanase Aspergillus Niger Wheat Natugrain .RTM.
BASF endo-1,4-.beta.-xylanase Aspergillus Niger TS/L Natuzyme
Bioproton endo-1,4-.beta.-xylanase Trichoderma longibrachiatum/
Trichoderma reesei Nutrase Xyla Nutrex endo-1,4-.beta.-xylanase
Bacillus subtilis Porzyme 8100 Danisco endo-1,4-.beta.-xylanase
Trichoderma longibrachiatum Porzyme 8300 Danisco
endo-1,4-.beta.-xylanase Trichoderma longibrachiatum Porzyme 9102
Danisco endo-1,4-.beta.-xylanase Trichoderma longibrachiatum
Porzyme Danisco endo-1,4-.beta.-xylanase Trichoderma 9310/Avizyme
longibrachiatum 1310 Porzyme tp100 Danisco endo-1,4-.beta.-xylanase
Trichoderma longibrachiatum Ronozyme AX DSM
endo-1,4-.beta.-xylanase Thermomyces lanuginosus gene expressed in
Aspergillus oryzae Ronozyme WX DSM/Novozymes
endo-1,4-.beta.-xylanase Thermomyces lanuginosus gene expressed in
Aspergillus oryzae Rovabio Excel Adisseo endo-1,4-.beta.-xylanase
Penicillium funiculosum Roxazyme G2 DSM/Novozymes
endo-1,4-.beta.-xylanase Trichoderma longibrachiatum Safizym X Le
Saffre endo-1,4-.beta.-xylanase Trichoderma longibrachiatum
Xylanase Lyven endo-1,4-.beta.-xylanase Trichoderma
longibrachiatum
[0254] In one embodiment the xylanase may be a xylanase comprising
(or consisting of) a polypeptide sequence shown herein as SEQ ID
No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ
ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10,
SEQ ID No. 11, or SEQ ID No. 12; or a variant, homologue, fragment
or derivative thereof having at least 75% identity (such as at
least 80%, 85%, 90%, 95%, 98% or 99% identity) with SEQ ID No. 1 or
SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No.
6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID
No. 11, or SEQ ID No. 12; or a polypeptide sequence which comprises
SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No.
5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID
No. 10, SEQ ID No. 11, or SEQ ID No. 12 with a conservative
substitution of at least one of the amino acids.
[0255] In one embodiment the xylanase may comprise a polypeptide
sequence shown herein as SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No.
3, or a variant, homologue, fragment or derivative thereof having
at least 98.5% (e.g. at least 98.8 or 99 or 99.1 or 99.5%) identity
with SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3
TABLE-US-00007 SEQ ID No. 1: QAADSINKLIKNKGKLYYGTITDPN
LLGVAKDTAIIKADFGAVTPENSGKWDATEPSQGKFNFGSFDQVVNFAQQ
NGLKVRGHTLVWHSQLPQWVKNINDKATLTKVIENHVTQVVGRYKGKIYA
WDVVNEIFEWDGTLRKDSHFNNVFGNDDYVGIAFRAARKADPNAKLYIND
YSLDSGSASKVTKGMVPSVKKWLSQGVPVDGIGSQTHLDPGAAGQIQGAL
TALANSGVKEVAITELDIRTAPANDYATVTKACLNVPKCIGITVWGVSDK
NSWRKEHDSLLFDANYNPKPAYTAVVNALR SEQ ID No. 2:
QAADSINKLIKNKGKLYYGTITDPNLLGVAKDTAIIKADFG
AVTPENSGKWDATEPSQGKFNFGSFDQVVNFAQQNGLKVRGHTLVWHSQL
PQWVKNINDKATLTKVIENHVTQVVGRYKGKIYAWDVVNEIFEWDGTLRK
DSHFNNVFGNDDYVGIAFRAARKADPNAKLYINDYSLDSGSASKVTKGMV
PSVKKWLSQGVPVDGIGSQTHLDPGAAGQIQGALTALANSGVKEVAITEL
DIRTAPANDYATVTKACLNVPKCIGITVWGVSDKNSWRKEHDSLLFDANY NPKPAYTAVVNALR
SEQ ID No. 3: QAADSINKLIKNKGKLYYGTITDPNLLGVAKDTAIIKADFGAVTPENSGK
WDATEPSQGKFNFGSFDQVVNFAQQNGLKVRGHTLVWHSQLPQWVKNIND
KATLTKVIENHVTQVVGRYKGKIYAWDVVNEIFEWDGTLRKDSHFNNVFG
NDDYVGIAFRAARKADPNAKLYINDYSLDSGSASKVTKGMVPSVKKWLSQ
GVPVDGIGSQTHLDPGAAGQIQGALTALANSGVKEVAITELDIRTAPAND
YATVTKACLNVPKCIGITVWGVSDKNSWRKEHDSLLFDANYNPKPAYTAV VNALR SEQ ID No.
4: QASDSINKLIKNKGKLYYGTITDPN
LLGVAKDTAIIKADFGAVTPENSGKWDATEPSQGKFNFGSFDQVVNFAQ
QNGLKVRGHTLVWHSQLPQWVKNINDKATLTKVIENHVTNVVGRYKGKI
YAWDVVNEIFDWDGTLRKDSHFNNVFGNDDYVGIAFRAARKADPNAKLY
INDYSLDSGSASKVTKGMVPSVKKWLSQGVPVDGIGSQTHLDPGAAGQI
QGALTALANSGVKEVAITELDIRTAPANDYATVTKACLNVPKCIGITVW
GVSDKNSWRKEHDSLLFDANYNPKAAYTAVVNALR SEQ ID No. 5:
QASDSINKLIKNKGKLYYGTITDPNLLGVAKDTAIIKADF
GAVTPENSGKWDATEPSQGKFNFGSFDQVVNFAQQNGLKVRGHTLVWHS
QLPQWVKNINDKATLTKVIENHVTNVVGRYKGKIYAWDVVNEIFDWDGT
LRKDSHFNNVFGNDDYVGIAFRAARKADPNAKLYINDYSLDSGSASKVT
KGMVPSVKKWLSQGVPVDGIGSQTHLDPGAAGQIQGALTALANSGVKEV
AITELDIRTAPANDYATVTKACLNVPKCIGITVWGVSDKNSWRKEHDSL
LFDANYNPKAAYTAVVNALR SEQ ID No. 6:
QASDSINKLIKNKGKLYYGTITDPNLLGVAKDTAIIKADFGAVTPENSGK
WDATEPSQGKFNFGSFDQVVNFAQQNGLKVRGHTLVWHSQLPQWVKNIND
KATLTKVIENHVTNVVGRYKGKIYAWDVVNEIFDWDGTLRKDSHFNNVFG
NDDYVGIAFRAARKADPNAKLYINDYSLDSGSASKVTKGMVPSVKKWLSQ
GVPVDGIGSQTHLDPGAAGQIQGALTALANSGVKEVAITELDIRTAPAND
YATVTKACLNVPKCIGITVWGVSDKNSWRKEHDSLLFDANYNPKAAYTAV VNALR SEQ ID No.
7: mvsfkylflaasalgalaAPVEVEESSWFNETALHEFAERAGTPSSTGWN
NGYYYSFVVTDNGGTVNYQNGNGGSYSVQWKDTGNFVGGKGWNPGSARTI
NYSGSFNPSGNAYLTVYGWTTNPLVEYYIVENYGTYNPGNGGTYRGSVYS
DGANYNIYTATRYNAPSIEGDKTFTQYWSVRQSKRTGGTVTTANHFNAWA
QLGMSLGTHNYQIVATEGYQSSGSSSITVY SEQ ID No. 8:
APVEVEESSWFNETALHEFAERAGTPSSTGWNNGYYYSFWTDNGGTVNYQ
NGNGGSYSVQWKDTGNFVGGKGWNPGSARTINYSGSFNPSGNAYLTVYGW
TTNPLVEYYIVENYGTYNPGNGGTYRGSVYSDGANYNIYTATRYNAPSIE
GDKTFTQYWSVRQSKRTGGTVTTANHFNAWAQLGMSLGTHNYQIVATEGY QSSGSSSITVY SEQ
ID No. 9: AGTPSSTGWNNGYYYSFWTDNGGTVNYQNGNGGSYSVQWKDTGNFVGGKG
WNPGSARTINYSGSFNPSGNAYLTVYGWTTNPLVEYYIVENYGTYNPGNG
GTYRGSVYSDGANYNIYTATRYNAPSIEGDKTFTQYWSVRQSKRTGGTVT
TANHFNAWAQLGMSLGTHNYQIVATEGYQSSGSSSITVY SEQ ID No. 10:
MVSFTSLLAAVSAVTGVMALPSAQPVDGMSVVERDPPTNVLDKRTQPTTG
TSGGYYFSFWTDTPNSVTYTNGNGGQFSMQWSGNGNHVGGKGWMPGTSRT
IKYSGSYNPNGNSYLAVYGWTRNPLIEYYIVENFGTYNPSSGGQKKGEVN
VDGSVYDIYVSTRVNAPSIDGNKTFQQYWSVRRNKRSSGSVNTGAHFQAW
KNVGLNLGTHDYQILAVEGYYSSGSASMTVSQ SEQ ID No. 11:
LPSAQPVDGMSVVERDPPTNVLDKRTQPTTGTSGGYYFSFWTDTPNSVTY
TNGNGGQFSMQWSGNGNHVGGKGWMPGTSRTIKYSGSYNPNGNSYLAVYG
WTRNPLIEYYIVENFGTYNPSSGGQKKGEVNVDGSVYDIYVSTRVNAPSI
DGNKTFQQYWSVRRNKRSSGSVNTGAHFQAWKNVGLNLGTHDYQILAVEG YYSSGSASMTVSQ
SEQ ID No. 12: TQPTTGTSGGYYFSFWTDTPNSVTYTNGNGGQFSMQWSGNGNHVGGKGWM
PGTSRTIKYSGSYNPNGNSYLAVYGWTRNPLIEYYIVENFGTYNPSSGGQ
KKGEVNVDGSVYDIYVSTRVNAPSIDGNKTFQQYWSVRRNKRSSGSVNTG
AHFQAWKNVGLNLGTHDYQILAVEGYYSSGSASMTVSQ
[0256] Preferably, the xylanase is present in the feedstuff in
range of about 500 XU/kg to about 16,000 XU/kg feed, more
preferably about 750 XU/kg feed to about 8000 XU/kg feed, and even
more preferably about 1000 XU/kg feed to about 4000 XU/kg feed
[0257] In one embodiment the xylanase is present in the feedstuff
at more than about 500 XU/kg feed, suitably more than about 600
XU/kg feed, suitably more than about 700 XU/kg feed, suitably more
than about 800 XU/kg feed, suitably more than about 900 XU/kg feed,
suitably more than about 1000 XU/kg feed.
[0258] In one embodiment the xylanase is present in the feedstuff
at less than about 16,000 XU/kg feed, suitably less than about 8000
XU/kg feed, suitably less than about 7000 XU/kg feed, suitably less
than about 6000 XU/kg feed, suitably less than about 5000 XU/kg
feed, suitably less than about 4000 XU/kg feed.
[0259] Preferably, the xylanase is present in the feed additive
composition in range of about 100 XU/g to about 320,000 XU/g
composition, more preferably about 300 XU/g composition to about
160,000 XU/g composition, and even more preferably about 500 XU/g
composition to about 50,000 XU/g composition, and even more
preferably about 500 XU/g composition to about 40,000 XU/g
composition.
[0260] In one embodiment the xylanase is present in the feed
additive composition at more than about 100 XU/g composition,
suitably more than about 200 XU/g composition, suitably more than
about 300 XU/g composition, suitably more than about 400 XU/g
composition, suitably more than about 500 XU/g composition.
[0261] In one embodiment the xylanase is present in the feed
additive composition at less than about 320,000 XU/g composition,
suitably less than about 160,000 XU/g composition, suitably less
than about 50,000 XU/g composition, suitably less than about 40,000
XU/g composition, suitably less than about 30000 XU/g
composition.
[0262] The xylanase activity can be expressed in xylanase units
(XU) measured as taught in the "Xylanase Activity Assay (XU)"
taught herein. See also Bailey, M. J. Biely, P. and Poutanen, K.,
Journal of Biotechnology, Volume 23, (3), May 1992, 257-270 the
teaching of which is incorporated herein by reference.
[0263] In one embodiment suitably the enzyme is classified using
the E.C. classification above, and the E.C. classification
designates an enzyme having that activity when tested in the
"Xylanase Activity Assay (XU)" taught herein for determining 1
XU.
[0264] In one embodiment the xylanase for use in the present
invention may have xylanase activity as determined using the
"Xylanase Activity Assay (ABX U/g)" taught herein.
[0265] Enzyme Activities and Assays
[0266] In one embodiment the feed additive composition may comprise
a DFM in combination with a xylanase and a .beta.-glucanase.
[0267] In one embodiment xylanase activity may be calculated using
the "Xylanase Activity Assay (XU)" taught herein.
[0268] In another embodiment the .beta.-glucanase activity may be
calculated using the ".beta.-glucanase Activity Assay (BGU)" taught
herein.
[0269] Suitably, the DFM in combination with a xylanase and a
.beta.-glucanase may be dosed as set out in the table below:
TABLE-US-00008 Dosage of constituent per g or per kg of final
feedstuff Xylanase (e.g. endo-1,4- 500-16000 XU/kg (preferably
2500-4000 .beta.-d-xylanase) XU/kg) activity .beta.-glucanase
activity 50-5000 BGU/kg (preferably 200-400 BGU/kg) DFM 1 .times.
10.sup.4-1 .times. 10.sup.9 CFU/g (preferably 5 .times. 10.sup.4-5
.times. 10.sup.8 CFU/g)
[0270] The enzyme activity presented in units may be calculated for
each enzyme as taught in the preceding sections.
[0271] In some embodiments the feed additive composition may
comprise a DFM in combination with a xylanase, a .beta.-glucanase
and a further fibre degrading enzyme as taught herein.
[0272] Suitably the DFM, xylanase, .beta.-glucanase and further
fibre degrading enzyme may be dosed as set out in the table
below:
TABLE-US-00009 Dosage of constituent per g or per kg of final
feedstuff Xylanase (e.g. endo-1,4- 500-16000 (preferably 2500-4000
XU/kg) .beta.-d-xylanase) activity .beta.-glucanase activity
100-2500 CMC U/kg (preferably 800-1000 CMC U/kg) DFM 1 .times.
10.sup.3-1 .times. 10.sup.9 CFU/g (preferably 5 .times. 10.sup.4-5
.times. 10.sup.8 CFU/g) Further fibre degrading >800 ABX U/kg
(preferably >1200 ABX U/kg) enzymes (e.g. of another >500
pNPG U/kg (preferably >800 pNPG xylanase and a U/kg)
beta-glucosidase)
[0273] In one embodiment preferably the feedstuff comprises the
following:
[0274] a xylanase at at least 1000 XU/kg to 5000 XU/kg (suitably at
at least 2000 XU/kg to 4500 XU/kg) of feed;
[0275] a .beta.-glucanase at at least 100 BGU/kg to 4000 BGU/kg
(suitably at at least 150 BGU/kg to 3000 BGU/kg); and
[0276] a DFM as taught herein at at least 50,000 CFU/g to 200,000
CFU/g (suitably at at least 70,000 CFU/g to 175,000 CFU/g) of
feed.
[0277] In another embodiment preferably the feedstuff comprises the
following:
[0278] a xylanase at at least 1000 XU/kg to 5000 XU/kg (suitably at
at least 2000 XU/kg to 4500 XU/kg) of feed;
[0279] a .beta.-glucanase at at least 100 BGU/kg to 4000 BGU/kg
(suitably at at least 150 BGU/kg to 3000 BGU/kg); and
[0280] a DFM as taught herein at at least 37,500 CFU/g to 100,000
CFU/g (suitably at at least 37,500 CFU/g to 75,000 CFU/g) of
feed.
[0281] In another embodiment preferably the feedstuff comprises the
following:
[0282] a xylanase at at least 1000 XU/kg to 5000 XU/kg (suitably at
at least 2000 XU/kg to 4500 XU/kg) of feed;
[0283] a .beta.-glucanase at at least 200-2000 CMC U/kg (suitably
at least 500-1500 CMC U/kg) of feed;
[0284] a DFM as taught herein at at least 50,000 CFU/g to 200,000
CFU/g (suitably at at least 70,000 CFU/g to 175,000 CFU/g) of feed;
and
[0285] a further fibre degrading enzyme mix comprising at least
800-3500 ABX U/kg (suitably at least 1000-2750 ABX U/g) of feed and
500-3000 pNPG U/kg (suitably at least 600-2000 pNPG U/kg) of
feed.
[0286] In another embodiment preferably the feedstuff comprises the
following:
[0287] a xylanase at at least 1000 XU/kg to 5000 XU/kg (suitably at
at least 2000 XU/kg to 4500 XU/kg) of feed;
[0288] a .beta.-glucanase at at least 200-2000 CMC U/kg (suitably
at least 500-1500 CMC U/kg) of feed;
[0289] a DFM as taught herein at at least 37,500 CFU/g to 100,000
CFU/g (suitably at at least 37,500 CFU/g to 75,000 CFU/g) of feed;
and
[0290] a further fibre degrading enzyme mix comprising at least
800-3500 ABX U/kg (suitably at least 1000-2750 ABX U/g) of feed and
500-3000 pNPG U/kg (suitably at least 600-2000 pNPG U/kg) of
feed.
[0291] In one embodiment the DFM may be dosed in accordance with
the number of units of xylanase present in the composition. In one
embodiment the DFM may be dosed in the range from
6.25.times.10.sup.1 CFU DFM: 1 XU enzyme to 2.times.10.sup.9 CFU
DFM: 1 XU enzyme; preferably in the range from 1.88.times.10.sup.4
CFU DFM: 1 XU enzyme to 1.0.times.10.sup.7 CFU DFM: 1 XU enzyme.
The DFM taught herein may be used in combination with a xylanase
and a .beta.-glucanase.
[0292] In another embodiment the DFM taught herein may be used in
combination with a xylanase, a .beta.-glucanase and a further fibre
degrading enzyme. In a preferred embodiment the further fibre
degrading enzyme may be a .beta.-glucosidase.
[0293] In one embodiment the xylanase for use in the present
invention may have xylanase activity as determined using the
"Xylanase Activity Assay (ABX U/g)" taught herein.
[0294] In a further embodiment the .beta.-glucanase for use in the
present invention may have .beta.-glucanase activity as determined
using the ".beta.-glucanase Activity Assay (CMC U/g)" taught
herein.
[0295] In a yet further embodiment the .beta.-glucosidase for use
in the present invention may have .beta.-glucosidase activity as
determined using the ".beta.-glucosidase Activity Assay (pNPG U/g)"
taught herein.
[0296] In one embodiment the DFM taught herein may be used in
combination with a xylanase and a .beta.-glucanase, wherein the
xylanase and .beta.-glucanase have the activities set out in the
tables below:
TABLE-US-00010 Range of activity in Units/g of each enzyme activity
in the composition Xylanase (e.g endo-1,4-.beta.-d-xylanase)
1500-6000 ABX U/g.sup.1 activity .beta.-glucanase activity 500-4000
CMC U/g.sup.2
TABLE-US-00011 Range of activity in Units/g of each enzyme activity
in the composition Xylanase 2000-6000 ABX (e.g. endo-1,4-.beta.-
U/g.sup.1 (preferably >3000 ABX u/g) d-xylanase) activity
.beta.-glucanase activity 1000-3500 CMC U/g.sup.2 (preferably about
2000-2600) CMC u/g)
[0297] In a preferred embodiment, the DFM taught herein may be used
in combination with a xylanase, a .beta.-glucanase and a
.beta.-glucosidase wherein the xylanase, .beta.-glucanase and
.beta.-glucosidase have the activities set out in the tables below:
.sup.1 One ABX unit is defined as the amount of enzyme required to
generate 1 .mu.mol of xylose reducing sugar equivalents per minute
at 50.degree. C. and pH 5.3..sup.2 One CMC unit of activity
liberates 1 .mu.mol of reducing sugars (expressed as glucose
equivalents) in one minute at 50.degree. C. and pH 4.8.
TABLE-US-00012 Range of activity in Units/g of each enzyme activity
in the composition Xylanase 1500-6000 ABX U/g.sup.1 (e.g.
endo-1,4-.beta.- d-xylanase) activity .beta.-glucanase activity
500-4000 CMC U/g.sup.2 .beta.-glucosidase activity 200-3500 pNPG
U/g.sup.3
TABLE-US-00013 Range of activity in Units/g of each enzyme activity
in the composition Xylanase 2000-6000 ABX (e.g. endo-1,4-.beta.-
U/g.sup.1 (preferably >3000 ABX U/g) d-xylanase) activity
.beta.-glucanase activity 1000-3500 CMC U/g.sup.2 (preferably about
2000-2600) CMC U/g) .beta.-glucosidase activity 300-3000 pNPG
U/g.sup.3 (preferably >2000 pNPG U/g)
[0298] In one embodiment the xylanase and .beta.-glucanase for use
in the present invention may comprise (or consist essentially of,
or consist of) more than about 3000 ABX u/g of xylanase activity
and about 2000-2600 CMC u/g of .beta.-glucanase activity,
respectively. .sup.1 One ABX unit is defined as the amount of
enzyme required to generate 1 .mu.mol of xylose reducing sugar
equivalents per minute at 50.degree. C. and pH 5.3..sup.2 One CMC
unit of activity liberates 1 .mu.mol of reducing sugars (expressed
as glucose equivalents) in one minute at 50.degree. C. and pH
4.8..sup.3 One pNPG unit denotes 1 .mu.mol of nitro-phenol
liberated from para-nitrophenyl-B-D-glucopyranoside per minute at
50.degree. C. and pH 4.8.
[0299] Suitably the xylanase, .beta.-glucanase and
.beta.-glucosidase for use in the present invention may comprise
(or consist essentially of, or consist of) more than about 3000 ABX
u/g of xylanase activity, about 2000-2600 CMC u/g of
.beta.-glucanase activity and more than about 2000 pNPG u/g of
.beta.-glucosidase activity, respectively.
[0300] In one embodiment the xylanase for use in the present
invention may comprise (or consist essentially of, or consist of)
at least 2000 ABX u/g xylanase activity (suitably at least 2500 ABX
u/g activity, suitably at least 3000 ABX u/g activity) as
determined using the "Xylanase Activity Assay (ABX U/g)".
[0301] Suitably, the xylanase for use in the present invention may
comprise (or consist essentially of, or consist of) about 2000 to
about 5000 ABX u/g xylanase activity (suitably at least about 2500
to about 4000 ABX u/g activity, suitably at least about 3000 to
about 4000 ABX u/g activity) as determined using the "Xylanase
Activity Assay (ABX U/g)".
[0302] In another embodiment the .beta.-glucanase for use in the
present invention may comprise (or consist essentially of, or
consist of) at least 1000 CMC u/g .beta.-glucanase activity
(suitably at least 1500 CMC u/g activity, suitably at least 2000
CMC u/g activity) as determined using the ".beta.-glucanase
Activity Assay (CMC U/g)".
[0303] Suitably, the .beta.-glucanase for use in the present
invention may comprise (or consist essentially of, or consist of)
about 600 to about 4000 CMC u/g .beta.-glucanase activity (suitably
at least about 1000 to about 3000 CMC u/g activity, suitably at
least about 1500 to about 2600 CMC u/g activity) as determined
using the "P3-glucanase Activity Assay (CMC U/g)".
[0304] In a further embodiment the .beta.-glucosidase for use in
the present invention may comprise (or consist essentially of or
consist of) at least 300 pNPG u/g .beta.-glucosidase activity
(suitably at least 500 pNPG u/g activity, suitably at least 1000
pNPG u/g activity or suitably at least 2000 pNPG u/g activity) as
determined using the ".beta.-glucosidase Activity Assay (pNPG
U/g)".
[0305] Suitably, the .beta.-glucosidase for use in the present
invention may comprise (or consist essentially of, or consist of)
about 200 to about 4000 pNPG u/g .beta.-glucosidase activity
(suitably at least about 300 to about 3000 pNPG u/g activity,
suitably at least about 1000 to about 3000 pNPG u/g activity or
suitably at least about 2000 to about 3000 pNPG u/g activity) as
determined using the ".beta.-glucosidase Activity Assay (pNPG
U/g)".
[0306] Suitably, the DFM taught herein may be used in combination
with a xylanase and a .beta.-glucanase comprising (or consisting
essentially of or consisting of) at least 2000 ABX u/g xylanase
activity (suitably at least 2500 ABX u/g activity, suitably at
least 3000 ABX u/g activity) as determined using the "Xylanase
Activity Assay (ABX U/g)"; and at least 1000 CMC u/g
.beta.-glucanase activity (suitably at least 1500 CMC u/g activity,
suitably at least 2000 CMC u/g activity) as determined using the
".beta.-glucanase Activity Assay (CMC U/g)".
[0307] Suitably, the DFM taught herein may be used in combination
with a xylanase, a .beta.-glucanase and a .beta.-glucosidase
comprising (or consisting essentially of, or consisting of) at
least 2000 ABX u/g xylanase activity (suitably at least 2500 ABX
u/g activity, suitably at least 3000 ABX u/g activity) as
determined using the "Xylanase Activity Assay (ABX U/g)"; and at
least 1000 CMC u/g .beta.-glucanase activity (suitably at least
1500 CMC u/g activity, suitably at least 2000 CMC u/g activity) as
determined using the ".beta.-glucanase Activity Assay (CMC U/g)";
and at least 300 pNPG u/g .beta.-glucosidase activity (suitably at
least 500 pNPG u/g activity, suitably at least 1000 pNPG u/g
activity or suitably at least 2000 pNPG u/g activity) as determined
using the ".beta.-glucosidase Activity Assay (pNPG U/g)".
[0308] In one embodiment the DFM taught herein may be used in
combination with a xylanase and a .beta.-glucanase comprising (or
consisting essentially of, or consisting of) about 2000 to about
5000 ABX u/g xylanase activity (suitably at least about 2500 to
about 4000 ABX u/g activity, suitably at least about 3000 to about
4000 ABX u/g activity) as determined using the "Xylanase Activity
Assay (ABX U/g)"; and about 600 to about 4000 CMC u/g
.beta.-glucanase activity (suitably at least about 1000 to about
3000 CMC u/g activity, suitably at least about 1500 to about 2600
CMC u/g activity) as determined using the ".beta.-glucanase
Activity Assay (CMC U/g)".
[0309] Suitably, the DFM taught herein may be used in combination
with a xylanase, a .beta.-glucanase and a .beta.-glucosidase
comprising (or consisting essentially of, or consisting of) about
2000 to about 5000 ABX u/g xylanase activity (suitably at least
about 2500 to about 4000 ABX u/g activity, suitably at least about
3000 to about 4000 ABX u/g activity) as determined using the
"Xylanase Activity Assay (ABX U/g)"; about 600 to about 4000 CMC
u/g .beta.-glucanase activity (suitably at least about 1000 to
about 3000 CMC u/g activity, suitably at least about 1500 to about
2600 CMC u/g activity) as determined using the ".beta.-glucanase
Activity Assay (CMC U/g)"; and about 200 to about 4000 pNPG u/g
.beta.-glucosidase activity (suitably at least about 300 to about
3000 pNPG u/g activity, suitably at least about 1000 to about 3000
pNPG u/g activity or suitably at least about 2000 to about 3000
pNPG u/g activity) as determined using the ".beta.-glucosidase
Activity Assay (pNPG U/g)".
[0310] "Xylanase Activity Assay (XU)"
[0311] The xylanase activity can be expressed in xylanase units
(XU) measured at pH 5.0 with AZCL-arabinoxylan (azurine-crosslinked
wheat arabinoxylan, Xylazyme 100 mg tablets, Megazyme) as
substrate. Hydrolysis by endo-(1-4)-.beta.-D-xylanase (xylanase)
produces water soluble dyed fragments, and the rate of release of
these (increase in absorbance at 590 nm) can be related directly to
enzyme activity. The xylanase units (XU) are determined relatively
to an enzyme standard (Danisco Xylanase, available from Danisco
Animal Nutrition) at standard reaction conditions, which are
40.degree. C., 10 min reaction time in Mcllvaine buffer, pH
5.0.
[0312] The xylanase activity of the standard enzyme is determined
as amount of released reducing sugar end groups from an
oat-spelt-xylan substrate per min at pH 5.3 and 50.degree. C. The
reducing sugar end groups react with 3, 5-Dinitrosalicylic acid and
formation of the reaction product can be measured as increase in
absorbance at 540 nm. The enzyme activity is quantified relative to
a xylose standard curve (reducing sugar equivalents). One xylanase
unit (XU) is the amount of standard enzyme that releases 0.5
.mu.mol of reducing sugar equivalents per min at pH 5.3 and
50.degree. C.
[0313] "Xylanase Activity Assay (ABX U/g)"
[0314] The xylanase activity can be expressed in acid birchwood
xylanase units (ABX U) measured at pH 5.3 with birchwood 4-O methyl
glucuronoxylan as substrate. Pipette 1.8 ml of 1% birchwood 4-O
methyl glucuronoxylan substrate solution into each test tube.
Incubate for 10-15 minutes, allowing to equilibrate at 50.degree.
C. Pipette 0.2 ml of enzyme dilution using positive displacement
pipettes or equivalent. Vortex to mix. Incubate each sample at
50.degree. C. for exactly 5 minutes. Add 3 ml of 1% 3,5
nitrosalicylic acid sodium salt (DNS) solution and mix. Cover the
tops of the test tubes with caps to prevent evaporation. Place test
tubes in a boiling bath for exactly 5 minutes. Cool test tubes for
10 minutes in ice/water bath. Incubate test tube for 10 minutes at
room temperature. Transfer test tube contents to cuvettes and
measure at 540 nm against deionised water. Correct the absorbance
for background colour by subtracting the corresponding enzyme
blank. The enzyme activity is quantified relative to a xylose
standard curve (reducing sugar equivalents).
[0315] One ABX unit is defined as the amount of enzyme required to
generate 1 .mu.mol of xylose reducing sugar equivalents per minute
at 50.degree. C. and pH 5.3.
[0316] ".beta.-Glucanase Activity Assay (CMC U/g)"
[0317] The .beta.-glucanase activity can be expressed in CMC units
measured at pH 4.8 with carboxylmethyl cellulose sodium salt (CMC)
as substrate. Pipette 1 ml of 1% carboxylmethyl cellulose sodium
salt (CMC) solution (prepared with 0.05M sodium acetate buffer)
into sample and blank tubes. Incubate tubes in a 50.degree. C.
water bath for 10 minutes. Pipette 1 ml of enzyme dilution at 15
second intervals to the sample tubes. Mix tubes after each
addition. After 10 minute, add 3 ml of 1% 3,5 dinitrosalicylic acid
sodium salt (DNS) in the same order and timing as the enzyme
addition to the sample tubes. Add 3 ml of DNS to the sample blank
tubes. After adding the DNS remove the test tubes to another rack
not in the 50.degree. C. water bath. Add 1 ml of diluted enzyme to
the corresponding sample blank. Cap the tubes and boil for exactly
5 minutes. Remove from the 100.degree. C. water bath and place in
an ice bath for 10 minutes. Leave at room temperature for 10-15
minutes. Transfer to 3 ml cuvettes. Using the reagent blank to zero
the spectrophotometer, each sample is read at 540 nm against
deionised water. The enzyme activity is quantified relative to a
glucose standard curve (reducing sugar equivalents).
[0318] One CMC unit of activity liberates 1 .mu.mol of reducing
sugars (expressed as glucose equivalents) in one minute at
50.degree. C. and pH 4.8.
[0319] ".beta.-Glucanase Activity Assay (BGU)"
[0320] The beta-glucanase activity can be expressed in
beta-glucanase units (BGU) measured at pH 5.0 with AZCL-glucan
(azurine-cross linked barley .beta.-glucan, Glucazyme 100 mg
tablets, Megazyme) as substrate. Hydrolysis by beta-glucanase
produces soluble dyed fragments, and the rate of release of these
(increase in absorbance at 590 nm) can be related directly to
enzyme activity. The beta-glucanase units (BGU) are determined
relatively to an enzyme standard (Multifect BGL, available from
Danisco Animal Nutrition) at standard reaction conditions, which
are 50.degree. C., 10 min reaction time in 0.1 M acetate buffer, pH
5.0.
[0321] The beta-glucanase activity of the standard enzyme is
determined as amount of released reducing sugar end groups from a
barley glucan substrate per min at pH 5.0 and 50.degree. C. The
reducing sugar end groups react with 3,5-Dinitrosalicylic acid and
formation of the reaction product can be measured as an increase in
absorbance at 540 nm. The enzyme activity is quantified relative to
a glucose standard curve (reducing sugar equivalents). One
beta-glucanase unit (BGU) is the amount of standard enzyme that
releases 2.4 .mu.mol of reducing sugar equivalents per min at pH
5.0 and 50.degree. C.
".beta.-Glucosidase Activity Assay (pNPG U/g)"
[0322] The .beta.-glucosidase activity can be expressed in pNPG
units measured at pH 4.8 with para-nitrophenyl-B-D-glucopyranoside
(pNPG) as substrate. Pipette 1 ml of 3%
nitrophenyl-beta-D-glucopyranoside (pNPG) solution (prepared with
0.05M sodium acetate buffer) into duplicate test tubes for each
sample and control. Place into 50.degree. C. water bath for 5
minutes.
[0323] Add 200 .mu.l of control or sample to their respective
duplicate tubes at intervals of 15-30 seconds. To the reagent blank
tube, add 200 .mu.l of sodium acetate buffer. Vortex each tube
after addition of sample. Let the tubes incubate for exactly 10
minutes. After the 10 minutes incubation, add 500 .mu.l of 1M
sodium carbonate solution to stop the reaction. Vortex each tube
after the addition and place the tube in a rack outside of the
water bath. Add 10 ml of milli-Q water to each tube and vortex to
mix. Using the reagent blank to zero the spectrophotometer, the
concentration of the 4-nitrophenol is measured by reading each
sample at 400 nm.
[0324] One pNPG unit denotes 1 .mu.mol of nitro-phenol liberated
from para-nitrophenyl-B-D-glucopyranoside per minute at 50.degree.
C. and pH 4.8.
[0325] Advantages
[0326] The interaction of DFMs with the xylanase and the
.beta.-glucanase (and optionally at least one further fibre
degrading enzyme) is complicated and without wishing to be bound by
theory, it is very surprising that we can see an increase in the
production of short chain fatty acids in the GIT of animals.
[0327] The combination of the specific DFMs taught herein with at
least one xylanase and at least one .beta.-glucanase (and
optionally at least one further fibre degrading enzyme) has been
found to be particularly advantageous in feedstuffs and/or in a
subject which is fed a feedstuff which is high in fibrous
by-products (e.g. from the biofuel and milling industries).
[0328] It has been surprisingly found that the nutritional value
and digestibility of feedstuffs comprising substantial quantities
(sometimes 30-60%) of fibrous by-products (having a high content of
non-starch polysaccharides, e.g. fibre) can be significantly
improved, as can the performance and weight gain of a subject fed
such feedstuffs.
[0329] One advantage of the present invention is the improvement of
feed conversion ratio (FCR) observed by using the combination of
the present invention.
[0330] Without wishing to be bound in theory the degradation of
dietary material derived from plant cell wall particles which is
high in non-starch polysaccharides (NSP) by xylanases can be
optimized for improved animal performance when combining xylanase
(e.g. endo-1,4-.beta.-d-xylanase) with one or more .beta.-glucanase
(and optionally in combination with one or more further fibre
degrading enzymes (e.g. a cellobiohydrolase (E.C. 3.2.1.176 and
E.C. 3.2.1.91), a .beta.-glucosidase (E.C. 3.2.1.21), a
.beta.-xylosidase (E.C. 3.2.1.37), a feruloyl esterase (E.C.
3.1.1.73), an .alpha.-arabinofuranosidase (E.C. 3.2.1.55), a
pectinase (e.g. an endopolygalacturonase (E.C. 3.2.1.15), an
exopolygalacturonase (E.C. 3.2.1.67) or a pectate lyase (E.C.
4.2.2.2)), or combinations thereof)) and one or more specific
direct fed-microbials (DFMs) selected for their capacity to produce
enzymes and/or their capacity of producing Short Chain Fatty Acids
(SCFA) from NSP fraction pentoses in anaerobic conditions and/or
their capacity to promote endogenous populations of fibrolytic
microflora in a subject's GIT and/or their capacity to degrade
C5-sugars.
[0331] The reason why this combination improves performance is that
the solubilisation of fibre, specifically hemicellulose, from the
diet is maximized in the gastro intestinal tract (GIT) of the
animals. This solubilisation of hemicellulose would not always be
sufficient to increase performance because C5-sugars released are
not an efficient source of energy for animals when they are
absorbed (Savory C. J. Br. J. Nut. 1992, 67: 103-114), but they are
a more efficient source of energy when converted into short chain
fatty acids (SCFA) either by microorganisms in the GIT or by
DFMs.
[0332] Therefore the energy value from plant products (e.g. wheat,
corn, oats, barley and cereals co-products (by-products) or mixed
grain diet readily accessible for monogastrics) can be optimized by
combining xylanase (e.g. endo-1,4-.beta.-d-xylanase) and
.beta.-glucanase (and optionally at least one other fibre degrading
enzyme (including but not limited to a cellobiohydrolase (E.C.
3.2.1.176 and E.C. 3.2.1.91), a .beta.-glucosidase (E.C. 3.2.1.21),
a .beta.-xylosidase (E.C. 3.2.1.37), a feruloyl esterase (E.C.
3.1.1.73), an .alpha.-arabinofuranosidase (E.C. 3.2.1.55), a
pectinase (e.g. an endopolygalacturonase (E.C. 3.2.1.15), an
exopolygalacturonase (E.C. 3.2.1.67) or a pectate lyase (E.C.
4.2.2.2)), or combinations thereof)) and specific DFMs that can
produce SCFAs from NSP fraction pentoses in anaerobic conditions
and/or that can modulate the microbial populations in the GIT to
increase SCFA production from the sugars released and/or that can
utilise C-5 sugars. The DFMs may adapt their metabolism to
synergistically increase the fibre hydrolysis in combination with
xylanase and .beta.-glucanase (and optionally at least one further
fibre degrading enzyme). Using DFMs that can produce (fibrolytic)
enzymes can provide additional benefits and maximize the benefits
of the added enzymes.
[0333] Specific DFMs selected for their enzymatic activities can be
considered as a glycan-driven bacterial food chain. The
specifically selected DFMs taught herein may preferentially utilize
dietary fibres, a trait that allows them to carry out the initial
glycan digestion steps to liberate shorter, more soluble
polysaccharides for other bacteria, e.g. other endogenous GIT
microflora. The specific DFMs have been selected for their
metabolism which adjusts according to the glycans released by
enzymes (e.g. xylanase and .beta.-glucanase (and optionally at
least one further fibre degrading enzyme)) to improve the efficacy
of the enzymes taught herein and the DFM(s) combination compared to
use of a combination of enzymes alone or the use of DFM(s)
alone.
[0334] Without wishing to be bound by theory, in the present
invention dietary material derived from plant cell wall particles
which is rich in source-specific glycans, such as cellulose,
hemicellulose and pectin (plant material) or glycosaminoglycans
enter the distal gut in particulate forms that are attacked by the
specific DFMs glycan degraders which are capable of directly
binding to these insoluble particles and digesting their glycan
components. After this initial degradation of glycan-containing
particles, more-soluble glycan fragments can be digested by
secondary glycan degraders present in the caecum, which contribute
to the liberated pool of short-chain fatty acid (SCFA) fermentation
products that is derived from both types of degraders. As SCFAs
arise from carbohydrate fermentation and/or protein fermentation
and deamination by the indigenous anaerobic microflora in the GIT,
SCFA concentration can be an index of the anaerobic-organism
population. SCFA may actually provide a number of benefits to the
host animal, acting as metabolic fuel for intestine, muscle,
kidney, heart, liver and brain tissue, and also affording
bacteriostatic and bacteriocidal properties against organisms such
as Salmonella and E. coli.
[0335] The nutritional value of fibre in non-ruminants can mainly
be derived through short chain fatty acids (SCFA) production via
fermentation of solubilized or degraded fibres by effective fibre
degrading enzymes (e.g. xylanases and .beta.-glucanase and/or a
further fibre degrading enzyme as taught herein). Feed xylanase
alone is not enough to use fibrous ingredients in animal
(especially non-ruminant) diets. A large array of chemical
characteristics exists among plant-based feed ingredients. Enzyme
application depends on the characteristics of the plant (feed)
material. By way of example only, in wheat grain arabinoxylans
predominates, however in wheat middlings (a co-product (by-product)
of wheat milling), the content of .beta.-glucan increases from 8
g.sup.-1 DM (in grain) to an excess of 26 g kg.sup.-1 DM. An enzyme
matrix containing a complex of xylanase and .beta.-glucanase (and
optionally at least one further fibre degrading enzyme) can improve
the nutritional value of feedstuffs high in co-product(s)
(by-product(s)) based diets.
[0336] SCFAs have different energy values and some can serve as
precursors of glucose and some can contribute to the maintenance of
intestinal integrity and health. The inventors have found that the
specific combinations taught herein preferentially move the
fermentation process in an animal's GIT towards the production of
more valuable/useful SCFA.
[0337] Without wishing to be bound by theory, the present inventors
have found that NSPs can be effectively degraded by a combination
of a DFM according to the present invention and a xylanase and a
.beta.-glucanase (and optionally at least one further fibre
degrading enzyme). In addition, it has been found that this
specific combination releases C-5 sugars which usually have only
marginal nutritional value to the animal. However, using
combinations as claimed herein it is possible to have
microorganisms in the GIT (either the DFM of the present invention)
or endogenous fibrolytic microflora (which are stimulated by the
combinations (of DFM) of the present invention) convert these C-5
sugars into useful and nutritionally valuable components, namely
short chain fatty acids. These short chain fatty acids can be
utilised by the animal. Thus the system improves the nutritional
value of a feedstuff for an animal.
[0338] Advantageously, the combination of a direct fed microbial, a
xylanase and a .beta.-glucanase (and optionally at least one
further fibre degrading enzyme) as taught herein surprisingly
increases fibre degradation in a feed additive composition, premix,
feed or feedstuff, which leads to improved performance of a
subject. In particular, the combination of the present invention
improves digestibility of a raw material in a feed resulting in an
increase in nutrient bioavailability (e.g. nutrient digestibility)
and metabolizable energy therein.
[0339] Formulation of the DFM with the Enzymes
[0340] The DFM of the present invention and the enzymes may be
formulated in any suitable way to ensure that the formulation
comprises viable DFMs and active enzymes.
[0341] In one embodiment the DFM and enzymes may be formulated as a
dry powder or a granule. The dry powder or granules may be prepared
by means known to those skilled in the art, such as in a
microingredients mixer.
[0342] For some embodiments the DFM and/or the enzyme(s) may be
coated, for example encapsulated. Suitably the DFM and enzymes may
be formulated within the same coating or encapsulated within the
same capsule. Alternatively one or two or three or four of the
enzymes may be formulated within the same coating or encapsulated
within the same capsule and the DFM could be formulated in a
coating separate to the one or more or all of the enzymes. In some
embodiments, such as where the DFM is capable of producing
endospores, the DFM may be provided without any coating. In such
circumstances, the DFM endospores may be simply admixed with one or
two or three or four enzymes. In the latter case, the enzymes may
be coated, e.g. encapsulated, for instance one or more or all of
the enzymes may be coated, e.g. encapsulated. The enzymes may be
encapsulated as mixtures (i.e. comprising one or more, two or more,
three or more or all) of enzymes or they may be encapsulated
separately, e.g. as single enzymes. In one preferred embodiment all
four enzymes may be coated, e.g. encapsulated, together.
[0343] In one embodiment the coating protects the enzymes from heat
and may be considered a thermoprotectant.
[0344] In one embodiment the feed additive composition is
formulated to a dry powder or granules as described in
WO2007/044968 (referred to as TPT granules) incorporated herein by
reference.
[0345] In some embodiments the DFM (e.g. DFM endospores for
example) may be diluted using a diluent, such as starch powder,
lime stone or the like.
[0346] In another embodiment the feed additive composition may be
formulated by applying, e.g. spraying, the enzyme(s) onto a carrier
substrate, such as ground wheat for example.
[0347] In one embodiment the feed additive composition according to
the present invention may be formulated as a premix. By way of
example only the premix may comprise one or more feed components,
such as one or more minerals and/or one or more vitamins.
[0348] In one embodiment the DFM and/or enzymes for use in the
present invention are formulated with at least one physiologically
acceptable carrier selected from at least one of maltodextrin,
limestone (calcium carbonate), cyclodextrin, wheat or a wheat
component, sucrose, starch, Na.sub.2SO.sub.4, Talc, PVA, sorbitol,
benzoate, sorbiate, glycerol, sucrose, propylene glycol,
1,3-propane diol, glucose, parabens, sodium chloride, citrate,
acetate, phosphate, calcium, metabisulfite, formate and mixtures
thereof.
[0349] Packaging
[0350] In one embodiment the feed additive composition and/or
premix and/or feed or feedstuff according to the present invention
is packaged.
[0351] In one preferred embodiment the feed additive composition
and/or premix and/or feed or feedstuff is packaged in a bag, such
as a paper bag.
[0352] In an alternative embodiment the feed additive composition
and/or premix and/or feed or feedstuff may be sealed in a
container. Any suitable container may be used.
[0353] By-Products
[0354] The animal feed industry has seen an increased feeding of
by-products, e.g. from biofuel processing, to animals (raising this
form of animal feed from 0-10% to the current extremes of 30-60%).
These diet cost savings have been a great opportunity for industry
to save on feed input costs, but come with a set of challenges as
well. The by-products are often high fibre (e.g. at least
approximately 40% fibre) products. Consequently the inclusion of
high-fibre by-product (e.g. DDGS) can have negative impact on
animal growth performance and carcass characteristics. In addition
to the negative effects on animal growth and carcass quality,
alterations in nutrient digestibility have implications for manure
(e.g. swine-manure) handling, storage and decomposition.
[0355] The term "by-product" as used herein means any fibrous plant
material, e.g. one which comprises at least approximately 20% or
30% fibre).
[0356] In one embodiment the term by-product means any by-product
of a high fibre feed material.
[0357] In one embodiment the by-product as referred to herein may
be selected from one or more of the following products: corn germ
meal, corn bran, Hominy feed, corn gluten feed, Distillers Dried
Grain Solubles (DDGS), Distillers Dried Grain (DDG), gluten meal,
wheat shorts, wheat middlings or combinations thereof.
[0358] In one embodiment the feedstuff of the present invention
comprises a fibrous by-product such as corn germ meal, corn bran,
Hominy feed, corn gluten feed, Distillers Dried Grain Solubles
(DDGS), Distillers Dried Grain (DDG), gluten meal, wheat shorts,
wheat middlings or combinations thereof.
[0359] In one embodiment the subject to which the DFM, xylanase and
.beta.-glucanase (and optionally at least one further fibre
degrading enzyme) combination of the present invention or feed
additive composition of the present invention is administered, is
also fed a feedstuff comprising a fibrous by-product such as corn
germ meal, corn bran, Hominy feed, corn gluten feed, Distillers
Dried Grain Solubles (DDGS), Distillers Dried Grain (DDG), gluten
meal, wheat shorts, wheat middlings or combinations thereof.
[0360] Breakdown or Degradation
[0361] The enzyme (or composition comprising the enzyme) of the
present invention or as disclosed herein may be used to breakdown
(degrade) insoluble arabinoxylan (AXinsol) or soluble arabinoxylan
(AXsol) or combinations thereof, or degradation products of
AXinsol. The term "breakdown" or "degrade" in synonymous with
hydrolyses.
[0362] Non-Starch Polysaccharides (NSPs)
[0363] A major part of common vegetable feed ingredients consists
of carbohydrates, making carbohydates a crucial factor in animal
production. Beside well digestible nutrients, such as starch and
sugars, the carbohydrate fraction of vegetable origin includes
indigestible (fibrous) components, such as cellulose,
hemicellulose, pectins, beta-glucans and lignin.
[0364] All of these poorly digestible components, excluding lignin,
are classified as a group referred to herein as non-starch
polysaccharides (NSPs). The NSP fraction is well known for the
anti-nutritional effects it can exert.
[0365] In one embodiment the term fibre may be used interchangeably
with the term NSPs.
[0366] Within the group of NSP, hemicellulose itself is a
heterogenous subgroup predominantly made up of xylans, arabinans,
galatans, glucans and mannans. Arabinoxylan is the principal
NSP-fraction in several of the most important feed raw materials,
including wheat and corn.
[0367] Arabinoxylan (AX)
[0368] The term "arabinoxylans" (AX) as used herein means a
polysaccharide consisting of a xylan backbone (1,4-linked xylose
units) with L-arabinofuranose (L-arabinose in its 5-atom ring form)
attached randomly by 1.alpha..fwdarw.2 and/or 1.alpha..fwdarw.3
linkages to the xylose units throughout the chain. Arabinoxylan is
a hemicellulose found in both the primary and secondary cell walls
of plants. Arabinoxylan can be found in the bran of grains such as
wheat, maize (corn), rye, and barley.
[0369] Arabinoxylan (AX) is found in close association with the
plant cell wall, where it acts as a glue linking various building
blocks of the plant cell wall and tissue, give it both structural
strength and rigidity.
[0370] Since xylose and arabinose (the constituents of
arabinoxylans) are both pentoses, arabinoxylans are usually
classified as pentosans.
[0371] AX is the principal Non Starch Polysaccharide (NSP)-fraction
in several of the most important feed raw material, including wheat
and corn.
[0372] Its abundance, location within vegetable material and
molecular structure cause AX to have a severe, negative impact on
feed digestibility, effectively reducing the nutritional value of
the raw materials in which it is present. This makes AX an
important anti-nutritional factor, reducing animal production
efficiency.
[0373] AXs can also hold substantial amounts of water (which can be
referred to as their water holding capacity)--this can cause
soluble arabinoxylans to result in (high) viscosity--which is a
disadvantage in many applications.
[0374] Water Insoluble Arabinoxylan (Axinsol)
[0375] Water-insoluble arabinoxylan (AXinsol) also known as
water-unextractable arabinoxylan (WU-AX) constitutes a significant
proportion of the dry matter of plant material.
[0376] In wheat AXinsol can account for 6.3% of the dry matter. In
wheat bran and wheat DDGS AXinsol can account for about 20.8% or
13.4% of the dry matter (w/w).
[0377] In rye AXinsol can account for 5.5% of the dry matter.
[0378] In corn AXinsol can account for 5.1% of the dry matter. In
corn DDGS AXinsol can account for 12.6% of the dry matter.
[0379] AXinsol causes nutrient entrapment in feed. Large quantities
of well digestible nutrients such as starch and proteins remain
either enclosed in clusters of cell wall material or bound to side
chains of the AX. These entrapped nutrients will not be available
for digestion and subsequent absorption in the small intestine.
[0380] Water-Soluble Arabinoxylan (Axsol)
[0381] Water-soluble arabinoxylan (AXsol) also known as water
extractable arabinoxylan (WE-AX) can cause problems in biofuel
production and/or malting and/or brewing and/or in feed as they can
cause increased viscosity due to the water-binding capacity of
AXsol.
[0382] In feed AXsol can have an anti-nutritional effect
particularly in monogastrics as they cause a considerable increase
of the viscosity of the intestinal content, caused by the
extraordinary water-binding capacity of AXsol. The increase
viscosity can affect feed digestion and nutrient use as it can
prevent proper mixing of feed with digestive enzymes and bile salts
and/or it slows down nutrient availability and absorption and/or it
stimulates fermentation in the hindgut.
[0383] In wheat AXsol can account for 1.8% of the dry matter. In
wheat bran and wheat DDGS AXsol can account for about 1.1% or 4.9%
of the dry matter (w/w).
[0384] In rye AXsol can account for 3.4% of the dry matter.
[0385] In barley AXsol can account for 0.4-0.8% of the dry
matter.
[0386] In corn AXsol can account for 0.1% of the dry matter. In
corn DDGS AXinsol can account for 0.4% of the dry matter.
[0387] In addition, however, to the amount of AXsol present in
plant material, when a xylanase solubilises AXinsol in the plant
material this can release pentosans and/or oligomers which
contribute to AXsol content of the plant material.
[0388] One significant advantage of some of the xylanases disclosed
herein is that they have the ability to both solubilise AXinsol as
well as to rapidly and efficiently breakdown the solubilised
oligomers and/or pentosans thus the enzymes are able to solubilise
AXinsol without increasing viscosity and/or decreasing
viscosity.
[0389] A breakdown of AXsol can decrease viscosity.
[0390] A breakdown of AXsol can release nutrients.
[0391] Viscosity
[0392] The present invention can be used to reduce viscosity in any
process where the water-binding capacity of AXsol causes an
undesirable increase in viscosity.
[0393] The present invention relates to reducing viscosity by
breaking down (degrading) AXsol or by breaking down (degrading) the
polymers and/or oligomers produced by solubilising AXinsol.
[0394] In the present invention a reduction in viscosity can be
calculated by comparing one sample comprising the xylanase of the
present invention (or taught herein) compared with another
comparable sample without the xylanase of the present invention (or
taught herein).
[0395] Comparing the viscosity reduction profiles of the xylanase
of the present invention with those of the market benchmark
xylanases demonstrates the enzyme performance. The aim is to
improve enzyme performance compared to the market benchmark. The
benchmark enzymes for the individual applications are provided in
the examples below
[0396] In one embodiment of the present invention the xylanases
taught herein are viscosity reducers.
[0397] Feed or Feedstuff
[0398] The enzyme or feed additive composition of the present
invention may be used as--or in the preparation of--a feed.
[0399] The term "feed" is used synonymously herein with
"feedstuff".
[0400] In one embodiment the feedstuff of the present invention
comprises high fibre feed material and/or at least one by-product
of the at least one high fibre feed material such as corn germ
meal, corn bran, Hominy feed, corn gluten feed, Distillers Dried
Grain Solubles (DDGS), Distillers Dried Grain (DDG), gluten meal,
wheat shorts, wheat middlings or combinations thereof.
[0401] In one embodiment the subject to which the DFM, xylanase and
.beta.-glucanase combination (optionally in combination a further
fibre degrading enzyme) of the present invention or feed additive
composition of the present invention is administered, is also fed a
feedstuff comprising a high fibre feed material and/or at least one
by-product of the at least one high fibre feed material such as
corn germ meal, corn bran, Hominy feed, corn gluten feed,
Distillers Dried Grain Solubles (DDGS), Distillers Dried Grain
(DDG), gluten meal, wheat shorts, wheat middlings or combinations
thereof.
[0402] Suitably, in one embodiment the cereal component of a
poultry subject's diet can be either wheat or barley with rye,
wheat middlings, wheat bran, oats, oats hulls whilst vegetable
components can be soybean meal with or without other protein
ingredients such as canola, rape seed meal, etc. provided that the
diet will contain wheat-barley as the main ingredients and
formulated to meet the nutrient requirements of the birds being
fed.
[0403] The feed according to the present invention may be in the
form of a solution or as a solid--depending on the use and/or the
mode of application and/or the mode of administration.
[0404] When used as--or in the preparation of--a feed--such as
functional feed--the enzyme or composition of the present invention
may be used in conjunction with one or more of: a nutritionally
acceptable carrier, a nutritionally acceptable diluent, a
nutritionally acceptable excipient, a nutritionally acceptable
adjuvant, a nutritionally active ingredient.
[0405] In a preferred embodiment the enzyme or feed additive
composition of the present invention is admixed with a feed
component to form a feedstuff.
[0406] The term "feed component" as used herein means all or part
of the feedstuff. Part of the feedstuff may mean one constituent of
the feedstuff or more than one constituent of the feedstuff, e.g. 2
or 3 or 4. In one embodiment the term "feed component" encompasses
a premix or premix constituents.
[0407] Preferably the feed may be a fodder, or a premix thereof, a
compound feed, or a premix thereof. In one embodiment the feed
additive composition according to the present invention may be
admixed with a compound feed, a compound feed component or to a
premix of a compound feed or to a fodder, a fodder component, or a
premix of a fodder.
[0408] The term fodder as used herein means any food which is
provided to an animal (rather than the animal having to forage for
it themselves). Fodder encompasses plants that have been cut.
[0409] The term fodder includes silage, compressed and pelleted
feeds, oils and mixed rations, and also sprouted grains and
legumes.
[0410] Fodder may be obtained from one or more of the plants
selected from: corn (maize), alfalfa (Lucerne), barley, birdsfoot
trefoil, brassicas, Chau moellier, kale, rapeseed (canola),
rutabaga (swede), turnip, clover, alsike clover, red clover,
subterranean clover, white clover, fescue, brome, millet, oats,
sorghum, soybeans, trees (pollard tree shoots for tree-hay), wheat,
and legumes.
[0411] The term "compound feed" means a commercial feed in the form
of a meal, a pellet, nuts, cake or a crumble. Compound feeds may be
blended from various raw materials and additives. These blends are
formulated according to the specific requirements of the target
animal.
[0412] Compound feeds can be complete feeds that provide all the
daily required nutrients, concentrates that provide a part of the
ration (protein, energy) or supplements that only provide
additional micronutrients, such as minerals and vitamins.
[0413] The main ingredients used in compound feed are the feed
grains, which include corn, wheat, wheat bran, soybeans, sorghum,
oats, and barley.
[0414] Suitably a premix as referred to herein may be a composition
composed of microingredients such as vitamins, minerals, chemical
preservatives, antibiotics, fermentation products, and other
essential ingredients. Premixes are usually compositions suitable
for blending into commercial rations.
[0415] Any feedstuff of the present invention may comprise one or
more feed materials selected from the group comprising a) cereals,
such as small grains (e.g., wheat, barley, rye, oats, triticale and
combinations thereof) and/or large grains such as maize or sorghum;
b) by products from cereals, such as corn germ meal, corn bran,
Hominy feed, corn gluten feed, Distillers Dried Grain Solubles
(DDGS), Distillers Dried Grain (DDG), gluten meal, wheat shorts,
wheat middlings or combinations thereof; c) protein obtained from
sources such as soya, sunflower, peanut, lupin, peas, fava beans,
cotton, canola, fish meal, dried plasma protein, meat and bone
meal, potato protein, whey, copra, sesame; d) oils and fats
obtained from vegetable and animal sources; e) minerals and
vitamins.
[0416] In one embodiment the feedstuff comprises or consists of
corn, DDGS (such as cDDGS), wheat, wheat bran or a combination
thereof.
[0417] In one embodiment the feed component may be corn, DDGS (e.g.
cDDGS), wheat, wheat bran or a combination thereof.
[0418] In one embodiment the feedstuff comprises or consists of
corn, DDGS (such as cDDGS) or a combination thereof.
[0419] In one embodiment a feed component may be corn, DDGS (such
as corn DDGS (cDDGS)) or a combination thereof.
[0420] A feedstuff of the present invention may contain at least
30%, at least 40%, at least 50% or at least 60% by weight corn and
soybean meal or corn and full fat soy, or wheat meal or sunflower
meal.
[0421] A feedstuff of the present invention may contain between
about 5 to about 40% corn DDGS.
[0422] For poultry--the feedstuff on average may contain between
about 7 to 12% corn DDGS. For swine (pigs)--the feedstuff may
contain on average 5 to 40% corn DDGS.
[0423] A feedstuff of the present invention may contain corn as a
single grain, in which case the feedstuff may comprise between
about 35% to about 85% corn.
[0424] In feedstuffs comprising mixed grains, e.g. comprising corn
and wheat for example, the feedstuff may comprise at least 10%
corn.
[0425] In addition or in the alternative, a feedstuff of the
present invention may comprise at least one high fibre feed
material and/or at least one by-product of the at least one high
fibre feed material to provide a high fibre feedstuff. Examples of
high fibre feed materials include: wheat, barley, rye, oats, by
products from cereals, such as corn gluten meal, wet-cake,
Distillers Dried Grain (DDG), Distillers Dried Grain with Solubles
(DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice
hulls, oat hulls, palm kernel, and citrus pulp. Some protein
sources may also be regarded as high fibre: protein obtained from
sources such as sunflower, lupin, fava beans and cotton.
[0426] In one embodiment the feedstuff of the present invention
comprises at least one high fibre material and/or at least one
by-product of the at least one high fibre feed material selected
from the group consisting of Distillers Dried Grain with Solubles
(DDGS)--particularly corn DDGS (cDDGS), wet-cake, Distillers Dried
Grain (DDG)--particularly corn DDG (cDDG), wheat bran, and wheat
for example.
[0427] In one embodiment the feedstuff of the present invention
comprises at least one high fibre material and/or at least one
by-product of the at least one high fibre feed material selected
from the group consisting of Distillers Dried Grain Solubles
(DDGS)--particularly cDDGS, wheat bran, and wheat for example.
[0428] In the present invention the feed may be one or more of the
following: a compound feed and premix, including pellets, nuts or
(cattle) cake; a crop or crop residue: corn, soybeans, sorghum,
oats, barley, copra, chaff, sugar beet waste; fish meal; meat and
bone meal; molasses; oil cake and press cake; oligosaccharides;
conserved forage plants: silage; seaweed; seeds and grains, either
whole or prepared by crushing, milling etc.; sprouted grains and
legumes; yeast extract.
[0429] The term feed in the present invention also encompasses in
some embodiments pet food. A pet food is plant or animal material
intended for consumption by pets, such as dog food or cat food. Pet
food, such as dog and cat food, may be either in a dry form, such
as kibble for dogs, or wet canned form. Cat food may contain the
amino acid taurine.
[0430] The term feed in the present invention also encompasses in
some embodiments fish food. A fish food normally contains macro
nutrients, trace elements and vitamins necessary to keep captive
fish in good health. Fish food may be in the form of a flake,
pellet or tablet. Pelleted forms, some of which sink rapidly, are
often used for larger fish or bottom feeding species.
[0431] Some fish foods also contain additives, such as beta
carotene or sex hormones, to artificially enhance the color of
ornamental fish.
[0432] The term feed in the present invention also encompasses in
some embodiment bird food. Bird food includes food that is used
both in birdfeeders and to feed pet birds. Typically bird food
comprises of a variety of seeds, but may also encompass suet (beef
or mutton fat).
[0433] As used herein the term "contacted" refers to the indirect
or direct application of the enzyme (or composition comprising the
enzyme) of the present invention to the product (e.g. the feed).
Examples of the application methods which may be used, include, but
are not limited to, treating the product in a material comprising
the feed additive composition, direct application by mixing the
feed additive composition with the product, spraying the feed
additive composition onto the product surface or dipping the
product into a preparation of the feed additive composition.
[0434] In one embodiment the feed additive composition of the
present invention is preferably admixed with the product (e.g.
feedstuff). Alternatively, the feed additive composition may be
included in the emulsion or raw ingredients of a feedstuff.
[0435] For some applications, it is important that the composition
is made available on or to the surface of a product to be
affected/treated. This allows the composition to impart one or more
of the following favourable characteristics: performance
benefits.
[0436] The enzyme (or composition comprising the enzyme) of the
present invention may be applied to intersperse, coat and/or
impregnate a product (e.g. feedstuff or raw ingredients of a
feedstuff) with a controlled amount of said enzyme.
[0437] Suitably the feed additive composition may be simply
administered to the subject at the same time as feeding the animal
a feedstuff.
[0438] Preferably, the enzyme (or composition comprising the
enzyme) of the present invention will be thermally stable to heat
treatment up to about 70.degree. C.; up to about 85.degree. C.; or
up to about 95.degree. C. The heat treatment may be performed for
up to about 1 minute; up to about 5 minutes; up to about 10
minutes; up to about 30 minutes; up to about 60 minutes. The term
thermally stable means that at least about 75% of the enzyme that
was present/active in the additive before heating to the specified
temperature is still present/active after it cools to room
temperature. Preferably, at least about 80% of the enzyme that is
present and active in the additive before heating to the specified
temperature is still present and active after it cools to room
temperature.
[0439] In a particularly preferred embodiment the enzyme (or
composition comprising the enzyme) of the present invention is
homogenized to produce a powder.
[0440] In an alternative preferred embodiment, the enzyme (or
composition comprising the enzyme) of the present invention is
formulated to granules as described in WO2007/044968 (referred to
as TPT granules) incorporated herein by reference.
[0441] In another preferred embodiment when the feed additive
composition is formulated into granules the granules comprise a
hydrated barrier salt coated over the protein core. The advantage
of such salt coating is improved thermo-tolerance, improved storage
stability and protection against other feed additives otherwise
having adverse effect on the enzyme.
[0442] Preferably, the salt used for the salt coating has a water
activity greater than 0.25 or constant humidity greater than 60% at
20.degree. C.
[0443] Preferably, the salt coating comprises a
Na.sub.2SO.sub.4.
[0444] The method of preparing an enzyme (or composition comprising
the enzyme) of the present invention may also comprise the further
step of pelleting the powder. The powder may be mixed with other
components known in the art. The powder, or mixture comprising the
powder, may be forced through a die and the resulting strands are
cut into suitable pellets of variable length.
[0445] Optionally, the pelleting step may include a steam
treatment, or conditioning stage, prior to formation of the
pellets. The mixture comprising the powder may be placed in a
conditioner, e.g. a mixer with steam injection. The mixture is
heated in the conditioner up to a specified temperature, such as
from 60-100.degree. C., typical temperatures would be 70.degree.
C., 80.degree. C., 85.degree. C., 90.degree. C. or 95.degree. C.
The residence time can be variable from seconds to minutes and even
hours.
[0446] Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1
minutes 2 minutes., 5 minutes, 10 minutes, 15 minutes, 30 minutes
and 1 hour.
[0447] It will be understood that the enzyme (or composition
comprising the enzyme) of the present invention is suitable for
addition to any appropriate feed material.
[0448] It will be understood by the skilled person that different
animals require different feedstuffs, and even the same animal may
require different feedstuffs, depending upon the purpose for which
the animal is reared.
[0449] Optionally, the feedstuff may also contain additional
minerals such as, for example, calcium and/or additional
vitamins.
[0450] Preferably, the feedstuff is a corn soybean meal mix.
[0451] In one embodiment, preferably the feed is not pet food.
[0452] In another aspect there is provided a method for producing a
feedstuff. Feedstuff is typically produced in feed mills in which
raw materials are first ground to a suitable particle size and then
mixed with appropriate additives. The feedstuff may then be
produced as a mash or pellets; the later typically involves a
method by which the temperature is raised to a target level and
then the feed is passed through a die to produce pellets of a
particular size. The pellets are allowed to cool. Subsequently
liquid additives such as fat and enzyme may be added. Production of
feedstuff may also involve an additional step that includes
extrusion or expansion prior to pelleting--in particular by
suitable techniques that may include at least the use of steam.
[0453] The feedstuff may be a feedstuff for a monogastric animal,
such as poultry (for example, broiler, layer, broiler breeders,
turkey, duck, geese, water fowl), swine (all age categories), a pet
(for example dogs, cats) or fish, preferably the feedstuff is for
poultry.
[0454] The feedstuff may be a feedstuff for a monogastric animal,
such as poultry (for example, broiler, layer, broiler breeders,
turkey, duck, geese, water fowl), swine (all age categories), a pet
(for example dogs, cats) or fish, preferably the feedstuff is for
poultry.
[0455] In one embodiment the feedstuff is not for a layer.
[0456] By way of example only a feedstuff for chickens, e.g.
broiler chickens may be comprises of one or more of the ingredients
listed in the table below, for example in the % ages given in the
table below:
TABLE-US-00014 Ingredients Starter (%) Finisher (%) Maize 46.2 46.7
Wheat Middlings 6.7 10.0 Maize DDGS 7.0 7.0 Soyabean Meal 48% CP
32.8 26.2 Animal/Vegetable Fat blend 3.0 5.8 L-Lysine HCl 0.3 0.3
DL-methionine 0.3 0.3 L-threonine 0.1 0.1 Salt 0.3 0.4 Limestone
1.1 1.1 Dicalcium Phosphate 1.2 1.2 Poultry Vitamins and
Micro-minerals 0.3 0.3
[0457] By way of example only the diet specification for chickens,
such as broiler chickens, may be as set out in the Table below:
TABLE-US-00015 Diet specification Crude Protein (%) 23.00 20.40
Metabolizable Energy Poultry 2950 3100 (kcal/kg) Calcium (%) 0.85
0.85 Available Phosphorus (%) 0.38 0.38 Sodium (%) 0.18 0.19 Dig.
Lysine (%) 1.21 1.07 Dig. Methionine (%) 0.62 0.57 Dig. Methionine
+ Cysteine (%) 0.86 0.78 Dig. Threonine (%) 0.76 0.68
[0458] By way of example only a feedstuff laying hens may be
comprises of one or more of the ingredients listed in the table
below, for example in the % ages given in the table below:
TABLE-US-00016 Ingredient Laying phase (%) Maize 10.0 Wheat 53.6
Maize DDGS 5.0 Soybean Meal 48% CP 14.9 Wheat Middlings 3.0 Soybean
Oil 1.8 L-Lysine HCl 0.2 DL-methionine 0.2 L-threonine 0.1 Salt 0.3
Dicalcium Phosphate 1.6 Limestone 8.9 Poultry Vitamins and
Micro-minerals 0.6
[0459] By way of example only the diet specification for laying
hens may be as set out in the Table below:
TABLE-US-00017 Diet specification Crude Protein (%) 16.10
Metabolizable Energy Poultry 2700 (kcal/kg) Lysine (%) 0.85
Methionine (%) 0.42 Methionine + Cysteine (%) 0.71 Threonine (%)
0.60 Calcium (%) 3.85 Available Phosphorus (%) 0.42 Sodium (%)
0.16
[0460] By way of example only a feedstuff for turkeys may be
comprises of one or more of the ingredients listed in the table
below, for example in the % ages given in the table below:
TABLE-US-00018 Phase 1 Phase 2 Phase 3 Phase 4 Ingredient (%) (%)
(%) (%) Wheat 33.6 42.3 52.4 61.6 Maize DDGS 7.0 7.0 7.0 7.0
Soyabean Meal 48% CP 44.6 36.6 27.2 19.2 Rapeseed Meal 4.0 4.0 4.0
4.0 Soyabean Oil 4.4 4.2 3.9 3.6 L-Lysine HCl 0.5 0.5 0.4 0.4
DL-methionine 0.4 0.4 0.3 0.2 L-threonine 0.2 0.2 0.1 0.1 Salt 0.3
0.3 0.3 0.3 Limestone 1.0 1.1 1.1 1.0 Dicalcium Phosphate 3.5 3.0
2.7 2.0 Poultry Vitamins and 0.4 0.4 0.4 0.4 Micro-minerals
[0461] By way of example only the diet specification for turkeys
may be as set out in the Table below:
TABLE-US-00019 Diet specification Crude Protein (%) 29.35 26.37
22.93 20.00 Metabolizable Energy Poultry 2.850 2.900 2.950 3.001
(kcal/kg) Calcium (%) 1.43 1.33 1.22 1.02 Available Phosphorus (%)
0.80 0.71 0.65 0.53 Sodium (%) 0.16 0.17 0.17 0.17 Dig. Lysine (%)
1.77 1.53 1.27 1.04 Dig. Methionine (%) 0.79 0.71 0.62 0.48 Dig.
Methionine + Cysteine (%) 1.12 1.02 0.90 0.74 Dig. Threonine (%)
1.03 0.89 0.73 0.59
[0462] By way of example only a feedstuff for piglets may be
comprises of one or more of the ingredients listed in the table
below, for example in the % ages given in the table below:
TABLE-US-00020 Ingredient Phase 1 (%) Phase 2 (%) Maize 20.0 7.0
Wheat 25.9 46.6 Rye 4.0 10.0 Wheat middlings 4.0 4.0 Maize DDGS 6.0
8.0 Soyabean Meal 48% CP 25.7 19.9 Dried Whey 10.0 0.0 Soyabean Oil
1.0 0.7 L-Lysine HCl 0.4 0.5 DL-methionine 0.2 0.2 L-threonine 0.1
0.2 L-tryptophan 0.03 0.04 Limestone 0.6 0.7 Dicalcium Phosphate
1.6 1.6 Swine Vitamins and Micro-minerals 0.2 0.2 Salt 0.2 0.4
[0463] By way of example only the diet specification for piglets
may be as set out in the Table below:
TABLE-US-00021 Diet specification Crude Protein (%) 21.50 20.00
Swine Digestible Energy (kcal/kg) 3380 3320 Swine Net Energy
(kcal/kg) 2270 2230 Calcium (%) 0.80 0.75 Digestible Phosphorus (%)
0.40 0.35 Sodium (%) 0.20 0.20 Dig. Lysine (%) 1.23 1.14 Dig.
Methionine (%) 0.49 0.44 Dig. Methionine + Cysteine (%) 0.74 0.68
Dig. Threonine (%) 0.80 0.74
[0464] By way of example only a feedstuff for grower/finisher pigs
may be comprises of one or more of the ingredients listed in the
table below, for example in the % ages given in the table
below:
TABLE-US-00022 Ingredient Grower/Finisher (%) Maize 27.5 Soyabean
Meal 48% CP 15.4 Maize DDGS 20.0 Wheat bran 11.1 Rice bran 12.0
Canola seed meal 10.0 Limestone 1.6 Dicalcium phosphate 0.01 Salt
0.4 Swine Vitamins and Micro-minerals 0.3 Lysine-HCl 0.2 Vegetable
oil 0.5
[0465] By way of example only the diet specification for
grower/finisher pigs may be as set out in the Table below:
TABLE-US-00023 Diet specification Crude Protein (%) 22.60 Swine
Metabolizable Energy (kcal/kg) 3030 Calcium (%) 0.75 Available
Phosphorus (%) 0.29 Digestible Lysine (%) 1.01 Dig. Methionine +
Cysteine (%) 0.73 Digestible Threonine (%) 0.66
[0466] Wet-Cake, Distillers Dried Grains (DDG) and Distillers Dried
Grain Solubles (DDGS)
[0467] Wet-cake, Distillers Dried Grains and Distillers Dried
Grains with Solubles are products obtained after the removal of
ethyl alcohol by distillation from yeast fermentation of a grain or
a grain mixture by methods employed in the grain distilling
industry.
[0468] Stillage coming from the distillation (e.g. comprising
water, remainings of the grain, yeast cells etc.) is separated into
a "solid" part and a liquid part.
[0469] The solid part is called "wet-cake" and can be used as
animal feed as such.
[0470] The liquid part is (partially) evaporated into a syrup
(solubles).
[0471] When the wet-cake is dried it is Distillers Dried Grains
(DDG).
[0472] When the wet-cake is dried together with the syrup
(solubles) it is Distillers Dried Grans with Solubles (DDGS).
[0473] Wet-cake may be used in dairy operations and beef cattle
feedlots.
[0474] The dried DDGS may be used in livestock, e.g. dairy, beef
and swine) feeds and poultry feeds.
[0475] Corn DDGS is a very good protein source for dairy cows.
[0476] Corn Gluten Meal
[0477] In one aspect, the by-product of corn may be corn gluten
meal (CGM).
[0478] CGM is a powdery by-product of the corn milling inductry.
CGM has utility in, for example, animal feed. It can be used as an
inexpensive protein source for feed such as pet food, livestock
feed and poultry feed. It is an especially good source of the amino
acid cysteine, but must be balanced with other proteins for
lysine.
[0479] Feed Additive Composition
[0480] The feed additive composition of the present invention
and/or the feedstuff comprising same may be used in any suitable
form.
[0481] The feed additive composition of the present invention may
be used in the form of solid or liquid preparations or alternatives
thereof. Examples of solid preparations include powders, pastes,
boluses, capsules, pellets, tablets, dusts, and granules which may
be wettable, spray-dried or freeze-dried. Examples of liquid
preparations include, but are not limited to, aqueous, organic or
aqueous-organic solutions, suspensions and emulsions.
[0482] In some applications, the feed additive compositions of the
present invention may be mixed with feed or administered in the
drinking water.
[0483] In one aspect the present invention relates to a method of
preparing a feed additive composition, comprising admixing a
xylanase, a .beta.-glucanase (and optionally at least one further
fibre degrading enzyme) and a DFM as taught herein with a feed
acceptable carrier, diluent or excipient, and (optionally)
packaging.
[0484] Premix
[0485] The feedstuff and/or feed additive composition may be
combined with at least one mineral and/or at least one vitamin. The
compositions thus derived may be referred to herein as a
premix.
[0486] Forms
[0487] The feed additive composition of the present invention and
other components and/or the feedstuff comprising same may be used
in any suitable form.
[0488] The feed additive composition of the present invention may
be used in the form of solid or liquid preparations or alternatives
thereof. Examples of solid preparations include powders, pastes,
boluses, capsules, pellets, tablets, dusts, and granules which may
be wettable, spray-dried or freeze-dried. Examples of liquid
preparations include, but are not limited to, aqueous, organic or
aqueous-organic solutions, suspensions and emulsions.
[0489] In some applications, DFM or feed additive compositions of
the present invention may be mixed with feed or administered in the
drinking water. In one embodiment the dosage range for inclusion
into water is about 1.times.10.sup.3 CFU/animal/day to about
1.times.10.sup.10 CFU/animal/day, and more preferably about
1.times.10.sup.7 CFU/animal/day.
[0490] Suitable examples of forms include one or more of: powders,
pastes, boluses, pellets, tablets, pills, capsules, ovules,
solutions or suspensions, which may contain flavouring or colouring
agents, for immediate-, delayed-, modified-, sustained-, pulsed- or
controlled-release applications.
[0491] By way of example, if the composition of the present
invention is used in a solid, e.g. pelleted form, it may also
contain one or more of: excipients such as microcrystalline
cellulose, lactose, sodium citrate, calcium carbonate, dibasic
calcium phosphate and glycine; disintegrants such as starch
(preferably corn, potato or tapioca starch), sodium starch
glycollate, croscarmellose sodium and certain complex silicates;
granulation binders such as polyvinyl pyrrolidone,
hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),
sucrose, gelatin and acacia; lubricating agents such as magnesium
stearate, stearic acid, glyceryl behenate and talc may be
included.
[0492] Examples of nutritionally acceptable carriers for use in
preparing the forms include, for example, water, salt solutions,
alcohol, silicone, waxes, petroleum jelly, vegetable oils,
polyethylene glycols, propylene glycol, liposomes, sugars, gelatin,
lactose, amylose, magnesium stearate, talc, surfactants, silicic
acid, viscous paraffin, perfume oil, fatty acid monoglycerides and
diglycerides, petroethral fatty acid esters,
hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.
[0493] Preferred excipients for the forms include lactose, starch,
a cellulose, milk sugar or high molecular weight polyethylene
glycols.
[0494] For aqueous suspensions and/or elixirs, the composition of
the present invention may be combined with various sweetening or
flavouring agents, colouring matter or dyes, with emulsifying
and/or suspending agents and with diluents such as water, propylene
glycol and glycerin, and combinations thereof.
[0495] Non-hydroscopic whey is often used as a carrier for DFMs
(particularly bacterial DFMs) and is a good medium to initiate
growth.
[0496] Bacterial DFM containing pastes may be formulated with
vegetable oil and inert gelling ingredients.
[0497] Fungal products may be formulated with grain by-products as
carriers.
[0498] In one embodiment preferably the feed additive composition
according to the present invention is not in the form of a
microparticle system, such as the microparticle system taught in
WO2005/123034.
[0499] Dosing
[0500] The DFM and/or feed additive composition according to the
present invention may be designed for one-time dosing or may be
designed for feeding on a daily basis.
[0501] The optimum amount of the composition (and each component
therein) to be used in the combination of the present invention
will depend on the product to be treated and/or the method of
contacting the product with the composition and/or the intended use
for the same.
[0502] The amount of DFM and enzymes used in the compositions
should be a sufficient amount to be effective and to remain
sufficiently effective in improving the performance of the animal
fed feed products containing said composition. This length of time
for effectiveness should extend up to at least the time of
utilisation of the product (e.g. feed additive composition or feed
containing same).
[0503] Combination with Other Components
[0504] The DFM and enzyme(s) for use in the present invention may
be used in combination with other components. Thus, the present
invention also relates to combinations. The DFM in combination with
the xylanase and a .beta.-glucanase (and optionally at least one
further fibre degrading enzyme) may be referred to herein as "the
feed additive composition of the present invention".
[0505] In a preferred embodiment "the feed additive composition of
the present invention" may comprise (or consist essentially of, or
consist of) DFM in combination with the xylanase and a
.beta.-glucanase and a further fibre degrading enzyme as taught
herein (e.g. suitably at least two, suitably at least three further
fibre degrading enzymes).
[0506] In a further preferred embodiment "the feed additive
composition of the present invention" may comprise (or consist
essentially of, or consist of) DFM in combination with the xylanase
and a .beta.-glucanase and a further fibre degrading enzyme as
taught herein (e.g. suitably at least four, suitably at least five
further fibre degrading enzymes).
[0507] The combination of the present invention comprises the feed
additive composition of the present invention (or one or more of
the constituents thereof) and another component which is suitable
for animal consumption and is capable of providing a medical or
physiological benefit to the consumer.
[0508] In one embodiment preferably the "another component" is not
a further enzyme or a further DFM.
[0509] The components may be prebiotics. Prebiotics are typically
non-digestible carbohydrate (oligo- or polysaccharides) or a sugar
alcohol which is not degraded or absorbed in the upper digestive
tract. Known prebiotics used in commercial products and useful in
accordance with the present invention include inulin
(fructo-oligosaccharide, or FOS) and transgalacto-oligosaccharides
(GOS or TOS). Suitable prebiotics include
palatinoseoligosaccharide, soybean oligosaccharide, alginate,
xanthan, pectin, locust bean gum (LBG), inulin, guar gum,
galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS),
non-degradable starch, lactosaccharose, lactulose, lactitol,
maltitol, maltodextrin, polydextrose (i.e. Litesse.RTM.), lactitol,
lactosucrose, soybean oligosaccharides, palatinose,
isomalto-oligosaccharides, gluco-oligosaccharides and
xylo-oligosaccharides.
[0510] In one embodiment the present invention relates to the
combination of the feed additive composition according to the
present invention (or one or more of the constituents thereof) with
a prebiotic. In another embodiment the present invention relates to
a feed additive composition comprising (or consisting essentially
of or consisting of) a DFM in combination with a xylanase, a
.beta.-glucanase, an amylase, a phytase, a protease and a
prebiotic.
[0511] The prebiotic may be administered simultaneously with (e.g.
in admixture together with or delivered simultaneously by the same
or different routes) or sequentially to (e.g. by the same or
different routes) the feed additive composition (or constituents
thereof) according to the present invention.
[0512] Other components of the combinations of the present
invention include polydextrose, such as Litesse.RTM., and/or a
maltodextrin and/or lactitol. These other components may be
optionally added to the feed additive composition to assist the
drying process and help the survival of DFM.
[0513] Further examples of other suitable components include one or
more of: thickeners, gelling agents, emulsifiers, binders, crystal
modifiers, sweeteners (including artificial sweeteners), rheology
modifiers, stabilisers, anti-oxidants, dyes, enzymes, carriers,
vehicles, excipients, diluents, lubricating agents, flavouring
agents, colouring matter, suspending agents, disintegrants,
granulation binders etc. These other components may be natural.
These other components may be prepared by use of chemical and/or
enzymatic techniques.
[0514] In one embodiment the DFM and/or enzymes may be
encapsulated. In one embodiment the feed additive composition
and/or DFM and/or enzymes is/are formulated as a dry powder or
granule as described in WO2007/044968 (referred to as TPT
granules)--reference incorporated herein by reference.
[0515] In one preferred embodiment the DFM and/or enzymes for use
in the present invention may be used in combination with one or
more lipids.
[0516] For example, the DFM and/or enzymes for use in the present
invention may be used in combination with one or more lipid
micelles. The lipid micelle may be a simple lipid micelle or a
complex lipid micelle.
[0517] The lipid micelle may be an aggregate of orientated
molecules of amphipathic substances, such as a lipid and/or an
oil.
[0518] As used herein the term "thickener or gelling agent" refers
to a product that prevents separation by slowing or preventing the
movement of particles, either droplets of immiscible liquids, air
or insoluble solids. Thickening occurs when individual hydrated
molecules cause an increase in viscosity, slowing the separation.
Gelation occurs when the hydrated molecules link to form a
three-dimensional network that traps the particles, thereby
immobilising them.
[0519] The term "stabiliser" as used here is defined as an
ingredient or combination of ingredients that keeps a product (e.g.
a feed product) from changing over time.
[0520] The term "emulsifier" as used herein refers to an ingredient
(e.g. a feed ingredient) that prevents the separation of emulsions.
Emulsions are two immiscible substances, one present in droplet
form, contained within the other. Emulsions can consist of
oil-in-water, where the droplet or dispersed phase is oil and the
continuous phase is water; or water-in-oil, where the water becomes
the dispersed phase and the continuous phase is oil. Foams, which
are gas-in-liquid, and suspensions, which are solid-in-liquid, can
also be stabilised through the use of emulsifiers.
[0521] As used herein the term "binder" refers to an ingredient
(e.g. a feed ingredient) that binds the product together through a
physical or chemical reaction. During "gelation" for instance,
water is absorbed, providing a binding effect. However, binders can
absorb other liquids, such as oils, holding them within the
product. In the context of the present invention binders would
typically be used in solid or low-moisture products for instance
baking products: pastries, doughnuts, bread and others.
[0522] "Carriers" or "vehicles" mean materials suitable for
administration of the DFM and/or enzymes and include any such
material known in the art such as, for example, any liquid, gel,
solvent, liquid diluent, solubilizer, or the like, which is
non-toxic and which does not interact with any components of the
composition in a deleterious manner.
[0523] In one embodiment the feed additive composition, premix,
feed or feedstuff of the present invention may be admixed with at
least one physiologically acceptable carrier selected from at least
one of maltodextrin, limestone (calcium carbonate), cyclodextrin,
wheat or a wheat component, sucrose, starch, Na.sub.2SO.sub.4,
Talc, PVA, sorbitol, benzoate, sorbiate, glycerol, sucrose,
propylene glycol, 1,3-propane diol, glucose, parabens, sodium
chloride, citrate, acetate, phosphate, calcium, metabisulfite,
formate and mixtures thereof.
[0524] Examples of excipients include one or more of:
microcrystalline cellulose and other celluloses, lactose, sodium
citrate, calcium carbonate, dibasic calcium phosphate, glycine,
starch, milk sugar and high molecular weight polyethylene
glycols.
[0525] Examples of disintegrants include one or more of: starch
(preferably corn, potato or tapioca starch), sodium starch
glycollate, croscarmellose sodium and certain complex
silicates.
[0526] Examples of granulation binders include one or more of:
polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),
hydroxypropylcellulose (HPC), sucrose, maltose, gelatin and
acacia.
[0527] Examples of lubricating agents include one or more of:
magnesium stearate, stearic acid, glyceryl behenate and talc.
[0528] Examples of diluents include one or more of: water, ethanol,
propylene glycol and glycerin, and combinations thereof.
[0529] The other components may be used simultaneously (e.g. when
they are in admixture together or even when they are delivered by
different routes) or sequentially (e.g. they may be delivered by
different routes).
[0530] Preferably, when the feed additive composition of the
present invention is admixed with another component(s), the DFM
remains viable.
[0531] In one embodiment preferably the feed additive composition
according to the present invention does not comprise chromium or
organic chromium
[0532] In one embodiment preferably the feed additive according to
the present invention does not contain sorbic acid.
[0533] Concentrates
[0534] The DFMs for use in the present invention may be in the form
of concentrates. Typically these concentrates comprise a
substantially high concentration of a DFM.
[0535] Feed additive compositions according to the present
invention may have a content of viable cells (colony forming units,
CFUs) which is in the range of at least 10.sup.4 CFU/g (suitably
including at least 10.sup.5 CFU/g, such as at least 10.sup.6 CFU/g,
e.g. at least 10.sup.7 CFU/g, at least 10.sup.8 CFU/g, such as at
least 10.sup.9 CFU/g) to about 10.sup.10 CFU/g (or even about
10.sup.11 CFU/g or about 1012 CFU/g).
[0536] When the DFM is in the form of a concentrate the feed
additive compositions according to the present invention may have a
content of viable cells in the range of at least 10.sup.9 CFU/g to
about 10.sup.12 CFU/g, preferably at least 10.sup.10 CFU/g to about
10.sup.12 CFU/g.
[0537] Powders, granules and liquid compositions in the form of
concentrates may be diluted with water or resuspended in water or
other suitable diluents, for example, an appropriate growth medium
such as milk or mineral or vegetable oils, to give compositions
ready for use.
[0538] The DFM or feed additive composition of the present
invention or the combinations of the present invention in the form
of concentrates may be prepared according to methods known in the
art.
[0539] In one aspect of the present invention the enzymes or feed
is contacted by a composition in a concentrated form.
[0540] The compositions of the present invention may be spray-dried
or freeze-dried by methods known in the art.
[0541] Typical processes for making particles using a spray drying
process involve a solid material which is dissolved in an
appropriate solvent (e.g. a culture of a DFM in a fermentation
medium). Alternatively, the material can be suspended or emulsified
in a non-solvent to form a suspension or emulsion. Other
ingredients (as discussed above) or components such as
anti-microbial agents, stabilising agents, dyes and agents
assisting with the drying process may optionally be added at this
stage.
[0542] The solution then is atomised to form a fine mist of
droplets. The droplets immediately enter a drying chamber where
they contact a drying gas. The solvent is evaporated from the
droplets into the drying gas to solidify the droplets, thereby
forming particles. The particles are then separated from the drying
gas and collected.
[0543] Subject
[0544] The term "subject", as used herein, means an animal that is
to be or has been administered with a feed additive composition
according to the present invention or a feedstuff comprising said
feed additive composition according to the present invention.
[0545] The term "subject", as used herein, means an animal.
Preferably, the subject is a mammal, bird, fish or crustacean
including for example livestock or a domesticated animal (e.g. a
pet).
[0546] In one embodiment the "subject" is livestock.
[0547] The term "livestock", as used herein refers to any farmed
animal. Preferably, livestock is one or more of cows or bulls
(including calves), poultry, pigs (including piglets), poultry
(including broilers, chickens and turkeys), birds, fish (including
freshwater fish, such as salmon, cod, trout and carp, e.g. koi
carp, and marine fish, such as sea bass), crustaceans (such as
shrimps, mussels and scallops), horses (including race horses),
sheep (including lambs).
[0548] In one embodiment the term livestock and/or poultry and/or
chickens does not include egg layers.
[0549] In another embodiment the "subject" is a domesticated animal
or pet or an animal maintained in a zoological environment.
[0550] The term "domesticated animal or pet or animal maintained in
a zoological environment" as used herein refers to any relevant
animal including canines (e.g. dogs), felines (e.g. cats), rodents
(e.g. guinea pigs, rats, mice), birds, fish (including freshwater
fish and marine fish), and horses.
[0551] Short Chain Fatty Acid (SCFA) Production
[0552] The term "short chain fatty acid" as used herein includes
volatile fatty acids as well as lactic acid. In one embodiment the
SCFA may be selected from the group consisting of: acetic acid,
propionic acid, butyric acid, isobutyric acid, valeric acid,
isovaleric acid, 2-methylbutyric acids and lactic acid, preferably
propionic acid and/or butyric acid.
[0553] In one embodiment the SCFA may be butyric acid and/or
propionic acid.
[0554] Short chain fatty acids (particularly volatile fatty acids,
e.g. propionic acid and butyric acid, and lactic acid) may be
analysed using the following method:
[0555] Chromatographic analysis of volatile fatty acids and lactic
acid, e.g. SCFAs, to be performed from simulation samples with
pivalic acid as internal standard as previously described (Ouwehand
et al., 2009 February; 101(3):367-75). Concentrations of acetic,
propionic, butyric, isobutyric, valeric, isovaleric,
2-methylbutyric acids, and lactic acid are determined.
[0556] Performance
[0557] As used herein, "animal performance" may be determined by
the feed efficiency and/or weight gain of the animal and/or by the
feed conversion ratio and/or by the digestibility of a nutrient in
a feed (e.g. amino acid digestibility) and/or digestible energy or
metabolizable energy in a feed and/or by nitrogen retention.
[0558] Preferably "animal performance" is determined by feed
efficiency and/or weight gain of the animal and/or by the feed
conversion ratio.
[0559] By "improved animal performance" it is meant that there is
increased feed efficiency, and/or increased weight gain and/or
reduced feed conversion ratio and/or improved digestibility of
nutrients or energy in a feed and/or by improved nitrogen retention
resulting from the use of feed additive composition of the present
invention in feed in comparison to feed which does not comprise
said feed additive composition.
[0560] Preferably, by "improved animal performance" it is meant
that there is increased feed efficiency and/or increased weight
gain and/or reduced feed conversion ratio.
[0561] As used herein, the term "feed efficiency" refers to the
amount of weight gain in an animal that occurs when the animal is
fed ad-libitum or a specified amount of food during a period of
time.
[0562] By "increased feed efficiency" it is meant that the use of a
feed additive composition according the present invention in feed
results in an increased weight gain per unit of feed intake
compared with an animal fed without said feed additive composition
being present.
[0563] Feed Conversion Ratio (FCR)
[0564] As used herein, the term "feed conversion ratio" refers to
the amount of feed fed to an animal to increase the weight of the
animal by a specified amount.
[0565] An improved feed conversion ratio means a lower feed
conversion ratio.
[0566] By "lower feed conversion ratio" or "improved feed
conversion ratio" it is meant that the use of a feed additive
composition in feed results in a lower amount of feed being
required to be fed to an animal to increase the weight of the
animal by a specified amount compared to the amount of feed
required to increase the weight of the animal by the same amount
when the feed does not comprise said feed additive composition.
[0567] Nutrient Digestibility
[0568] Nutrient digestibility as used herein means the fraction of
a nutrient that disappears from the gastro-intestinal tract or a
specified segment of the gastro-intestinal tract, e.g. the small
intestine. Nutrient digestibility may be measured as the difference
between what is administered to the subject and what comes out in
the faeces of the subject, or between what is administered to the
subject and what remains in the digesta on a specified segment of
the gastro intestinal trace, e.g. the ileum.
[0569] Nutrient digestibility as used herein may be measured by the
difference between the intake of a nutrient and the excreted
nutrient by means of the total collection of excreta during a
period of time; or with the use of an inert marker that is not
absorbed by the animal, and allows the researcher calculating the
amount of nutrient that disappeared in the entire gastro-intestinal
tract or a segment of the gastro-intestinal tract. Such an inert
marker may be titanium dioxide, chromic oxide or acid insoluble
ash. Digestibility may be expressed as a percentage of the nutrient
in the feed, or as mass units of digestible nutrient per mass units
of nutrient in the feed.
[0570] Nutrient digestibility as used herein encompasses starch
digestibility, fat digestibility, protein digestibility, and amino
acid digestibility.
[0571] Energy digestibility as used herein means the gross energy
of the feed consumed minus the gross energy of the faeces or the
gross energy of the feed consumed minus the gross energy of the
remaining digesta on a specified segment of the gastro-intestinal
tract of the animal, e.g. the ileum. Metabolizable energy as used
herein refers to apparent metabolizable energy and means the gross
energy of the feed consumed minus the gross energy contained in the
faeces, urine, and gaseous products of digestion. Energy
digestibility and metabolizable energy may be measured as the
difference between the intake of gross energy and the gross energy
excreted in the faeces or the digesta present in specified segment
of the gastro-intestinal tract using the same methods to measure
the digestibility of nutrients, with appropriate corrections for
nitrogen excretion to calculate metabolizable energy of feed.
[0572] Nitrogen Retention
[0573] Nitrogen retention as used herein means as subject's ability
to retain nitrogen from the diet as body mass. A negative nitrogen
balance occurs when the excretion of nitrogen exceeds the daily
intake and is often seen when the muscle is being lost. A positive
nitrogen balance is often associated with muscle growth,
particularly in growing animals.
[0574] Nitrogen retention may be measured as the difference between
the intake of nitrogen and the excreted nitrogen by means of the
total collection of excreta and urine during a period of time. It
is understood that excreted nitrogen includes undigested protein
from the feed, endogenous proteinaceous secretions, microbial
protein, and urinary nitrogen.
[0575] Carcass Yield and Meat Yield
[0576] The term carcass yield as used herein means the amount of
carcass as a proportion of the live body weight, after a commercial
or experimental process of slaughter. The term carcass means the
body of an animal that has been slaughtered for food, with the
head, entrails, part of the limbs, and feathers or skin removed.
The term meat yield as used herein means the amount of edible meat
as a proportion of the live body weight, or the amount of a
specified meat cut as a proportion of the live body weight.
[0577] Weight Gain
[0578] The present invention further provides a method of
increasing weight gain in a subject, e.g. poultry or swine,
comprising feeding said subject a feedstuff comprising a feed
additive composition according to the present invention.
[0579] An "increased weight gain" refers to an animal having
increased body weight on being fed feed comprising a feed additive
composition compared with an animal being fed a feed without said
feed additive composition being present.
[0580] Other Properties
[0581] In one embodiment the feed additive composition, feed,
feedstuff or method according to the present invention may not
modulate (e.g. improve) the immune response of the subject.
[0582] In a further embodiment the feed additive composition, feed,
feedstuff or method according to the present invention may not
improve survival (e.g. reduce mortality) of the subject.
[0583] In a preferred embodiment the feed additive composition,
feed, feedstuff or method according to the present invention may
not modulate (e.g. improve) the immune response or improve survival
(e.g. reduce mortality) of the subject.
[0584] Probiotic
[0585] For some applications, it is believed that the DFM in the
composition of the present invention can exert a probiotic culture
effect. It is also within the scope of the present invention to add
to the composition of the present invention further probiotic
and/or prebiotics.
[0586] Here, a prebiotic is:
[0587] "a non-digestible food ingredient that beneficially affects
the host by selectively stimulating the growth and/or the activity
of one or a limited number of beneficial bacteria".
[0588] The term "probiotic culture" as used herein defines live
microorganisms (including bacteria or yeasts for example) which,
when for example ingested or locally applied in sufficient numbers,
beneficially affects the host organism, i.e. by conferring one or
more demonstrable health benefits on the host organism. Probiotics
may improve the microbial balance in one or more mucosal surfaces.
For example, the mucosal surface may be the intestine, the urinary
tract, the respiratory tract or the skin. Whilst there are no lower
or upper limits for probiotic intake, it has been suggested that at
least 10.sup.6-10.sup.12, preferably at least 10.sup.6-10.sup.10
preferably 10.sup.8-10.sup.9, cfu as a daily dose will be effective
to achieve the beneficial health effects in a subject.
[0589] Isolated
[0590] In one aspect, preferably the enzyme used in the present
invention is in an isolated form. The term "isolated" means that
the enzyme is at least substantially free from at least one other
component with which the enzyme is naturally associated in nature
and as found in nature. The enzyme of the present invention may be
provided in a form that is substantially free of one or more
contaminants with which the substance might otherwise be
associated. Thus, for example it may be substantially free of one
or more potentially contaminating polypeptides and/or nucleic acid
molecules.
[0591] Purified
[0592] In one aspect, preferably the enzyme and/or DFM according to
the present invention is in a purified form. The term "purified"
means that the enzyme and/or DFM is present at a high level. The
enzyme and/or DFM is desirably the predominant component present in
a composition. Preferably, it is present at a level of at least
about 90%, or at least about 95% or at least about 98%, said level
being determined on a dry weight/dry weight basis with respect to
the total composition under consideration.
[0593] It is envisaged within the scope of the present invention
that the embodiments of the invention can be combined such that
combinations of any of the features described herein are included
within the scope of the present invention. In particular, it is
envisaged within the scope of the present invention that any of the
therapeutic effects of the bacteria may be exhibited
concomitantly.
[0594] Amino Acid Sequences
[0595] The scope of the present invention also encompasses amino
acid sequences of enzymes having the specific properties as defined
herein.
[0596] As used herein, the term "amino acid sequence" is synonymous
with the term "polypeptide" and/or the term "protein". In some
instances, the term "amino acid sequence" is synonymous with the
term "peptide". In some instances, the term "amino acid sequence"
is synonymous with the term "enzyme".
[0597] The amino acid sequence may be prepared/isolated from a
suitable source, or it may be made synthetically or it may be
prepared by use of recombinant DNA techniques.
[0598] Preferably the amino acid sequence when relating to and when
encompassed by the per se scope of the present invention is not a
native enzyme. In this regard, the term "native enzyme" means an
entire enzyme that is in its native environment and when it has
been expressed by its native nucleotide sequence.
[0599] Sequence Identity or Sequence Homology
[0600] The present invention also encompasses the use of sequences
having a degree of sequence identity or sequence homology with
amino acid sequence(s) of a polypeptide having the specific
properties defined herein or of any nucleotide sequence encoding
such a polypeptide (hereinafter referred to as a "homologous
sequence(s)"). Here, the term "homologue" means an entity having a
certain homology with the subject amino acid sequences and the
subject nucleotide sequences. Here, the term "homology" can be
equated with "identity".
[0601] The homologous amino acid sequence and/or nucleotide
sequence should provide and/or encode a polypeptide which retains
the functional activity and/or enhances the activity of the
enzyme.
[0602] The term "nucleotide sequence" in relation to the present
invention includes genomic DNA, cDNA, synthetic DNA, and RNA.
Preferably it means DNA, more preferably cDNA sequence coding for
the present invention.
[0603] In the present context, in some embodiments a homologous
sequence is taken to include an amino acid or a nucleotide sequence
which may be at least 97% identical, preferably at least 98 or 99%
identical to the subject sequence.
[0604] In some embodiments a homologous sequence is taken to
include an amino acid or a nucleotide sequence which may be at
least 85% identical, preferably at least 90 or 95% identical to the
subject sequence.
[0605] Typically, the homologues will comprise the same active
sites etc. as the subject amino acid sequence for instance.
Although homology can also be considered in terms of similarity
(i.e. amino acid residues having similar chemical
properties/functions), in the context of the present invention it
is preferred to express homology in terms of sequence identity.
[0606] In one embodiment, a homologous sequence is taken to include
an amino acid sequence or nucleotide sequence which has one or
several additions, deletions and/or substitutions compared with the
subject sequence.
[0607] In one embodiment the present invention relates to a protein
whose amino acid sequence is represented herein or a protein
derived from this (parent) protein by substitution, deletion or
addition of one or several amino acids, such as 2, 3, 4, 5, 6, 7,
8, 9 amino acids, or more amino acids, such as 10 or more than 10
amino acids in the amino acid sequence of the parent protein and
having the activity of the parent protein.
[0608] Typically, the homologues will comprise the same sequences
that code for the active sites etc. as the subject sequence.
Although homology can also be considered in terms of similarity
(i.e. amino acid residues having similar chemical
properties/functions), in the context of the present invention it
is preferred to express homology in terms of sequence identity.
[0609] Homology comparisons can be conducted by eye, or more
usually, with the aid of readily available sequence comparison
programs. These commercially available computer programs can
calculate % homology between two or more sequences.
[0610] % homology may be calculated over contiguous sequences, i.e.
one sequence is aligned with the other sequence and each amino acid
in one sequence is directly compared with the corresponding amino
acid in the other sequence, one residue at a time. This is called
an "ungapped" alignment. Typically, such ungapped alignments are
performed only over a relatively short number of residues.
[0611] Although this is a very simple and consistent method, it
fails to take into consideration that, for example, in an otherwise
identical pair of sequences, one insertion or deletion will cause
the following amino acid residues to be put out of alignment, thus
potentially resulting in a large reduction in % homology when a
global alignment is performed. Consequently, most sequence
comparison methods are designed to produce optimal alignments that
take into consideration possible insertions and deletions without
penalising unduly the overall homology score. This is achieved by
inserting "gaps" in the sequence alignment to try to maximise local
homology.
[0612] However, these more complex methods assign "gap penalties"
to each gap that occurs in the alignment so that, for the same
number of identical amino acids, a sequence alignment with as few
gaps as possible--reflecting higher relatedness between the two
compared sequences--will achieve a higher score than one with many
gaps. "Affine gap costs" are typically used that charge a
relatively high cost for the existence of a gap and a smaller
penalty for each subsequent residue in the gap. This is the most
commonly used gap scoring system. High gap penalties will of course
produce optimised alignments with fewer gaps. Most alignment
programs allow the gap penalties to be modified. However, it is
preferred to use the default values when using such software for
sequence comparisons.
[0613] Calculation of maximum % homology therefore firstly requires
the production of an optimal alignment, taking into consideration
gap penalties. A suitable computer program for carrying out such an
alignment is the Vector NTI (Invitrogen Corp.). Examples of
software that can perform sequence comparisons include, but are not
limited to, the BLAST package (see Ausubel et al 1999 Short
Protocols in Molecular Biology, 4th Ed--Chapter 18), BLAST 2 (see
FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999
177(1): 187-8 and tatiana@ncbi.nlm.nih.gov), FASTA (Altschul et al
1990 J. Mol. Biol. 403-410) and AlignX for example. At least BLAST,
BLAST 2 and FASTA are available for offline and online searching
(see Ausubel et al 1999, pages 7-58 to 7-60).
[0614] Although the final % homology can be measured in terms of
identity, the alignment process itself is typically not based on an
all-or-nothing pair comparison. Instead, a scaled similarity score
matrix is generally used that assigns scores to each pairwise
comparison based on chemical similarity or evolutionary distance.
An example of such a matrix commonly used is the BLOSUM62
matrix--the default matrix for the BLAST suite of programs. Vector
NTI programs generally use either the public default values or a
custom symbol comparison table if supplied (see user manual for
further details). For some applications, it is preferred to use the
default values for the Vector NTI package.
[0615] Alternatively, percentage homologies may be calculated using
the multiple alignment feature in Vector NTI (Invitrogen Corp.),
based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp
PM (1988), Gene 73(1), 237-244).
[0616] Once the software has produced an optimal alignment, it is
possible to calculate % homology, preferably % sequence identity.
The software typically does this as part of the sequence comparison
and generates a numerical result.
[0617] Should Gap Penalties be used when determining sequence
identity, then preferably the following parameters are used for
pairwise alignment:
TABLE-US-00024 FOR BLAST GAP OPEN 0 GAP EXTENSION 0
TABLE-US-00025 FOR CLUSTAL DNA PROTEIN WORD SIZE 2 1 K triple GAP
PENALTY 15 10 GAP EXTENSION 6.66 0.1
[0618] In one embodiment, CLUSTAL may be used with the gap penalty
and gap extension set as defined above.
[0619] Suitably, the degree of identity with regard to an amino
acid sequence is determined over at least 20 contiguous amino acid
residues, preferably over at least 30 contiguous residues,
preferably over at least 40 contiguous residues, preferably over at
least 50 contiguous residues, preferably over at least 60
contiguous residues, preferably over at least 100 contiguous
residues.
[0620] Suitably, the degree of identity with regard to amino acid
sequence may be determined over the whole sequence taught
herein.
[0621] The sequences may also have deletions, insertions or
substitutions of amino acid residues which produce a silent change
and result in a functionally equivalent substance. Deliberate amino
acid substitutions may be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or the amphipathic nature of the residues as long as the
secondary binding activity of the substance is retained. For
example, negatively charged amino acids include aspartic acid and
glutamic acid; positively charged amino acids include lysine and
arginine; and amino acids with uncharged polar head groups having
similar hydrophilicity values include leucine, isoleucine, valine,
glycine, alanine, asparagine, glutamine, serine, threonine,
phenylalanine, and tyrosine.
[0622] Conservative substitutions may be made, for example
according to the Table below. Amino acids in the same block in the
second column and preferably in the same line in the third column
may be substituted for each other:
TABLE-US-00026 ALIPHATIC Non-polar G A P I L V Polar - uncharged C
S T M N Q Polar - charged D E K R AROMATIC H F W Y
[0623] The present invention also encompasses homologous
substitution (substitution and replacement are both used herein to
mean the interchange of an existing amino acid residue, with an
alternative residue) that may occur i.e. like-for-like substitution
such as basic for basic, acidic for acidic, polar for polar etc.
Non-homologous substitution may also occur i.e. from one class of
residue to another or alternatively involving the inclusion of
unnatural amino acids such as ornithine (hereinafter referred to as
Z), diaminobutyric acid ornithine (hereinafter referred to as B),
norleucine ornithine (hereinafter referred to as O), pyriylalanine,
thienylalanine, naphthylalanine and phenylglycine.
[0624] Replacements may also be made by unnatural amino acids
include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino
acids*, lactic acid*, halide derivatives of natural amino acids
such as trifluorotyrosine*, p-Cl-phenylalanine*,
p-Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*,
.beta.-alanine*, L-.alpha.-amino butyric acid*, L-.gamma.-amino
butyric acid*, L-.alpha.-amino isobutyric acid*, L-.epsilon.-amino
caproic acid.sup.#, 7-amino heptanoic acid*, L-methionine
sulfone.sup.#*, L-norleucine*, L-norvaline*,
p-nitro-L-phenylalanine*, L-hydroxyproline.sup.#, L-thioproline*,
methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*,
pentamethyl-Phe*, L-Phe (4-amino).sup.#, L-Tyr (methyl)*, L-Phe
(4-isopropyl)*, L-Tic
(1,2,3,4-tetrahydroisoquinoline-.beta.-carboxyl acid)*,
L-diaminopropionic acid # and L-Phe (4-benzyl)*. The notation * has
been utilised for the purpose of the discussion above (relating to
homologous or non-homologous substitution), to indicate the
hydrophobic nature of the derivative whereas # has been utilised to
indicate the hydrophilic nature of the derivative, #* indicates
amphipathic characteristics.
[0625] Variant amino acid sequences may include suitable spacer
groups that may be inserted between any two amino acid residues of
the sequence including alkyl groups such as methyl, ethyl or propyl
groups in addition to amino acid spacers such as glycine or
.beta.-alanine residues. A further form of variation, involves the
presence of one or more amino acid residues in peptoid form, will
be well understood by those skilled in the art. For the avoidance
of doubt, "the peptoid form" is used to refer to variant amino acid
residues wherein the .alpha.-carbon substituent group is on the
residue's nitrogen atom rather than the .alpha.-carbon. Processes
for preparing peptides in the peptoid form are known in the art,
for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 and
Horwell D C, Trends Biotechnol. (1995) 13(4), 132-134.
[0626] In one embodiment the xylanase for use in the present
invention may comprise a polypeptide sequence herein with a
conservative substitution of at least one of the amino acids.
[0627] Suitably there may be at least 2 conservative substitutions,
such as at least 3 or at least 4 or at least 5.
[0628] Suitably there may be less than 15 conservative
substitutions, such as less than 12, less than 10, or less than 8
or less than 5.
EXAMPLES
Example 1: Responses of Broiler Chickens Fed Wheat-Based Diets
Containing Xylanase, .beta.-Glucanase and Direct Fed Microbials
[0629] Material and Methods
[0630] The use of animals and experimental protocol was approved by
the Institutional Animal Experiment Committee. A diet was
formulated to be balanced for energy and nutrients for young
broiler chicks (0-21 days of life) (Table 1, Diet I). The cereal
component of the diet was either wheat, barley, rye, wheat
middlings, wheat bran or combinations thereof whilst the protein
component was soybean meal and the source of fat was rapeseed oil.
No synthetic antimicrobials or anti-coccidial drugs were included,
and the diet was supplied as a mash.
[0631] The basal diet was divided into portions and the respective
enzymes and DFMs added to constitute experimental diets identified
in Table 2.
[0632] Each supplement was provided in a premix and added to the
mixer during diet preparation. Diets containing the DFM were mixed
first and the mixer was flushed between each diet to prevent cross
contamination. Samples were collected from each treatment diet from
the beginning, middle, and end of each batch and blended together
to confirm enzyme activities and DFM presence in feed before
commencement of the animal trial. Additional samples from each
treatment diet were retained and stored until required at
-20.degree. C..+-.2.degree. C. for analysis.
[0633] Male broiler (Ross 308) chicks were obtained as day-olds
from a commercial hatchery. The chicks were individually weighed
and allocated to 32 brooder cages (8 chicks per cage) so that the
average bird weight per cage was similar. The 4 dietary treatments
(Table 2) were then randomly assigned to 8 cages each. On day 12,
the birds were transferred to grower cages. The space allocation
per bird in brooder and grower cages was 530 and 640 cm.sup.2,
respectively. The brooder and grower cages were housed in
environmentally controlled rooms. The temperature was maintained at
31.degree. C. in the first week and then gradually reduced to
22.degree. C. by the end of third week. The birds received 20 hours
fluorescent illumination and, allowed free access to the diets and
water. The diets were offered from d 0 to 21. Body weights were
recorded at weekly intervals throughout the 21-d experimental
period. Mortality was recorded daily. The data were analyzed using
the GLM procedure of SAS.
TABLE-US-00027 TABLE 1 Diet composition of broiler wheat-basal
diets (% as fed) Ingredients Diet I Diet II Diet III Wheat 44.9
43.9 44.36 Wheat middlings 3.00 2.83 -- Barley 10.0 10.0 -- Rye --
5.00 -- Wheat bran -- -- 22.8 Soybean Meal 30.9 29.3 23.9 Fat 5.89
4.25 -- Rapeseed oil -- -- 4.5 L-Lysine HCl 0.40 0.32 0.59
DL-Methionine 0.34 0.24 0.23 L-Threonine 0.19 0.10 0.25 Sodium
Bicarbonate -- 0.20 -- Salt 0.17 0.23 0.36 Limestone 1.69 1.32 1.00
Monocalcium Phosphate 1.55 1.00 1.61 Trace minerals/vitamins premix
0.50 1.00 0.40 Titanium dioxide -- 0.30 -- Calculated Provisions
Crude protein, % 22.1 21.8 21.8 Metabolizable energy, MJ/kg 12.7
11.60 11.63 Calcium, % 1.05 0.88 0.88 Available Phosphorous, % 0.50
0.38 0.38 Digestible Lysine, % 1.27 1.15 1.15 Digestible Methionine
% 0.63 0.51 0.51
TABLE-US-00028 TABLE 2 Identification of treatments ID
Description.sup.1 1 Control, no additive 2 NC + Xylanase (2500
XU/kg) 3 NC + Xylanase (2500 XU/kg) + .beta.-glucanase (200 BGU/kg)
4 NC + Xylanase (2500 XU/kg) + .beta.-glucanase (200 BGU/kg) +
DFM.sup.2(7.5e+04) .sup.1The enzymes (xylanase (Danisco Xylanase an
endo-1,4-.beta.-D-xylanase (E.C. 3.2.1.8)) and .beta.-glucanase
Axtra .RTM.XB) are commercial products supplied by Danisco Animal
nutrition. .sup.2A three-strain Bacillus based direct fed
microbial, selected for the ability to secrete enzymes supplied by
Danisco Animal nutritionas equal proportions of strains AGTP BS918
(NRRL B-50508), AGTP BS3BP5 (NRRL B-50510) and AGTP BS1013 (NRRL
B-50509).
[0634] Results
TABLE-US-00029 TABLE 3 Effects of xylanase, .beta.-glucanase and a
bacillus based direct fed microbials on growth performance of a
young broiler chick. Body weight at 21 days, g Body weight gain, g
1 863.8b 827.4b 2 897.0ab 860.4ab 3 899.6ab 863.4ab 4 906.3a 869.6a
Std. error 16.98 16.92 N.B. Different letters following the values
show statistical differences (P .ltoreq. 0.10) between values in
that column
[0635] Chicks fed combination of xylanase, a fibre degrading enzyme
(.beta.-glucanase) and a bacillus based DFM grew faster than
control and numerically better than chicks fed enzymes only diets.
The body weight at 21 days and the body weight gain was numerically
better in chicks fed three way combinations of xylanase,
.beta.-glucanase and DFM relative to the control.
II. Nutrients and Energy Retention/Digestibility
[0636] Material and Methods
[0637] The use of animals and experimental protocol was approved by
the Institutional Animal Experiment Committee. A wheat-barley based
diet was formulated to be balanced for energy and nutrients for
young broiler chicks (0-21 days of life) (Table 1, Diet II).
Titanium dioxide was included at 0.30% to allow determination of
dietary component retention. No synthetic antimicrobials or
anti-coccidial drugs were included, and the diet was supplied as a
mash. The basal diet was divided into portions and the respective
enzymes and DFMs added to constitute experimental diets identified
in Table 4. Each supplement was pre-mixed and the mixer was flushed
to prevent cross contamination of treated diets. Samples were
collected from each treatment diet from the beginning, middle, and
end of each batch and blended together to confirm enzyme activities
and DFM presence in feed before commencement of the animal trial.
Additional samples from each treatment diet are retained and stored
until required at -20.degree. C..+-.2.degree. C. for analysis.
TABLE-US-00030 TABLE 4 Identification of treatments ID
Description.sup.1 1 Control, no additive 2 NC + Xylanase (2500
XU/kg) 3 NC + Xylanase (2500 XU/kg) + .beta.-glucanase (200
XBGU/kg) 4 NC + Xylanase (2500 XU/kg) + .beta.-glucanase (200
BGU/kg) + DFM.sup.2((7.5e+04) .sup.1The enzymes (xylanase (Danisco
Xylanase an endo-1,4-.beta.-D-xylanase (E.C. 3.2.1.8)) and
.beta.-glucanase (Axtra .RTM. XB)) are commercial products supplied
by Danisco Animal nutrition. .sup.2A three-strain Bacillus based
direct fed microbials, selected for their ability to secrete
enzymes supplied by Danisco Animal nutrition as equal proportions
of strains AGTP BS918 (NRRL B-50508), AGTP BS3BP5 (NRRL B-50510)
and AGTP BS1013 (NRRL B-50509).
[0638] The study involved a cage trial, which was conducted to
obtain excreta samples for energy and nutrients digestibility
measurements. Day-old male broiler chicks (Ross 308) were obtained
from a commercial hatchery. The chicks were individually weighed
upon arrival and stratified by body weight and allocated to 30
cages (five chicks per cage) so that the average bird weight per
cage was similar. The four dietary treatments were then randomly
assigned to six replicate cages. The trial was conducted from day 0
to 21 during which the birds had free access to their assigned
dietary treatments and water. The brooder and room temperatures
were set at 32 and 29.degree. C., respectively, during the first
week. Thereafter, heat supply in the brooder was switched off and
room temperature was maintained at 29.degree. C. throughout the
experiment. Light was provided for 24 h throughout the experiment.
On days 17, 18, 19 and 20, samples of excreta were collected and
stored frozen at -20.degree. C. for the determination of energy and
nutrients retention/digestibility. Care was taken during the
collection of excreta samples to avoid contamination from feathers
and other foreign materials. Excreta samples were pooled within a
cage mixed well using a blender and two representative samples per
cage were taken. The samples were freeze-dried. Dried samples were
ground to pass through a 0.5 mm sieve and stored in airtight
plastic containers at -4.degree. C. until chemical analyses.
Samples of diets and excreta were analyzed for dry matter, crude
protein (as nitrogen), gross energy, fat (as hexane extracts) and
neutral detergent fibre according to AOAC official methods of
analysis). Titanium (digestibility marker) was analyzed according
to the procedures described by Lomer et al. (2000, Analyst
125:2339-2343). Retention/Digestibility was calculated using the
standard procedures (Adeola, O. 2001. Digestion and balance
techniques in pigs. Pages 903-916 in Swine Nutrition, 2nd ed. A. J.
Lewis, and L. L. Southern, ed. CRC Press, Washington, D.C.). Data
were analyzed using the General Linear Models procedure of SAS
(2004).
[0639] Results
TABLE-US-00031 TABLE 5 Effects of xylanase, a fibre degrading
enzyme and a bacillus based direct fed microbials on nutrients
retention/digestibility and energy metabolizability in a young
broiler chick. Apparent retention/digestibility, % Neutral Dry
Crude detergent Treatment matter protein Fat fibre ME, kcal/kg 1
67.4d 62.2c 78.3c 29.0c 2875c 2 71.2b 64.7b 81.5b 37.1a 3033b 3
70.9c 63.8bc 82.9b 33.1b 3040b 4 73.9a 68.8a 86.0a 38.9a 3154a Std.
error 0.06 0.70 0.71 1.06 2.92 N.B. Different letters following the
values show statistical differences (P .ltoreq. 0.10) between
values in that column
[0640] A combination of xylanase, .beta.-glucanase and a bacillus
based direct fed microbial improved utilization of dietary energy
young broiler compared to either, the control or xylanase alone or
a combination of xylanase and .beta.-glucanase (Table 5). This
could be linked increased retention of energy yielding nutrients
such as fibre, fat and nitrogen (Table 5). The enhanced fat
retention due to the three way combinations is noteworthy and could
be linked to enhanced digestion and absorption of dietary fat and
also production and absorption of short chain fatty acids from
fermentation. The observed benefits of the three way combination of
xylanase, .beta.-glucanase, bacillus/propionic DFM better in energy
and nutrients utilization could also be speculatively linked to
improved gut health and function through positive microbiota
modulation and gut digestive/absorptive function.
III. Lactic Acid Production in the Caecum
[0641] Materials and Methods
[0642] In Vitro Simulation of Chicken Caecum
[0643] A chicken caecum model was developed from an earlier
described human colon in vitro system (Makivuokko et al. 2006;
Nutrition and Cancer 52:94-104, Makelainen et al. 2009;
International Dairy Journal 19:675-683). This caecum in vitro model
is comprised of four connected vessels inoculated with fresh caecal
microbes. A wheat-wheat bran based basal diet was formulated to be
balanced for energy and nutrients for young broiler chicks (Table
1, Diet III). No synthetic antimicrobials or anti-coccidial drugs
were included in the basal diet. The basal diet was divided into
portions and the respective enzymes and DFMs added to constitute
experimental diets identified in Table 6. The different feeds
underwent a simulated digestion of the upper gastrointestinal tract
before they were fed to the in vitro caecum system during a 5-hour
simulation. The vessels model the caecum compartments of the
chicken, each having the same pH (6.25). Chromatographic analysis
of lactic acid from the caecal simulation samples was performed
with pivalic acid as internal standard in a similar matter as
previously described (Ouwehand et al. 2009; The British Journal of
Nutrition 101:367-375).
TABLE-US-00032 TABLE 6 Identification of treatments ID Description
1 Xylanase.sup.1 (2500 XU/kg) 2 Xylanase + FDE mix.sup.2 3 Xylanase
(2500 XU/kg) + FDE mix.sup.2 + DFM.sup.3 .sup.1Danisco xylanase,
Danisco Animal Nutrition .sup.2ACCELLERASE .RTM. TRIO .TM. enzyme
complex contains a potent combination of multiple enzyme activities
including .beta.-glucanases (200 CMC U/kg), xylanases (e.g.
endoxylanases - endo-1,4-.beta.-D-xylanase (E.C. 3.2.1.8))(>1200
ABX U/kg) and .beta.-glucosidases (>800 pNPG U/kg) supplied by
DuPont Industrial Bioscences. .sup.3a three-strain Bacillus based
direct fed microbial selected for their ability to secrete enzymes
supplied by Danisco Animal Nutrition as equal proportions of
strains AGTP BS918 (NRRL B-50508), AGTP BS3BP5 (NRRL B-50510) and
AGTP BS1013 (NRRL B-50509).
[0644] Results
TABLE-US-00033 TABLE 7 Effects of xylanase, a mixture of fibre
degrading enzymes and a direct fed microbial on lactic acid
production in a chicken cecum Treatment Lactic acid, .mu.mol/ml 1
17.51b 2 19.67b 3 42.23a SEM 7.525 N.B. Different letters following
the values show statistical differences (P .ltoreq. 0.10) between
values in that column
[0645] The combination of xylanase+a mix of other fibre degrading
enzymes+bacillus based direct fed microbials increased the caecal
lactic acid production compared with single, enzyme or enzyme
combinations alone. Lactic acid is produced by lactic acid
bacteria, in which lactobacilli and streptococci predominate; these
bacteria are known to have health-promoting properties in the gut
(Walter, 2008; Applied and Environmental Microbiology 74:
4985-4996). Lactic acid has antibacterial effects on pathogens such
as E. coli and Salmonella species (Nout et al. 1989; International
Journal of Food Microbiology 8, 351-361), and lactobacilli can
inhibit adhesion of E. coli to the intestines (Hillman et al. 1994;
Journal of Applied Microbiology 76: 294-300.). High concentrations
of lactic acid due to a three way combination of xylanase, fibre
degrading enzymes and direct fed microbial should therefore reflect
an increased population and activity of these gut health related
microbes.
IV. Caecal Microbial Population
[0646] Materials and Methods
[0647] Broiler chickens are assigned to pens based on initial body
weight and experimental diets randomly allocated using a recognized
experimental design. The birds are allowed free access to
experimental diets for a period between day 0 to 21.
[0648] Excreta samples are collected daily from day d18 to d20 and
stored at -20.degree. C. On d 21, the birds are euthanized by
cervical dislocation, and contents of caeca obtained and stored
frozen at -20.degree. C. for determination of caecal VFA.
[0649] DNA Extraction:
[0650] 0.2 g of caecal digesta suspended in PBS, and then further
isolated by a bead beating step and then automatically with MagMax
using a commercial kit, MagMAX.TM. Total Nucleic Acid Isolation Kit
(Applied biosystems). The amount of isolated DNA was determined by
using a Nanodrop ND-1000 Full-spectrum UV/Vis Spectrophotometer
(Wilmington, Del., USA). Flow cytometry utilised as previously
described (Apajalahti et al. 2002, Appl Environ Microbiol 68(10):
4986-4995) for enumeration of total or specific bacteria from the
samples.
[0651] PCR Procedures:
[0652] Isolated DNA is analysed by qPCR (quantitative polymerase
chain reaction) using a applied biosystem. Specific primers are
used to detect specifically interesting microbial genus as
described in 3.
TABLE-US-00034 TABLE 8 References where genus specific primers can
be found for the quantification by qPCR of digesta microbial
population Reference from which specific primers are Genus of
interest obtained Enterobacteriaceae Matsuda et al. (2007), Appl
Environ Microbiol 73(1): 32-39 Propionibacterium Peng et al. 2011
Lactobacillus Heilig et al (2002) Appl Environ Microbiol 68:
114-123, Walter et al (2001) Appl Environ Microbiol 67: 2578-2585
Ruminococcus Rinttila et al (2004), J Appl Microbiol 97, 1166-1177,
Mosoni et al. J Appl Microbiol, 2007, 103: 2676-85
http://www.ncbi.nlm.nih.gov/pubmed/18045448 Fibrobacter MvDonald et
al. 2008. Environ. Microbiol. 1: 1310-1319 Roseburia Makivuokko et
al. 2010. Beneficial Microbes, 1; 131-137 Faecalibacterium Rinttila
et al. J Appl Microbiol, 2004, 97, 1166-1177 Bacteroides Mulugeta
et al., 2012. J Environ Manage. 2012 Jul. 30; 103: 95-101.
[0653] The combination of xylanase+(mannanase or
.beta.-Glucanase)+DFMs induces a shift in caecal microbial
population in favour of Lactobacillus and/or other specific groups
known as fibrolytic bacteria: Ruminococcus, Bacteroides,
Roseburia.
Example 2: Effect of 2 Xylanases and Other Fibre Degrading Enzymes
(FDE-Mix) and DFM (Bacillus Based Direct Fed Microbial;
Lactobacillus Based Direct Fed Microbials when Fed Singly or in
Combination on Growth Performance and Cecal Volatile Fatty Acids in
Young Broiler Chickens Fed Corn-Based Diets
[0654] Experiment 1
[0655] Material and Methods
[0656] The use of animals and experimental protocol is approved by
the institutional Animal Experiment Committee. The basal diet, as
fed, is formulated to be balanced for energy and protein, and to
match the requirements for growing birds of this age and genotype
(Table 9). The cereal component of the diet is corn, and protein
component can be soybean meal with or without other protein
ingredients such as canola, rape seed meal, etc. Corn co-products
such as DDGS or corn gem meal or corn gluten feed can be included
either singly or in combination provided that the diet is
formulated to meet the nutrient requirements of the birds being
fed. No synthetic antimicrobials or anti-coccidial drugs are
included, and the diet is supplied as a mash. A common
digestibility marker (Titanium dioxide, chromic oxide or celite) is
included at 3 g/kg to allow determination of digestibility of
dietary components. The basal diet is divided into portions and the
respective enzymes and DFMs added to constitute experimental diets
identified in Table 10. Each supplement is pre-mixed and the mixer
is flushed to prevent cross contamination of treated diets. Samples
are collected from each treatment diet from the beginning, middle,
and end of each batch and blended together to confirm enzyme
activities and DFM presence in feed before commencement of the
animal trial. Additional samples from each treatment diet are
retained and stored until required at -20.degree. C..+-.2.degree.
C. for analysis.
TABLE-US-00035 TABLE 9 Composition of the corn basal diet (%, as
fed) for broilers d 0-21 Diet I Diet II Corn 54.7 58.2 Corn DDGS
11.0 -- Rapeseed meal -- 16.2 Soybean Meal 28.9 19.4 Fat 1.00 --
Rapeseed oil -- 2.11 L-Lysine HCl 0.43 0.50 DL-Methionine 0.27 0.17
L-Threonine 0.11 0.16 Sodium Bicarbonate 0.20 -- Salt 0.22 0.35
Limestone 1.53 0.70 Monocalcium phosphate 0.56 1.90 Vitamin/mineral
premix 1.00 0.40 Calculated provisions Crude protein, % 21.1 21.1
Metabolizable energy, MJ/kg 11.5 11.6 Calcium 0.89 0.89 Digestible
phosphorous, % 0.28 0.28 Digestible Lysine, % 1.15 1.15 Digestible
Methionine, % 0.55 0.55
TABLE-US-00036 TABLE 10 Experimental diets identification Treatment
Description 1 Control, basal (NC) 2 NC + Xylanase.sup.1 1 3 NC +
Xylanase 1 + FDE mix.sup.4 4 NC + Xylanase 1 + Bacillus DFM.sup.2 5
NC + Xylanase 1 + Lactobacillus DFM.sup.3 6 NC + Xylanase 1 + FDE
mix.sup.4 + Bacillus DFM.sup.2 7 NC + Xylanase 1 + FDE mix.sup.4 +
Lactobacillus DFM.sup.3 8 NC + Xylanase.sup.1 2 9 NC + Xylanase 2 +
FDE mix.sup.4 10 NC + Xylanase 2 + Bacillus DFM.sup.2 11 NC +
Xylanase 2 + Lactobacillus DFM.sup.3 12 NC + Xylanase 2 + FDE
mix.sup.4 + Bacillus DFM.sup.2 13 NC + Xylanase 2 + FDE mix.sup.4 +
Lactobacillus DFM.sup.3 .sup.1Xylanases (e.g.
endo-1,4-.beta.-D-xylanase (E.C. 3.2.1.8) from two different origin
organisms .sup.2Bacillus DFM selected as an enzyme producing strain
.sup.3Lactobacillus DFM known to be a C5 sugar-fermenting strain; a
short-chain fatty acid-producing strain; a fibrolytic, endogenous
microflora-promoting strain; or combinations thereof .sup.4FDE mix:
Combination of fibre degrading enzyme activities including
beta-glucanase, beta-glucosidase, beta-xylosidase and/or
alpha-arabinofuranosidase
[0657] Broiler chickens are assigned to pens based on initial body
weight and experimental diets randomly allocated using a recognized
experimental design. The birds are allowed free access to
experimental diets for a period between day 0 to 21. The body
weight (BW), feed intake (FI) and mortalities are recorded to
calculate body weight gain (BWG), feed conversion ratio (FCR) and
feed conversion efficiency (FCE).
[0658] Excreta samples are collected daily from day d18 to d20 and
stored at -20.degree. C. for determination of nutrients and fibre
retention, and AME and AMEn contents. On d 21, the birds are
euthanized by cervical dislocation, and contents of ileum (from
Meckel's diverticulum to approximately 1 cm above the ileal-cecal
junction) and ceca obtained and stored frozen at -20.degree. C. for
determination of ileal digestibility of components and cecal
VFA.
[0659] Daily excreta samples are pooled for each cage and
oven-dried at 60.degree. C., whereas ileal digesta samples were
pooled on cage/pen basis and freeze-dried. Samples of the diets,
excreta and ileal digesta are finely ground and thoroughly mixed
for analysis. All samples are analyzed for dry matter, nitrogen,
fat and gross energy according to A.O.A.C. (2005) procedures.
Soluble and insoluble non-starch polysaccharides are assayed in
diets and excreta according to Englyst et al. (1988) whereas
neutral detergent fibre, neutral detergent insoluble nitrogen are
assayed according to the methods of Tilley and Terry (1962).
Digestibility marker is analyzed according to standard procedure of
selected marker.
[0660] Chromatographic analysis of volatile fatty acids and lactic
acid, e.g. SCFAs, to be performed from simulation samples with
pivalic acid as internal standard in a similar matter as previously
described (Ouwehand et al., 2009 February; 101(3):367-75).
Concentrations of acetic, propionic, butyric, isobutyric, valeric,
isovaleric, 2-methylbutyric acids, and lactic acid are
determined.
[0661] Coefficient of ileal apparent digestibility and coefficient
of apparent retention of components are calculated according to
Adeola et al., 2010 (Poult Sci. 2010 September; 89(9):1947-54).
[0662] The cage (pen) is the experimental unit. ANOVA is conducted
using the General Linear Models of SAS (SAS Inst. Inc., Cary,
N.C.). When F-ratios indicate significance, treatment means are
separated.
[0663] Results
[0664] Treated groups fed the whole combination: xylanase plus a
secondary fibre degrading enzyme(s) and a DFM (Bacillus or LB),
have higher BWG (g/bird/day), and/or a lower FCR (g BW gain/g feed
intake) and/or better nutrients, energy and fibre
digestibility/retention than either the control, or these additives
fed alone or in two-way combination.
[0665] The combination of xylanases (xylanase 1 and/or 2)+an FDE
mix+DFMs significantly increases the ileal and/or caecal total VFA
and the concentration of butyric acid or propionic acid in the
ileal and/or caecal digesta of broilers.
II. Growth Performance
[0666] Experiment I
[0667] Materials and Methods
[0668] The use of animals and experimental protocol was approved by
the Institutional Animal Experiment Committee. A corn/soy based
diet was formulated to be balanced for energy and nutrients for
young broiler chicks (0-21 days of life) (Table 9, Diet I). No
synthetic antimicrobials or anti-coccidial drugs were included, and
the diet was supplied as a mash. The basal diet was divided into
portions and the respective enzymes and DFMs added to constitute
experimental diets identified in Table 11.
TABLE-US-00037 TABLE 11 Treatments identification used in
experiment I ID Description 1 Negative Control, no additive (NC) 2
NC + Xylanase.sup.a 1 3 NC + Xylanase 1 + B-glucanase.sup.a 4 NC +
Xylanase 1 + Bacillus DFM.sup.b 5 NC + Xylanase 1 + B-glucanase +
Bacillus DFM 6 NC + Xylanase 2 .sup.c 7 NC + Xylanase 2 +
B-glucanase 8 NC + Xylanase 2 + Bacillus DFM 9 NC + Xylanase 2 +
B-glucanase + Bacillus DFM .sup.aThe enzymes (xylanase (Danisco
Xylanase an endo-1,4-.beta.-D-xylanase (E.C. 3.2.1.8)) and
.beta.-glucanase (Axtra .RTM. XB)) are commercial products supplied
by Danisco Animal nutrition .sup.bThree-strain Bacillus based DFM
(equal proportions of strains AGTP BS918 (NRRL B-50508), AGTP
BS3BP5 (NRRL B-50510) and AGTP BS1013 (NRRL B-50509)), selected for
their ability to secrete enzymes .sup.c FveXyn4 xylanase (an
endo-1,4-.beta.-D-xylanase (E.C. 3.2.1.8)) shown as SEQ ID No. 3
herein (also described in PCT/CN2012/079650 which is incorporated
herein by reference), Danisco Animal Nutrition.
[0669] All supplements were provided in a premix which was added to
the mixer during diet preparation. Diets containing the DFM were
mixed first and the mixer was flushed between each diet to prevent
cross contamination. Samples were collected from each treatment
diet from the beginning, middle, and end of each batch and blended
together to confirm enzyme activities and DFM presence in feed
before commencement of the animal trial. Additional samples from
each treatment diet were retained and stored until required at
-20.degree. C..+-.2.degree. C. for analysis. Male broiler
(Hubbard-Cobb) chicks were obtained as day-olds from a commercial
hatchery. On day 0 the chicks were individually weighed and
allocated to 72 cages (8 chicks per cage) so that the average bird
weight per cage was similar. The 9 dietary treatments (Table 11)
were then randomly assigned to 8 cages each. The cages were housed
in environmentally controlled rooms. The temperature was maintained
at 31.degree. C. in the first week and then gradually reduced to
22.degree. C. by the end of third week. The birds received 20 hours
fluorescent illumination and, allowed free access to the diets and
water for the duration of the study. Body weights and feed intake
were recorded the beginning and end of the 21-d experimental
period. Mortality was recorded daily. Feed conversion ratios were
calculated by dividing total feed intake by weight gain of live
plus dead birds. Data was analysed using the General Linear Models
of SAS (SAS Inst. Inc., Cary, N.C.). When F-ratios indicate
significance, treatment means are separated.
[0670] Results, Experiment I
TABLE-US-00038 TABLE 12 Effects of xylanase, .beta.-glucanase and a
bacillus based direct fed microbials on growth performance of a
young broiler chick. Body Weight Gain Feed Intake Feed (g) (g)
Conversion (g/g) 1 652.5.sup.d 980.8 1.498.sup.a 2 670.6.sup.bc
982.3 1.465.sup.bc 3 673.6.sup.abc 978.3 1.452.sup.cde 4
681.7.sup.ab 982.3 1.441.sup.def 5 688.2.sup.a 977.2 1.420.sup.f 6
665.7.sup.cd 985.4 1.477.sup.ab 7 671.2.sup.bc 982.4 1.464.sup.bcd
8 677.0.sup.abc 979.0 1.446.sup.cde 9 684.5.sup.ab 981.5
1.430.sup.ef Std. error 6.4 11.5 0.009 N.B. Different letters
following the values show statistical differences (P .ltoreq. 0.10)
between values in that column
[0671] Treated groups fed the whole combination:
xylanase+.beta.-glucanase+Bacillus DFM combination had higher BWG
(g/bird/day), and lower FCR (g BW gain/g feed intake) than either
the control, or these additives fed alone or in two-way combination
(Table 12). This was the case when both Xylanase 1 and Xylanase 2
were administered.
[0672] Experiment II
[0673] Materials and Methods
[0674] The use of animals and experimental protocol was approved by
the Institutional Animal Experiment Committee. A corn/soy based
diet was formulated to be balanced for energy and nutrients for
young broiler chicks (0-21 days of life) (Table 9, Diet I). No
synthetic antimicrobials or anti-coccidial drugs were included, and
the diet was supplied as a mash. The basal diet was divided into
portions and the respective enzymes and DFMs added to constitute
experimental diets identified in Table 13.
TABLE-US-00039 TABLE 13 Treatments identification for Experiment II
ID Description 1 Negative Control, no additive (NC) 2 NC + Xylanase
1 (2500 XU/kg) 3 NC + Xylanase 1 (2500 XU/kg) + .beta.-glucanase
(200 BGU/kg) 4 NC + Xylanase 1 (2500 XU/kg) + Enterococcus DFM 5 NC
+ Xylanase 1 (2500 XU/kg) + .beta.-glucanase (200 BGU/kg) +
Enterococcus DFM 6 NC + Xylanase 2 (2500 XU/kg) 7 NC + Xylanase 2
(2500 XU/kg) + .beta.-glucanase (200 BGU/kg) 8 NC + Xylanase 2
(2500 XU/kg) + Enterococcus DFM 9 NC + Xylanase 2 (2500 XU/kg) +
.beta.-glucanase (200 BGU/kg) + Enterococcus DFM .sup.aThe enzymes
(xylanase (Danisco Xylanase an endo-1,4-.beta.-D-xylanase (E.C.
3.2.1.8)) and .beta.-glucanase (Axtra .RTM. XB)) are commercial
products supplied by Danisco Animal nutrition .sup.bEnterococcus
based DFM (Enterococcus faecium ID7 (referred to as Lactococcus
lactis ID7 in granted U.S. Pat. No. 7,384,628 and deposited at the
ATCC depository as PTA-6103 and later reclassified as Enterococcus
faecium ID7)), .sup.cFveXyn4 xylanase (an
endo-1,4-.beta.-D-xylanase (E.C. 3.2.1.8)) shown as SEQ ID No. 3
herein (also described in PCT/CN2012/079650 which is incorporated
herein by reference), Danisco Animal Nutrition
[0675] All supplements were provided in a premix which was added to
the mixer during diet preparation. Diets containing the DFM were
mixed first and the mixer was flushed between each diet to prevent
cross contamination. Samples were collected from each treatment
diet from the beginning, middle, and end of each batch and blended
together to confirm enzyme activities and DFM presence in feed
before commencement of the animal trial. Additional samples from
each treatment diet were retained and stored until required at
-20.degree. C..+-.2.degree. C. for analysis. Male broiler
(Hubbard-Cobb) chicks were obtained as day-olds from a commercial
hatchery. On day 0 the chicks were individually weighed and
allocated to 72 cages (8 chicks per cage) so that the average bird
weight per cage was similar. The 9 dietary treatments (Table 13)
were then randomly assigned to 8 cages each. The cages were housed
in environmentally controlled rooms. The temperature was maintained
at 31.degree. C. in the first week and then gradually reduced to
22.degree. C. by the end of third week. The birds received 20 hours
fluorescent illumination and, allowed free access to the diets and
water for the duration of the study. Body weights were recorded the
beginning and end of the 21-d experimental period. Mortality was
recorded daily. The data were analyzed using the GLM procedure of
SAS.
[0676] Results, Experiment II
TABLE-US-00040 TABLE 14 Effects of xylanase, .beta.-glucanase and
an Enterococcus based direct fed microbials on growth performance
of a young broiler chick. Body Weight Gain (g) 1 652.5.sup.c 2
670.6.sup.ab 3 673.6.sup.ab 4 677.7.sup.ab 5 684.4.sup.a 6
665.7.sup.bc 7 671.2.sup.ab 8 673.7.sup.ab 9 682.4.sup.a Std. Error
6.5 N.B. Different letters following the values show statistical
differences (P .ltoreq. 0.10) between values in that column
[0677] There was a numerical improvement in broiler body weight
gain, when the combination of
xylanase+.beta.-glucanase+Enterococcus DFM was supplemented on top
of xylanase+.beta.-glucanase or xylanase+Enterococcus DFM (Table
14).
III. Volatile Fatty Acid Production in the Caecum
[0678] Materials and Methods
[0679] A corn-soybean meal-rapeseed meal based basal diet was
formulated to be balanced for energy and nutrients for young
broiler chicks (Table 9, Diet II). No synthetic antimicrobials or
anti-coccidial drugs were included in the basal diet. The basal
diet was divided into portions and the respective enzymes and DFMs
added to constitute experimental diets identified in Table 15.
Subsequent procedures were similar to the ones described for
Example 1, part III. followed. Chromatographic analysis of volatile
fatty acids from simulation samples (see Example 1, part III) was
performed with pivalic acid as internal standard in a similar
matter as previously described (Ouwehand et al. 2009; The British
Journal of Nutrition 101: 367-375 the teaching of which is
incorporated herein by reference). Concentrations of acetic,
propionic, butyric, isobutyric, valeric, isovaleric, and
2-methylbutyric acids were determined.
TABLE-US-00041 TABLE 15 Treatments identification ID Description 1
Control 2 Xylanase (2500 XU/kg) 3 Xylanase (2500 XU/kg) +
.beta.-glucanase (200 BGU/kg) 4 Xylanase (2500 XU/kg) +
.beta.-glucanase (200 BGU/kg) + DFM )7.5e+04).sup.1 .sup.1A
three-strain Bacillus based direct fed microbial (equal proportions
of strains AGTP BS918 (NRRL B-50508), AGTP BS3BP5 (NRRL B-50510)
and AGTP BS1013 (NRRL B-50509)), selected for their ability to
secrete enzymes supplied by Danisco Animal Nutrition. The enzymes
(xylanase (Danisco Xylanase an endo-1,4-.beta.-D-xylanase (E.C.
3.2.1.8)) and .beta.-glucanase (Axtra .RTM. XB)) are commercial
products supplied by Danisco Animal nutrition
[0680] Results
TABLE-US-00042 TABLE 16 Effects of xylanase, .beta.-glucanase and a
direct fed microbial on acetic and butyric and total volatile fatty
acids (VFA) production in chicken cecum Concentration, .mu.mol/ml
Acetic Butyric VFA 1 61.36b 6.06c 68.59b 2 112.6ab 33.0b 148.3ab 3
133.6ab 43.5ab 181.3ab 4 164.6a 59.0a 227.2a Pooled std. error
27.40 5.26 35.99 N.B. Different letters following the values show
statistical differences (P .ltoreq. 0.10) between values in that
column
[0681] The combination of xylanase+.beta.-glucanase+direct fed
microbials increased the caecal acetic acid, butyric acid and
volatile fatty acid (VFA) production compared with single DFM,
enzymes or enzyme combinations alone (Table 16). Volatile fatty
acids can provide significant amount of energy to the chicken.
Butyric acid is also known to improve gastrointestinal health and
reduced incidence of colon cancer in humans (Brons et al., 2002,
Trends Food Science and Technology 13:251-261 which is incorporated
herein by reference).
Example 3: Effect of Xylanase and Other Fibrolytic Enzymes
(.beta.-Glucanase or Fibre Degrading Enzyme Mix (FDE-Mix)) and DFM
(Bacillus Based Direct Fed Microbial) when Fed Singly or in
Combination on Growth Performance and Nutrients Digestibility in
Pigs (25 to 60 kg) Fed Mixed Grains-Based Diets
[0682] Material and Methods
[0683] The use of animals and experimental protocol is approved by
the Animal Experiment Committee. The basal diet, as fed, is
formulated to be balanced for energy and protein, and to match the
requirements for growing pigs of this age and genotype (Table 17).
The major ingredients composition (type and inclusion levels) in
the basal diet can vary as shown in table 17 provided that the diet
is formulated to meet the nutrient requirements of the pigs being
fed. A common digestibility marker (Titanium dioxide, chromic oxide
or celite) is included at 3 g/kg to allow determination of
digestibility of dietary components. No synthetic antimicrobials or
anti-coccidial drugs are included, and the diet is supplied as a
mash. The basal diet is divided into portions which are then
treated with the enzymes and DFMs identified in Table 18. During
feed mixing, the mixer is flushed to prevent cross contamination of
diet. Samples are collected from each treatment diet from the
beginning, middle, and end of each batch and blended together to
confirm enzyme activities and DFM presence in feed. Samples from
each treatment diet are retained during mixing and stored at
-20.degree. C. until required.
TABLE-US-00043 TABLE 17 Examples of basal diet composition for pigs
20 to 60 kg body weight (%, as fed) Diet I Diet II Corn 45.4 9.50
Wheat -- 25.0 Barley -- 25.0 corn DDGS 25.0 10.0 Corn germ meal
15.0 -- Wheat middlings/rice bran -- 7.00 Soybean Meal 10.0 10.0
Canola Meal -- 9.00 Fat 0.56 1.23 Molasses -- -- L-Lysine HCl 0.59
0.47 DL-methionine 0.02 0.02 L-threonine 0.13 0.09 L-tryptophan --
0.01 Salt 0.46 0.54 Limestone 1.16 0.63 Dicalcium Phosphate 0.39
1.12 Vitamin and mineral premix 1.00 0.10 Inert marker
digestibility marker 0.30 0.30 Crude protein, % 19.1 18.3
Digestible energy, MJ/kg 13.8 13.6 Digestible lysine, % 1.03 0.98
Calcium, % 0.66 0.66 Digestible phosphorous, % 0.31 0.31
TABLE-US-00044 TABLE 18 Experimental diets identification Treatment
Description 1 Control, basal (NC) 2 NC + xylanase 3 NC + xylanase +
.beta.-Glucanase 4 NC + xylanase + FDE mix.sup.1 5 NC + xylanase +
Bacillus DFM.sup.2 6 NC + xylanase + FDE mix + Bacillus DFM.sup.2 7
NC + xylanase + .beta.-Glucanase + Bacillus DFM.sup.2 .sup.1FDE
mix: Combination of fibre degrading enzyme activities including
beta-glucanase, beta-glucosidase, beta-xylosidase and/or
alpha-arabinofuranosidase .sup.2Bacillus DFM selected as an enzyme
producing strain
[0684] The experiment is planned and conducted to correspond to
growing phase (.ltoreq.25 to .about.60 kg body weight). The
experimental diets are fed for 42 days of 6 weeks. A group of
female and male pigs close to the target initial body are procured
from the same herd (genetics). Upon arrival pigs are weighed and
allotted to the dietary treatments using a recognised experimental
design such that each treatment has a minimum of 8 replicate pens.
The body weight and feed intake are monitored weekly for
calculation of feed conversion efficiency of gain efficiency
corrected for mortalities. Fresh grab fecal samples are collected
in week 3 and 6 to allow for calculation of dietary component
digestibility.
[0685] Growing barrows (initial body weight of 30 kg) are equipped
with a T-cannula in the distal ileum for the purpose of the
experiment. Pigs are housed in individual pens (1.2.times.1.5 m) in
an environmentally controlled room. Each pen was equipped with a
feeder and a nipple drinker and had fully slatted concrete floors.
The experiment is designed and conducted to give a minimum of 6
replicates per treatment. All pigs are fed at a level of 3 times
their maintenance energy requirement (106 kcal ME per kg.sup.0.75;
NRC, 1998), and provided at two equal portions at 0800 and 1700 h.
Animals are allowed free access to water through a bowl-type
drinker. Pig weights are recorded at the beginning and at the end
of each period and the amount of feed supplied each day are
recorded. Experimental period lasts for 15 d. The initial 10 days
of each period are considered an adaptation period to the diet.
Fresh grab fecal samples are collected on d 11 to 13 and Ileal
digesta are collected for 8 h on d 14 and 15 using standard
operating procedures. For ileal digesta collection, a plastic bag
is attached to the cannula barrel and digesta flowing into the bag
collected. Bags are removed whenever they are filled with
digesta--or at least once every 30 min and immediately frozen at
-20.degree. C.
[0686] Fecal and ileal samples are thawed, mixed within animal and
diet, and a sub-sample collected for chemical analysis. A sample of
basal diet is also collected and analyzed. Digesta samples were
lyophilized and finely ground prior to chemical analysis. Fecal
samples are dried in an oven and finely ground for analysis. All
samples were analyzed for dry matter, digestibility marker, gross
energy, crude protein, fat and neutral detergent fibre according to
standard procedures (AOAC, 2005).
[0687] The values for apparent ileal and total digestibility of
energy and nutrients are calculated as described previously (Stein
et al., 2007. J. Anim. Sci. 85:172-180). The pen is the
experimental unit. Data are subjected the MIXED procedures of
SAS.
[0688] Results
[0689] Treated groups fed the whole combination: xylanase plus a
secondary fibre degrading enzyme (.beta.-Glucanase or FDE-mix) and
a DFM (Bacillus based direct fed microbial), have higher BWG,
and/or a lower FCR (g BW gain/g feed intake) and/or high
digestibility of nutrients and/or energy and/or dry matter and/or
fibre.
Example 4: Effects of Xylanase, .beta.-Glucanase and a Propionic
Acid Producing Strain of Bacteria Based Direct Fed Microbials on
Nutrients Retention/Digestibility and Energy Metabolizability in a
Young Broiler Chick
[0690] Composition of the Wheat Based Experimental Diets Used in
Example 4
TABLE-US-00045 TABLE 19 Diet composition of broiler wheat-basal
diets (% as fed) Ingredients % Wheat 43.9 Wheat middlings 2.83
Barley 10.0 Rye 5.00 Soybean Meal 29.3 Fat 4.25 L-Lysine HCl 0.32
DL-Methionine 0.24 L-Threonine 0.10 Sodium Bicarbonate 0.20 Salt
0.23 Limestone 1.32 Monocalcium Phosphate 1.00 Trace
minerals/vitamins premix 1.00 Titanium dioxide 0.30 Calculated
Provisions Crude protein, % 21.8 Metabolizable energy, MJ/kg 11.60
Calcium, % 0.88 Available Phosphorous, % 0.38 Digestible Lysine, %
1.15 Digestible Methionine % 0.51
[0691] Material and Methods
[0692] The use of animals and experimental protocol was approved by
the Institutional Animal Experiment Committee. A wheat-barley based
diet was formulated to be balanced for energy and nutrients for
young broiler chicks (0-21 days of life) (Table 19). Titanium
dioxide was included at 0.30% to allow determination of dietary
component retention. No synthetic antimicrobials or anti-coccidial
drugs were included, and the diet was supplied as a mash. The basal
diet was divided into portions and the respective enzymes and DFMs
added to constitute experimental diets identified in Table 20. Each
supplement was pre-mixed and the mixer was flushed to prevent cross
contamination of treated diets. Samples were collected from each
treatment diet from the beginning, middle, and end of each batch
and blended together to confirm enzyme activities and DFM presence
in feed before commencement of the animal trial. Additional samples
from each treatment diet are retained and stored until required at
-20.degree. C..+-.2.degree. C. for analysis.
TABLE-US-00046 TABLE 20 Identification of treatments ID Description
1 Control, no additive 2 NC + Xylanase (2500 XU/kg) 3 NC + Xylanase
(2500 XU/kg) + .beta.-glucanase (200 BGU/kg) 4 NC + Xylanase (2500
XU/kg) + .beta.-glucanase (200 BGU/kg) + DFM.sup.1 (7.5e+04)
.sup.1Propionic acid producing strains based direct fed microbials
(Propionibacterium acidipropionici P169 PTA-5271, Omni-Bos .RTM.
P169). The enzymes (xylanase (Danisco Xylanase an
endo-1,4-.beta.-D-xylanase (E.C. 3.2.1.8)) and .beta.-glucanase
(Axtra .RTM. XB)) are commercial products supplied by Danisco
Animal nutrition.
[0693] The study involved a cage trial, which was conducted to
obtain excreta samples for energy and nutrients digestibility
measurements. Day-old male broiler chicks (Ross 308) were obtained
from a commercial hatchery. The chicks were individually weighed
upon arrival and stratified by body weight and allocated to 30
cages (five chicks per cage) so that the average bird weight per
cage was similar. The four dietary treatments were then randomly
assigned to six replicate cages. The trial was conducted from day 0
to 21 during which the birds had free access to their assigned
dietary treatments and water. The brooder and room temperatures
were set at 32 and 29.degree. C., respectively, during the first
week. Thereafter, heat supply in the brooder was switched off and
room temperature was maintained at 29.degree. C. throughout the
experiment. Light was provided for 24 h throughout the experiment.
On days 17, 18, 19 and 20, samples of excreta were collected and
stored frozen at -20.degree. C. for the determination of energy and
nutrients retention/digestibility. Care was taken during the
collection of excreta samples to avoid contamination from feathers
and other foreign materials. Excreta samples were pooled within a
cage mixed well using a blender and two representative samples per
cage were taken. The samples were freeze-dried. Dried samples were
ground to pass through a 0.5 mm sieve and stored in airtight
plastic containers at -4.degree. C. until chemical analyses.
Samples of diets and excreta were analyzed for dry matter, crude
protein (as nitrogen), gross energy, fat (as hexane extracts) and
neutral detergent fibre according to AOAC official methods of
analysis). Titanium (digestibility marker) was analyzed according
to the procedures described by Lomer et al. (2000, Analyst
125:2339-2343), which is incorporated herein by reference.
Retention/Digestibility was calculated using the standard
procedures (Adeola, O. 2001. Digestion and balance techniques in
pigs. Pages 903-916 in Swine Nutrition, 2nd ed. A. J. Lewis, and L.
L. Southern, ed. CRC Press, Washington, D.C. which is incorporated
herein by reference). Data were analyzed using the General Linear
Models procedure of SAS (2004).
[0694] Results
TABLE-US-00047 TABLE 21 Effects of xylanase, a fibre degrading
enzyme and a propionic acid producing strain of bacteria based
direct fed microbials on nutrients retention/digestibility and
energy metabolizability in a young broiler chick. Apparent
retention/digestibility, % Treatment Dry matter Fat ME, kcal/kg 1
67.4d 78.3c 2875c 2 71.2b 81.5b 3033b 3 70.9c 82.9b 3040b 4 72.7a
86.1a 3160a Std. error 0.06 0.89 15.1 N.B. Different letters
following the values show statistical differences (P .ltoreq. 0.10)
between values in that column
[0695] A combination of xylanase, .beta.-glucanase and a bacillus
based direct fed microbial improved utilization of dietary energy
compared to either, the control or xylanase alone or a combination
of xylanase and .beta.-glucanase (Table 20). This could be linked
increased retention of energy yielding nutrients in the dry matter
such as fat (Table 20). The enhanced fat retention due to the three
way combinations is noteworthy and could be linked to enhanced
digestion and absorption of dietary fat and also production and
absorption of short chain fatty acids from fermentation. The
observed benefits of the three way combination of xylanase,
.beta.-glucanase, bacillus/propionic DFM better in energy and
nutrients utilization could also be speculatively linked to
improved gut health and function through positive microbiota
modulation and gut digestive/absorptive function.
Example 5: Responses of Broiler Chicken when Fed Corn-Based Diets
Containing Xylanase, Other Fibre Degrading Enzymes and Propionic
Acid Producing Direct Fed Microbials
[0696] Composition of the Experimental Diets Used in Example 5
TABLE-US-00048 TABLE 22 Diet composition of broiler corn-basal
diets (% as fed) Composition (%) Corn 54.7 Corn DDGS 11.0 Soybean
Meal 28.9 Fat 1.00 L-Lysine HCl 0.43 DL-Methionine 0.27 L-Threonine
0.11 Sodium Bicarbonate 0.20 Salt 0.22 Limestone 1.53 Monocalcium
phosphate 0.56 Vitamin/mineral premix 1.00 Calculated provisions
Crude protein, % 21.1 Metabolizable energy, MJ/kg 11.5 Calcium 0.89
Digestible phosphorous, % 0.28 Digestible Lysine, % 1.15 Digestible
Methionine, % 0.55
[0697] Materials and Methods
[0698] The use of animals and experimental protocol was approved by
the Institutional Animal Experiment Committee. A corn based diet
was formulated to be balanced for energy and nutrients for young
broiler chicks (0-21 days of life) (Table 22). No synthetic
antimicrobials or anti-coccidial drugs were included, and the diet
was supplied as a mash. The basal diet was divided into portions
and the respective enzymes and DFMs added to constitute
experimental diets identified in Table 23. Each supplement was
pre-mixed and the mixer was flushed to prevent cross contamination
of treated diets. Samples were collected from each treatment diet
from the beginning, middle, and end of each batch and blended
together to confirm enzyme activities and DFM presence in feed
before commencement of the animal trial. Additional samples from
each treatment diet are retained and stored until required at
-20.degree. C..+-.2.degree. C. for analysis.
TABLE-US-00049 TABLE 23 Treatments identification ID Description 1
Control 2 Xylanase (2500 XU/kg).sup.1 3 Xylanase + FDE mix.sup.2 4
Xylanase (2500 XU/kg) + FDE mix + DFM.sup.3(7.5e+04) .sup.1Danisco
xylanase, Danisco Animal nutrition .sup.2ACCELLERASE .RTM. TRIO
.TM. enzyme complex contains a potent combination of multiple
enzyme activities including .beta.-glucanases (200 CMC U/kg),
xylanases (e.g. endoxylanases, endo-1,4-.beta.-D-xylanase (E.C.
3.2.1.8)) (>1200 ABX U/kg), and .beta.-glucosidases (>800
pNPG U/kg) (DuPont Industrial Bioscences). .sup.3Propionic acid
producing strains based direct fed microbials (Propionibacterium
acidipropionici P169 PTA-5271, Omni-Bos .RTM. P169)
[0699] Day old chicks were procured from a commercial hatchery and
upon arrival the birds were weighed and tagged for identification
and allocated into six blocks by body weight, and randomly allotted
to 4 treatments (Table 23) within a block with ten birds per pen in
a randomized completed block design. From d 1 and were also allowed
ad libitum access to clean drinking water. The chicks were weighed
on days 0 and 21 and their weights were recorded, feed consumption
was also monitored and documented on chick weigh days. The chicks
were monitored daily and variations in their appearance or
behaviour were recorded. At the end of each feeding period,
parameters such as weight gain, feed intake, feed conversion ratio,
feed efficiency, and mortality were determined. Data were analyzed
as a randomized complete block design using the GLM procedure of
SAS software (SAS Institute, Inc. 2006).
[0700] Results
TABLE-US-00050 TABLE 24 Effects of xylanase, a mixture of other
fibre degrading enzymes and a propionic based direct fed microbials
on growth performance of a young broiler chick. Body weight at Body
weight Feed Feed conversion 21 days, g gain, g intake, g
efficiency, g/g 1 830.4 783.5 1006.6 1.284a 2 804.2 757.3 964.0
1.273ab 3 817.6 770.7 983.3 1.275ab 4 813.2 766.4 953.9 1.245b Std.
error 12.96 11.82 23.37 0.017 N.B. Different letters following the
values show statistical differences (P .ltoreq. 0.10) between
values in that column
[0701] Chicks fed combination of xylanase, a mixture of other fibre
degrading enzymes and a propionic based DFM had better FCR than
control and numerically better than chicks fed enzymes only diets
(Table 24).
Example 6: Effects of Xylanase and .beta.-Glucanase without or with
Bacillus Strains Based Direct Fed Microbial on Growth Performance,
Microbial Counts and Nutrients Digestibility in Growing Finishing
Pigs
[0702] Composition of the Experimental Diets Used in Example 6
TABLE-US-00051 TABLE 25 Diet composition of growing pig feed (20-60
kg body weight) (% as fed) Diet I Diet II Corn 45.4 42.3 Wheat --
5.00 corn DDGS 25.0 20 Corn germ meal 15.0 -- Wheat middlings/rice
bran -- 3.00 Soybean Meal 10.0 19.8 Canola Meal -- 2.00 Fat 0.56
2.00 Molasses -- 3.00 L-Lysine HCl 0.59 0.24 DL-methionine 0.02
0.02 L-threonine 0.13 -- Salt 0.46 0.30 Limestone 1.16 1.18
Dicalcium Phosphate 0.39 0.45 Vitamin and mineral premix 1.00 0.30
Inert marker digestibility marker 0.30 0.30 Calculated provisions
Crude protein, % 19.1 19.2 Digestible energy, MJ/kg 13.8 14.6
Digestible lysine, % 1.03 0.91 Calcium, % 0.66 0.72 Digestible
phosphorous, % 0.31 0.33
[0703] Materials and Methods
[0704] Two experiments were conducted to evaluate growth
performance, fecal microbial counts and digestibility effects of a
xylanase and .beta.-glucanase enzyme blend fed without or with
bacillus strains based direct fed microbial in growing finishing
pigs. The Institutional Animal Care and Use Committee approved the
use of the pigs and relevant welfare guidelines for the Country
were used. A total of 42 pigs ([ Yorkshire.times.Landrace].times.
eDuroc) housed in groups of two were used in experiment 1 and 72
pigs of the same breed housed in groups of three were used in
experiment 2. Each pen had smooth transparent plastic sides and
plastic-covered expanded metal sheet flooring in a
temperature-controlled room (22.+-.2.degree. C.).
[0705] Respective basal diets were formulated to meet the NRC
nutrient recommendations for swine (NRC, 1998 Table 25 diet I for
experiment 1 and diet II for experiment 2). In each experiment, one
batch of the basal diet is manufactured and split into two portions
and each portion subsequently mixed with additives identified in
Table 26.
TABLE-US-00052 TABLE 26 Identification of Treatments ID Description
1 Control 2 Xylanase (4000 XU/kg) + .beta.-glucanase (360 BGU/kg) 3
Xylanase(4000 U/kg) + .beta.-glucanase(360 U/kg) +
DFM.sup.1(3.0e+08) .sup.1a three-strain Bacillus based direct fed
microbial (equal proportions of strains AGTP BS918 (NRRL B-50508),
AGTP BS3BP5 (NRRL B-50510) and AGTP BS1013 (NRRL B-50509)),
selected for their ability to secrete enzymes supplied by Danisco
Animal Nutrition. The enzymes (xylanase (Danisco Xylanase an
endo-1,4-.beta.-D-xylanase (E.C. 3.2.1.8)) and .beta.-glucanase
(Axtra .RTM. XB)) are commercial products supplied by Danisco
Animal nutrition
[0706] The treatments identified in table 26, were allocated to 7
and 8 replicate pens in experiment 1 and 2, respectively. Pen
allocation to the treatments was randomized based on pig body
weight at the start of the experiment. Body weight and Feed intake
were recorded on a weekly basis and used to calculate feed
conversion ratio. Pigs were offered the experimental diets for 42
days in both experiments. Feed and water were freely available at
all times during experimentation. In experiment 2, fresh fecal
samples were collected on days, 38, 39 and 40 for determination of
nutrients, energy and fibre digestibility as well as fecal
microbial counts. One gram of the composite fecal sample from each
pen was diluted with 9 mL of 1% peptone broth (Becton, Dickinson
and Co., Franklin Lakes, N.J.) and then homogenized. Viable counts
of bacteria in the fecal samples were then conducted by plating
serial 10-fold dilutions (in 1% peptone solution) onto MacConkey
agar plates (Difco Laboratories, Detroit, Mich.) and lactobacilli
medium III agar plates (Medium 638, DSMZ, Braunschweig, Germany) to
isolate the E. coli and Lactobacillus, respectively. The
lactobacilli medium III agar plates were then incubated for 48 h at
39.degree. C. under anaerobic conditions. The MacConkey agar plates
were incubated for 24 h at 37.degree. C. The E. coli and
Lactobacillus colonies were counted immediately after removal from
the incubator. Before chemical analysis, the fecal samples were
thawed and dried at 60.degree. C. for 72 h, after which they were
finely ground to a size that could pass through a 1-mm screen. All
feed and fecal samples were, then, analyzed for dry matter, gross
energy and acid detergent fibre following the procedures outlined
by the AOAC (Official Methods of Analysis). Chromium (digestibility
marker) was analyzed following the method described by Williams et
al. 1962, J. Anim. Sci. 59:381-389, which is incoporporated herein
by reference. Digestibility was calculated using standard
procedures (Adeola, O. 2001. Digestion and balance techniques in
pigs. Pages 903-916 in Swine Nutrition, 2nd ed. A. J. Lewis, and L.
L. Southern, ed. CRC Press, Washington, D.C.--the teaching of which
is incorporated herein by reference). The growth performance data
(BW, ADFI, ADG and FCR) were subjected to the GLM procedures of SAS
with treatments, experiment and interactions as effects in the
model. Initial analysis revealed interactions were not significant
and as such dropped in further analysis, subsequently treatments
main effects are presented. The microbial count data were log
transformed and along with digestibility subjected to one-way anova
using the GLM procedures of SAS.
[0707] Results
TABLE-US-00053 TABLE 27 Effects of xylanase and .beta.-glucanase
without or with bacillus strains based direct fed microbial on
growth performance in growing finishing pigs Initial Final Daily
Feed Feed Treat- body body gain, intake, conversion ments weight,
kg weight, kg grams/day grams/day efficiency, g/g 1 17.4 50.6b
719.4b 1411.1a 1.967 2 17.5 51.7ab 743.9ab 1431.3ab 1.942 3 17.4
52.6a 764.1a 1471.5a 1.922 Std. err. 0.35 0.81 14.00 19.20 0.041
N.B. Different letters following the values show statistical
differences (P .ltoreq. 0.10) between values in that column
TABLE-US-00054 TABLE 28 Effects of xylanase and .beta.-glucanase
without or with bacillus strains based direct fed microbial on dry
matter, nitrogen, fibre and energy digestibility (%) in growing
finishing pigs Treatments Dry matter Nitrogen Acid detergent fibre
Energy 1 80.4b 77.4b 44.2b 79.3b 2 80.8b 77.8b 44.6b 78.6b 3 82.0a
79.4a 56.1a 80.5a Std. err. 0.41 0.51 1.65 0.47 N.B. Different
letters following the values show statistical differences (P
.ltoreq. 0.10) between values in that column
[0708] A combination of xylanase, .beta.-glucanase and a direct fed
microbial containing either bacillus improved growing pig growth
performance and utilization of dietary nutrients and energy
compared to either, the control or enzyme only (Tables 27 &
28). Three way combinations were also seen to result in more fibre
degradation and promoted proliferation of lactobacillus bacteria in
the gut (FIG. 1).
Example 7: Effects of Xylanase, Other Fibre Degrading Enzymes and
Direct Fed Microbials on Short Chain Fatty Acids Production in
Swine Hind Gut
[0709] Composition of the Experimental Diets Used in Example 7
TABLE-US-00055 TABLE 29 Diet composition of growing pig feed (20-60
kg body weight) (% as fed) Diet I Diet II Corn 45.7 9.50 Wheat --
25.3 Barley -- 25.0 corn DDGS 25.0 10.0 Corn germ meal 15.0 --
Wheat middlings/rice bran -- 7.00 Soybean Meal 10.0 10.0 Canola
Meal -- 9.00 Fat 0.56 1.23 Molasses -- -- L-Lysine HCl 0.59 0.47
DL-methionine 0.02 0.02 L-threonine 0.13 0.09 L-tryptophan -- 0.01
Salt 0.46 0.54 Limestone 1.16 0.63 Dicalcium Phosphate 0.39 1.12
Vitamin and mineral premix 1.00 0.10 Calculated chemical
concentration Crude protein, % 19.1 18.3 Digestible energy, MJ/kg
13.8 13.6 Digestible lysine, % 1.03 0.98 Calcium, % 0.66 0.66
Digestible phosphorous, % 0.31 0.31 Neutral detergent fibre, % 23.8
21.8 Dry matter, % 89.7 90.8
[0710] Materials and Methods
[0711] In order to establish a swine hindgut model, a method was
adapted from (Boisen and Fernandez 1997, Animal Feed Science and
Technology 68: 277-286 the teaching of which is incorporated herein
by reference) to generate swine ileal effluent in vitro. In brief,
1.35 kg of complete mash feed (corn and wheat based, details see
table 29) was combined with 3.00 L of phosphate buffer (0.1 M, pH
6) and 1.20 L of 0.2 M HCl in a 3 gallon bucket with a re-sealable
lid. The pH was adjusted to 2 using 10 M HCl or NaOH. Then 120 mL
of a pre-prepared Pepsin solution (250 mg of Pepsin (Sigma-Aldrich,
Inc., St. Louis, Mo.) per mL of water) was added. The bucket was
sealed and incubated at 39.degree. C. for 2 hours with shaking in
order to simulate stomach digestion. For small intestine digestion
simulation, 1.20 L phosphate buffer (0.2 M, pH 6.8) and 600 mL of
0.6 M NaOH were added to the solution and the pH adjusted to 6.8
using 10 M NaOH or HCl as before. After neutralization, 120 mL of
pre-prepared pancreatin solution (1000 mg Pancreatin
(Sigma-Aldrich) per mL of water) were added, the bucket sealed and
incubated at 39.degree. C. for 4 hours with shaking. Following the
incubation, the liquid was filtered off using a double layered and
twice folded in half brew bag (Jumbo Nylon Coarse, LD Carlson
Company, Kent, Ohio). The remaining slurry was homogenized and
divided into portions of 128 g, each weighed into separate 250 mL
Pyrex bottles. The bottles were subsequently stored at -20.degree.
C. As inoculant for large bowl microbiota, cecal content was
collected from 12 grower pigs. Contents were homogenized, mixed
with 10% glycerol and 14 g aliquots weighed into 15 mL conicals.
Conicals were then sealed and stored at -80.degree. C.
[0712] Swine hindgut simulation experiments were performed in
duplicate runs, each with 1 control and 3 treatments (Table 30).
Each treatment was tested in triplicate. For each in vitro swine
hindgut fermentation trial, a total of 24 Pyrex bottles with
simulated ileal effluent and one 15 mL conical with cecal content
were used. Bottles were thawed overnight and 240 mL sterile 0.1 M
phosphate buffer solution (pH 6) with 4 g/L mucin (Sigma-Aldrich)
added to each bottle, similar to methods described in (Christensen
et al. 1999, Journal of the Science of Food and Agriculture 79,
755-762) and Aristoteli and Willcox, 2003, Infection and immunity
71: 5565-5575) the teaching of these documents being incorporated
herein by reference. The inoculant was thawed for 30 minutes at
room temperature while Pyrex bottles were pre-warmed at 39.degree.
C. for 30 minutes in a shaking water bath, then treatments in 1 mL
1% peptone solution and 450 .mu.L 0.1 M phosphate buffer (see table
30) were added.
TABLE-US-00056 TABLE 30 Treatments tested for swine in vitro
hindgut fermentation* Treatment 3 Control Treatment 1 Treatment 2
Treatment Basal Control with Treatment 1 with 2 + direct- diet
Xylanase fibre degrading fed microbial Experiment only .sup.#
enzyme .sup..dagger. enzyme .sup..dagger-dbl. (DFM) .sup.+ 1 CC NGX
Accel. P169 2 CC NGX Accel. Bacillus 3 CW Y5 Accel. P169 4 CW Y5
Axtra .RTM. XB Bacillus 5 CW Y5 Accel. Bacillus *enzyme and
direct-fed microbial products were included at a rate similar to
500 g per metric ton in feed inclusion, each experiment was
performed in duplicate runs, treatments were measured in triplicate
in each run; .sup.# Basal diet is either corn control diet (CC) or
wheat control diet (CW), as described in table 29; .sup..dagger.
Xylanase is either Y5 (Danisco Xylanase an
endo-1,4-.beta.-D-xylanase (E.C. 3.2.1.8)) or NGX (FveXyn4 (an
endo-1,4-.beta.-D-xylanase (E.C. 3.2.1.8)) shown as SEQ ID No. 3
herein (also described in PCT/CN2012/079650 which is incorporated
herein by reference), Danisco Animal Nutrition) with a guaranteed
activity of 4000 XU/kg of feed; .sup..dagger-dbl. Fibre degrading
enzyme is either Accel. (Accelerase Trio, ACCELLERASE .RTM. TRIO
.TM. enzyme complex contains a combination of multiple enzyme
activities including .beta.-glucanases (200 CMC U/kg), xylanases
(e.g. endoxylanases, endo-1,4-.beta.-D-xylanase (E.C. 3.2.1.8))
(>1200 ABX U/kg) and .beta.-glucosidases (>800 pNPG U/kg)
(DuPont Industrial Bioscences) enzyme mix or Axtra .RTM. XB
.beta.-glucanase with a guaranteed activity of 360 BGU of
.beta.-glucanase/kg of feed. .sup.+ Direct-fed microbial is either
Bacillus based (equal proportions of strains AGTP BS918 NRRL
B-50508, AGTP BS1013 NRRL B-50509 and AGTP BS3BP5 NRRL B-50510)
with a guaranteed activity of 3.0 .times. 10.sup.8 cfu per gram of
product, or Propionibacterium acidipropionici P169 PTA-5271
Omni-Bos .RTM. P169 with a guaranteed activity of 2.1 .times.
10.sup.9 cfu per gram of product.
[0713] Bottles were flushed with CO.sub.2 gas for 30 seconds while
250 .mu.L of cecal inoculant were added (based on Coles et al.
2005, Animal Feed Science and Technology 123: 421-444 the teaching
of which is incorporated herein by reference) and a 10 mL baseline
sample was collected, baseline pH determined and sample stored at
-20.degree. C. Bottles were capped, gently mixed and placed into a
shaking water bath at 39.degree. C. and 160 rpm. After 12 h,
another 10 mL sample was collected, pH determined and sample stored
at -20.degree. C. For volatile fatty acid (VFA) quantification by
high-performance liquid chromatography (HPLC) samples were thawed
and centrifuged at 16.1 rad for 20 minutes, and the supernatant
filtered through a 0.22 .mu.m mixed cellulose ester membrane
(Milex-GS, EMD Millipore Corp., Billerica, Mass.). Of the filtrate,
20 .mu.L was injected into a Waters Alliance 2695 Separations
Module (Waters Corp., Milford, Mass.) equipped with a Shodex SH-G
guard column (Waters) and 300.times.7.8 mm Aminex HPX-87H column
(Biorad Laboratories, Inc., Hercules, Calif.). An isocratic method
was applied with a mobile phase consisting of 16.8 mM phosphoric
acid in water/acetonitrile (98:2, v/v) at 0.525 mL/min flow rate
and 35.degree. C. column temperature. Volatile fatty acids were
detected using a Waters 2996 photo diode array (PDA) detector
(Waters) at 211 nm absorption. Instrument control, data
acquisition, and data processing were achieved with Waters Empower
3 software (Waters). Volatile fatty acids were quantified using
standard curves generated from high grade (.gtoreq.99.9%) reagents
(Sigma Aldrich, St. Louis, Mo.). Linear dilutions of standards in
16.8 mM phosphoric acid in water/acetonitrile (98:2, v/v) were
prepared at 6 concentrations ranging from 0.05% to 2.0%.
Concentration of acetic acid, propionic acid, butyric acid,
iso-butyric acid, valeric acid, iso-valeric acid (the sum of which
is presented as total VFA) and lactic acid were determined.
Statistical analysis for each experiment was performed as one-way
ANOVA blocked by run using GLM procedure of SPSS (version 17, SPSS
Inc., Chicago, Ill.). Significance was declared for P.ltoreq.0.10,
treatment means were separated using Duncan's multiple range
test.
[0714] Results
[0715] In wheat based diets, a significant increase in total VFA
and lactic acid production after 12 h of swine hindgut simulation
was observed when NGX xylanase, Accelerase Trio fibre degrading
enzyme mix and a DFM were added and compared to control without
supplementation (Table 31, experiment 1 and 2). Usage of
Propionibacterium acidipropionici P169 based DFM further
significantly increased propionate levels and had a greater
acidification of simulated colonic content in the combination
treatment compared to the control (Table 31, experiment 1). In corn
based diets, the combination treatment of Y5 xylanase, Accelerase
Trio fibre degrading enzyme and DFM significantly increased
butyrate levels compared to control after 12 h simulated swine
hindgut fermentation, with an additional increase of total VFA when
Propionibacterium acidipropionici P169 based DFM was used (Table
31, experiment 3 and 5). Replacement of Accelerase Trio enzyme mix
with Axtra.RTM. XB .beta.-glucanase and usage of Bacillus based DFM
in corn diet with Y5 resulted in significant increase of total VFA
and lactate compared to control treatment (Table 31, experiment
4).
TABLE-US-00057 TABLE 31 Mean abundance of Propionate, Butyrate,
total volatile fatty acids (VFA) and lactate (% as is), as well as
pH differences comparing to baseline samples after 12 h of swine
hindgut fermentation in vitro. Trt# Treatment Propionate Butyrate
Total VFA Lactate .DELTA. pH Experiment 1 1 Corn control (CC)
0.010.sup.b 0.457.sup.b 1.193.sup.b 2.452.sup.c 2 CC + NGX
0.010.sup.b 0.501.sup.b 1.254.sup.ab 2.462.sup.bc 3 CC + NGX +
Accel. 0.022.sup.ab NS 0.624.sup.ab 1.360.sup.ab 2.472.sup.ab 4 CC
+ NGX + Accel. + P169 0.056.sup.a 0.865.sup.a 1.595.sup.a
2.485.sup.a SEM 0.008 0.073 0.086 0.005 Experiment 2 1 Corn control
(CC) 0.531.sup.b 1.341.sup.b 2 CC + NGX 0.605.sup.ab 1.433.sup.b 3
CC + NGX + Accel. NS NS 0.607.sup.ab 1.406.sup.b NS 4 CC + NGX +
Accel. + 0.773.sup.a 1.681.sup.a Bacillus SEM 0.049 0.087
Experiment 3 1 Wheat control (CW) 0.145.sup.b 0.685.sup.b 2 CW + Y5
0.154.sup.ab 0.706.sup.ab 3 CW + Y5 + Accel. NS 0.155.sup.ab
0.713.sup.ab NS NS 4 CW + Y5 + Accel. + P169 0.163.sup.a
0.731.sup.a SEM 0.006 0.011 Experiment 4 1 Wheat control (CW)
0.7720.sup.b 1.970.sup.b 2 CW + Y5 0.8258.sup.ab 1.993.sup.ab 3 CW
+ Y5 + Axtra .RTM. XB NS NS 0.7885.sup.ab 2.014.sup.ab NS 4 CW + Y5
+ Axtra .RTM. XB + 0.8600.sup.a 2.027.sup.a Bacillus SEM 0.031
0.021 Experiment 5 1 Wheat control (CW) 0.0627.sup.b 2 CW + Y5
0.0930.sup.ab 3 CW + Y5 + Accel. NS 0.1242.sup.ab NS NS NS 4 CW +
Y5 + Accel. + Bacillus 0.1728.sup.a SEM 0.031 .sup.a,bvalues with
differing superscripts within a column are significantly different
at P .ltoreq. 0.10; NS, not significant; SEM, standard error of the
mean; treatment details see Table 26
Example 8 Effects of Xylanase, Other Fibrolytic Enzymes and Direct
Fed Microbials on Swine Hindgut Fibre Degradation
[0716] Composition of the Experimental Diet Used in Example 8
TABLE-US-00058 TABLE 32 Diet composition of growing pig feed (20-60
kg body weight) (% as fed) Diet Corn 9.50 Wheat 25.0 Barley 25.0
corn DDGS 10.0 Corn germ meal -- Wheat middlings 7.00 Soybean Meal
10.0 Canola Meal 9.00 Fat 1.23 L-Lysine HCl 0.47 DL-methionine 0.02
L-threonine 0.09 L-tryptophan 0.01 Salt 0.54 Limestone 0.63
Dicalcium Phosphate 1.12 Vitamin and mineral premix 0.10 Inert
marker digestibility marker 0.30 Calculated chemical concentration
Crude protein, % 18.3 Digestible energy, MJ/kg 13.6 Digestible
lysine, % 0.98 Calcium, % 0.66 Digestible phosphorous, % 0.31
Neutral detergent fibre, % 21.8 Dry matter, % 90.8
[0717] To demonstrate disappearance of dry matter (DM) and
degradation of fibre, ileal effluents were generated and hindgut
fermentation set up as described in example 7. In brief, the wheat
based diet (CW, see Table 32) was used as control without any
treatment, as well as CW in addition with Y5 xylanase (Treatment
1), CW with Y5 and Accelerase Trio fibre degrading enzyme mix
(Treatment 2), CW with Y5, Accelerase in combination with a three
strain Bacillus direct-fed microbial (Treatment 3), details to
enzyme and DFM treatments see Table 33.
TABLE-US-00059 TABLE 33 Identification of Treatments* Control
Treatment 1 Treatment 2 Treatment 3 Basal Control with Treatment 1
with Treatment diet Xylanase fibre degrading 2 + direct- only
.sup.# enzyme .sup..dagger. enzyme .sup..dagger-dbl. fed microbial
(DFM) .sup.+ CW Y5 Accel. Bacillus *enzyme and direct-fed microbial
products were included at a rate similar to 500 g per metric ton in
feed inclusion, each experiment was performed in duplicate runs,
treatments were measured in triplicate in each run; .sup.# Basal
diet is a wheat control diet (CW), as described in table 32;
.sup..dagger. Xylanase is Y5 (Danisco Xylanase an
endo-1,4-.beta.-D-xylanase (E.C. 3.2.1.8)) with a guaranteed
activity of 4000 XU/kg of feed; .sup..dagger-dbl. Fibre degrading
enzyme is either Accel. (Accelerase Trio, ACCELLERASE .RTM. TRIO
.TM. enzyme complex contains a potent combination of multiple
enzyme activities including .beta.-glucanases (200 CMC U/kg),
xylanases (e.g. endoxylanases, endo-1,4-.beta.-D-xylanase (E.C.
3.2.1.8)) (>1200 ABX U/kg) and .beta.-glucosidases (>800 pNPG
U/kg) (DuPont Industrial Bioscences) the enzyme mix was dosed to
ensure a guaranteed activity of 360 BGU of .beta.-glucanase/kg of
feed. .sup.+ Direct-fed microbial is either Bacillus based (equal
proportions of strains AGTP BS918 NRRL B-50508, AGTP BS1013 NRRL
B-50509 and AGTP BS3BP5 NRRL B-50510) with a guaranteed activity of
3.0 .times. 10.sup.8 cfu per gram of product, or Propionibacterium
acidipropionici P169 PTA-5271 Omni-Bos .RTM. P169 with a guaranteed
activity of 2.1 .times. 10.sup.9 cfu per gram of product.
[0718] Treatment effects on DM and fibre disappearance. At 0 and 48
hours of the experiment, liquid was filtered off and remaining
solids were collected and send for approximate nutrient analysis of
dry matter (DM), acid and neutral detergent fibre (ADF and NDF,
respectively), the latter were generated on DM basis according to
methods described in (Association of Analytical Chemists (AOAC)
2007, 18.sup.th edition. AOAC, Washington, D.C.). Data was
calculated as percent disappearance, statistical analysis was
performed as one-way ANOVA blocked by run using GLM procedure of
SPSS (version 17, SPSS Inc., Chicago, Ill.). Significance was
declared for P.ltoreq.0.10, treatment means were separated using
Duncan's multiple range test.
[0719] Results
[0720] In the tested wheat based diet, the combination treatment
with Y5 Xylanase, Accelerase Trio fibre degrading enzyme mix and
three Bacillus based DFM had the greatest disappearance of DM, ADF
and NDF compared to CW without any enzyme and DFM supplementation
(Table 34).
TABLE-US-00060 TABLE 34 Percent disappearance of dry matter, acid
and neutral detergent fibre during 48 h swine hindgut fermentation
in vitro Trt# Treatment .DELTA. DM (%) .DELTA. ADF (%) .DELTA. NDF
(%) 1 Wheat control (CW) 3.95 .sup.b 2.31 .sup.b 4.27 .sup.b 2 CW +
Y5 3.97 .sup.b 3.26 .sup.ab 5.87 .sup.ab 3 CW + Y5 + Accel. 4.19
.sup.ab 3.46 .sup.ab 5.77 .sup.ab 4 CW + Y5 + Accel. + 4.57 .sup.a
3.66 .sup.a 7.37 .sup.a Bacillus SEM 0.17 .sup. 0.31 0.88 .sup.a, b
values with differing superscripts within a column are
significantly different at P .ltoreq. 0.10; SEM, standard error of
the mean; treatment details see Table 7.2.
[0721] All publications mentioned in the above specification are
herein incorporated by reference. Various modifications and
variations of the described methods and system of the present
invention will be apparent to those skilled in the art without
departing from the scope and spirit of the present invention.
Although the present invention has been described in connection
with specific preferred embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
described modes for carrying out the invention which are obvious to
those skilled in biochemistry and biotechnology or related fields
are intended to be within the scope of the following claims.
Sequence CWU 1
1
201328PRTArtificial SequenceXylanase sequence 1Met Lys Leu Ser Ser
Phe Leu Tyr Thr Ala Ser Leu Val Ala Ala Ile1 5 10 15Pro Thr Ala Ile
Glu Pro Arg Gln Ala Ala Asp Ser Ile Asn Lys Leu 20 25 30Ile Lys Asn
Lys Gly Lys Leu Tyr Tyr Gly Thr Ile Thr Asp Pro Asn 35 40 45Leu Leu
Gly Val Ala Lys Asp Thr Ala Ile Ile Lys Ala Asp Phe Gly 50 55 60Ala
Val Thr Pro Glu Asn Ser Gly Lys Trp Asp Ala Thr Glu Pro Ser65 70 75
80Gln Gly Lys Phe Asn Phe Gly Ser Phe Asp Gln Val Val Asn Phe Ala
85 90 95Gln Gln Asn Gly Leu Lys Val Arg Gly His Thr Leu Val Trp His
Ser 100 105 110Gln Leu Pro Gln Trp Val Lys Asn Ile Asn Asp Lys Ala
Thr Leu Thr 115 120 125Lys Val Ile Glu Asn His Val Thr Gln Val Val
Gly Arg Tyr Lys Gly 130 135 140Lys Ile Tyr Ala Trp Asp Val Val Asn
Glu Ile Phe Glu Trp Asp Gly145 150 155 160Thr Leu Arg Lys Asp Ser
His Phe Asn Asn Val Phe Gly Asn Asp Asp 165 170 175Tyr Val Gly Ile
Ala Phe Arg Ala Ala Arg Lys Ala Asp Pro Asn Ala 180 185 190Lys Leu
Tyr Ile Asn Asp Tyr Ser Leu Asp Ser Gly Ser Ala Ser Lys 195 200
205Val Thr Lys Gly Met Val Pro Ser Val Lys Lys Trp Leu Ser Gln Gly
210 215 220Val Pro Val Asp Gly Ile Gly Ser Gln Thr His Leu Asp Pro
Gly Ala225 230 235 240Ala Gly Gln Ile Gln Gly Ala Leu Thr Ala Leu
Ala Asn Ser Gly Val 245 250 255Lys Glu Val Ala Ile Thr Glu Leu Asp
Ile Arg Thr Ala Pro Ala Asn 260 265 270Asp Tyr Ala Thr Val Thr Lys
Ala Cys Leu Asn Val Pro Lys Cys Ile 275 280 285Gly Ile Thr Val Trp
Gly Val Ser Asp Lys Asn Ser Trp Arg Lys Glu 290 295 300His Asp Ser
Leu Leu Phe Asp Ala Asn Tyr Asn Pro Lys Pro Ala Tyr305 310 315
320Thr Ala Val Val Asn Ala Leu Arg 3252313PRTArtificial
SequenceXylanase sequence 2Ile Pro Thr Ala Ile Glu Pro Arg Gln Ala
Ala Asp Ser Ile Asn Lys1 5 10 15Leu Ile Lys Asn Lys Gly Lys Leu Tyr
Tyr Gly Thr Ile Thr Asp Pro 20 25 30Asn Leu Leu Gly Val Ala Lys Asp
Thr Ala Ile Ile Lys Ala Asp Phe 35 40 45Gly Ala Val Thr Pro Glu Asn
Ser Gly Lys Trp Asp Ala Thr Glu Pro 50 55 60Ser Gln Gly Lys Phe Asn
Phe Gly Ser Phe Asp Gln Val Val Asn Phe65 70 75 80Ala Gln Gln Asn
Gly Leu Lys Val Arg Gly His Thr Leu Val Trp His 85 90 95Ser Gln Leu
Pro Gln Trp Val Lys Asn Ile Asn Asp Lys Ala Thr Leu 100 105 110Thr
Lys Val Ile Glu Asn His Val Thr Gln Val Val Gly Arg Tyr Lys 115 120
125Gly Lys Ile Tyr Ala Trp Asp Val Val Asn Glu Ile Phe Glu Trp Asp
130 135 140Gly Thr Leu Arg Lys Asp Ser His Phe Asn Asn Val Phe Gly
Asn Asp145 150 155 160Asp Tyr Val Gly Ile Ala Phe Arg Ala Ala Arg
Lys Ala Asp Pro Asn 165 170 175Ala Lys Leu Tyr Ile Asn Asp Tyr Ser
Leu Asp Ser Gly Ser Ala Ser 180 185 190Lys Val Thr Lys Gly Met Val
Pro Ser Val Lys Lys Trp Leu Ser Gln 195 200 205Gly Val Pro Val Asp
Gly Ile Gly Ser Gln Thr His Leu Asp Pro Gly 210 215 220Ala Ala Gly
Gln Ile Gln Gly Ala Leu Thr Ala Leu Ala Asn Ser Gly225 230 235
240Val Lys Glu Val Ala Ile Thr Glu Leu Asp Ile Arg Thr Ala Pro Ala
245 250 255Asn Asp Tyr Ala Thr Val Thr Lys Ala Cys Leu Asn Val Pro
Lys Cys 260 265 270Ile Gly Ile Thr Val Trp Gly Val Ser Asp Lys Asn
Ser Trp Arg Lys 275 280 285Glu His Asp Ser Leu Leu Phe Asp Ala Asn
Tyr Asn Pro Lys Pro Ala 290 295 300Tyr Thr Ala Val Val Asn Ala Leu
Arg305 3103305PRTTrichoderma reesei 3Gln Ala Ala Asp Ser Ile Asn
Lys Leu Ile Lys Asn Lys Gly Lys Leu1 5 10 15Tyr Tyr Gly Thr Ile Thr
Asp Pro Asn Leu Leu Gly Val Ala Lys Asp 20 25 30Thr Ala Ile Ile Lys
Ala Asp Phe Gly Ala Val Thr Pro Glu Asn Ser 35 40 45Gly Lys Trp Asp
Ala Thr Glu Pro Ser Gln Gly Lys Phe Asn Phe Gly 50 55 60Ser Phe Asp
Gln Val Val Asn Phe Ala Gln Gln Asn Gly Leu Lys Val65 70 75 80Arg
Gly His Thr Leu Val Trp His Ser Gln Leu Pro Gln Trp Val Lys 85 90
95Asn Ile Asn Asp Lys Ala Thr Leu Thr Lys Val Ile Glu Asn His Val
100 105 110Thr Gln Val Val Gly Arg Tyr Lys Gly Lys Ile Tyr Ala Trp
Asp Val 115 120 125Val Asn Glu Ile Phe Glu Trp Asp Gly Thr Leu Arg
Lys Asp Ser His 130 135 140Phe Asn Asn Val Phe Gly Asn Asp Asp Tyr
Val Gly Ile Ala Phe Arg145 150 155 160Ala Ala Arg Lys Ala Asp Pro
Asn Ala Lys Leu Tyr Ile Asn Asp Tyr 165 170 175Ser Leu Asp Ser Gly
Ser Ala Ser Lys Val Thr Lys Gly Met Val Pro 180 185 190Ser Val Lys
Lys Trp Leu Ser Gln Gly Val Pro Val Asp Gly Ile Gly 195 200 205Ser
Gln Thr His Leu Asp Pro Gly Ala Ala Gly Gln Ile Gln Gly Ala 210 215
220Leu Thr Ala Leu Ala Asn Ser Gly Val Lys Glu Val Ala Ile Thr
Glu225 230 235 240Leu Asp Ile Arg Thr Ala Pro Ala Asn Asp Tyr Ala
Thr Val Thr Lys 245 250 255Ala Cys Leu Asn Val Pro Lys Cys Ile Gly
Ile Thr Val Trp Gly Val 260 265 270Ser Asp Lys Asn Ser Trp Arg Lys
Glu His Asp Ser Leu Leu Phe Asp 275 280 285Ala Asn Tyr Asn Pro Lys
Pro Ala Tyr Thr Ala Val Val Asn Ala Leu 290 295
300Arg3054328PRTArtificial SequenceXylanase sequence 4Met Lys Leu
Ser Ser Phe Leu Tyr Thr Ala Ser Leu Val Ala Ala Ile1 5 10 15Pro Thr
Ala Ile Glu Pro Arg Gln Ala Ser Asp Ser Ile Asn Lys Leu 20 25 30Ile
Lys Asn Lys Gly Lys Leu Tyr Tyr Gly Thr Ile Thr Asp Pro Asn 35 40
45Leu Leu Gly Val Ala Lys Asp Thr Ala Ile Ile Lys Ala Asp Phe Gly
50 55 60Ala Val Thr Pro Glu Asn Ser Gly Lys Trp Asp Ala Thr Glu Pro
Ser65 70 75 80Gln Gly Lys Phe Asn Phe Gly Ser Phe Asp Gln Val Val
Asn Phe Ala 85 90 95Gln Gln Asn Gly Leu Lys Val Arg Gly His Thr Leu
Val Trp His Ser 100 105 110Gln Leu Pro Gln Trp Val Lys Asn Ile Asn
Asp Lys Ala Thr Leu Thr 115 120 125Lys Val Ile Glu Asn His Val Thr
Asn Val Val Gly Arg Tyr Lys Gly 130 135 140Lys Ile Tyr Ala Trp Asp
Val Val Asn Glu Ile Phe Asp Trp Asp Gly145 150 155 160Thr Leu Arg
Lys Asp Ser His Phe Asn Asn Val Phe Gly Asn Asp Asp 165 170 175Tyr
Val Gly Ile Ala Phe Arg Ala Ala Arg Lys Ala Asp Pro Asn Ala 180 185
190Lys Leu Tyr Ile Asn Asp Tyr Ser Leu Asp Ser Gly Ser Ala Ser Lys
195 200 205Val Thr Lys Gly Met Val Pro Ser Val Lys Lys Trp Leu Ser
Gln Gly 210 215 220Val Pro Val Asp Gly Ile Gly Ser Gln Thr His Leu
Asp Pro Gly Ala225 230 235 240Ala Gly Gln Ile Gln Gly Ala Leu Thr
Ala Leu Ala Asn Ser Gly Val 245 250 255Lys Glu Val Ala Ile Thr Glu
Leu Asp Ile Arg Thr Ala Pro Ala Asn 260 265 270Asp Tyr Ala Thr Val
Thr Lys Ala Cys Leu Asn Val Pro Lys Cys Ile 275 280 285Gly Ile Thr
Val Trp Gly Val Ser Asp Lys Asn Ser Trp Arg Lys Glu 290 295 300His
Asp Ser Leu Leu Phe Asp Ala Asn Tyr Asn Pro Lys Ala Ala Tyr305 310
315 320Thr Ala Val Val Asn Ala Leu Arg 3255313PRTArtificial
SequenceXylanase sequence 5Ile Pro Thr Ala Ile Glu Pro Arg Gln Ala
Ser Asp Ser Ile Asn Lys1 5 10 15Leu Ile Lys Asn Lys Gly Lys Leu Tyr
Tyr Gly Thr Ile Thr Asp Pro 20 25 30Asn Leu Leu Gly Val Ala Lys Asp
Thr Ala Ile Ile Lys Ala Asp Phe 35 40 45Gly Ala Val Thr Pro Glu Asn
Ser Gly Lys Trp Asp Ala Thr Glu Pro 50 55 60Ser Gln Gly Lys Phe Asn
Phe Gly Ser Phe Asp Gln Val Val Asn Phe65 70 75 80Ala Gln Gln Asn
Gly Leu Lys Val Arg Gly His Thr Leu Val Trp His 85 90 95Ser Gln Leu
Pro Gln Trp Val Lys Asn Ile Asn Asp Lys Ala Thr Leu 100 105 110Thr
Lys Val Ile Glu Asn His Val Thr Asn Val Val Gly Arg Tyr Lys 115 120
125Gly Lys Ile Tyr Ala Trp Asp Val Val Asn Glu Ile Phe Asp Trp Asp
130 135 140Gly Thr Leu Arg Lys Asp Ser His Phe Asn Asn Val Phe Gly
Asn Asp145 150 155 160Asp Tyr Val Gly Ile Ala Phe Arg Ala Ala Arg
Lys Ala Asp Pro Asn 165 170 175Ala Lys Leu Tyr Ile Asn Asp Tyr Ser
Leu Asp Ser Gly Ser Ala Ser 180 185 190Lys Val Thr Lys Gly Met Val
Pro Ser Val Lys Lys Trp Leu Ser Gln 195 200 205Gly Val Pro Val Asp
Gly Ile Gly Ser Gln Thr His Leu Asp Pro Gly 210 215 220Ala Ala Gly
Gln Ile Gln Gly Ala Leu Thr Ala Leu Ala Asn Ser Gly225 230 235
240Val Lys Glu Val Ala Ile Thr Glu Leu Asp Ile Arg Thr Ala Pro Ala
245 250 255Asn Asp Tyr Ala Thr Val Thr Lys Ala Cys Leu Asn Val Pro
Lys Cys 260 265 270Ile Gly Ile Thr Val Trp Gly Val Ser Asp Lys Asn
Ser Trp Arg Lys 275 280 285Glu His Asp Ser Leu Leu Phe Asp Ala Asn
Tyr Asn Pro Lys Ala Ala 290 295 300Tyr Thr Ala Val Val Asn Ala Leu
Arg305 3106305PRTArtificial SequenceXylanase sequence 6Gln Ala Ser
Asp Ser Ile Asn Lys Leu Ile Lys Asn Lys Gly Lys Leu1 5 10 15Tyr Tyr
Gly Thr Ile Thr Asp Pro Asn Leu Leu Gly Val Ala Lys Asp 20 25 30Thr
Ala Ile Ile Lys Ala Asp Phe Gly Ala Val Thr Pro Glu Asn Ser 35 40
45Gly Lys Trp Asp Ala Thr Glu Pro Ser Gln Gly Lys Phe Asn Phe Gly
50 55 60Ser Phe Asp Gln Val Val Asn Phe Ala Gln Gln Asn Gly Leu Lys
Val65 70 75 80Arg Gly His Thr Leu Val Trp His Ser Gln Leu Pro Gln
Trp Val Lys 85 90 95Asn Ile Asn Asp Lys Ala Thr Leu Thr Lys Val Ile
Glu Asn His Val 100 105 110Thr Asn Val Val Gly Arg Tyr Lys Gly Lys
Ile Tyr Ala Trp Asp Val 115 120 125Val Asn Glu Ile Phe Asp Trp Asp
Gly Thr Leu Arg Lys Asp Ser His 130 135 140Phe Asn Asn Val Phe Gly
Asn Asp Asp Tyr Val Gly Ile Ala Phe Arg145 150 155 160Ala Ala Arg
Lys Ala Asp Pro Asn Ala Lys Leu Tyr Ile Asn Asp Tyr 165 170 175Ser
Leu Asp Ser Gly Ser Ala Ser Lys Val Thr Lys Gly Met Val Pro 180 185
190Ser Val Lys Lys Trp Leu Ser Gln Gly Val Pro Val Asp Gly Ile Gly
195 200 205Ser Gln Thr His Leu Asp Pro Gly Ala Ala Gly Gln Ile Gln
Gly Ala 210 215 220Leu Thr Ala Leu Ala Asn Ser Gly Val Lys Glu Val
Ala Ile Thr Glu225 230 235 240Leu Asp Ile Arg Thr Ala Pro Ala Asn
Asp Tyr Ala Thr Val Thr Lys 245 250 255Ala Cys Leu Asn Val Pro Lys
Cys Ile Gly Ile Thr Val Trp Gly Val 260 265 270Ser Asp Lys Asn Ser
Trp Arg Lys Glu His Asp Ser Leu Leu Phe Asp 275 280 285Ala Asn Tyr
Asn Pro Lys Ala Ala Tyr Thr Ala Val Val Asn Ala Leu 290 295
300Arg3057229PRTArtificial SequenceXylanase sequence 7Met Val Ser
Phe Lys Tyr Leu Phe Leu Ala Ala Ser Ala Leu Gly Ala1 5 10 15Leu Ala
Ala Pro Val Glu Val Glu Glu Ser Ser Trp Phe Asn Glu Thr 20 25 30Ala
Leu His Glu Phe Ala Glu Arg Ala Gly Thr Pro Ser Ser Thr Gly 35 40
45Trp Asn Asn Gly Tyr Tyr Tyr Ser Phe Trp Thr Asp Asn Gly Gly Thr
50 55 60Val Asn Tyr Gln Asn Gly Asn Gly Gly Ser Tyr Ser Val Gln Trp
Lys65 70 75 80Asp Thr Gly Asn Phe Val Gly Gly Lys Gly Trp Asn Pro
Gly Ser Ala 85 90 95Arg Thr Ile Asn Tyr Ser Gly Ser Phe Asn Pro Ser
Gly Asn Ala Tyr 100 105 110Leu Thr Val Tyr Gly Trp Thr Thr Asn Pro
Leu Val Glu Tyr Tyr Ile 115 120 125Val Glu Asn Tyr Gly Thr Tyr Asn
Pro Gly Asn Gly Gly Thr Tyr Arg 130 135 140Gly Ser Val Tyr Ser Asp
Gly Ala Asn Tyr Asn Ile Tyr Thr Ala Thr145 150 155 160Arg Tyr Asn
Ala Pro Ser Ile Glu Gly Asp Lys Thr Phe Thr Gln Tyr 165 170 175Trp
Ser Val Arg Gln Ser Lys Arg Thr Gly Gly Thr Val Thr Thr Ala 180 185
190Asn His Phe Asn Ala Trp Ala Gln Leu Gly Met Ser Leu Gly Thr His
195 200 205Asn Tyr Gln Ile Val Ala Thr Glu Gly Tyr Gln Ser Ser Gly
Ser Ser 210 215 220Ser Ile Thr Val Tyr2258211PRTArtificial
SequenceXylanase sequence 8Ala Pro Val Glu Val Glu Glu Ser Ser Trp
Phe Asn Glu Thr Ala Leu1 5 10 15His Glu Phe Ala Glu Arg Ala Gly Thr
Pro Ser Ser Thr Gly Trp Asn 20 25 30Asn Gly Tyr Tyr Tyr Ser Phe Trp
Thr Asp Asn Gly Gly Thr Val Asn 35 40 45Tyr Gln Asn Gly Asn Gly Gly
Ser Tyr Ser Val Gln Trp Lys Asp Thr 50 55 60Gly Asn Phe Val Gly Gly
Lys Gly Trp Asn Pro Gly Ser Ala Arg Thr65 70 75 80Ile Asn Tyr Ser
Gly Ser Phe Asn Pro Ser Gly Asn Ala Tyr Leu Thr 85 90 95Val Tyr Gly
Trp Thr Thr Asn Pro Leu Val Glu Tyr Tyr Ile Val Glu 100 105 110Asn
Tyr Gly Thr Tyr Asn Pro Gly Asn Gly Gly Thr Tyr Arg Gly Ser 115 120
125Val Tyr Ser Asp Gly Ala Asn Tyr Asn Ile Tyr Thr Ala Thr Arg Tyr
130 135 140Asn Ala Pro Ser Ile Glu Gly Asp Lys Thr Phe Thr Gln Tyr
Trp Ser145 150 155 160Val Arg Gln Ser Lys Arg Thr Gly Gly Thr Val
Thr Thr Ala Asn His 165 170 175Phe Asn Ala Trp Ala Gln Leu Gly Met
Ser Leu Gly Thr His Asn Tyr 180 185 190Gln Ile Val Ala Thr Glu Gly
Tyr Gln Ser Ser Gly Ser Ser Ser Ile 195 200 205Thr Val Tyr
2109189PRTArtificial SequenceXylanase sequence 9Ala Gly Thr Pro Ser
Ser Thr Gly Trp Asn Asn Gly Tyr Tyr Tyr Ser1 5 10 15Phe Trp Thr Asp
Asn Gly Gly Thr Val Asn Tyr Gln Asn Gly Asn Gly 20 25 30Gly Ser Tyr
Ser Val Gln Trp Lys Asp Thr Gly Asn Phe Val Gly Gly 35 40 45Lys Gly
Trp Asn Pro Gly Ser Ala Arg Thr Ile Asn Tyr Ser Gly Ser 50 55 60Phe
Asn Pro Ser Gly Asn Ala Tyr Leu Thr Val Tyr Gly Trp Thr Thr65 70 75
80Asn Pro Leu Val Glu Tyr Tyr Ile Val Glu Asn Tyr Gly Thr Tyr Asn
85 90 95Pro Gly Asn Gly Gly Thr Tyr Arg Gly Ser Val
Tyr Ser Asp Gly Ala 100 105 110Asn Tyr Asn Ile Tyr Thr Ala Thr Arg
Tyr Asn Ala Pro Ser Ile Glu 115 120 125Gly Asp Lys Thr Phe Thr Gln
Tyr Trp Ser Val Arg Gln Ser Lys Arg 130 135 140Thr Gly Gly Thr Val
Thr Thr Ala Asn His Phe Asn Ala Trp Ala Gln145 150 155 160Leu Gly
Met Ser Leu Gly Thr His Asn Tyr Gln Ile Val Ala Thr Glu 165 170
175Gly Tyr Gln Ser Ser Gly Ser Ser Ser Ile Thr Val Tyr 180
18510232PRTArtificial SequenceXylanase sequence 10Met Val Ser Phe
Thr Ser Leu Leu Ala Ala Val Ser Ala Val Thr Gly1 5 10 15Val Met Ala
Leu Pro Ser Ala Gln Pro Val Asp Gly Met Ser Val Val 20 25 30Glu Arg
Asp Pro Pro Thr Asn Val Leu Asp Lys Arg Thr Gln Pro Thr 35 40 45Thr
Gly Thr Ser Gly Gly Tyr Tyr Phe Ser Phe Trp Thr Asp Thr Pro 50 55
60Asn Ser Val Thr Tyr Thr Asn Gly Asn Gly Gly Gln Phe Ser Met Gln65
70 75 80Trp Ser Gly Asn Gly Asn His Val Gly Gly Lys Gly Trp Met Pro
Gly 85 90 95Thr Ser Arg Thr Ile Lys Tyr Ser Gly Ser Tyr Asn Pro Asn
Gly Asn 100 105 110Ser Tyr Leu Ala Val Tyr Gly Trp Thr Arg Asn Pro
Leu Ile Glu Tyr 115 120 125Tyr Ile Val Glu Asn Phe Gly Thr Tyr Asn
Pro Ser Ser Gly Gly Gln 130 135 140Lys Lys Gly Glu Val Asn Val Asp
Gly Ser Val Tyr Asp Ile Tyr Val145 150 155 160Ser Thr Arg Val Asn
Ala Pro Ser Ile Asp Gly Asn Lys Thr Phe Gln 165 170 175Gln Tyr Trp
Ser Val Arg Arg Asn Lys Arg Ser Ser Gly Ser Val Asn 180 185 190Thr
Gly Ala His Phe Gln Ala Trp Lys Asn Val Gly Leu Asn Leu Gly 195 200
205Thr His Asp Tyr Gln Ile Leu Ala Val Glu Gly Tyr Tyr Ser Ser Gly
210 215 220Ser Ala Ser Met Thr Val Ser Gln225 23011213PRTArtificial
SequenceXylanase sequence 11Leu Pro Ser Ala Gln Pro Val Asp Gly Met
Ser Val Val Glu Arg Asp1 5 10 15Pro Pro Thr Asn Val Leu Asp Lys Arg
Thr Gln Pro Thr Thr Gly Thr 20 25 30Ser Gly Gly Tyr Tyr Phe Ser Phe
Trp Thr Asp Thr Pro Asn Ser Val 35 40 45Thr Tyr Thr Asn Gly Asn Gly
Gly Gln Phe Ser Met Gln Trp Ser Gly 50 55 60Asn Gly Asn His Val Gly
Gly Lys Gly Trp Met Pro Gly Thr Ser Arg65 70 75 80Thr Ile Lys Tyr
Ser Gly Ser Tyr Asn Pro Asn Gly Asn Ser Tyr Leu 85 90 95Ala Val Tyr
Gly Trp Thr Arg Asn Pro Leu Ile Glu Tyr Tyr Ile Val 100 105 110Glu
Asn Phe Gly Thr Tyr Asn Pro Ser Ser Gly Gly Gln Lys Lys Gly 115 120
125Glu Val Asn Val Asp Gly Ser Val Tyr Asp Ile Tyr Val Ser Thr Arg
130 135 140Val Asn Ala Pro Ser Ile Asp Gly Asn Lys Thr Phe Gln Gln
Tyr Trp145 150 155 160Ser Val Arg Arg Asn Lys Arg Ser Ser Gly Ser
Val Asn Thr Gly Ala 165 170 175His Phe Gln Ala Trp Lys Asn Val Gly
Leu Asn Leu Gly Thr His Asp 180 185 190Tyr Gln Ile Leu Ala Val Glu
Gly Tyr Tyr Ser Ser Gly Ser Ala Ser 195 200 205Met Thr Val Ser Gln
21012188PRTArtificial SequenceXylanase sequence 12Thr Gln Pro Thr
Thr Gly Thr Ser Gly Gly Tyr Tyr Phe Ser Phe Trp1 5 10 15Thr Asp Thr
Pro Asn Ser Val Thr Tyr Thr Asn Gly Asn Gly Gly Gln 20 25 30Phe Ser
Met Gln Trp Ser Gly Asn Gly Asn His Val Gly Gly Lys Gly 35 40 45Trp
Met Pro Gly Thr Ser Arg Thr Ile Lys Tyr Ser Gly Ser Tyr Asn 50 55
60Pro Asn Gly Asn Ser Tyr Leu Ala Val Tyr Gly Trp Thr Arg Asn Pro65
70 75 80Leu Ile Glu Tyr Tyr Ile Val Glu Asn Phe Gly Thr Tyr Asn Pro
Ser 85 90 95Ser Gly Gly Gln Lys Lys Gly Glu Val Asn Val Asp Gly Ser
Val Tyr 100 105 110Asp Ile Tyr Val Ser Thr Arg Val Asn Ala Pro Ser
Ile Asp Gly Asn 115 120 125Lys Thr Phe Gln Gln Tyr Trp Ser Val Arg
Arg Asn Lys Arg Ser Ser 130 135 140Gly Ser Val Asn Thr Gly Ala His
Phe Gln Ala Trp Lys Asn Val Gly145 150 155 160Leu Asn Leu Gly Thr
His Asp Tyr Gln Ile Leu Ala Val Glu Gly Tyr 165 170 175Tyr Ser Ser
Gly Ser Ala Ser Met Thr Val Ser Gln 180 1851319DNAArtificial
SequencePrimer 28Fmisc_feature(11)..(11)n is a, c, g, or t
13gagtttgatc ntggctcag 191418DNAArtificial SequencePrimer
519Rmisc_feature(3)..(3)n is a, c, g, or tmisc_feature(8)..(8)n is
a, c, g, or t 14gtnttacngc ggckgctg 181510DNAArtificial
SequencePrimer 1 15ggtgcgggaa 101610DNAArtificial SequencePrimer 2
16gtttcgctcc 101710DNAArtificial SequencePrimer 3 17gtagacccgt
101810DNAArtificial SequencePrimer 4 18aagagcccgt
101910DNAArtificial SequencePrimer 5 19aacgcgcaac
102010DNAArtificial SequencePrimer 6 20cccgtcagca 10
* * * * *
References