U.S. patent application number 13/072131 was filed with the patent office on 2011-07-21 for enzymes for reduced immunological stress.
This patent application is currently assigned to CHEMGEN CORPORATION. Invention is credited to David M. Anderson, Humg-Yu Hsiao, Lin Liu.
Application Number | 20110177195 13/072131 |
Document ID | / |
Family ID | 38218427 |
Filed Date | 2011-07-21 |
United States Patent
Application |
20110177195 |
Kind Code |
A1 |
Anderson; David M. ; et
al. |
July 21, 2011 |
ENZYMES FOR REDUCED IMMUNOLOGICAL STRESS
Abstract
Compositions suitable for oral administration to an animal
comprising at least one immune stress-reducing enzyme in an amount
effective to decrease the level of positive acute phase protein in
an animal, increase the level of negative acute phase protein in an
animal, and/or improve animal growth performance is provided, as
are methods using such compositions. The compositions include
animal feed compositions, liquid compositions other than animal
feed, and solid compositions other than animal feed.
Inventors: |
Anderson; David M.;
(Rockville, MD) ; Hsiao; Humg-Yu; (Rockville,
MD) ; Liu; Lin; (Rockville, MD) |
Assignee: |
CHEMGEN CORPORATION
|
Family ID: |
38218427 |
Appl. No.: |
13/072131 |
Filed: |
March 25, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11610572 |
Dec 14, 2006 |
7914782 |
|
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13072131 |
|
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60750339 |
Dec 15, 2005 |
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Current U.S.
Class: |
426/2 ;
426/61 |
Current CPC
Class: |
A23K 50/75 20160501;
A61P 1/14 20180101; A61P 31/12 20180101; A61K 38/47 20130101; A61P
37/06 20180101; A61P 31/00 20180101; A61P 37/02 20180101; C12N
9/2488 20130101; C12Y 302/01039 20130101; C12Y 302/01006 20130101;
A61K 9/0056 20130101; A61P 31/04 20180101; C12N 9/244 20130101;
A23K 20/189 20160501; A61P 37/00 20180101; C12Y 302/01078 20130101;
A61P 33/00 20180101; C12N 9/2494 20130101; A61P 31/10 20180101 |
Class at
Publication: |
426/2 ;
426/61 |
International
Class: |
A23K 1/165 20060101
A23K001/165; A23K 1/18 20060101 A23K001/18 |
Claims
1-30. (canceled)
31. A composition suitable for oral administration to an animal
comprising an immune stress-reducing enzyme in an orally acceptable
carrier, wherein said composition is selected from the group
consisting of: (i) an animal feed comprising at least 20 IU of said
immune stress-reducing enzyme/kg feed; (ii) a liquid composition
other than an animal feed comprising at least 40,000 IU of said
immune stress-reducing enzyme/L; and (iii) a solid composition
other than an animal feed comprising at least 40,000 IU of said
immune stress-reducing enzyme/kg, wherein said immune stress
reducing enzyme is selected from the group consisting of
.beta.-glucosidase, xyloglucanase, DNAases, non-specific nucleases,
RNAse L, dsRNA specific adenosine deaminase, CG specific
restriction endonuclease, N-glycanases, endo enzymes, PNGases,
.alpha.-1,2-fucosidase, .alpha.-1,3-1,4-fucosidase,
.beta.-1,4-galactosidase, endo-.beta.-N-acetylglucosaminidase F
(endo F), peptide-N--(N-acetyl-beta-glucosaminyl)asparagine amidase
F (PNGase F), PNGase A, endo-.beta.-N-acetylglucosaminidase H
(endoH), endo D, endo C, .alpha.-N-acetylgalacosaminidase,
.beta.-1,3-galactosidase, endo-N-acyl-neuraminidase (endo N),
.alpha.-2,3-neuraminidase, .alpha.-2,6-neuraminidase,
.alpha.-2,8-neuraminidase, .beta.-N-acetylhexosaminidase,
endo-.beta.-N-galactosidase, endo-.alpha.-N-acetylglactosaminidase,
arabinogalactanase, .alpha.-mannanase, sphingomyelinase, chitinase,
chitin deacetylase, N-acetylglucosaminidase, phosphatidylserine
decarboxylase, sulfatase, .beta.-galactosidase, arabinanase,
hyaluronidase, .alpha.-arabinofuranosidase, chondroitinase,
glucocerebrosidase, methyl esterase, ferulic acid esterase,
furuloyl esterase, acetyl esterase, carbohydrate deacetylase,
phosphorylcholine hydrolase, acid phosphatase, phosphorylcholine
esterase and phosphorylcholine phosphatase.
32. The composition of claim 31, wherein said composition is an
animal feed comprising at least 20 IU of said immune
stress-reducing enzyme/kg feed.
33. The composition of claim 31, wherein said composition is a
liquid composition other than an animal feed comprising at least
40,000 IU of said immune stress-reducing enzyme/L.
34. The composition of claim 31, wherein said composition is a
solid composition other than an animal feed comprising at least
40,000 IU of said immune stress-reducing enzyme/kg.
35. The composition of claim 31, wherein the composition is a solid
composition other than an animal feed comprising at least 80,000 IU
of said immune stress-reducing enzyme/kg.
36. The composition of claim 31, wherein the composition is a solid
composition other than an animal feed comprising at least 160,000
IU of said immune stress-reducing enzyme/kg.
37. The composition of claim 31, wherein the composition is an
animal feed that comprises an ingredient that induces an immune
response in the animal and wherein said immune stress-reducing
enzyme degrades said ingredient.
38. The composition of claim 37, wherein said ingredient is a
non-pathogenic molecule that displays a molecular pattern that is
recognized by the animal's innate immune system, and that is
degraded by said immune stress reducing enzyme.
39. The composition of claim 37, wherein said ingredient is an
antigen displayed by a pathogenic microorganism.
40. The composition of claim 31, wherein the immune stress-reducing
enzyme is selected from the group consisting of DNAase,
non-specific nucleases, RNAse, RNAse L, CG specific restriction
endonuclease, phosphatidylserine decarboxylase, hyaluronidase,
chitinase, xyloglucanase and arabinanase.
41. The composition of claim 40, wherein the immune stress-reducing
enzyme is selected from the group consisting of chitinase,
xyloglucanase and arabinanase.
42. The composition according to claim 41, wherein the composition
is selected from the group consisting of (i) a composition
comprising 1,4-.beta.-mannanase and chitanase; (ii) a composition
comprising 1,4-.beta.-mannanase and xyloglucanase; and (iii) a
composition comprising 1,4-.beta.-mannanase and arabinanase.
43. A method of improving animal growth performance and/or reducing
immune stress in an animal, comprising orally administering to said
animal a composition according to claim 31.
44. The method of claim 43, wherein said animal is administered an
ingredient that induces an immune response in the animal and
wherein said composition comprises at least one immune
stress-reducing enzyme that degrades said ingredient.
45. The method of claim 44, wherein said ingredient and said immune
stress-reducing enzyme are administered in the same
composition.
46. The method of claim 45, wherein said composition is an animal
feed.
47. The method of claim 44, wherein said ingredient is a
non-pathogenic molecule that displays a molecular pattern that is
recognized by the animal's innate immune system, and that is
degraded by said immune stress reducing enzyme.
48. The method of claim 44, wherein said ingredient is an antigen
displayed by a pathogenic microorganism.
49. A method of preventing or treating infection associated with a
pathogenic microorganism that displays an antigen, comprising
orally administering to an animal in need thereof a composition
according to claim 31, wherein the composition comprises at least
one immune stress-reducing enzyme that degrades said antigen.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit under 35 U.S.C.
.sctn.119(e) of the filing date of U.S. provisional application
60/750,339, filed Dec. 15, 2006, the entire contents of which are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention provides compositions and methods for
reducing immunological stress and improving animal growth
performance. In particular, the invention provides compositions
comprising enzymes that are effective to reduce immunological
stress or that are effective to treat or prevent infection or that
are effective to improve animal growth performance. The invention
also provides methods using the compositions.
BACKGROUND
[0003] An animal may experience immunological stress for a number
of reasons, including exposure to an antigen that is recognized by
the animal's immune system. An antigen may trigger an immune
response that is an adaptive immune response or that is an innate
immune response. When an immune response is triggered, the animal
experiences immunological stress as its immune system responds to
the perceived threat. Often, immunological stress hampers animal
growth performance.
[0004] Acute phase proteins (APP) are a group of blood proteins
whose blood concentration changes when an animal is experiencing
stress, such as infection, inflammation, surgical trauma, or other
internal or external challenges. See, e.g., Murata et al., Vet. J.
168: 28 (2004). APP are believed to play a role in an animal's
innate immune response. For example, APP may be involved in
restoring homeostasis and restraining microbial growth until an
acquired immunity is developed.
[0005] APP include "negative" proteins whose concentration
decreases with stress, and "positive" proteins whose concentration
increases with stress. See, e.g., Murata et al., supra. Negative
APP include albumin and transferrin. Positive APP include proteins
synthesized by hepatocytes upon stimulation by pro-inflammatory
cytokines and released into the bloodstream, such as haptoglobin,
C-reactive protein, serum amyloid A, ceruloplasmin, fibrinogen, and
.alpha.-1-acid glycoprotein (AGP). Extra-hepatic production of APP
also has been reported for most mammalian species. Id.
Pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor
necrosis factor .alpha. (TNF-.alpha.) are believed to be the major
mediators of APP synthesis in the liver. Inflammation, infection or
tissue injury triggers cytokine release by defense-oriented cells,
thereby inducing APP synthesis. The induction of positive APP also
is associated with a decrease in the synthesis of negative APP.
Id.
[0006] Methods of quantifying APP have been established, and
circulating APP concentration (e.g., serum levels of APP) has been
correlated to the severity of the animal's condition. Id. Thus, APP
concentration can be used as an indicator of an animal's immune
stress level.
[0007] An animal's immune system may recognize antigens that do not
pose a real threat to the animal's health, such as plant- and
animal-derived ingredients in animal feed compositions. These
antigens may trigger an immune response, such as an innate immune
response, thereby causing the animal to experience immunological
stress. This stress response can be identified and monitored via
serum APP concentration.
[0008] Even when the immune-triggering antigen did not pose a real
threat to the animal's health, the stress response can have a
detrimental effect. This may be observed as a decrease in feed
efficiency, a decrease in weight gain rate or decrease in weight,
an increase in susceptibility to infection, or an increase in body
temperature, for example.
[0009] The use of antibodies, such as anti-phospholipase A2
antibodies, to reduce gastrointestinal inflammation in animals has
been described. See, e.g., U.S. Pat. No. 6,383,485. Feed
compositions have been described that comprise a hemicellulase
capable of degrading .beta.-mannan-containing hemicellulose (e.g.,
a .beta.-mannanase-type hemicellulase), such as
endo-1,4-.beta.-mannanase, or a phospholipase, such as
phospholipase A2, for improved feed efficiency. See, e.g., WO
97/41739, U.S. Pat. No. 6,162,473, and U.S. Pat. No. 6,183,739.
[0010] Likewise described have been feed compositions comprised of
an enzyme, such as PI-PLC, that cleaves a linkage, thereby to
effect release of a cell-surface protein or carbohydrate, for the
treatment or prevention of digestive tract infection. See, e.g., WO
01/41785. Walsh et al., J. Anim. Sci. 73: 1074 (1995), discuss feed
compositions comprising glucanase enzymes that cleave a mixed link
glucan substrate, such as 1,4-.beta.-glucanase which cleaves mixed
.beta.-1,3, .beta.-1,4-substrates. In our tests, however, neither
PI-PLC nor 1,4-.beta.-glucanase displayed immune-stress reducing
activity.
[0011] There has been no description heretofore of a feed
composition comprised of an enzyme that is other than a
.beta.-mannanase-type hemicellulase or a phospholipase and that is
present in an amount effective to reduce immunological stress.
[0012] Accordingly, there is a need for compositions and
methodology for reducing immunological stress in animals.
SUMMARY OF THE INVENTION
[0013] One embodiment provides a composition suitable for oral
administration to an animal comprising an immune stress-reducing
enzyme in an orally acceptable carrier. The composition is selected
from the group consisting of: (i) an animal feed comprising an
amount of the enzyme effective to decrease the level of positive
acute phase protein in the animal, increase the level of negative
acute phase protein in the animal, and/or improve animal growth
performance; (ii) a liquid composition other than an animal feed
comprising at least 40,000 IU enzyme/L; and (iii) a solid
composition other than an animal feed comprising at least 40,000 IU
enzyme/kg. The enzyme is other than a .beta.-mannanase-type
hemicellulase or phospholipase, and, if the enzyme comprises
1,3-.beta.-glucanase, the composition is selected from the group
consisting of (i) an animal feed comprising at least 20 IU
1,3-.beta.-glucanase/kg feed; (ii) a liquid composition other than
an animal feed comprising at least 155,000 IU
1,3-.beta.-glucanase/L and (iii) a solid composition other than an
animal feed comprising at least 300,000 IU
1,3-.beta.-glucanase/kg.
[0014] In one embodiment, the composition is an animal feed
comprising at least 20 IU enzyme/kg feed. In another embodiment,
the composition is a solid composition other than an animal feed
comprising at least 80,000 IU enzyme/kg, or at least 160,000 IU
enzyme/kg.
[0015] In one embodiment, the composition is an animal feed that
comprises an ingredient that induces an immune response in the
animal and the enzyme comprises an enzyme that degrades said
ingredient. In one embodiment, the ingredient is an antigen
displayed by a pathogenic microorganism.
[0016] In one embodiment, the enzyme comprises
1,3-.beta.-glucanase. In one embodiment, the enzyme comprises
1,3-.beta.-glucanase and the composition is selected from the group
consisting of (i) an animal feed comprising at least 30 IU
1,3-.beta.-glucanase/kg feed; (ii) a liquid composition other than
an animal feed comprising at least 230,000 IU
1,3-.beta.-glucanase/L and (iii) a solid composition other than an
animal feed comprising at least 450,000 IU
1,3-.beta.-glucanase/kg.
[0017] Another embodiment provides a composition suitable for oral
administration to an animal comprising two or more immune
stress-reducing enzymes, wherein the composition comprises at least
one immune stress-reducing enzyme other than 1,4-.beta.-mannanase
and 1,3-.beta.-glucanase. The composition is selected from the
group consisting of: (i) an animal feed comprising an amount of
said immune stress-reducing enzymes effective to decrease the level
of positive acute phase protein in said animal, increase the level
of negative acute phase protein in said animal, and/or improve
animal growth performance; (ii) a liquid composition other than an
animal feed comprising at least one immune stress-reducing enzyme
in an amount of at least 40,000 IU enzyme/L; and (iii) a solid
composition other than an animal feed comprising at least one
immune stress-reducing enzyme in an amount of at least 40,000 IU
enzyme/kg.
[0018] In one embodiment, the composition is an animal feed
comprising at least one immune stress-reducing enzyme in an amount
of at least 20 IU enzyme/kg feed. In another embodiment, the
composition is a solid composition other than an animal feed
comprising at least one immune stress-reducing enzyme in an amount
of at least 80,000 IU enzyme/kg, or at least 160,000 IU
enzyme/kg.
[0019] In specific embodiments, the composition is selected from
the group consisting of (i) a composition comprising
1,4-.beta.-mannanase and chitanase; (ii) a composition comprising
1,4-.beta.-mannanase and xyloglucanase; (iii) a composition
comprising 1,4-.beta.-mannanase and arabinanase; (iv) a composition
comprising 1,3-.beta.-glucanase and chitanase; (v) a composition
comprising 1,3-.beta.-glucanase and xyloglucanase; (vi) a
composition comprising 1,3-.beta.-glucanase and arabinanase and
(vii) a composition comprising 1,4-.beta.-mannanase,
1,3-.beta.-glucanase and arabinanase.
[0020] Another embodiment provides a composition suitable for oral
administration to an animal comprising 1,4-.beta.-mannanase and
1,3-.beta.-glucanase. The composition is selected from the group
consisting of (i) an animal feed comprising 1,4-.beta.-mannanase
and at least 20 IU 1,3-.beta.-glucanase/kg feed, (ii) a liquid
composition other than an animal feed comprising
1,4-.beta.-mannanase and at least 155,000 IU 1,3-.beta.-glucanase/L
and (iii) a solid composition other than an animal feed comprising
1,4-.beta.-mannanase and at least 300,000 IU
1,3-.beta.-glucanase/kg. In one embodiment, the composition is
selected from the group consisting of (i) an animal feed comprising
1,4-.beta.-mannanase and at least 30 IU 1,3-.beta.-glucanase/kg
feed; (ii) a liquid composition other than an animal feed
comprising 1,4-.beta.-mannanase and at least 230,000 IU
1,3-.beta.-glucanase/L and (iii) a solid composition other than an
animal feed comprising 1,4-.beta.-mannanase and at least 450,000 IU
1,3-.beta.-glucanase/kg. In one embodiment, the composition further
comprises one or more additional immune stress-reducing
enzymes.
[0021] Another embodiment provides a method of improving animal
growth performance and/or reducing immune stress in an animal,
comprising orally administering to the animal any of the
compositions described above.
[0022] In one embodiment, the animal is administered an ingredient
that induces an immune response in the animal and the composition
comprises at least one immune stress-reducing enzyme that degrades
the ingredient. In one embodiment, the ingredient and enzyme are
administered in the same composition. In one embodiment, the
composition is an animal feed. In one embodiment, the ingredient is
an antigen displayed by a pathogenic microorganism.
[0023] Another embodiment provides a method of preventing or
treating infection associated with a pathogenic microorganism that
displays an antigen, comprising orally administering to an animal
in need thereof any of the compositions described above, wherein
the composition comprises at least one immune stress-reducing
enzyme that degrades the antigen.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 shows the best curve fit (and underlying polynomial
equation) for calculating the concentration of chicken
.alpha.-1-acid glycoprotein (AGP) in plasma samples of test
chickens with data obtained in Example 1.
[0025] FIG. 2 is a Box and Wisker plot graphically showing the AGP
levels in chicken serum from test chickens, as described in Example
2. The range of the data is represented by the vertical lines. The
box represents the range of the data within one standard deviation
of the mean. The horizontal line indicates the data mean.
[0026] FIG. 3 shows the AGP levels of serum from chickens receiving
one of several different feeds, including feeds in accordance with
the invention and prior art feeds, as described in Example 3.
[0027] FIG. 4 shows the best curve fit (and underlying polynomial
equation) for calculating the concentration of AGP in plasma
samples of test turkeys with data obtained in Example 8.
DETAILED DESCRIPTION
[0028] As used in the following discussion, the terms "a" or "an"
should be understood to encompass one or more, unless otherwise
specified.
[0029] As used herein, the term "animal" refers to any animal,
including humans and other animals, including companion animals
such as dogs and cats, livestock, such as cows and other ruminants,
buffalo, horses, pigs, sheep, fowl (e.g., chicken, ducks, turkeys,
and geese) and aquaculture animals (e.g., fish and shrimp and
eels).
[0030] In the present description, the phrases "enzyme that
degrades an antigen" and "enzyme that degrades an ingredient" mean
that the enzyme converts the antigen or ingredient to a form that
is not recognized by the animal's immune system. The ability of an
enzyme to degrade an antigen or ingredient can be identified by
measuring the animal's serum APP concentration, whereby a decrease
in the serum concentration of positive APP, or an increase in the
serum concentration of negative APP, indicates that the enzyme has
degraded the antigen or ingredient.
[0031] As noted above, the term "APP" include "negative" proteins
whose concentration decreases with stress, and "positive" proteins
whose concentration increases with stress. The invention includes
compositions and methods that increase the concentration of
negative acute phase proteins whose concentration typically
decreases with stress, as well as compositions and methods that
decrease the concentration of positive acute phase proteins whose
concentrations typically increase with stress. For convenience, in
the discussion that follows, the invention is exemplified with
reference to the effect of the compositions and methods on positive
acute phase proteins. Thus, the term "APP" in the discussion that
follows generally refers to any one or more positive acute phase
proteins associated with an animal's stress response. It should be
understood that the compositions and methods described herein as
decreasing the concentration of "APP" (referring to positive acute
phase proteins) also are useful for increasing the concentration of
negative acute phase proteins.
[0032] One aspect of the invention relates to a composition
comprising an enzyme that is effective to reduce the immunological
stress experienced by an animal. For convenience, these enzymes are
referred to herein as "immune stress-reducing" enzymes. As used
herein, the term "immune stress-reducing enzyme" means any enzyme
that degrades an antigen or molecular pattern that is recognized by
the animal's immune system, e.g., an antigen or molecular pattern
that triggers an immune response, thereby causing the animal to
experience immunological stress. The term "molecular pattern" as
used herein includes general molecular patterns that are bound by
receptors in the context of the innate immune system, such as
molecular patterns that are usually associated with pathogens.
[0033] In accordance with one embodiment, the immune
stress-reducing enzyme is not a .beta.-mannanase-type
hemicellulase. In one accordance with that embodiment, the immune
stress-reducing enzyme is not endo-1,4-.beta.-D-mannanase. In
accordance with another embodiment, the enzyme is not a
phospholipase. In accordance with another embodiment, the immune
stress-reducing enzyme is not 1,4-.beta.-D-glucanase. In accordance
with another embodiment, the immune stress-reducing enzyme is not
PI-PLC. In accordance with another embodiment, the immune
stress-reducing enzyme is not a .beta.-mannanase-type hemicellulase
or a phospholipase. In accordance with yet another embodiment, the
immune stress-reducing enzyme is not a .beta.-mannanase-type
hemicellulase, is not 1,4-.beta.-glucanase, and is not a
phospholipase. In accordance with a further embodiment, the immune
stress-reducing enzyme is not a .beta.-mannanase-type
hemicellulase, is not 1,4-.beta.-glucanase, is not a phospholipase,
and is not PI-PLC.
[0034] While not wanting to be bound by any theory, the present
inventors believe that the immune stress-reducing enzyme's
degradation of the antigen or molecular pattern inhibits or reduces
the immune response triggered by the antigen or molecular pattern,
thereby reducing the animal's immunological stress. The reduction
in immunological stress can be identified and monitored by
measuring the animal's serum APP concentration, using methods known
in the art for quantifying APP. Examples of such methods are
referenced in Murata, et al., supra, and are described and
referenced in Hulten et al., Vet. Microbial. 95: 75 (2003) and Holt
et al., supra, as well as in Example 1 below.
[0035] In a related embodiment, the invention provides methods for
reducing immunological stress in an animal that comprise
administering to the animal a composition comprising an amount of
an immune stress-reducing enzyme effective to reduce the level of
APP in the animal.
[0036] A number of different positive acute phase proteins have
been identified, including .alpha.-1-acid glycoprotein (AGP),
ceruloplasmin (Cp), proteins of the collectin family (e.g., lung
surfactant proteins, conglutinin and mannan-binding lectin),
fibrinogen (Fb), C-reactive protein (CRP), haptoglobin, protease
inhibitors (e.g., .alpha.-1-antitrypsin,
.alpha.-1-antichymotrypsin, and .alpha.-2-macroglobulin) and serum
amyloid-A (SAA). Other potential APP include
lipopolysaccharide-binding protein (LPB), phospholipid-binding
proteins such as annexins and Major Acute Phase Protein (MAP).
Murata, et al., supra. Serum concentrations of any one of these or
other APP can be used to identify, assess and monitor enzyme
activity in accordance with the invention.
[0037] Different APP may play more significant roles in the stress
responses of different animals. For example, AGP is known to be
clinically important in cattle, and is associated with infection in
pigs, dogs, cats and chicken (including hens). Cp has been reported
to be an indicator of infection in cattle, horses, and chickens.
CRP has been identified in ruminants, horses, pigs, dogs, and cats,
although it has not been demonstrated that CRP is an APP in cattle.
CRP has been shown to be associated with infection in horses and
pigs. Fb is a reliable indicator of inflammation, bacterial
infection or surgical trauma in cattle and sheep, and is associated
with infection in horses. Hp is an APP in a number of production
and companion animals, including ruminants such as cattle, sheep,
pigs, horses, and dogs. SAA has been associated with inflammation
and infection in cattle and with infection in horses, pigs,
companion animals such as dogs, and chicken. An increase in SAA
milk levels has been found in cows and ewes with mastitis. Serum
LBP has been associated with infection in cattle, as has local
levels of annexins (on the surfaces of secretory epithelia in lungs
of infected cattle). MAP is reported to be an indicator of
infection in pigs. Additionally, while transferrin is usually
considered a negative acute phase protein, it appears to play a
role as a positive acute phase protein in chickens. Murata, et al.,
supra; Holt et al., Poultry Sci. 81: 1295-1300 (2002). Others also
have reported that SAA and Hp, as well as CRP and MAP, are
associated with infection in pigs. Hulten et al., supra.
[0038] In some embodiments, the compositions of the invention
comprise an amount of immune stress-reducing enzyme that is
effective to decrease the serum concentration of APP in an animal.
The amount may vary depending on the animal and the immune
stress-reducing enzyme, and can readily be determined by those
skilled in the art using methods known in the art. For example, an
animal's serum APP levels can be measured prior and subsequent to
administration of the enzyme, or serum APP levels of equivalent
treated and control animals can be compared. (In this regard, it
may be advantageous to compare treated and control animals of the
same age, as APP levels may change with age. For example, we have
found that serum AGP levels increase with chicken age.) A decrease
in serum APP concentration associated with administration of the
enzyme indicates that an effective amount of enzyme was
administered.
[0039] In other embodiments, the compositions of the invention
comprise an amount of immune stress-reducing enzyme that is
effective to improve animal growth performance (also referred to as
"live performance," particularly in the field of poultry). As used
herein, the phrase "animal growth performance" includes any
parameter that reflects animal growth, including feed conversion,
water absorption, feces water content, uniformity of body weight
within a flock or group of animals, livability, and mortality.
While not wanting to be bound by any theory, it is believed that,
under some conditions, the effect of the immune stress-reducing
enzyme on APP concentration is masked by factors such as immune
stress-inducing factors, such as the presence of a low-level
infection in a group of animals or stressful living conditions.
Under such conditions, the immune stress-reducing enzyme may
nevertheless be effective to improve animal growth performance.
Thus, animal growth performance is an alternative measure of the
effectiveness of the compositions and methods of the present
invention.
[0040] The composition may be any composition suitable for
administration to an animal. In one embodiment, the composition is
suitable for oral administration. In one specific embodiment, the
composition that is suitable for oral administration is generally
recognized as safe for oral administration to an animal. In another
specific embodiment, the composition that is suitable for oral
administration contains only ingredients, and amounts of said
ingredients, that are generally recognized as safe for oral
administration to an animal. In another specific embodiment, the
composition that is suitable for oral administration does not
contain any ingredients, or amounts of said ingredients, that are
not generally recognized as safe for oral administration to an
animal. In another specific embodiment, the composition that is
suitable for oral administration contains only ingredients, and
amounts of said ingredients, that are allowed, or that are not
prohibited, for oral administration to an animal. In another
specific embodiment, the composition that is suitable for oral
administration does not contain any ingredients, or amounts of said
ingredients, that are not allowed, or that are prohibited, for oral
administration to an animal.
[0041] In some embodiments, the composition comprises an orally
acceptable carrier for the enzyme. As used herein, "orally
acceptable carrier" includes any physiologically acceptable carrier
suitable for oral administration. Orally acceptable carriers
include without limitation animal feed compositions, aqueous
compositions, and liquid and solid compositions suitable for use in
animal feed products and/or for oral administration to an animal.
Suitable carriers are known in the art, and include those described
in U.S. Pat. No. 6,780,628.
[0042] In some embodiments, the composition is an animal feed. As
used herein, the term "animal feed" has its conventional meaning in
the field of animal husbandry. For example, animal feed includes
edible materials which are consumed by livestock for their
nutritional value. Animal feed includes feed rations, e.g.,
compositions that meet an animal's nutritional requirements, and
also include compositions that do not meet an animal's nutritional
requirements.
[0043] In specific examples of such an embodiment, the amount of
enzyme is at least about 50,000 international units (IU) per U.S.
ton of feed, at least about 60,000 IU per ton of feed, at least
about 70,000 IU per ton of feed, at least about 80,000 IU per ton
of feed, at least about 90,000 IU per ton of feed, at least about
100,000 IU per ton of feed, at least about 200,000 IU per ton of
feed, or at least about 500,000 IU per ton of feed, or higher.
[0044] In other specific examples, the invention provides an animal
feed comprising an amount of immune stress-reducing enzyme of at
least about 20 IU/kg feed, such as at least 20 IU/kg feed, at least
at 25 IU/kg feed, at least at 30 IU/kg feed, at least at 35 IU/kg
feed, at least at 40 IU/kg feed, at least at 45 IU/kg feed, at
least 50 IU/kg feed, or more. While not wanting to be bound by any
theory, it is believed that an animal feed comprising an amount of
immune stress-reducing enzyme of at least about 20 IU/kg feed will
be effective to decrease the level of positive acute phase protein
in said animal, increase the level of negative acute phase protein
in said animal, and/or improve animal growth performance.
[0045] Thus, in some embodiments, the invention provides an animal
feed comprising an amount of immune stress-reducing enzyme
effective to decrease the level of positive acute phase protein in
the animal, increase the level of negative acute phase protein in
the animal, and/or improve animal growth performance.
[0046] The feed composition may be prepared by methods known in the
art. For example, immune stress-reducing enzyme can be added to the
other feed ingredients at any stage during the manufacturing
process, as deemed to be appropriate by those skilled in the art.
In one embodiment, the enzyme is provided as a solution, such as a
liquid enzyme concentrate that is added to other feed ingredients
during the manufacturing process. Alternatively, an
enzyme-containing solution is sprayed on to a substantially final
form of the animal feed. In another embodiment, the enzyme is
provided as a solid composition (such as a powder), such as a solid
composition that is added to other feed ingredients during the
manufacturing process. Exemplary methods for manufacturing
enzyme-containing feed are described in WO 97/41739.
[0047] In some embodiments, the composition is other than an animal
feed. For example, the composition may be a liquid composition
other than an animal feed or a solid composition other than an
animal feed. Such compositions may be suitable for direct
administration to an animal or may be used as a feed additive
(e.g., added to feed prior to feeding) or a feed supplement
(including supplements that are diluted with other feed components
prior to feeding and supplements that are offered to an animal on a
free choice, separate basis). Examples of a liquid composition
other than an animal feed include liquid enzyme concentrates,
including liquid enzyme concentrates that are typically diluted or
combined with other ingredients prior to oral administration to an
animal.
[0048] In embodiments where the composition is a liquid composition
other than an animal feed, such as an enzyme solution, the liquid
composition or solution may comprise at least about 40,000
international units (IU) per liter of solution, such as at least
40,000 IU/L, at least 50,000 IU/L, at least 60,000 IU/L, at least
70,000 IU/L, at least 80,000 IU/L, at least 90,000 IU/L, at least
100,000 IU/L, at least about 500,000 IU/L, at least about 600,000
IU/L, at least about 700,000 IU/L, at least about 800,000 IU/L, at
least about 900,000 IU/L, at least about 1,000,000 IU/L, at least
about 2,000,000 IU/L, or at least about 5,000,000 IU/L.
[0049] In some embodiments, an amount of liquid composition other
than an animal feed, such as about 500 mL solution, is applied to
or combined with an amount of feed, such as to a ton of feed, to
arrive at feed formulations with enzyme levels described above. In
other embodiments, an amount of liquid composition other than an
animal feed is applied to or combined with an amount of feed to
prepare an animal feed with an amount of enzyme effective to
decrease the level of positive acute phase protein in the animal,
increase the level of negative acute phase protein in the animal,
and/or improve animal growth performance.
[0050] It is believed that currently available liquid enzyme
concentrate compositions (other than the 1,3-.beta.-glucanase
compositions discussed below) that are suitable for oral
administration comprise much less than at least about 40,000 IU/L
of an immune stress-reducing enzyme, if any at all, and are not
effective to decrease the level of positive acute phase protein,
increase the level of negative acute phase protein, and/or improve
animal growth performance, when used in accordance with their
instructions.
[0051] In embodiments where the composition is a solid composition
other than an animal feed, the composition may comprise at least
about 40,000 IU/kg, such as at least 40,000 IU/kg, at least 50,000
IU/kg, at least 60,000 IU/kg, at least 70,000 IU/kg, at least
80,000 IU/kg, at least 90,000 IU/kg, at least 100,000 IU/kg, at
least 120,000 IU/kg, at least 140,000 IU/kg, at least 160,000
IU/kg, at least 180,000 IU/kg, at least 200,000 IU/kg, or more.
[0052] In some embodiments, an amount of a solid composition other
than an animal feed is applied to or combined with an amount of
feed to arrive at feed formulations with enzyme levels described
above. In other embodiments, an amount of solid composition other
than an animal feed is combined with an amount of feed to prepare
an animal feed with an amount of enzyme effective to decrease the
level of positive acute phase protein in the animal, increase the
level of negative acute phase protein in the animal, and/or improve
animal growth performance.
[0053] It is believed that currently available solid enzyme powder
compositions that are suitable for oral administration comprise
much less than at least about 40,000 IU/kg of an immune
stress-reducing enzyme, if any at all, and are not effective to
decrease the level of positive acute phase protein, increase the
level of negative acute phase protein, and/or improve animal growth
performance, when used in accordance with their instructions.
[0054] As conventional in the art, the term "IU" or "international
unit" refers to an amount of enzyme that will catalyse the
transformation of 1 micromole of the substrate per minute under
conditions that are optimal for the enzyme. Weight equivalents to
international units of immune stress-reducing enzymes are known in
the art and can be determined using standard assays. Exemplary
standard assays for representative immune stress-reducing enzymes
are outlined below.
[0055] In one embodiment, the enzyme is expressed by a plant that
is used in animal feed. For example, corn can be genetically
engineered to express an immune stress-reducing enzyme and the
resulting genetically modified corn product can be used in feed.
Production also can be effected with other genetically modified or
classically modified systems such as bacteria, e.g., E. coli,
Bacillus sp., Lactobacillus; yeast, e.g., Pichia, Yarrow,
Saccharomyces, Schizosaccharomyces (e.g., Schizosaccharomyces pomb,
Hansenula, Kluyveromyces, Candida), and other fungus, such as
Aspergillus, Rhizopus, Tricoderma, Humicola, Penicillium, and
Humicola.
[0056] In accordance with another embodiment, the immune
stress-reducing enzyme is provided in a capsule or tablet from for
oral ingestion. The invention also encompasses embodiments where
the enzyme is administered by other routes, such as intravenously,
peritoneally, or subcutaneously, as a component of a composition
formulated for such administration in accordance with known
pharmacological practices.
[0057] An animal's immune system may recognize as an antigen or
molecular pattern certain ingredients of a feed composition that do
not pose a real threat to the animal's health. Nonetheless, the
ingredient triggers an immune response that causes the animal to
experience immunological stress, and that can be identified and
monitored by an increase in the serum concentration of one or more
APP. While not wanting to be bound by any theory, the present
inventors believe that this "unnecessary and counterproductive"
immune response may involve pattern recognition receptors (PRR),
such as those involved in the innate immune system.
[0058] The innate immune system provides an immune response that
does not depend on specific antigen recognition. See, e.g., Tosi,
J. Allergy Clin. Immunol. 116: 241 (2005). One aspect of the innate
immune system involves PRR, which recognize and bind
pathogen-associated molecular patterns, transducing immune response
signals. See, e.g., Fabrick et al., J. Biol. Chem. 279: 26605
(2004). Examples of PRR include Toll-like receptors (TLR) that
recognize a range of molecular patterns and generate intracellular
signals for activation of a range of host responses. See, e.g.,
Tosi, supra; Blach-Olszewska, Arch. Immunol. Ther. Exp. 53: 245
(2005). PRR/TLR have been identified that recognize mannose (e.g.,
Blach-Olszewska, supra), 1,3-.beta.-glucan (e.g., Rice et al., J.
Leukoc. Biol. 72:140 (2002)), lipopolysaccharide and
phosphorylcholine (e.g., Baumgarth et al., Semin. Immunopathol. 26:
347 (2005)), lipoteichoic acid, phenol-soluble modulin, muramyl
dipeptide and peptidoglycan (e.g., Fournier et al., Clin.
Microbiol. Rev. 18: 521 (2005). Immunomodulatory receptors for
mannan (e.g., Klabunde et al., Parasitol. Res. 88: 113 (2002)
(mannan-binding lectin)), and N-acetyl-D-glucosamine and
N-acetyl-D-mannosamine (e.g., Hansen et al., J. Immunol. 169: 5726
(2002)). TLRs for double stranded RNA (e.g., Bell et al., Proc.
Nat'l Acad. Sci. USA 102: 10976 (2005)) and DNA with methylation
patterns that differ from endogenous DNA (e.g., Huang at al., J.
Immunol. 175: 3964 (2005); Normemacher et al., Infect. Immun. 71:
850 (2003)) also have been identified.
[0059] While these molecular patterns are associated with
pathogenic microorganisms (e.g., bacteria, viruses, fungi and
protozoa) they also are presented by some non-pathogenic molecules,
such as animal feed ingredients. An innate immune response to
non-pathogenic molecules presenting these molecular patterns
unnecessarily subjects an animal to immunological stress, and may
detrimentally impact the animal's feed efficiency, slow the
animal's rate of weight gain or result in weight loss, make the
animal more susceptible to infection, increase the animal's body
temperature, or otherwise have a negative impact on the animal's
health or food energy (calorie) utilization efficiency. The innate
immune response resulting from MBL (mannose-binding lectin)
function, for example, induces powerful responses. It has been
shown that mutation of one of the mannose binding protein's genes
in mice paradoxically allows survival from a normally lethal acute
septic peritonitis challenge (Takahashi, K. et al., Microbes
Infect. 4 (8): 773-784, 2002). The immune stress from aggressive
innate immune response is more lethal than the infection in this
case.
[0060] .beta.-mannan is a component of soybean products and
soybean-based animal feeds. High molecular weight forms of
.beta.-mannan present in animal feed can trigger an "unnecessary
and counterproductive" innate immune response, thereby subjecting
the animal to immunological stress. The present inventors found
that this immunological stress can be reduced or prevented using a
.beta.-mannanase-type hemicellulase, endo-1,4-.beta.-D-mannanase,
an enzyme which degrades .beta.-mannans (e.g. .beta.-galactomannan,
.beta.-glucomannan), thereby reducing or preventing the immune
response to .beta.-mannan. As shown in the Examples below, the
reduction in immunological stress is reflected in a decrease in
serum APP concentration.
[0061] .alpha.-mannanase, which degrades .alpha.-mannan, is useful
as an immune-stress reducing enzyme in accordance with the
invention. .alpha.-mannan is not considered to be a hemicellulose
because it does not share characteristic properties of
hemicelluloses.
[0062] In the field of industrial enzymes, the term "hemicellulase"
has been used as a trade name for .beta.-mannanase. Likewise,
patents and publications co-authored by the inventors use the term
"hemicellulase" to refer to .beta.-mannanase, including
endo-1,4-.beta.-D-mannanase. See, e.g., U.S. Pat. No. 6,162,473. In
other contexts, the term "hemicellulase" may be broader,
encompassing glucanases and xylanases in addition to mannanase, as
explained below.
[0063] The term "hemicellulose" was coined to describe carbohydrate
plant material obtained by extraction with a dilute alkaline
solution that is hydrolyzed more easily than cellulose. See, e.g.,
Schulze, E., Berichte der Deutschen Botanischen Gesellschaf, 24:
2277 (1891); Schulze, E., Z. Physiol. Chem. 16: 387 (1892). Since
then, "hemicellulose" has come to specify water insoluble plant
polysaccharides associated with cellulose, other than pectin and
starches and polysaccharides in plant sap, that are soluble in
dilute alkali solutions. See, e.g., Whisler et al.,
"Hemicelluloses," in IV POLYSACCHARIDE CHEMISTRY 112 (Academic
Press, 1953). Xylan, .beta.-mannans and galactans are generally
considered to be hemicelluloses, although some .beta.-mannans, like
Locust bean gum and guar gum galacotmannans are readily soluble.
Softwood trees have a lot of .beta.-mannans associated with their
cellulose and hardwoods have a lot of xylans.
[0064] In contrast to hemicelluloses, .alpha.-mannan is associated
with fungal cells walls, such as Saccharomyces, is not a structural
component of wood, and is uniformly found in eucaryotic
glycoproteins that are generally soluble in water. Thus,
.alpha.-mannan is not considered to be a hemicellulose, and
.alpha.-mannanase is not a hemicellulase. .alpha.-mannanase is
useful as an immune stress-reducing enzyme in accordance with the
present invention because it degrades .alpha.-mannans that are
recognized by an animal's immune system, but that are not pathogen
associated. The innate immune system is sensitive to mannan because
polymers containing mannose are found on the surface of many
pathogens.
[0065] Other feed ingredients that may be recognized by an animal's
immune system include .beta.-1,3-glucan (a common structural
component of plant materials), N-linked glycoprotein complexes
(found, for example, in soybean products), double-stranded RNA from
plants, animals or microbes, and DNA from microbes, plants or
animals with a foreign (non-endogenous) methylation pattern. Thus,
in accordance with one embodiment, the invention provides a
composition comprising one or more immune stress-reducing enzymes
that degrade one or more of these or other feed ingredients. In a
related embodiment, the invention provides methods for reducing
immunological stress in an animal that comprise administering to
the animal a composition comprising an effective amount of such an
enzyme or enzymes. Specific examples of immune stress-reducing
enzymes and the antigens they degrade are set forth in the
following table. The invention encompasses compositions that
comprise other immune stress-reducing enzymes that degrade the same
or different antigens, as well as the use of such other enzymes to
reduce immunological stress.
TABLE-US-00001 ANTIGENS ENZYMES .alpha.-mannan .alpha.-mannanase
.alpha.-mannosidase .beta.-mannans .beta.-mannanase hemicellulase
(.beta.-mannanase type) 1,4-.beta.-mannanase
endo-1,4-.beta.-D-mannanase .beta.-1,3-glucans 1,3-.beta.-glucanase
Endo-1,3-.beta.-glucanase (EC 3.2.1.39) .beta.-glucosidase double
stranded RNA non-specific nuclease non-capped mRNA RNAse L 3pRNA
dsRNA specific adenosine deaminase DNA DNAase non-specific nuclease
CG specific restriction endonuclease N-linked glycoproteins
carbohydrases (e.g., asialoglycoprotein) N-glycanases endo enzymes
PNGases phosphocholine in sphingomyelin sphingomyelinase
N-acetlyglucosamine containing chitinase polymer, (e.g., chitin)
(EC 9 3.2.1.14) chitin deacetylase carbohydrate deacetylase
N-acetylglucosaminidase phosphatidylserine phosphatidylserine
decarboxylase phospholipase C phospholipase D sulfated
galactoside-saccharide sulfatase .beta.-galactoside
.beta.-galactosidase xyloglucan xyloglucanase (EC 3.2.1.15)
lipoarabinomannan (LAM) arabinanase arabinogalactan (AG) hyaluronan
(hyaluronic acid) hyaluronidase (EC 3.2.1.35) arabinogalactan and
other .alpha.-arabinofuranosidase arabino-modifided carbohydrates
chondroitin sulfate chondroitinase glucocerebrosides
glucocerebrosidase methyl esters of carbohydrates methyl esterase
ferulic acid esterified ferulic acid esterase carbohydrates
furuloyl esterase acetylated carbohydrate polymer acetyl esterase
carbohydrate deacetylase
[0066] In accordance with some embodiments, the invention provides
a composition comprising two or more immune stress-reducing
enzymes. In one embodiment, at least one of the two or more enzymes
is not 1,4-.beta.-mannanase or 1,3-.beta.-glucanase. In another
embodiment, a composition comprises 1,4-.beta.-mannanase and
1,3-.beta.-glucanase.
[0067] In one specific embodiment, the composition is an animal
feed comprising 1,4-.beta.-mannanase and at least about 20 IU
1,3-.beta.-glucanase/kg feed, such as at least 20 IU/kg feed, at
least 25 IU/kg feed, at least 30 IU/kg feed, at least 35 IU/kg
feed, at least 40 IU/kg feed, at least 45 IU/kg feed, at least 50
IU/kg feed, or more, of 1,3-.beta.-glucanase.
[0068] In another specific embodiment, the composition is a liquid
composition other than an animal feed comprising
1,4-.beta.-mannanase and at least about 155,000 IU
1,3-.beta.-glucanase/L, such as at least 155,000 IU/L, at least
230,000 IU/L, at least 300,000 IU/L, at least 380,000 IU/L, or
more, of 1,3-.beta.-glucanase.
[0069] In another specific embodiment, the composition is a solid
composition other than an animal feed comprising
1,4-.beta.-mannanase and at least about 300,000 IU
1,3-.beta.-glucanase/kg, such as at least 300,000 IU/kg, at least
450,000 IU/kg, at least 600,000 IU/kg, at least 750,000 IU/kg, at
least 900,000 IU/kg, or more, of 1,3-.beta.-glucanase.
[0070] In another embodiment, a composition comprises
1,4-.beta.-mannanase and xyloglucanase. In another embodiment, a
composition comprises 1,3-.beta.-glucanase and xyloglucanase. In
another embodiment, a composition comprises 1,4-.beta.-mannanase
and chitinase. In another embodiment, a composition comprises
1,3-.beta.-glucanase and chitanase. In another embodiment, a
composition comprises 1,4-.beta.-mannanase and arabinanase. In
another embodiment, a composition comprises 1,3-.beta.-glucanase
and arabinanase. In another embodiment, a composition comprises
1,4-.beta.-mannanase, 1,3-.beta.-glucanase and arabinanase.
[0071] It will be understood that these combinations are exemplary
only, and the invention includes compositions comprising other
combinations of immune stress-reducing enzymes. For example, the
invention includes compositions comprising any one or more of the
immune stress-reducing enzymes listed above and/or discussed below
and 1,4-.beta.-mannanase.
[0072] Immune stress caused by a feed ingredient may not always be
an innate immune system response. It is well known that a certain
small percentage of infants fed soy protein-based human
milk-replacer formula develop a strong detrimental
immunological-based intestinal reaction (see the report from the
Committee on nutrition, American Academy of Pediatrics, Pediatrics
101 (1): p 148, (1998)). N-linked Gycoproteins in soy, for example
.beta.-conglycinin, some times referred to as 7S globulin (Ogawa T,
et al., Biosci. Biotechnol. Biochem. 59(5):831-833, 1995; Burks A
W, et al., J Pediatr. Gastroenterol. Nutr. 8(2):195-203, 1989) can
be strong antigens and are recognized as having anti-nutritional
qualities. .beta.-conglycinin is deliberately removed from soy
protein isolate preparations used for nutritional supplements
despite its contribution to total protein. Hydrolysis destroys the
antigenicity. In addition, we have found that an enriched 7S soy
glycoprotein fraction used in feeding roosters was less well
digested than another less glycosylated soy protein fraction.
[0073] Examples of suitable enzymes for degrading carbohydrates in
N-linked glycoproteins include .alpha.-fucosidases such as
.alpha.-1,2-fucosidase and .alpha.-1,3-1,4-fucosidase,
.alpha.-mannosidases such as .alpha.-1,6-mannosidase,
.alpha.-1,2-mannosidase, and .alpha.-1,3-mannosidase,
.beta.-1,4-galactosidase, endo-.beta.-N-acetylglucosaminidase F
(endo F), peptide-N-(N-acetyl-beta-glucosaminyl)asparagine amidase
F (PNGase F), PNGase A, endo-.beta.-N-acetylglucosaminidase H
(endoH), endo D, endo C, .alpha.-N-acetylgalactosaminidase,
.beta.-1,3-galactosidase, endo-N-acyl-neuraminidase (endo N),
.alpha.-2,3-neuraminidase, .alpha.-2,6-neuraminidase,
.alpha.-2,8-neuraminidase, 3-N-acetylhexosaminidase,
endo-.beta.-N-galactosidase, endo-.alpha.-N-acetylglactosaminidase,
endo-.alpha.-1,6-D-mannanase, arabinogalactanase,
.alpha.-galactosidase, .beta.-galactosidase.
[0074] These enzymes are known in the art and some are available
from commercial sources. Alternatively, immune stress-reducing
enzymes can be obtained from microorganisms that produce enzymes,
such as bacteria, fungi and yeast. Additionally, the enzymes can be
obtained using recombinant technology methods known in the art, by,
for example, genetically engineering a host cell to produce an
enzyme, e.g., causing transcription and translations of a gene
encoding the enzyme. The amino acid sequences of a number of the
enzymes set forth above are known in the art. Using those sequences
or known nucleotide sequences encoding those sequences, those
skilled in the art can design suitable genes for recombinant
expression of the enzymes. Additionally or alternatively, a
nucleotide sequence encoding a known immune stress-reducing enzyme
can be used to probe a DNA library to identify other nucleotide
sequences encoding immune stress-reducing enzymes. As known in the
art, such a DNA library can be derived from a defined organism or
population of organisms, or can be obtained from natural sources
and thus represent DNA from microorganisms that are difficult to
culture.
[0075] In embodiments where the composition comprises a combination
of enzymes, the enzyme may be produced individually, by separate
organisms, or two or more of the enzymes may be produced by a
single organism. For example, a single organism can be
recombinantly engineered to produce two or more enzymes by methods
known in the art.
[0076] As discussed above, an animal's immune system recognizes a
number of different molecular patterns displayed by pathogenic
microorganisms, including lipopolysaccharide (associated with, for
example, gram negative bacteria), bacterial flagella containing the
conserved protein flagellin, peptidoglycan (associated with, for
example, gram positive bacteria), lipotechoic acid (associated
with, for example, gram positive bacteria) is bound by the C-type
lectin L-Ficolin, (Lynch, N. J., et al., J. Immunology 172:
1198-1202, 2004), phosphorylcholine (associated with, for example,
gram positive and gram negative bacteria), DNA (such as bacterial
DNA with CpG non-methylated motifs, see Van Uden and Raz, J Allergy
Clin Immunol. 104(5):902-10, 1999.), and double-stranded RNA and
3pRNA (Hornung, et. al., Science 314: 994-997, 2006). The immune
response to these molecules includes an increase in serum APP.
[0077] Other pathogenic molecular patterns include
N-acetylglucosamine containing molecules and N-acetylmannosamine
containing molecules. The exact binding specificity of all
collectins (mannose-binding lectin is a collectin or C-type lectin)
may not be known, but binding to a number of different bacterial
pathogens is observed by for example, H-ficolin,
surfactant-associated protein A (SP-A), and conglutinin. Compounds
like N-acetylglucosamine and N-acetylmannosamine can inhibit the
binding and thus are presumed to be part of the pattern recognition
binding specificity (Haurum, J. S., et al., Biochemical J. 293 (3):
873-878, 1993).
[0078] Examples of other antigens and molecular patterns that can
be targeted for enzyme degradation in accordance with the invention
include bacterial lipoproteins (Hacker, H. et al., J. Exper. Med.
192 (4): 595-600, 2000); .beta.-1,3-glucan binding by the collectin
Dectin-1 (Adachi, Y., et al., Infection and Immunity 72 (7):
4159-4171, 2004); flagellin (which bind the TLF5) (Honko, A. N.,
and Mizel, S. B., Immunol. Res. 33 (1): 83-101, 2005); fucosyl
glycoconjugates; .alpha.-Gal-ceramide; fibrinogen; heparin sulfate;
sulfated gal-saccharide; chitosan, N-acetylglucosamine;
asialoglycoprotein; and .beta.-galactosides.
[0079] The class of receptors called scavenger receptors (SR) are
structurally related to some of the innate immune response
receptors, and may create immune stress. It is believed that SR are
involved in the recycle and clean up of apoptosis or otherwise
damaged cells. The scavenger receptors (SR) expressed by
macrophages and dendritic cells are also receptors for the innate
immune system. Moreover, some SR recognize pathogens and some
innate immune receptors are shown to be important for apoptosis.
Thus, in accordance with one embodiment of the invention, the
molecular pattern binding targets of SR are targeted for enzyme
degradation.
[0080] One such SR molecular pattern-binding target is oxidized low
density lipoprotein (LDL). The receptors called LOX-1 (Peiser, L.,
et al., Current Opinion in Immunology 14:123-128, 2002)
SR-PSOX/CXCL-16 (Fukumoto, N., et al., J. Immunol. 173(3):
1620-1627, 2004) and CD36 (Bruni, F., et al., Clin. Appl. Thromb.
Hemost. 119(4): 417-28, 2005) bind oxidized-LDL that may be present
in some feeds, particularly feeds containing animal by-product
meals such as blood meals.
[0081] Another SR molecular pattern binding target is
phosphatidlyserine (PS) and lyso phosphatidlyserine (lyso PS). SR
for PS include SR-PSOX/CXCL-16 and other PS receptors (Schlegel, R.
A. and Williamson, P., Cell Death Differ. 8 (6): 545-548, 2001).
Exposure to phosphatidylserine phospholipids may lead to
inflammatory responses and phosphatidylserine phospholipids are
believed to present in most feeds at some level.
[0082] Hyaluronan is abundant in extracellular fluids in animals,
but is also recognized by innate immune/scavenger system
mechanisms, for example, in wound healing. See, e.g., Jameson, et
al., J. Expt. Medicine 210 (8): 1269-1279, 2005. Chicken combs are
a commercial source of hyaluronan, typically used in the purified
form of hyaluronic acid. Thus, poultry meal made from byproducts of
meat processing can contain hyaluronan, often in abundant amounts.
Hyaluronidase (EC 3.2.1.35), which degrades hyaluronan and
hyaluronic acid, is useful as an immune-stress reducing enzyme in
accordance with the invention, particularly in the context of
animals that are fed poultry meal. For example, hyaluronidase is
useful in reducing immune stress associated with feeding poultry
meal.
[0083] Enzymes that degrade any of these molecular patterns thereby
inhibit or reduce the immune response, thus reducing the animal's
immunological stress. For example, DNAases and non-specific
nucleases are known that degrade double-stranded RNA and bacterial
DNA. Restriction endonuclease enzymes specific for methylated CG
motifs in non-mammalian DNA are known. Enzymes that degrade
phosphorylcholine include phosphorylcholine hydrolyase, alkaline
phosphatase, acid phosphatase, phosphorylcholine esterase, and
phosphorylcholine phosphatase.
[0084] This stress reduction can be identified and monitored by
measuring the level of serum APP, as described above, with
decreased serum APP concentrations reflecting reduced immunological
stress.
[0085] As noted above, the composition comprises an amount of
immune stress-reducing enzyme that is effective to decrease the
level of acute phase protein in the animal. This amount may vary
from animal to animal, and from enzyme to enzyme, but readily can
be determined by those skilled in the art, for example, by
measuring APP levels, as described above. For example, an animal's
serum APP levels can be measured prior and subsequent to
administration of the enzyme, or serum APP levels of treated and
control animals can be compared. In embodiments where the effective
amount is assessed by measuring serum APP levels prior and
subsequent to administration of the enzyme, the subsequent
measurement can be made from at least about one day to at least
about several days or longer after initial administration of the
enzyme. A decrease in serum APP concentration associated with
administration of the enzyme indicates that an effective amount of
enzyme was administered. It should be understood, however, that APP
levels generally decrease as the animal's adaptive immune response
takes effect.
[0086] In accordance with some embodiments, the present invention
provides composition comprising 1,3-.beta.-glucanase in an amount
effective to effective to decrease the level of positive acute
phase protein in said animal, increase the level of negative acute
phase protein in said animal, and/or improve animal growth
performance. In one specific embodiment, the composition is an
animal feed comprising at least about 20 IU 1,3-.beta.-glucanase/kg
feed, such as at least 20 IU/kg feed, at least 25 IU/kg feed, at
least 30 IU/kg feed, at least 35 IU/kg feed, at least 40 IU/kg
feed, at least 45 IU/kg feed, at least 50 IU/kg feed, or more, of
1,3-.beta.-glucanase. In another specific embodiment, the
composition is a liquid composition other than an animal feed
comprising at least about 155,000 IU 1,3-.beta.-glucanase/L, such
as at least 155,000 IU/L, at least 230,000 IU/L, at least 300,000
IU/L, at least 380,000 IU/L, or more, of 1,3-.beta.-glucanase. In
another specific embodiment, the composition is a solid composition
other than an animal feed comprising at least about 300,000 IU
glucanase/kg, such as at least 300,000 IU/kg, at least 450,000
IU/kg, at least 600,000 IU/kg, at least 750,000 IU/kg, at least
900,000 IU/kg, or more, of 1,3-.beta.-glucanase.
[0087] In some animal feed embodiments where the enzyme comprises
1,3-.beta.-glucanase, the enzyme may be present in amount that is
at least about 100,000 IU per ton feed.
[0088] These amounts are much higher than the 1,3-.beta.-glucanase
content of commercial feed enzyme additives and commercially
available feeds, which the present inventors have analyzed and
found to provide at most from about 10,000 IU/ton feed, about
72,500 IU/L non-feed liquid composition, or about 150,000 IU/kg
non-feed solid composition. The present inventors do not believe
that 10,000 IU/ton feed 1,3-.beta.-glucanase would be effective to
reduce APP, and confirmed this belief experimentally. The present
inventors also determined experimentally that commercial products
such as Avizyme.RTM. (Danisco A/S, Langebrogade 1, Dk-1001,
Copenhagen, Denmark) and Rovobio (Adisseo France SAS, 42, Avenue
Aristide Briand, BP100, 92164 Antony Cedex,) and commercial feeds
comprising standard amounts of .beta.-1,3-1,4-glucanase
(Brewzyme.TM. BG plus, Dyadic International, 140 Intracoastal
Pointe Drive, Suite 404, Jupiter, Fla. 33477-5094), xylanase
(Multifect.RTM. XL, Genencor International, Inc., 925 Page Mill
Road, Palo Alto, Calif.), PI-PLC (ChemGen Corp., 211 Perry Parkway,
Gaithersburg, Md.) and amylase (Amylase FRED, Genencor
International, Inc., 925 Page Mill Road, Palo Alto, Calif.) do not
reduce APP. See Example 3 below and FIG. 3. In the cases where
1,3-.beta.-glucanase activity is present, it is within the low
ranges noted above and not effective to reduce AGP.
[0089] In another embodiment, the immune stress-reducing enzyme is
provided as a component of a composition that also comprises the
antigen or molecular pattern containing compounds that are degraded
by the enzyme. For example, the invention includes an animal feed
comprising .beta.-1,3-glucan and a 1,3-.beta.-glucanase; an animal
feed comprising DNA or double-stranded RNA and a DNAase or and
non-specific nucleases; an animal feed comprising an N-linked
glycoprotein and an endo- or exo-carbohydrase, N-glycanase, or
PNGase, or any of the other enzymes set forth above. Other suitable
combinations of antigens and immune stress-reducing enzymes will be
apparent to those skilled in the art, and are encompassed by the
invention.
[0090] In this embodiment, it is expected that serum APP levels
will remain elevated as long as the composition is administered.
Thus, if the effective amount of immune stress-reducing enzyme is
assessed by measuring serum APP levels prior and subsequent to
administration of the enzyme, the subsequent measurement can be
made days or weeks after initial administration of the enzyme.
[0091] As noted above, the invention includes methods of reducing
immune stress in an animal, comprising administering to the animal
a composition comprising an immune stress-reducing enzyme in an
amount effective to decrease the level of acute phase protein in
the animal. The composition may be any composition described above,
including an oral composition, such as animal feed, a liquid
composition other than an animal feed, or a solid composition other
than an animal feed. The animal may be any animal, including a
human, and may be a healthy animal or an animal suffering from
infection or other disease or condition.
[0092] The invention also includes methods of improving animal
growth performance, comprising administering to the animal a
composition comprising an immune stress-reducing enzyme. In some
embodiments, the composition comprises an amount of immune
stress-reducing enzyme effective to improve animal growth
performance. The composition may be any composition described
above, including an oral composition, an animal feed, a liquid
composition other than an animal feed, or a solid composition other
than an animal feed. The animal may be any animal, including a
human, and may be a healthy animal or an animal suffering from
infection or other disease or condition.
[0093] In one embodiment, the enzyme is expressed by a plant that
is used in animal feed. For example, corn can be genetically
engineered to express an immune stress-reducing enzyme and the
resulting genetically modified corn product can be used in
feed.
[0094] In one embodiment, the animal is administered the immune
stress-reducing enzyme and also is administered the antigen (e.g.,
the pattern-containing molecule degraded by the enzyme). The enzyme
and antigen may be administered separately or simultaneously as
part of the same or different compositions. In one embodiment, the
animal is administered a feed comprising the antigen or pattern
containing molecule, and is separately administered a composition
comprising the immune stress-reducing enzyme. In another
embodiment, the animal is administered a feed comprising the
antigen or pattern containing molecule and a feed supplement
comprising the enzyme. In another embodiment, the animal is
administered a feed comprising both the antigen and the enzyme.
[0095] Another aspect of the invention provides compositions and
methods for reducing immunological stress by preventing and
treating infection caused by pathogenic microorganisms. Sometimes
animals consume compositions, such as water or animal feed, that
comprise pathogenic microorganisms (e.g., bacteria, viruses, fungi
and protozoa), or are otherwise exposed to such pathogens. The
present invention provides compositions comprising an enzyme that
degrades pathogenic microorganism's key components (i.e., a
"pathogenic component"), in an amount effective to decrease
infection and therefore the level of APP expressed in the animal
responding to the infection. The composition is useful for reducing
immunological stress through preventing or minimizing the infection
thereby decreasing the immunological stress caused directly by the
pathogen. In one particular aspect of this embodiment, the
invention provides a method preventing and treating digestive tract
infection.
[0096] By degrading pathogen components, enzymes may also treat or
prevent infection. That is, because a pathogenic component is
degraded, the pathogen could lose its ability to infect the host.
This decrease in actual infection would result in reduced immune
stress and reduction in serum APP by a different mechanism than
described above, but in practice indistinguishable in terms of the
observed APP reduction. There are at least three scenarios where
enzymatic treatment could have a positive result. If the pathogen
molecular structure degraded by the enzyme is involved in binding
of the pathogen to the host cells, the first step required for
infection, or any other key step necessary for successful
infection, then enzymatic treatment could help. Alternatively, the
binding structure on the host cell might be modified. For example a
number of bacterial and protozoan pathogens have been shown in
interact with proteoglycans on the eukaryotic host cells surface,
particularly sulfated proteoglycans (Flekenstein, J. M. et al.,
Infection and Immunity 70 (3): 1530-1537, 2002). The application of
enzymes such as heparinase, and N-acetylglucosamine-4-sulfatase, or
arlysulfatases could reduce the interaction and infection.
[0097] In a second scenario the pathogen molecular structure
degraded could be a toxin that disrupts the target cell's metabolic
functions. In a third scenario, the pathogenic component degraded
by the enzyme might be involved in the pathogen's mechanism to
evade the host immune response. Numerous immune response evasion
mechanisms have evolved in pathogens ranging from mimicking the
host ells outer appearance to inhibiting immune response, for
example complement reactions or apoptosis. The reduction or
prevention of infection also can be assessed by measuring serum
APP, with higher APP levels being associated with infection.
[0098] Enzymes that degrade pathogenic components, such as those
described above, are known in the art. For example, an
endosialidase derived from a bacteriophage was shown to prevent the
lethality of E. coli K1 systemic infection of rats by degrading the
PSA (polysialic acid) capsule on the bacteria surface. Although
degrading the capsular carbohydrate has no effect on the viability
of the E. coli in vitro, loss of capsule in vivo allows recognition
and control of the infection by the host immune system eliminating
lethality (Mushtaq, N., et al. Antimicrobial Agents and
ChemoTherapy 48(5):1503-1508, 2004). The PSA capsule allows the E.
coli surface to look like a host cell thus evading host innate
immune responses. Another known enzyme useful in the present
invention is heparinase I (Neutralase.TM., Ibex Technologies,
Canada). Many enzymes are available from commercial sources or can
be obtained from microorganisms that produce enzymes, such as
bacteria, and fungi including yeast, or can be produced
recombinantly, as discussed above.
[0099] Desired enzymes can be produced by recombinant DNA
techniques when the gene coding for the enzyme is known. The
advancement of rapid DNA sequencing methodology has resulted in
large public databases of proteins and their gene coding sequences,
such as the NCBI Genbank. Using rapid sequencing technology from,
for example, 454 Life Sciences (454 Life Sciences, 20 Commercial
Street, Branford, Conn. 06405), a typical bacterial genome can be
sequenced in four hours. A previously unknown gene of new desired
enzyme from the genome can be obtained by probing the genome using,
for example, previously identified coding sequences from the same
type or similar types of enzymes described in commercial or public
databases, using readily available computer programs such as Blast.
Those skilled in the art can identify DNA in the genome that has a
threshold level of homology to the known sequence and other
properties of a gene-coding region, and then isolate and amplify
the gene using, for example, polymerase chain reaction (PCR)
technology. The gene can then be expressed in a host and its
desired protein enzymatic properties can be confirmed.
[0100] If a desired enzyme activity is not previously known, then
it can be located using standard microbiology enrichment techniques
selecting for growth on the substrate. Microbes using the substrate
as the sole carbon or nitrogen source must express enzymes capable
of degrading the target compound. In order to develop economical
production, one has the choice to improve the production of that
enzyme using classical mutation/selection or enrichment methods
with the producing microorganism, or through recombinant DNA
expression methods well known in the art.
[0101] The composition comprising an immune stress-reducing enzyme
that degrades a pathogenic microorganism may be any composition
suitable for administration to an animal, including compositions
suitable for oral administration to an animal, as described above.
As noted above, the composition may comprise an amount of enzyme
that is effective to decrease the level of positive acute phase
protein (or increase the level of negative acute phase protein) in
the animal and/or improve animal growth performance. This amount
may vary from animal to animal, and from enzyme to enzyme, but
readily can be determined by those skilled in the art, for example,
by measuring APP levels and/or monitoring animal growth
performance, as described above and as known in the art.
[0102] In one embodiment, the immune stress-reducing enzyme that
targets a pathogenic antigen is provided as a component of an
animal feed. In one example of this embodiment, the amount of
enzyme is at least about 100,000 IU/ton feed.
[0103] In another embodiment, the immune stress-reducing enzyme
that targets a pathogenic antigen is provided as a component of a
composition that also comprises the pathogenic antigen. For
example, the invention includes an animal feed comprising (A) a
pathogenic microorganism displaying an antigen such as
lipopolysaccharide, peptidoglycan, lipotechoic acid,
phosphorylcholine, double-stranded RNA and DNA and (B) and enzyme
that degrades the antigen. Pathogenic organisms can find the way
into feed due to the inherent nature of unsanitary conditions
caused by the dense growth of animals in production situations.
[0104] As noted above, the invention includes methods of reducing
immune stress in an animal and/or of improving animal growth
performance, comprising administering to the animal a composition
comprising an immune stress-reducing enzyme. In one embodiment, the
animal is administered the immune stress-reducing enzyme that
degrades a pathogenic antigen and also is administered the
pathogenic antigen. The enzyme and antigen may be administered
separately or simultaneously as part of the same or different
compositions. In one embodiment, the animal is administered a feed
comprising the antigen, and is separately administered a
composition comprising the enzyme. In another embodiment, the
animal is administered a feed comprising the antigen and a feed
supplement comprising the enzyme. In another embodiment, the animal
is administered a feed comprising both the antigen and the
enzyme.
[0105] The following examples further illustrate the invention, but
the invention is not limited to the specifically exemplified
embodiments.
Example 1
[0106] An animal feed comprising hemicellulase
(endo-1,4-.beta.-mannanase) was prepared and administered to
chickens and AGP levels were measured, as described in more detail
below.
[0107] A total of 4000 one-day-old male Cobb.times.Cobb chicks were
allocated at random to 8 experimental treatments, and each
treatment was replicated 10 times: [0108] Experimental Design: 8
Treatments [0109] Total No. of pens: 80 [0110] Total No. of
Treatments: 8 [0111] No. of birds per pen: 50 [0112] No. of pens
per Treatment: 10 [0113] No. of birds per Treatment: 500
[0114] Two of the eight treatments comprised stress-reducing
enzymes in accordance with the invention: Treatment 3 (mannanase in
the form of evaporated whole cell broth of B. lentus fermentation
applied at approximately 100 MU/ton in the basal diets) and
Treatment 6 (mannanase in the form of cell-free centrifuged
supernatant of B. lentus fermentation broth applied at
approximately 30 MU/ton in the basal diets). Treatment 8 was a
control with no added enzyme. (1 MU=4000 IU)
[0115] The basal meal feed batches were divided evenly in eight
parts and each was sprayed with the appropriate amount of the test
materials. Starter and Grower feeds contained 90 g/ton Cobon.TM.
(an anticoccidial drug of the ionophore type) plus 50 g/ton
BMD.RTM. (antibiotic). Finisher feeds were non-medicated.
[0116] Starter Diets were offered to all birds from day-old until
21 days of age, Grower Diets from 22-35 days, and Finisher Diets
from 36-42 days. The diets and water were provided ad libitum. The
diets were presented to the birds as crumbles/pellets during all
feeding periods. Tap water was used as drinking water and supplied
by an internal water system network.
TABLE-US-00002 Composition and Analyses of the Basal Experimental
Diets Ingredients Starter Grower Finisher Corn 60.3851 67.6864
72.1098 Soybean meal (48.5% CP) 34.5066 27.8363 23.3785 Fat AV
Blend 1.0516 0.9915 1.1389 Dicalcium phosphate 1.761 1.2682 1.3021
Limestone flour 1.3192 1.383 1.26 Sodium chloride 0.3299 0.3304
0.3305 DL Methionine 0.2135 0.0793 0.0552 L-lys.hcl 0.008 -- --
Choline chloride 70% 0.05 0.05 0.05 Vitamin premix 0.25 0.25 0.25
Mineral premix 0.075 0.075 0.075 Coban, g/ton 90 90 -- BMD, g/ton
50 50 -- Calculated Analyses.sup.2 ME.sub.n poultry (kcals/kg)
3080.0 3150.0 3200.0 Dry matter, % 88.9169 88.9236 88.9054 Crude
protein, % 22.0 19.3 17.5 Crude fibre, % 2.8813 2.8176 2.7632 Fat,
%, 3.6777 3.8291 4.0981 Calcium, % 1.0 0.9 0.85 Total phosphorus, %
0.7088 0.5967 0.5877 Available phosphorus, % 0.45 0.35 0.35 Sodium,
% 0.18 0.18 0.18 Lysine, % 1.2 1.0152 0.8948 Methionine + Cysteine,
% 0.92 0.72 0.65 Threonine, % 0.8821 0.7657 0.6932 Tryptophan, %
0.2938 0.2489 0.2185
[0117] Two birds from each of the ten pens in Treatments 3, 6 and 8
were randomly selected for blood analysis at the end of the 42 days
after weighing, for a total of 20 birds out of the 500 per
treatment. Samples were collected onto ice in blood collection
tubes containing anti-coagulant heparin, and plasma was obtained by
centrifugation.
[0118] Blood plasma samples were assayed for chicken .alpha.-1-acid
glycoprotein using an immunodiffusion based assay kit from
Cardiotech Services, Inc. (Louisville, Ky.). Serum samples taken
from the two birds/pen were added to the test plates (5 .mu.L per
well) and to some wells standard pure AGP was added at
concentrations ranging up to 1000 .mu.g/mL. Precipitin rings were
measured using a precipitin ring measurement scale to the nearest
0.1 mm diameter.
[0119] A polynomial equation was used to provide the best curve fit
with the data and to allow the rapid calculation of the
concentration of AGP in the plasma samples as shown in FIG. 1.
[0120] The measurements of precipitin ring diameters for all the
recovered chicken serum samples and the calculated AGP
concentration for each bird is shown in the table below. The birds
fed mannanase on average have a very statistically significant
decrease in the average AGP concentration compared to the control
birds.
TABLE-US-00003 42 Day Blood Samples (y = AGP ug/ML; x = ring
measurement in mm) Treatment 3 Treatment 8 Treatment 6 (Mannanase)
(control) (Mannanase) X y x y X y 5.3 225.0 6 317.7 5.3 225.0 5.3
225.0 6 317.7 5.7 276.2 4.9 178.6 6.1 332.1 5.8 289.7 5.2 212.9 5.2
212.9 5.1 201.2 5.8 289.7 7.4 546.9 5.4 237.4 5.4 237.4 5.4 237.4
5.7 276.2 5.4 237.4 6.2 346.9 5.5 250.0 5.9 303.6 5.9 303.6 6.1
332.1 5.6 262.9 6 317.7 5.4 237.4 5.9 303.6 6.2 346.9 5.2 212.9 5.2
212.9 6.1 332.1 5 189.7 5.4 237.4 5.6 262.9 6.1 332.1 5.3 225.0 5.9
303.6 5.5 250.0 5.1 201.2 6.3 361.9 5.5 250.0 5.3 225.0 6.2 346.9 6
317.7 5.3 225.0 7.5 565.5 5.4 237.4 5.7 276.2 7.4 546.9 5.6 262.9
5.3 225.0 7.5 565.5 4.4 127.2 5.7 276.2 7.5 565.5 5 189.7 6.1 332.1
AVE 238.5 373.1 250.3 SD 35.8 115.6 51.3 CV 14.99 30.97 20.50 T
Test p vs. 8 2.94E-05 T Test vs. 8 0.000193
Example 2
[0121] Another experiment using hemicellulase
(endo-1,4-.beta.-mannanase) was conducted. In this experiment,
groups of chickens (10 pens each, with 50 birds per pen) were fed
one of four diets:
[0122] Treatment 1 (control): Feed comprising BMD antibiotic
sprayed post-pelleting with a control formulation, and 35% sorbitol
with brown food dye, applied at 100 ml/ton feed.
[0123] Treatment 2 (control): Feed without BMD sprayed
post-pelleting with a control formulation comprising 35% sorbitol
with brown food dye, applied at 100 ml/ton feed.
[0124] Treatment 3: Feed sprayed post-pelleting with a formulation
comprising hemicellulase (endo-1,4-.beta.-mannanase) derived from
B. lentus, applied at 100 ml/ton feed.
[0125] Treatment 4: Feed formulated with a powder composition
(added into the mixer prior to pelleting) comprising hemicellulase
(endo-1,4-.beta.-mannanase) derived from B. lentus at 454 g of
composition added/ton feed to provide 100 MU/ton of feed. (1
MU=4000 IU).
[0126] The chickens were 1 day old at the start of the
experiment.
[0127] The diets were provided ad libitum. Starter (days 0-21),
grower (days 21-35) and finisher (days 35-42) feeds with the
following compositions were used as the base feeds:
TABLE-US-00004 Expected Amounts Nutrient Analysis Starter Grower
Finisher ME POUL KCAL 2960.0 3020.0 3080.0 Crude Protein 22.0 19.4
17.5 Fat % 3.1439 3.0647 3.4503 Calcium % 0.9 0.8 0.8 T phos 0.7032
0.6315 0.515 A Phos 0.45 0.39 0.35 Sodium 0.18 0.18 0.18 Lysine %
1.205 1.0302 0.9014 Methionine 0.5446 0.3838 0.3435 Met + Cys 0.92
0.72 0.65
TABLE-US-00005 Added Ingredients Ingredient Starter Grower Finisher
Limestone 0.8291 0.7674 0.9012 Salt 0.2696 0.2698 0.2702 D-L Meth
0.1963 0.0682 0.0532 Choline Chloride 70% 0.0500 0.0500 0.0500
Dical P 1.6869 1.4088 1.2295 Fat 0.6517 0.4751 0.8162 Corn 59.3480
67.4725 70.9038 Soybean meal 33.5934 27.1132 22.4010 Poultry
By-Product 3.0 3.0 3.0 meal Vitamin 0.25 0.25 0.25 Mineral 0.075
0.075 0.075 Salinomycin 0.05 0.05 0.05
[0128] On day 21 approximately 3 ml of blood were collected from 3
birds per pen (30 per group). Blood was placed into a heparinized
tube and lightly mixed. Tubes were slowly centrifuged and then
serum was removed. Serum samples were placed into tubes with caps
and labeled with pen number. Serum was frozen for subsequent AGP
analysis, as described in Example 1 above. The immunodiffusion
rings used to quantitate chicken .alpha.1 acid glycoprotein are
easily measured, highly reproducible and exhibit a coefficient of
variation of 4% or less.
[0129] The 21 day average results of 30 birds per treatment are
shown in the table below and graphically in FIG. 2. It can be seen
that leaving the antibiotic (BMD) out of the diet creates a large
and significant increase in the plasma AGP level (compare Treatment
1 and Treatment 2). The addition of either hemicellulase
(endo-1,4-.beta.-mannanase) formulation into the no-BMD diet
(Treatments 3 and 4) restored the AGP to the level seen with
antibiotic use, indicating a significant reduction of immunological
stress.
TABLE-US-00006 Treatment 1 2 3 4 AGP Avg. 214.35 267.99 220.28
233.09 SD 62.16 82.42 68.58 67.73 CV 29.00 30.76 31.13 29.06 TTest
P 0.003055 0.008952 0.03919 vs. Trt. 1 Trt. 2 Trt. 2 TTest P
0.234892 vs. Trt. 3 TTest P 0.363305 0.134383 vs. Trt. 1 Trt. 1
[0130] The growth performance of the chickens also was assessed,
with the results summarized in the table below.
TABLE-US-00007 Growth Performance Wt. Wt. adj. CV of P FCR.sup.1 P
val. gain P val. FCR.sup.2 P val. ID.sup.3 val. Day 21 T1 1.394
0.150 0.693 0.016 1.379 0.017 13.81 0.33 T2 1.412 0.657 1.424 14.77
T3 1.404 0.605 0.673 0.197 1.404 0.248 14.03 0.46 T4 1.407 0.740
0.670 0.459 1.410 0.551 14.40 0.69 Day 42 T1 1.776 0.006 2.102
0.189 1.772 0.007 11.23 0.19 T2 1.813 2.073 1.820 10.42 T3 1.770
0.001 2.131 0.088 1.756 0.005 9.97 0.49 T4 1.761 0.0001 2.060 0.572
1.772 0.003 10.38 0.95 .sup.1FCR = Feed conversion .sup.2Wt. Adj.
FCR = weight adjusted feed conversion .sup.3CV of ID = coefficient
of variation in individual weights
[0131] Thus, both feed conversion and weight adjusted feed
conversion were improved at 21 days with statistical significance
in chickens receiving .beta.-mannanase. This indicates that the
reduction in serum AGP can translate into real significance for
animal performance.
Example 3
[0132] The ability of other enzymes commonly used in animal feed
were assessed for their possible effect on AGP. Commercial type
chicken starter rations (low metabolic energy) were compounded with
feedstuffs commonly used in the United States. These rations (in
mash or crumble form) were fed ad libitum from the date of chick
arrival until Day 21 of the study. Experimental treatment feeds
were prepared from this basal starter feed. Treatment feeds were
mixed to assure a uniform distribution of respective test
article.
TABLE-US-00008 Composition and Analyses of the Basal Experimental
Diets Ingredients Corn 59.398 Soybean meal (48.5% CP) 33.5934 Fat
AV Blend 0.6517 Dicalcium phosphate 1.6869 Limestone flour 0.8291
Sodium chloride 0.2696 DL Methionine 0.1963 Poultry By Product Meal
3.0 Choline chloride 70% 0.05 Vitamin premix 0.25 Mineral premix
0.075 LO ME ME.sub.n poultry (kcals/kg) 2960 Crude protein, % 22.0
Crude fibre, % 2.8899 Fat, %, 3.1439 Calcium, % 0.9 Total
phosphorus, % 0.7032 Available phosphorus, % 0.45 Sodium, % 0.18
Lysine, % 1.205 Methionine + Cysteine, % 0.92 Threonine, %
0.8266
BMD 50 g/t and Salinomycin 60 g/t were added to all feeds.
TABLE-US-00009 Enzyme Preparations Sample enzyme assay data Stock
vol Diluent Notes Use Level 3 -- n.d. 20 plus food 20 mL/100 Kg
coloring 6 Adessio, Rovabio 10 10 20 mL/100 Kg Rovabio commercial
Excel LC product 7 Danisco, Avizyme Solid -- 100 g/100 Kg Avizyme
commercial product (1500 product Granular) 8 PI-PLC 106 U/mL 50 --
1.0 mL/Kg 9 Genencor 20 mL/100 Kg Amylase FRED 10 Genencor 20
mL/100 Kg Multifect XL 11 Dyadic 20 mL/100 Kg Brewzyme BG 12
Hemicell 1092 MU/L 13 12 20 mL/100 Kg 17 -- n.d. 20 plus food 20
mL/100 Kg coloring (1 MU = 4000 IU)
TABLE-US-00010 Enzyme Preparations Further Information Endo-1,3
.beta.-glucanase Sample Main Activity Use Level minor activity* 3
-- 6 Endo-1,4-.beta.- Endo-1,4-.beta.-Xylanase 9139 IU/ton Xylanase
350 AXC 350 AXC U/Ml 63,350 AXC U/ton Endo .beta.-1,4- Endo
.beta.-1,4- .beta.-glucanase .beta.-glucanase 500 AGL U/mL 90,500
AGL U/ton 7 Amylase 1.0 kg per ton -- xylanase protease 8 PI-PLC
96,188 IU/ton or -- 106,000 IU/L 24 MU/ton 9 Amylase 4700 MU/L 1.88
.times. 10.sup.6 IU/ton or -- 470 MU/ton 10 Endo-1,4-.beta.-
900,000 IU/ton or -- Xylanase 225 MU/ton 4500 MU/L 11 Endo
.beta.-1,4-1,3- 634,400 IU/ton or 1040 IU/ton glucanase 159 MU/ton
1586 MU/L 12 Endo-1,4 .beta.- 400,000 IU/ton or 580 IU/ton
mannanase 100 MU/ton 1092 MU/L 17 -- ChemGen MU = 4000 IU; AXC -
xylanase units defined by Adisseo; AGL - glucanase units defined by
Adisseo; *approximate level measured by ChemGen Corp. by reducing
sugar assay
[0133] Feed and water were available ad libitum throughout the
trial. On Day 15, birds in treatment 17 were orally inoculated with
a mixed inoculum containing approximately 30,000 oocysts E.
acervulina per bird, 2,500 oocysts of E. maxima per bird, and
25,000 oocysts E. tenella per bird. Coccidial oocyst inoculation
procedures are described in SPR SOP: IN1.002.
[0134] Means for cage weight gain, feed consumption, and feed
conversion are determined. The results are set forth below. Only
animals receiving sample 17 were infected.
TABLE-US-00011 21 Day Growth Data Vs. Treatment Avg. 3 Live Enzyme
TTEST AGP Wt. level per Sample P = Treatment ave Gain Conv. metric
Ton 3 -- control 170.52 0.624 1.438 none 6 0.2076 Rovabio 186.55
0.626 1.395 100 mL 7 0.2770 Avizyme 160.44 0.633 1.426 1.0 kg 8
0.1263 PI-PLC 196.35 0.650 1.406 106,000 IU 9 0.3962 Amylase 164.05
0.622 1.434 10 0.3783 Xylanase 175.89 0.593 1.444 11 0.2647
Glucanase 182.00 0.629 1.396 12 0.0178 Hemicell 138.22 0.645 1.421
102 MU 17 0.0043 control - 252.04 0.564 1.507 none infected
[0135] We found that commercial feeds comprising standard amounts
of amylase, 1,3-glucanase, 1,4-glucanase, xylanase and PI-PLC did
not reduce AGP levels. Indeed, only hemicellulase
(endo-1,4-.beta.-mannanase) showed a significant effect on AGP
levels. Additionally, comparing treatment 1 and 17 clearly shows
that AGP is a highly responsive APP in chickens because infection
increased the AGP level 82 .mu.g/mL. See also FIG. 3.
Example 4
[0136] Test animal feed comprising 1,3-.beta.-glucan is formulated
to include 1,3-.beta.-glucanase at a concentration of 400,000 IU
(100 ChemGen MU) per ton feed. The test animal feed is administered
to test chickens, while control chickens receive the same animal
feed (comprising 1,3-.beta.-glucan) without 1,3-.beta.-glucanase.
After 21 and 42 days on this regimen, blood serum AGP levels are
assessed as described above. Chickens receiving the
enzyme-formulated animal feed have significantly lower levels of
AGP than control animals. The test chickens also exhibit greater
feed efficiency and improved weight gain as compared to control
chickens.
Example 5
[0137] Test animal feed comprising a source of bacterial DNA (e.g.
Biolys.RTM. Lysine or other fermentation product containing cell
products) is formulated to include non-specific nuclease derived
from the Cyanobacterium Anabaena sp. 7120 (NucA), one of the most
active non-specific nucleases known (Meiss, G. et al., Eur. J.
Biochem. 251(3): 924-934, 1998). The enzyme is added at a
concentration of 1.times.10.sup.7 Kunitz Units enzyme/kg feed or
approximately 1 mg (pure basis) per kg of feed. The test animal
feed is administered to test chickens, while control chickens
receive the same animal feed (comprising bacterial DNA) without
non-specific nuclease. After 21 days or 42 days on this regimen,
blood serum AGP levels are assessed as described above. Chickens
receiving the enzyme-formulated animal feed have significantly
lower levels of AGP than control animals.
Example 6
[0138] Test animal feed comprising meat and bone meal, blood meal
or other animal derived by-product is formulated to include
phosphatidylserine decarboxylase at a concentration of 400,000
IU/ton of feed. The test animal feed is administered to test
chickens, while control chickens receive the same animal feed
without phosphatidylserine decarboxylase. After 21 days or 42 days
on this regimen, blood serum AGP levels are assessed as described
above. Chickens receiving the enzyme-formulated animal feed have
significantly lower levels of AGP than control animals.
Example 7
[0139] Test animal feed soy meal or other plant derived meal is
formulated to include an .alpha.-mannanase and/or
1,3-.beta.-glucanase enzymes derived from B. lentus, each at a
concentration of 400,000 IU/ton of feed. The test animal feed is
administered to test chickens, while control chickens receive the
same animal feed without .alpha.-mannanase or 1,3-.beta.-glucanase.
After 21 days or 42 days on this regimen, blood serum AGP levels
are assessed as described above. Chickens receiving the
enzyme-formulated animal feed have significantly lower levels of
AGP than control animals.
Example 8
[0140] In this example Hemicell.RTM. mannanase added to feed (a
conventional corn-soybean diet) was shown to reduce al acid
glycoprotein (AGP) in turkey serum while also improving live growth
performance. The experiment consisted of 48 pens of 11 tom turkeys
(initial placement). The six treatments were replicated in 8
blocks, randomized within blocks of six pens each:
TABLE-US-00012 No. Birds/Treatment 88 No. Reps/Treatment 8 Total
Treatments 6 Total No. Pens 48 Total No. Birds 528
[0141] One treatment that comprised stress-reducing enzymes in
accordance with the invention, Treatment 1 (commercial
Hemicell.RTM. with 100 MU/ton of feed) was analyzed for AGP. (1
MU=4000 IU) Treatment 2 was a control feed without added
enzyme.
[0142] Feed was mixed to assure uniform distribution of basal feeds
among treatments. All enzymes were mixed (sprayed on) to assure a
uniform distribution of test enzymes and to assure similar feed
condition between treatments. Each time treatment feed was made, a
sample from the beginning, middle, and end of each treatment feed
were mixed to form a composite sample. One sample was taken from
the composite for each treatment, and for enzyme level
verification.
[0143] The turkey diets fed in this study to Treatments 1 and 2 are
described in detail below in the following tables. Tables show the
composition of components, the calculated nutrient levels and
finally some measured nutrient values with the returned feeds. The
diets are representative of what might be used in a commercial
turkey growing operation and thus the diet is adjusted several
times throughout the 20-week period. Diet compositions were changed
at 6, 9, 12, 15 and 18 weeks.
[0144] The diet compositions at each period are slightly different
for Treatments 1 and 2. It is well known that Hemicell.RTM.
mannanase has the effect of increasing the effective energy content
of feeds (see U.S. Pat. No. 6,162,473). For that reason, the diets
of Treatment 1 are formulated with fewer calories than the diets of
Treatment 2 in order to minimize growth difference between
Treatments 1 and 2 for the purpose of this study.
TABLE-US-00013 Ingredient composition and calculated nutrient
levels, 0-9 weeks Period: 0-6 weeks 6-9 weeks Treatment 1 Treatment
1 with with Ingredient (%) Hemicell .RTM. Treatment 2 Hemicell
.RTM. Treatment 2 Corn 46.77 45.39 53.14 51.79 Soybean meal 37.15
37.40 29.30 29.50 Poultry meal 9.00 9.00 9.00 9.00 Poultry Fat 1.50
2.65 3.50 4.65 Limestone 1.20 1.20 1.25 1.25 Dical phosphate 18.5
2.70 2.70 2.35 2.35 Salt 0.325 0.325 0.315 0.32 DL Methionine 0.315
0.315 0.245 0.25 L-Lysine-HCl 0.41 0.405 0.335 0.34 Vitamin pre-mix
0.25 0.25 0.25 0.25 Trace minerals 0.075 0.075 0.075 0.075 Choline
Cl 60% 0.135 0.135 0.085 0.085 Copper sulfate 0.05 0.05 0.05 0.05
Coban 60 g/lb 0.055 0.055 0.05 0.05 BMD 50 g/lb 0.05 0.05 0.05 0.05
Hemicell .RTM. 0.0125 0.0 0.0125 0.0 Crude protein (%) 28.00 28.00
24.5 24.5 ME (Kcal/lb) 1323 1323 1408 1407 Calcium (%) 1.484 1.484
1.462 1.462 A. Phosphorus (%) 0.797 0.797 0.764 0.763 Lysine (%)
1.794 1.793 1.502 1.501 Met + Cys (%) 1.179 1.177 1.018 1.021
TABLE-US-00014 Ingredient composition and calculated nutrient
levels, 9-15 weeks Period: 9-12 weeks 12-15 weeks Treatment 1
Treatment 1 Ingredient (%) with Hemicell .RTM. Treatment 2 with
Hemicell .RTM. Treatment 2 Corn 56.88 55.55 62.45 61.05 Soybean
meal 24.55 24.75 21.15 21.40 Poultry meal 9.00 9.00 7.00 7.00
Poultry Fat 5.00 6.22 5.00 6.15 Limestone 1.20 1.20 1.15 1.15 Dical
phosphate 18.5 1.95 1.95 1.75 1.75 Salt 0.32 0.32 0.32 0.32 DL
Methionine 0.22 0.22 0.30 0.30 L-Lysine-HCl 0.315 0.315 0.42 0.42
Vitamin pre-mix 0.25 0.25 0.25 0.25 Trace minerals 0.075 0.075
0.075 0.075 Choline Cl 60% 0.085 0.085 0.015 0.015 Copper sulfate
0.05 0.05 0.05 0.05 Coban 60 g/lb 0.05 0.05 0.00 0.00 BMD 50 g/lb
0.05 0.05 0.05 0.05 Hemicell 0.0125 0.0 0.0125 0.0 Crude protein
(%) 22.5 22.5 20.0 20.0 ME (Kcal/lb) 1469 1469 1490 1490 Calcium
(%) 1.35 1.35 1.19 1.19 A. Phosphorus (%) 0.681 0.681 0.59 0.59
Lysine (%) 1.350 1.350 1.298 1.298 Met + Cys (%) 0.940 0.940 0.95
0.95
TABLE-US-00015 Ingredient composition and calculated nutrient
levels, 15-20 weeks Period: 15-18 weeks 18-20 weeks Treatment 1
with Treatment 1 with Ingredient (%) Hemicell .RTM. Treatment 2
Hemicell .RTM. Treatment 2 Corn 67.25 65.85 70.60 69.15 Soybean
meal 17.90 18.15 15.60 15.85 Poultry meal 5.00 5.00 4.00 4.00
Poultry Fat 6.00 7.15 6.50 7.70 Limestone 1.00 1.00 0.85 0.85 Dical
phosphate 1.50 1.50 1.23 1.23 18.5 Salt 0.33 0.33 0.34 0.34 DL
Methionine 0.205 0.205 0.193 0.193 L-Lysine-HCl 0.340 0.340 0.235
0.235 Vitamin pmx 0.25 0.25 0.25 0.25 Trace minerals 0.075 0.075
0.075 0.075 Choline Cl 60% 0.02 0.02 0.02 0.02 Copper sulfate 0.05
0.05 0.05 0.05 Coban 60 g/lb 0.00 0.00 0.00 0.00 BMD 50 g/lb 0.05
0.05 0.05 0.05 Hemicell 0.0125 0.0 0.0125 0.0 Crude protein (%)
17.5 17.5 16.0 16.0 ME (Kcal/lb) 1539 1539 1570 1570 Calcium (%)
0.982 0.982 0.82 0.82 A. Phosphorus 0.490 0.490 0.41 0.41 (%)
Lysine (%) 1.10 1.10 0.93 0.93 Met + Cys (%) 0.791 0.791 0.74
0.74
TABLE-US-00016 Diet analysis with returned feeds, 0-12 weeks
Treatment 1 with Hemicell .RTM. Treatment 2 Nutrient Calculated
Analyzed Calculated Analyzed 0-3 weeks Protein 28 27.46 28 27.57
Fat 4.2 4.03 5.3 4.99 Calcium 1.48 1.38 1.48 1.45 T. Phosphorus
1.02 0.96 1.02 1.08 Hemicell units 100 70.1 0 8.9 3-6 weeks Protein
28 25.90 28 27.17 Fat 4.2 4.01 5.3 5.16 Calcium 1.48 1.63 1.48 1.52
T. Phosphorus 1.02 1.15 1.02 1.12 Hemicell units 100 99.3 0 8.9 6-9
weeks Protein 24.5 23.42 24.5 24.17 Fat 6.3 6.33 7.4 7.00 Calcium
1.46 1.69 1.46 1.65 T. Phosphorus 0.96 1.09 0.96 1.06 Hemicell
units 100 138.6 0 14.4 9-12 weeks Protein 22.5 22.61 22.5 23.61 Fat
7.9/8.0 7.91 9.0 9.02 Calcium 1.35 1.37 1.35 1.32 T. Phosphorus
0.86 0.93 0.86 0.90 Hemicell units 100 83.9 0 12.0
TABLE-US-00017 Diet analysis with returned feeds, 12-20 weeks
Treatment 1 with Hemicell .RTM. Treatment 2 Nutrient Calculated
Analyzed Calculated Analyzed 12-15 weeks Protein 20 20.49 20 21.09
Fat 7.79 8.25 8.89 9.03 Calcium 1.19 1.16 1.19 1.09 T. Phosphorus
0.76 0.78 0.76 0.77 Hemicell units 100 90.5 0 13.6 15-18 weeks
Protein 17.5 17.20 17.5 16.24 Fat 8.68 8.00 9.78 9.21 Calcium 0.98
0.96 0.98 0.94 T. Phosphorus 0.65 0.68 0.65 0.70 Hemicell units 100
98.6 0 5.6 18-20 weeks Protein 16.0 15.95 16.0 15.14 Fat 9.14 8.93
10.29 10.39 Calcium 0.82 0.79 0.82 0.87 T. Phosphorus 0.57 0.61
0.57 0.65 Hemicell units 100 113.4 0 13.6
Glycoprotein Measurement:
[0145] Blood was obtained at the end of the trial from four birds
per pen selected at random from treatments 1, 2, and 5. The blood
was collected into tubes containing EDTA anticoagulant, mixed then
centrifuged to precipitate the whole cells.
[0146] Turkey AGP test plates were obtained from Cardiotech
Services (Louisville, Ky.). The AGP test is an immunodiffusion
based test. Equal volumes of test or serum samples were added into
the immunodiffusion plate wells as recommended by the manufacturer,
then after two days incubation at room temperature, the diameter of
the resulting immunoprecipitation rings was measured.
[0147] A sample of purified turkey AGP standard provided in the kit
was tested at several concentrations to make a standard curve as
shown in FIG. 4. A polynomial curve fit equation developed from the
standards was used to calculate the turkey plasma AGP level in the
test samples.
TABLE-US-00018 Calculation of AGP levels and statistical analysis
with Students T test Hemicell mannanase (Treatment 1) Control
(Treatment 2) mm AGP outlier mm AGP outlier 5.2 231.5 5.7 304.5 4.8
181.6 5.1 218.4 5 205.7 5.6 288.9 5 205.7 5.3 245.1 5 205.7 5.1
218.4 5.1 218.4 5.1 218.4 5.3 245.1 5.9 337.3 6.1 372.3 5 205.7 5.8
320.6 6.3 409.6 4.7 170.2 7.5 687.1 4.9 193.4 6.45 439.1 5 205.7
5.4 259.2 5.2 231.5 5.1 218.4 5.5 273.8 5.3 245.1 5.7 304.5 5.1
218.4 5.1 218.4 5.6 288.9 4.7 170.2 5.6 288.9 5 205.7 6.05 363.3
4.5 148.5 5.6 288.9 5.3 245.1 5.4 259.2 5.2 231.5 5 205.7 4.4 138.3
5 205.7 4.3 128.4 5.2 231.5 6.35 419.3 5.4 259.2 4.9 193.4 4.8
181.6 5.65 296.6 5 205.7 5.2 231.5 5.2 231.5 5 205.7 5.4 259.2 4.8
181.6 4.9 193.4 5.2 231.5 5.1 218.4 7.5 687.1 4.2 118.9 5 205.7 6.5
449.3 Ave 226.4 260.5 Std. 63.4 74.6 Deviation CV 28.0 28.6 T Test
P value 0.0284 Outliers >2 std. deviation from mean removed
[0148] The average plasma AGP for the enzyme treated group was
significantly less than the untreated control. For this analysis,
one outlier was removed from the analysis from each group. These
may be birds that experienced an unusual amount of stress due to
injury or infection. The reduced AGP caused by enzyme feeding was
correlated with statistically significant improved live bird
performance as shown below in Treatment 1 (mannanase) vs. Treatment
2.
TABLE-US-00019 140 Day Growth Results Live CV in Mort. Feed Feed
Wt. weight Pen Weight Mort. # Weight Consumed Conv. Gain 140 d
Hemicell 3 200.25 0 0 487.35 2.434 18.147 5.766 7 193.3 0 0 479.55
2.481 17.512 7.426 14 143.95 3 10.817 389.05 2.514 17.934 6.857 21
164.1 2 24.205 448.70 2.383 18.172 4.411 29 185.8 1 0.975 448.90
2.403 18.521 6.092 36 161.1 2 16.49 419.90 2.364 17.839 8.352 37
182.1 1 7.611 443.70 2.339 18.150 5.944 47 182.55 1 13.115 449.05
2.295 18.195 3.405 Avg. 445.78 2.402 18.059 6.032 T Test P val 0.05
0.01 0.05 Vs. Trt 2 Control 5 144.1 2 13.085 389.00 2.475 15.950
14.518 12 196.1 0 0 486.70 2.482 17.766 5.288 13 178.7 1 14.65
466.25 2.411 17.810 9.717 23 141.95 3 21.912 389.40 2.376 17.684
8.012 28 190.2 0 0 482.10 2.535 17.231 9.331 35 194.5 0 0 486.25
2.500 17.622 9.722 42 159.25 2 10.814 409.40 2.407 17.635 5.054 48
176.65 1 9.05 459.10 2.472 17.605 4.772 Avg. 446.03 2.457 17.413
8.302
[0149] Birds receiving feed with mannanase had greater average
weight gain by 3.7%, decreased feed conversion by 2.3% and decrease
in the CV (coefficient of variation=std. deviation/mean) of body
weight uniformity. The reduction in immune stress as indicated by
reduced AGP serum levels correlated with several measurements of
growth improvement.
Example 9
[0150] In this example 1,4-.beta.-mannanase from B. lentus,
1,3-.beta.-glucanase from B. lentus, and a combination of the two
enzymes were added to feed (a conventional corn-soybean diet). Each
enzyme treatment improved live growth performance in 6 week old
Nicholas 700 female turkeys, with results achieved by the
combination being unexpectedly greater than results achieved with
treatments using only one of the enzymes.
[0151] The experiment used 80 pens of 40 female turkeys. The
treatments were replicated in ten (10) blocks, with eight
treatments (seven enzyme treatments and one negative control)
randomized within each block.
[0152] Treatment 3 used a composition comprising an immune
stress-reducing enzyme in accordance with the invention,
1,4-.beta.-mannanase at 100 MU/ton of feed. Treatment 6 also used a
composition comprising an immune stress-reducing enzyme in
accordance with the invention, 1,3-.beta.-glucanase at 60 MU/ton of
feed. Treatment 8 used a combination composition in accordance with
the invention, comprising 1,4-.beta.-mannanase at 100 MU/ton of
feed and 1,3-.beta.-glucanase at 60 MU/ton of feed. Treatment 1 was
a control feed without added enzyme. (1 MU=4000 IU).
[0153] Feed was mixed to assure uniform distribution of basal feeds
among treatments. All enzymes were mixed (sprayed on) to assure a
uniform distribution of test enzymes and to assure similar feed
condition between treatments. Each time a treatment feed was made,
a sample from the beginning, middle, and end of each treatment feed
were mixed to fowl a composite sample. One sample was taken from
the composite for each treatment, and for enzyme level
verification.
[0154] The turkey diets fed in this study are typical commercial
turkey feeds. The diets are representative of what might be used in
a commercial turkey growing operation and thus the diet was
adjusted after 3 weeks. The growth results for Treatments 1, 3, 6
and 8 are shown in the table below.
TABLE-US-00020 6-week Growth Parameters Average Weight- Mortal-
Live Adjusted ity Weight Feed Feed Treatment (%) (lbs)
Conversion.sup.1 Conversion.sup.2 T1 Control 1.75.sup.A 5.406.sup.A
1.500.sup.A 1.520.sup.A T3 1,4-.beta.-mannanase 1.50.sup.A
5.484.sup.ABC 1.495.sup.AB 1.502.sup.AB (100 MU/ton) T6
1,3-.beta.-glucanase 1.50.sup.A 5.540.sup.C 1.467.sup.C 1.465.sup.C
(60 MU/ton) T8 1,4-.beta.-mannanase 3.25.sup.A 5.718.sup.D
1.420.sup.D 1.389.sup.D (100 MU/ton) and 1,3-.beta.-glucanase (60
MU/ton) (1 MU = 4000 IU) Note 1: Feed Conversion is mortality
corrected. Note 2: Weight-adjusted feed conversion for each
treatment is calculated as follows: (a) the average live weight of
the entire test is subtracted from the average live weight for the
treatment, resulting in Quantity A; (b) Quantity A is divided by 6,
resulting in Quantity B; (c) Quantity B is subtracted from the feed
conversion resulting in the weight-adjusted feed conversion for the
treatment. Statistics shown are for LSD test; P < 0.05.
[0155] In comparison to Treatment 1 (control), Turkey hens
receiving Treatment 3 (feed with 1,4-.beta.-mannanase) had a
numerically improved average live weight and a numerically improved
(decreased) feed conversion. Similarly, Turkey hens receiving
Treatment 6 (feed with 1,3-.beta.-glucanase) had a
statistically-significant improved average live weight and a
statistically-significant improved (decreased) feed conversion.
[0156] Surprisingly, Turkey hens receiving Treatment 8 (feed with a
combination of 1,4-.beta.-mannanase and 1,3-.beta.-glucanase) had
an unusually large statistically significant improved average live
weight and an unusually large statistically significant improved
(decreased) feed conversion. The results observed with Treatment 8
were greater than could be explained by an additive effect of the
two enzymes administered individually. Thus, the combination
treatment comprising 1,4-.beta.-mannanase and 1,3-.beta.-glucanase
produced an unexpectedly large improvement in growth
performance.
Example 10
[0157] In this example, 1,3-.beta.-glucanase from B. lentus,
xyloglucanase from B. lentus and a combination of the two enzymes
were added to feed (a conventional corn-soybean diet). Each enzyme
treatment improved live growth performance in 35-day old male
broiler chickens, with results achieved by the combination being
greater than results achieved with treatments using only one of the
enzymes.
[0158] The experiment used 49 pens of 44 Cobb.times.Cobb male
chickens. The treatments were replicated in seven blocks, with
seven treatments (six enzyme treatments and one negative control)
randomized within each block.
[0159] Treatment 4 used a composition comprising an immune
stress-reducing enzyme in accordance with the invention,
1,3-.beta.-glucanase at 70 MU/ton of feed. Treatment 5 also used a
composition comprising an immune stress-reducing enzyme in
accordance with the invention, xyloglucanase at 100 MU/ton of feed.
Treatment 6 used a combination composition in accordance with the
invention, comprising xyloglucanase at 100 MU/ton of feed and
1,3-.beta.-glucanase at 60 MU/ton of feed. Treatment 1 was a
control feed without added enzyme. (1 MU=4000 IU).
[0160] Feed was mixed to assure uniform distribution of basal feeds
among treatments. All enzymes were mixed (sprayed on) to assure a
uniform distribution of test enzymes and to assure similar feed
condition between treatments. Each time a treatment feed was made,
a sample from the beginning, middle, and end of each treatment feed
were mixed to form a composite sample. One sample was taken from
the composite for each treatment, and for enzyme level
verification.
[0161] The diets fed in this study are typical broiler chicken
feeds. The diets are representative of what might be used in a
commercial broiler growing operation and thus the diet was adjusted
after 3 weeks. The growth results for Treatments 1, 4, 5, and 6
after 35 days of growth are shown in the table below:
TABLE-US-00021 35-day Growth Parameters Average Weight- Live
Adjusted Mortality Weight Feed Feed Treatment (%) (lbs)
Conversion.sup.1 Conversion.sup.2 T1 Control 3.25.sup.A 4.347.sup.A
1.675.sup.A 1.690.sup.A T4 1,3-.beta.-glucanase 3.25.sup.A
4.433.sup.AB 1.651.sup.AB 1.651.sup.AB (70 MU/ton) T5 Xyloglucanase
2.92.sup.A 4.415.sup.AB 1.646.sup.AB 1.650.sup.AB (100 MU/ton) T6
Xyloglucanase 4.55.sup.A 4.461.sup.AB 1.643.sup.AB 1.639.sup.AB
(100 MU/ton) And 1,3-.beta.-glucanase (70 MU/ton) (1 MU = 4000 IU)
Note .sup.1Feed Conversion is mortality corrected. Note
.sup.2Weight-adjusted feed conversion for each treatment is
calculated as described above. Statistics shown are for LSD test; P
< 0.05.
[0162] In comparison to Treatment 1 (control), the chickens
receiving Treatment 4 (1,3-.beta.-glucanase) had a numerically
improved average live weight and a numerically improved (decreased)
feed conversion. Similarly, the chickens receiving Treatment 5
(feed with xyloglucanase) had a numerically improved average live
weight and a numerically improved (decreased) feed conversion.
[0163] Chickens receiving Treatment 6 (a combination of
1,3-.beta.-glucanase and xyloglucanase) achieved improvements in
their average live weight and feed conversions greater than the
effect observed when the enzymes were administered individually.
Thus, the combination treatment comprising 1,3-.beta.-glucanase and
xyloglucanase achieved a significant improvement in growth
performance.
Example 11
[0164] Reduction of Chicken Serum APP by Application of Enzymes in
Feed
[0165] Chicken broilers were grown from 1 to 14 days and fed a
typical corn-soybean starter diet (as shown in the Diet Composition
table below) with various enzymes added (as summarized in the
Enzyme table below). Sample sizes for each enzyme type included
three cages with eight birds per cage.
TABLE-US-00022 Diet Composition Component Percent Corn 7.35% CP
53.98 Soybean meal 47.2 CP 39.03 Soy oil 3.0 Limestone 1.307
Dicalcium phosphate 1.735 Salt (NaCl) 0.331 DL Methionine 0.186
Vitamin premix 0.25 Choline chloride 60% 0.05 Copper Sulfate
0.05
TABLE-US-00023 Enzymes P val Enzyme mG/L T Treatment Enzyme 1
Enzyme 2 3 AGP Test A -- -- -- 268.1 B 1,3-.beta.-galactanase
1,4-.beta.-mannanase -- 281.4 (46,495 IU/ton) (85,312 IU/ton) C
1,3-.beta.-galactanase 1,4-.beta.-mannanase -- 266.6 (9,8911
IU/ton) (181,488 IU/ton) D 1,4-.beta.-galactanase
1,4-.beta.-mannanase -- 261.7 (81,046 IU/ton) (174,889 IU/ton) E
1,4-.beta.-galactanase 1,4-.beta.-mannanase -- 261.7 (114,418
IU/ton) (246,902 IU/ton) F xylanase 1,4-.beta.-mannanase -- 257.8
(95,225 IU/ton) (381,142 IU/ton) G chitinase 1,4-.beta.-mannanase
-- 263.8 (5,218 IU/ton) (27,016 IU/ton) H chitinase
1,4-.beta.-mannanase -- 220.2 0.040 (5,218 IU/ton) (205,807 IU/ton)
I 1,3-.beta.-glucanase 1,4-.beta.-mannanase -- 236.9 0.125 (127,042
IU/ton) (181,488 IU/ton) J xylanase 1,4-.beta.-mannanase esterase
234.5 0.083 (126,758 IU/ton) (362,976 IU/ton)
[0166] The "esterase" in Treatment J is an uncharacterized enzyme
from a B. lentus gene in the same operon with xylanase. The
substrate for this enzyme was not identified. Its assignment as an
"esterase" is based on the similarity of the DNA sequence of this
gene to other known esterase genes. The esterase activity was not
determined, but would be similar to the xylanase level if the two
proteins have similar specific activities.
[0167] The 1,4-.beta.-galactanase and 1,3-galactanase were measured
using a reducing sugar assay with pectin substrate.
[0168] At 14 days, blood serum was collected from all birds and
samples were analyzed for .alpha.1-acid glycoprotein (AGP) content
as described above. The average level of .alpha.-1-acid
glycoprotein from each treatment group is shown in the table
above.
[0169] As reflected in the table, relative to Treatment A (no
enzyme), Treatments H, I and J resulted in reduction of serum
AGP.
[0170] Treatment H (chitinase plus 1,4-.beta.-mannanase) yielded
significant results after only two weeks of growth. Although the
amounts of enzymes were at levels that did not show a response in
other Treatments (compare to Treatment G with a comparable amount
of chitinase and Treatments E and F with a comparable amounts of
1,4-.beta.-mannanase), the combination of chitinase and
1,4-.beta.-mannanase resulted in significant AGP reduction, that
could not be predicted from the results obtained when only one
enzyme was used.
[0171] Treatment I (1,3-.beta.-glucanase and 1,4-.beta.-mannanase)
yielded notable results, although not clearly statistically
significant in this experiment (P value of 0.125). In other tests
of longer duration, treatment with 1,3-.beta.-glucanase and
1,4-.beta.-mannanase did have a significant effect on AGP
level.
[0172] Comparing Treatment J (xylanase, 1,4-.beta.-mannanase,
esterase; P=0.083 vs Treatment A control) to Treatment F
(xylanase+1,4-.beta.-mannanase, without "esterase") reveals that
Treatment J yielded a notable effect, where Treatment F did not
show AGP reduction.
Example 12
[0173] This example tests the hypothesis that altering a feed
composition to include ingredients that stimulate the innate immune
system will increase serum APP levels.
[0174] A 21-day chicken broiler test was performed using a basal
corn-soybean diet with a soy-oil high-energy diet as control. To
obtain test diets, the control diet was modified to contain
practical materials suspected to have immune stimulatory components
while maintaining the same approximate equivalent nutritional
value. The test diets included the following variations:
Corn/soy/soy oil control AV blend oil (animal vegetable blend oil)
inclusion; soy lecithin inclusion; poultry meal inclusion; DDGS
(distillers grains and solubles by-product from ethanol
manufacture) inclusion at 5% with soy hull; DDGS inclusion at 15%
w/o soy hull.
[0175] The diets are described in more detail in the tables
below.
[0176] The AV blend and soy lecithin are expected to contain
phospholipids comprising the innate immune system stimulator
phosphatidlyserine. The poultry meal may contain
phosphatidlyserine, hyaluronan, and various microbial stimulators
derived from the offal or secondary microbial growth that could
occur before processing. The DDGS is expected to contain abundant
yeast residue, including cell walls comprising .alpha.-mannan,
1,3-.beta.-glucan and chitin, as well as potentially stimulating
non-fermented carbohydrate polymers from the original fermentation
substrate.
TABLE-US-00024 Diet Compositions Percent Composition Component Diet
#1 Diet #2 Diet #3 Diet #4 Diet #5 Diet 6 Corn 56.9707 56.2985
56.2985 59.878 47.7939 49.3523 7.35% CP Soy meal 36.4392 36.5403
36.5403 29.048 29.0293 29.0284 48.5% CP Soy oil 2.5279 0 0 1.7221
3.1922 2.6831 AV blend 0 3.0975 0 0 0 0 Soy lecithin 0 0 3.0975 0 0
0 Poultry 0 0 0 5.0 0 0 BPM.sup.a 65% Soy hulls.sup.d 0 0 0 1.0559
1.0586 0 DDGS.sup.b 0 0 0 0 15 15 Limestone 1.3129 1.3118 1.3118
1.2224 1.4158 1.4293 Dicalcium 1.7527 1.7544 1.7544 1.766 1.5615
1.5576 phosphate Salt 0.3312 0.3315 0.3315 0.2313 0.1416 0.1407
DL-methionine 0.2404 0.241 0.241 0.2276 0.2419 0.2392 L-lysine HCl
0 0 0 0.0131 0.1402 0.1402 Vitamin 0.25 0.25 0.25 0.25 0.25 0.25
premix 0.25% Mineral 0.075 0.075 0.075 0.075 0.075 0.075 PMX 0.075%
Choline 0.05 0.05 0.05 0.05 0.05 0.05 chloride 60% Copper sulfate
0.05 0.05 0.05 0.05 0.05 0.05 .sup.apoultry BPM (by product meal);
.sup.bDDGS (distiller dry grain and solubles); .sup.cAV blend
(animal vegetable blend oil); .sup.daddition of soy hulls to the
poultry meal diet equalizes the mannan content to compensate for
reduced soy meal
[0177] For each diet, chicken broilers (Cobb.times.Cobb) were grown
in three Petersime battery cages with eight birds per cage (0.631
sq. ft. per bird). After 21 days, the serum AGP levels of each bird
was analyzed as described in previous examples.
[0178] The modified diets showed clear evidence of innate immune
system stimulation based on significant increases in serum AGP at
21 days, as shown in the following table. The data also underscores
the opportunities to reduce immune stress caused by diet components
in accordance with the invention, such as by the use of
compositions comprising enzymes that degrade immune stress inducing
ingredients.
TABLE-US-00025 Diet Addition Mg/L AGP T Test (P value vs. 1) 1
Control 221.9 2 AV blend 301.2 0.01882 3 Lecithin 309.0 0.01052 4
Poultry Meal 265.8 0.08613 5 DDGS w/hull 307.6 0.00002 6 DDGS 386.7
0.00275
Example 13
[0179] This example demonstrates the efficacy of compositions
comprising 1,3-.beta.-glucanase in reducing immune stress
associated with 1,3-.beta.-glucan, which is present in feedstuffs
and, by virtue of its association with fungal cell walls, is a
molecular pattern apparently recognized universally by the innate
immune system of animals. The results show that, like
1,4-.beta.-mannanase, 1,3-.beta.-glucanase reduces serum levels of
APP and improves animal growth performance.
[0180] Chicken broilers (Cobb.times.Cobb) were grown from day 1 to
21 on the typical low fat corn/soybean meal diet shown in the table
below. In two cases, the diets were supplemented by uniformly
spraying liquid enzyme concentrate solutions prepared from B.
lentus fermentations to apply either 400,000 IU/ton
1,4-.beta.-mannanase or 264,000 IU/ton 1,3-.beta.-glucanase. (In
this case, a ton represents 2000 lbs. or 907.4 kg.)
TABLE-US-00026 Diet Composition Component Diet % Corn 7.35% CP
59.5757 Soy meal 48.5% CP 36.0474 Soy oil 0.321 Limestone 1.3175
Dicalcium phosphate 1.7461 Salt 0.3294 DL-methionine 0.2378 Vitamin
premix 0.25% 0.25 Mineral PMX 0.075% 0.075 Choline chloride 60%
0.05 Copper sulfate 0.05
[0181] For each diet type, birds were grown in three Petersime.RTM.
battery cages with eight birds per cage (0.631 sq. ft. per bird).
After 21 days, serum AGP levels of each bird was analyzed as
described in previous examples. Bird weights and feed consumed were
determined utilizing standard procedures and feed conversion was
calculated. The results are shown in the following table
[0182] WAFC (weight adjusted feed conversion) is calculated as
follows:
WAFC=FC-2.204*((W-Wa)/3)
where FC=weight of feed consumed/weight gained Wa=average weight of
all birds in the trial W=average live weight gain per cage
TABLE-US-00027 T Test mg/L P vs. Treatment AGP contr. WAFC P value
Control 255.4 1.47 a (no enzyme) 1,4-.beta.-mannanase 184.1 0.011
1.39 ab 1,3-.beta.-glucanase 157.7 0.001 1.26 c
[0183] Both 1,4-.beta.-mannanase and 1,3-.beta.-glucanase reduced
the serum levels of .alpha.1-acid glycoprotein (AGP). Both enzyme
treatments reduced the weight adjusted feed conversion, and the
reduction in the 1,3-.beta.-glucanase fed group was statistically
significant.
Example 14
[0184] A chicken broiler trial was conducted in Petersine.RTM.
battery cages with the feed and methods described in Example 13
above, except with different enzyme treatments, as summarized in
the table below.
[0185] Lyticase, a crude 1,3-.beta.-glucanase product obtained by
fermentation of Arthrobacter luteus, was obtained from the Sigma
Chemical Company, St. Louis Mo. Lyticase activity was determined by
the reducing sugar method described below and 60 MU/ton (equivalent
to 240,000 IU/ton) was applied. According to the manufacturer, this
product also contains other activities, including chitinase
activity, that was not measured.
TABLE-US-00028 Treatment Enzyme (s) Dose (MU/ton) AGP (mg/L) 1 none
0 215.5 2 1,3-.beta.-glucanase 3 213.7 3 1,3-.beta.-glucanase 15
199.4 4 1,3-.beta.-glucanase 30 185.5 5 1,3-.beta.-glucanase 60
201.0 6 1,3-.beta.-glucanase 60 189.2 1,4-.beta.-mannanase 100 7
1,3-.beta.-glucanase 75 194.9 8 1,3-.beta.-glucanase 90 180.7 9
Lyticase 60 165.2 10 Xyloglucanase 100 162.2 (1 MU = 4000 IU)
[0186] Increased levels of 1,3-.beta.-glucanase resulted in an
increased effect on AGP level (e.g., a dose response) up to about
30 MU/ton (120,000 IU/ton). Providing this type of animal feed with
about 30 MU/ton (120,000 IU/ton) 1,3-.beta.-glucanase is expected
to reduce immune stress, as reflected in a reduced level of serum
AGP and/or improved animal growth performance.
[0187] The results also show that xyloglucanase was effective at
reducing serum AGP levels. Xyloglucanase (EC 3.2.1.151) is a
1,4-.beta.-glucanase with specificity for xyloglucan, a structural
polymer in plants.
[0188] With the exception of the Lyticase, all of the enzymes used
in this example were produced by B. lentus. Lyticase is produced by
A. luteus which has been reclassified as Cellulosimicrobium
cellulans. Fermentation of A. luteus has been shown to produce
multiple forms of 1,3-.beta.-glucanase. See, e.g., (Ferrer, P.
Microb Cell Factories 5:10, 2006, published online 2006 Mar. 17.
doi: 10.1186/1475-2859-5-10). The results above show that Lyticase
reduced the chicken serum AGP at least as well as the B. lentus
1,3-.beta.-glucanase preparation, indicating that the source of the
enzyme is not important. That is, enzymes from any source can be
used in accordance with the invention. It also is possible that
chitinase (reported by Sigma to be present in Lyticase) may have
improved the performance of the Lyticase treatment.
Example 15
[0189] The following assays can be used to assess enzyme
activity
(I) Xyloglucanase
[0190] Xyloglucanase activity can be assayed using the following
protocol:
[0191] DNS reagent: 10 g/L NaOH, 2 g/L phenol, 10 g/L
dinitrosalicylic acid, 1200 g/L potassium sodium tartrate
tetrahydrate is prepared daily. Immediately before use, 0.5 g/L
anhydrous sodium sulfite is added.
[0192] Standard Solutions and Standard Curve: A series of
D-(+)-mannose standard solutions dissolved in water in the
concentration range of 0.1 to 0.5 g/Liter are prepared. 0.6 mL of
each mannose standard (in duplicate or triplicate) is added to 1.5
mL DNS working solution in 13.times.100 mm glass tubes. A sample
with a 0.6 mL aliquot of water can be used as a reagent blank to
zero the spectrophotometer. The solutions are heated in a boiling
water bath for 5 minutes, cooled to ambient temperature and
absorbance is read at 550 nm. The expected result is a linear dose
response between 0.20 and 1.2 O.D. units. The slope of the standard
curve (O.D 550/g/L mannose) is calculated from the linear portion
of the curve only. With this slope, the value of the g/L of
reducing sugar is determined in the enzyme reactions.
[0193] Xyloglucan Substrate: Xyloglucan (Tamarind) is obtained from
Megazyme International Ireland Ltd., Bray, Co., Ireland is
dissolved at 5 g/L in 50 mM Tris buffer, pH 7.5 with 0.05%
glucose.
[0194] Reaction conditions: 0.25 mL of 5 g/L xyloglucan substrate
is used with a 0.05 mL enzyme dilution in 50 mM Tris buffer, and
the reaction mixture is incubated at 40.degree. C. 0.75 mL DNS
reagent is added to stop the reaction, and the stopped reaction
mixture is heated in boiling water bath for five minutes and then
cooled prior to reading absorbance at 550 nm. A zero time point
with enzyme solution is used to determine the background level.
[0195] Calculation: A ChemGen xyloglucanase MU is defined as the
ability to produce 0.72 grams of reducing sugar per minute (using
pure mannose, a reducing sugar, as standard). One ChemGen MU is
equivalent to 4000 IU. In other words, one CG U is equivalent to
250 IU (IU=1.0 .mu.mole/minute).
(II) B-1,3-glucanase
[0196] B-1,3-glucanase activity can be assayed using the following
protocol:
[0197] This assay uses the same DNS reagent, standard solutions,
standard curve, and enzyme unit calculation and dilution amount as
described above for the xyloglucanase assay. The buffer used is a
50 mM MOPS (4-Morpholinepropanesulfonic Acid, FW=209.26) buffer at
pH 6.5.
[0198] CM pachyman Substrate: Carboxymethyl Pachyman (CM Pachyman,
CMP) is obtained from Megazyme International Ireland Ltd., Bray,
Co., Ireland. CMP substrate is prepared at 5 g/L by slowly adding
CMP into a fast stirring 50 mM MOPS buffer solution (pH 6.5) at
about 90.degree. C. The enzyme powder is well-dispersed, and the
vessel is covered or sealed tightly, while the suspension is heated
slowly to boiling and simmered for 30 minutes with stirring on a
heated-stir plate, to obtain a well-hydrated gel with no small
clumps of non-hydrated gel visible in the solution. The solution is
cooled to room temperature, stored at 4.degree. C. when not in use,
and mixed well prior to use after storage.
[0199] Reaction conditions: 0.25 mL of 5 gL CM Pachyman substrate
is used with 0.05 mL enzyme dilution in MOPS buffer and the
reaction mixture is incubated at 40.degree. C. for various times,
up to 45 minutes. 0.75 mL DNS reagent is added to stop the
reaction. The stopped reaction mixture is heated in boiling water
bath for five minutes and then cooled prior to reading absorbance
at 550 nm. A zero time point with enzyme solution is used to
determine the background level.
(III) Chitinase
[0200] Chitinase activity can be determined using the fluorogenic
chitin substrate described in Thompson et al., Appl. Environ.
Microbiol. 67: 4001-008 (2001),
4-methylumbelliferyl-beta-D-N,N',N'',N'''-tetraacetylchitotetraoside.
The substrate is dissolved in DMSO at 2.5 mM.
[0201] In an exemplary assay, 20 .mu.L chitin substrate (2.5 mM) is
used with 150 .mu.L tris (20.0 mM, pH 7.5). The substrate mixture
is placed in a black 98 well microtiter plate and pre-heated to
37.degree. C. for 10 minutes. Multiple replicaes of reactions are
started by addition of 30 .mu.L diluted enzyme and incubation is
continued at 37.degree. C. Individual reactions are stopped at 2,
4, 6, 8 and 10 minutes with 50 .mu.L 3 MNa.sub.2CaO.sub.3.
Fluorescence is read in a microtiter plate reader (Fluoroscan II)
using excitation 355 nm band pass filter and emission 460 nm band
pass filter wavelengths. The enzyme is diluted such that the
4-methylumbelliferone is produced at a linear rate for the term of
the reaction and within the range of a standard curve produced
under conditions identical to the enzyme assay but without enzyme
and substrate present. The release of one micromole of
4-methylumbelliferone per minute is defined as one IU. A standard
curve is made with several concentrations between zero and
1.times.10.sup.4 micromole 4-methylumbelliferone in 200 .mu.L
reaction buffer solution followed by addition of 50 .mu.L 3 M
Na.sub.2CaO.sub.3.
[0202] While the invention has been described and exemplified in
sufficient detail for those skilled in this art to make and use it,
various alternatives, modifications, and improvements should be
apparent without departing from the spirit and scope of the
invention. The examples provided herein are representative of
preferred embodiments, are exemplary, and are not intended as
limitations on the scope of the invention. Modifications therein
and other uses will occur to those skilled in the art. These
modifications are encompassed within the spirit of the invention
and are defined by the scope of the claims.
[0203] It will be readily apparent to a person skilled in the art
that varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention.
[0204] All patents and publications mentioned in the specification
are indicative of the levels of those of ordinary skill in the art
to which the invention pertains. All patents and publications are
herein incorporated by reference to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference.
* * * * *