U.S. patent application number 13/985863 was filed with the patent office on 2014-08-21 for feed additive composition.
This patent application is currently assigned to DUPONT NUTRITION BIOSCIENCES APS. The applicant listed for this patent is Luis Fernando Romero Millan. Invention is credited to Luis Fernando Romero Millan.
Application Number | 20140234279 13/985863 |
Document ID | / |
Family ID | 43881323 |
Filed Date | 2014-08-21 |
United States Patent
Application |
20140234279 |
Kind Code |
A1 |
Millan; Luis Fernando
Romero |
August 21, 2014 |
FEED ADDITIVE COMPOSITION
Abstract
A feed additive composition comprising a direct fed microbial in
combination with a protease, a xylanase, an amylase and a phytase,
and a method for improving the performance of a subject or for
improving digestibility of a raw material in a feed (e.g. nutrient
digestibility, such as amino acid digestibility), or for improving
nitrogen retention, or for avoiding the negative effects of
necrotic enteritis or for improving feed conversion ratio (FCR) or
for improving weight gain in a subject or for improving feed
efficiency in a subject or for modulating (e.g. improving) the
immune response of the subject or for promoting the growth of
beneficial bacteria in the gastrointestinal tract of a subject,
which method comprising administering to a subject a direct fed
microbial in combination with a protease, a xylanase, an amylase
and a phytase.
Inventors: |
Millan; Luis Fernando Romero;
(Wiltshire, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Millan; Luis Fernando Romero |
Wiltshire |
|
GB |
|
|
Assignee: |
DUPONT NUTRITION BIOSCIENCES
APS
Copenhagen
DK
|
Family ID: |
43881323 |
Appl. No.: |
13/985863 |
Filed: |
January 19, 2012 |
PCT Filed: |
January 19, 2012 |
PCT NO: |
PCT/GB12/50124 |
371 Date: |
August 15, 2013 |
Current U.S.
Class: |
424/93.41 ;
424/93.4; 424/93.44; 424/93.45; 424/93.46; 424/93.462; 424/93.48;
424/94.2; 426/2; 426/63 |
Current CPC
Class: |
A61P 1/04 20180101; A61K
35/741 20130101; C12N 1/20 20130101; A61K 38/47 20130101; C12R
1/125 20130101; A23K 10/18 20160501; C12Y 304/00 20130101; A61K
35/742 20130101; A23K 40/30 20160501; A61P 43/00 20180101; C12Y
302/01 20130101; A61K 38/48 20130101; A61P 33/02 20180101; C12Y
301/03 20130101; A23K 20/189 20160501; A61K 38/465 20130101; A23K
50/75 20160501; A23K 40/10 20160501 |
Class at
Publication: |
424/93.41 ;
426/63; 426/2; 424/93.4; 424/93.45; 424/93.44; 424/93.46;
424/93.48; 424/93.462; 424/94.2 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A61K 38/46 20060101 A61K038/46; A61K 38/48 20060101
A61K038/48; A61K 38/47 20060101 A61K038/47; A23K 1/165 20060101
A23K001/165; A23K 1/00 20060101 A23K001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 18, 2011 |
GB |
1102857.8 |
Claims
1. A feed additive composition comprising a direct fed microbial in
combination with a protease, a xylanase, an amylase and a
phytase.
2. A feed additive composition according to claim 1 wherein the
direct fed microbial is an antipathogen direct fed microbial.
3. A feed additive composition according to claim 1 or claim 2
wherein the direct fed microbial is a viable bacterium.
4. A feed additive composition according to any one of claims 1 to
3 wherein the direct fed microbial comprises a bacterium from one
or more of the following genera: Lactobacillus, Lactococcus,
Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc,
Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium and
Megasphaera and combinations thereof.
5. A feed additive composition according to any one of the
preceding claims wherein the direct fed microbial comprises a
bacterium from one or more of the following species: Bacillus
subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens,
Enterococcus, Enterococcus spp, and Pediococcus spp, Lactobacillus
spp, Bifidobacterium spp, Lactobacillus acidophilus, Pediococsus
acidilactici, Lactococcus lactis, Bifidobacterium bifidum,
Propionibacterium thoenii, Lactobacillus farciminus, lactobacillus
rhamnosus, Clostridium butyricum, Bifidobacterium animalis ssp.
animalis, Lactobacillus reuteri, Bacillus cereus, Lactobacillus
salivarius ssp. salivarius, Megasphaera elsdenii, Propionibacteria
sp and combinations thereof.
6. A feed additive composition according to any one of the
preceding claims wherein the direct fed microbial is one or more of
the following strains: Bacillus subtilis strains 3A-P4 (PTA-6506);
15A-P4 (PTA-6507); 22C-P1 (PTA-6508); 2084 (NRRL B-500130); LSSA01
(NRRL-B-50104); BS27 (NRRL B-50105); BS 18 (NRRL B-50633); and BS
278 (NRRL B-50634).
7. A feed additive composition according to any one of the
preceding claims wherein the direct fed microbial is in the form of
an endospore.
8. A feed additive composition according any one of the preceding
claims wherein the xylanase is an endo-1,4-.beta.-d-xylanase or a
1,4 .beta.-xylosidase preferably an endo-1,4-.beta.-d-xylanase.
9. A feed additive composition according to any one of the
preceding claims wherein the xylanase is from Bacillus,
Trichoderma, Thermomyces, Aspergillus, Penicillium and
Humicola.
10. A feed additive composition according to any one of the
preceding claims wherein the protease is a subtilisin, a
bacillolysin, an alkaline serine protease, a keratinase or a
Nocardiopsis protease.
11. A feed additive composition according to any one of the
preceding claims wherein the phytase is a 6-phytase or a
3-phytase.
12. A feed additive composition according to claim 11 wherein the
phytase is a 6-phytase.
13. A feed additive composition according to any one of the
preceding claims wherein the phytase is an E. coli phytase or a
Buttiauxella phytase or Hafnia phytase or a Citrobacter phytase or
a Aspergillus phytase or a Penicillium phytase or a Trichoderma
phytase or a Hansenula phytase.
14. A feed additive composition according to any one of the
preceding claims wherein the amylase is selected from one or more
of the group consisting of: an .alpha.-amylase, a G4-forming
amylase, a .beta.-amylase and a .gamma.-amylases.
15. A feed additive composition according to claim 14 wherein the
amylase is an .alpha.-amylase.
16. A feed additive composition according to any one of the
preceding claims wherein the amylase is from Bacillus
licheniformis, B. amyloliquefaciens, Trichoderma spp. or
Aspergillus spp.
17. A feed additive composition according to any one of the
preceding claims wherein the phytase is present at a dosage of
between 200 FTU/g feed additive composition and 10000 FTU/g feed
additive composition.
18. A feed additive composition according to any one of the
preceding claims wherein the amylase is present at a dosage of
between 50 AU/g feed additive composition and 20000 AU/g feed
additive composition.
19. A feed additive composition according to any one of the
preceding claims wherein the xylanase is present at a dosage of
between 500 XU/g feed additive composition and 40000 XU/g feed
additive composition.
20. A feed additive composition according to any one of the
preceding claims wherein the protease is present at a dosage of
1000 PU/g feed additive composition and 60000 PU/g feed additive
composition.
21. A feed additive composition according to any one of the
preceding claims wherein the DFM is present at a dosage of
3.75.times.10.sup.7 CFU/g feed additive composition and
1.times.10.sup.11 CFU/g feed additive composition.
22. A method for improving the performance of a subject or for
improving digestibility of a raw material in a feed (e.g. nutrient
digestibility, such as amino acid digestibility), or for improving
nitrogen retention, or for improving the subjects resistance to
necrotic enteritis or for improving feed conversion ratio (FCR) or
for improving body weight gain in a subject or for improving feed
efficiency in a subject or for modulating (e.g. improving) the
immune response of the subject, or for promoting the growth of
beneficial bacteria in the gastrointestinal tract of a subject or
for reducing populations of pathogenic bacteria in the
gastrointestinal tract of a subject, or for reducing nutrient
excretion in manure, which method comprising administering to a
subject a direct fed microbial in combination with a protease, a
xylanase, an amylase and a phytase.
23. A method according to claim 22 comprising administering the
feed additive composition according to any one of claims 1-21.
24. A method according to claim 22 or 23 wherein the direct fed
microbial is an antipathogen direct fed microbial.
25. A method according to any one of claims 22-24 wherein the
direct fed microbial is a viable bacteria.
26. A method according to any one of claims 22 to 25 wherein the
direct fed microbial comprises a bacterium from one or more of the
following genera: Lactobacillus, Lactococcus, Streptococcus,
Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium,
Propionibacterium, Bifidobacterium, Clostridium and Megasphaera and
combinations thereof.
27. A method according to any one of claims 22-26 wherein the
direct fed microbial comprises a bacterium from one or more of the
following species: Bacillus subtilis, Bacillus licheniformis,
Bacillus amyloliquefaciens, Enterococcus, Enterococcus spp, and
Pediococcus spp, Lactobacillus spp, Bifidobacterium spp,
Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus
lactis, Bifidobacterium bifidum, Propionibacterium thoenii,
Lactobacillus farciminus, lactobacillus rhamnosus, Clostridium
butyricum, Bifidobacterium animalis ssp. animalis, Lactobacillus
reuteri, Bacillus cereus, Lactobacillus salivarius ssp. salivarius,
Megasphaera elsdenii, Propionibacteria sp and combinations
thereof.
28. A method according to any one of claims 22-27 wherein the
direct fed microbial is one or more of the following strains:
Bacillus subtilis strains 3A-P4 (PTA-6506); 15A-P4 (PTA-6507);
22C-P1 (PTA-6508); 2084 (NRRL B-500130); LSSA01 (NRRL-B-50104);
BS27 (NRRL B-50105); BS 18 (NRRL B-50633); and BS 278 (NRRL
B-50634).
29. A method according to any one of claims 22-28 wherein the
direct fed microbial is in the form of an endospore.
30. A method according to any one of claims 22-29 wherein the
xylanase is an endo-1,4-.beta.-d-xylanase or a 1,4
.beta.-xylosidase preferably an endo-1,4-.beta.-d-xylanase.
31. A method according to any one of claims 22-30 wherein the
xylanase is from Bacillus, Trichoderma, Thermomyces, Aspergillus,
Penicillium and Humicola.
32. A method according to any one of claims 22-31 wherein the
protease is a subtilisin, a bacillolysin, an alkaline serine
protease or a keratinase or a Nocardiopsis protease.
33. A method according to any one of claims 22-32 wherein the
phytase is a 6-phytase or a 3-phytase.
34. A method according to claim 33 wherein the phytase is a
6-phytase.
35. A method according to any one of claims 22-34 wherein the
phytase is an E. coli phytase or a Buttiauxella phytase or Hafnia
phytase or Citrobacter phytase or a Aspergillus phytase or a
Penicillium phytase or a Trichoderma phytase or a Hansenula
phytase.
36. A method according to any one of claims 22-35 wherein the
amylase is selected from one or more of the group consisting of: an
.alpha.-amylase, a G4-forming amylase, a .beta.-amylase and a
7-amylases.
37. A method according to any one of claims 22-36 wherein the
amylase is an .alpha.-amylase.
38. A method according to any one of claims 22-37 wherein the
amylase is from Bacillus licheniformis, B. amyloliquefaciens,
Trichoderma spp. or Aspergillus spp.
39. Use of a direct fed microbial in combination with a protease, a
xylanase, an amylase and a phytase for improving the performance of
a subject or for improving digestibility of a raw material in a
feed (e.g. nutrient digestibility, such as amino acid
digestibility) or for improving nitrogen retention) or for
improving the subject's resistance to of necrotic enteritis or for
improving feed conversion ratio (FCR) or for improving weight gain
in a subject or for improving feed efficiency in a subject or for
modulating (e.g. improving) the immune response of the subject or
for promoting the growth of beneficial bacteria in the
gastrointestinal tract of a subject or for reducing populations of
pathogenic bacteria in the gastrointestinal tract of a subject, or
for reducing nutrient excretion in manure.
40. Use according to claim 39 wherein the feed additive composition
according to any one of claims 1-21 is used.
41. Use according to any one of claims 39-40 wherein the direct fed
microbial is an antipathogen direct fed microbial.
42. Use according to any one of claims 39-41 wherein the direct fed
microbial is a viable bacterium.
43. Use according to any one of claims 39-42 wherein the direct fed
microbial comprises a bacterium from one or more of the following
genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus,
Pediococcus, Enterococcus, Leuconostoc, Carnobacterium,
Propionibacterium, Bifidobacterium, Clostridium and Megasphaera and
combinations thereof.
44. Use according to any one of claims 39-43 wherein the direct fed
microbial comprises a bacterium from one or more of the following
species: Bacillus subtilis, Bacillus licheniformis, Bacillus
amyloliquefaciens, Enterococcus, Enterococcus spp, and Pediococcus
spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillus
acidophilus, Pediococsus acidilactici, Lactococcus lactis,
Bifidobacterium bifidum, Propionibacterium thoenii, Lactobacillus
farciminus, lactobacillus rhamnosus, Clostridium butyricum,
Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri,
Bacillus cereus, Lactobacillus salivarius ssp. salivarius,
Megasphaera elsdenii, Propionibacteria sp and combinations
thereof.
45. Use according to any one of claims 39-44 wherein the direct fed
microbial is one or more of the following strains: Bacillus
subtilis strains 3A-P4 (PTA-6506); 15A-P4 (PTA-6507); 22C-P1
(PTA-6508); 2084 (NRRL B-500130); LSSA01 (NRRL-B-50104); BS27 (NRRL
B-50105); BS 18 (NRRL B-50633); and BS 278 (NRRL B-50634).
46. Use according to any one of claims 39-45 wherein the direct fed
microbial is in the form of an endospore.
47. Use according to any one of claims 39-46 wherein the xylanase
is an endo-1,4-.beta.-d-xylanase or a 1,4 .beta.-xylosidase
preferably an endo-1,4-.beta.-d-xylanase.
48. Use according to any one of claims 39-47 wherein the xylanase
is from Bacillus, Trichoderma, Thermomyces, Aspergillus,
Penicillium and Humicola.
49. Use according to any one of claims 39-48 wherein the protease
is a subtilisin, a bacillolysin, an alkaline serine protease or a
keratinase or a Nocardiopsis protease.
50. Use according to any one of claims 39-49 wherein the phytase is
a 6-phytase or a 3-phytase.
51. Use according to claim 50 wherein the phytase is a
6-phytase.
52. Use according to any one of claims 39-51 wherein the phytase is
an E. coli phytase or a Buttiauxella phytase or Hafnia phytase or
Citrobacter phytase or a Aspergillus phytase or a Penicillium
phytase or a Trichoderma phytase or a Hansenula phytase.
53. Use according to any one of claims 39-52 wherein the amylase is
selected from one or more of the group consisting of: an
.alpha.-amylase, a G4-forming amylase, a .beta.-amylase and a
.gamma.-amylases.
54. Use according to any one of claims 39-53 wherein the amylase is
an .alpha.-amylase.
55. Use according to any one of claims 39-54 wherein the amylase is
from Bacillus licheniformis, B. amyloliquefaciens, Trichoderma spp.
or Aspergillus spp.
56. A kit comprising a direct fed microbial, a protease, a
xylanase, an amylase, a phytase and instructions for
administration.
57. A kit according to claim 56 wherein said kit comprises the feed
additive composition according to any one of claims 1-21.
58. A kit according to any one of claims 56-57 wherein the direct
fed microbial is an antipathogen direct fed microbial.
59. A kit according to any one of claims 56-58 wherein the direct
fed microbial is a viable bacterium.
60. A kit according to any one of claims 56-59 wherein the direct
fed microbial comprises a bacterium from one or more of the
following genera: Lactobacillus, Lactococcus, Streptococcus,
Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium,
Propionibacterium, Bifidobacterium, Clostridium and Megasphaera and
combinations thereof.
61. A kit according to any one of claims 56-60 wherein the direct
fed microbial comprises a bacterium from one or more of the
following species: Bacillus subtilis, Bacillus licheniformis,
Bacillus amyloliquefaciens, Enterococcus, Enterococcus spp, and
Pediococcus spp, Lactobacillus spp, Bifidobacterium spp,
Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus
lactis, Bifidobacterium bifidum, Propionibacterium thoenii,
Lactobacillus farciminus, lactobacillus rhamnosus, Clostridium
butyricum, Bifidobacterium animalis ssp. animalis, Lactobacillus
reuteri, Bacillus cereus, Lactobacillus salivarius ssp. salivarius,
Megasphaera elsdenii, Propionibacteria sp and combinations
thereof.
62. A kit according to any one of claims 56-61 wherein the direct
fed microbial is one or more of the following strains: Bacillus
subtilis strains 3A-P4 (PTA-6506); 15A-P4 (PTA-6507); 22C-P1
(PTA-6508); 2084 (NRRL B-500130); LSSA01 (NRRL-B-50104); BS27 (NRRL
B-50105); BS 18 (NRRL B-50633); and BS 278 (NRRL B-50634).
63. A kit according to any one of claims 56-62 wherein the direct
fed microbial is in the form of an endospore.
64. A kit according to any one of claims 56-63 wherein the xylanase
is an endo-1,4-.beta.-d-xylanase or a 1,4 .beta.-xylosidase
preferably an endo-1,4-.beta.-d-xylanase.
65. A kit according to any one of claims 56-64 wherein the xylanase
is from Bacillus, Trichoderma, Thermomyces, Aspergillus,
Penicillium and Humicola.
66. A kit according to any one of claims 56-65 wherein the protease
is a subtilisin, a bacillolysin, an alkaline serine protease or a
keratinase or a Nocardiopsis protease.
67. A kit according to any one of claims 56-66 wherein the phytase
is a 6-phytase or a 3-phytase.
68. A kit according to claim 67 wherein the phytase is a
6-phytase.
69. A kit according to any one of claims 56-68 wherein the phytase
is an E. coli phytase or a Buttiauxella phytase or Hafnia phytase
or Citrobacter phytase or an Aspergillus phytase or a Penicillium
phytase or a Trichoderma phytase or a Hansenula phytase.
70. A kit according to any one of claims 56-69 wherein the amylase
is selected from one or more of the group consisting of: an
.alpha.-amylase, a G4-forming amylase, a .beta.-amylase and a
.gamma.-amylases.
71. A kit according to any one of claims 56-70 wherein the amylase
is an .alpha.-amylase.
72. A kit according to any one of claims 56-71 wherein the amylase
is from Bacillus licheniformis or, B. amyloliquefaciens,
Trichoderma spp. or Aspergillus spp.
73. A method of preparing a feed additive composition, comprising
admixing a direct fed microbial with a protease, a xylanase, an
amylase and a phytase and (optionally) packaging.
74. A feed comprising a feed additive composition according to any
one of claims 1-21.
75. A feed according to claim 74 wherein the phytase is present at
a dosage of between 400 FTU/kg feed and 1000 FTU/kg feed.
76. A feed according to claim 74 or claim 75, wherein the amylase
is present at a dosage of between 100 AU/kg feed and 2000 AU/kg
feed.
77. A feed according to any one of claims 74-76 wherein the
xylanase is present at a dosage of between 1000 XU/kg feed and 4000
XU/kg feed.
78. A feed according to any one of claims 74-77 wherein the
protease is present at a dosage of between 2000 PU/kg feed to 6000
PU/kg feed.
79. A feed according to any one of claims 74-77 wherein the DFM is
present at a dosage of 7.5.times.10.sup.4 CFU/kg feed and
1.times.10.sup.7 CFU/kg feed.
80. A method of preparing a feedstuff comprising admixing a feed
component with a feed additive composition according to any one of
claims 1-21.
81. A premix comprising a feed additive composition comprising a
direct fed microbial in combination with a protease, a xylanase, an
amylase and a phytase, and at least one mineral and/or at least one
vitamin.
82. A premix comprising a feed additive composition according to
any one of claims 1-21 in combination with at least one mineral
and/or at least one vitamin.
83. A feed additive composition according to any one of claims 1-21
for preventing and/or treating coccidiosis and/or necrotic
enteritis in a subject.
84. A method of preventing and/or treating necrotic enteritis
and/or coccidiosis wherein an effective amount of a feed additive
composition according to any one of claims 1-21 is administered to
a subject.
85. A feed additive composition or feed or kit or method or use or
premix as defined generally herein with reference to the Figures
and the Examples.
Description
FIELD OF INVENTION
[0001] The present invention relates to methods for improving feed
compositions using a direct fed microbial in combination with a
specific combination of enzymes, and to a feed additive composition
comprising a direct fed microbial in combination with a specific
combination of enzymes. The present invention further relates to
uses and kits.
BACKGROUND OF THE INVENTION
[0002] Supplemental enzymes are used as additives to animal feed,
particularly poultry and swine feeds, as a means to improve
nutrient utilization and production performance characteristics.
Enzyme blends are available to improve the nutritional value of
diets containing soybean meal, animal protein meals, or high fibre
food by-products.
[0003] The concept of direct fed microbials (DFM) involves the
feeding of beneficial microbes to animals, such as dairy cattle
when they are under periods of stress (disease, ration changes,
environmental or production challenges). Probiotics is another term
for this category of feed additives. Probiotics or DFM have been
shown to improve animal performance in controlled studies. DFM
including direct fed bacteria and or yeast-based products.
[0004] Although combinations of DFMs with some enzymes have been
contemplated, the interaction between DFMs and enzyme has never
been fully understood. The present invention relates to novel
specific combinations which surprisingly significantly improve
production performance characteristics of animals.
SUMMARY OF INVENTION
[0005] A seminal finding of the present invention is that a DFM in
combination with a protease, xylanase, amylase and phytase has
significant beneficial effects on the performance of an animal.
[0006] In particular, a seminal finding of the present invention is
that a DFM in combination with a protease, xylanase, amylase and
phytase has significant beneficial effects on the performance of an
animal, including improving one or more of the following: feed
conversion ratio (FCR), ability to digest a raw material (e.g.
nutrient digestibility, such as amino acid digestibility), nitrogen
retention, survival, carcass yield, growth rate, weight gain, feed
efficiency animals resistance to necrotic enteritis, immune
response of the subject, or the growth of beneficial bacteria in
the gastrointestinal tract of a subject.
[0007] Another surprising effect of the present invention is that
it can reduce nutrient excretion in manure (e.g. reduce nitrogen
and phosphorus) content of a subject's manure.
[0008] In one aspect, the present invention provides a feed
additive composition comprising (or consisting essentially of or
consisting of) a direct fed microbial in combination with a
protease, a xylanase, an amylase and a phytase.
[0009] In another aspect, the present invention provides a method
for improving the performance of a subject or for improving
digestibility of a raw material in a feed (e.g. nutrient
digestibility, such as amino acid digestibility), or for improving
nitrogen retention, or for avoiding the negative effects of
necrotic enteritis or for improving feed conversion ratio (FCR) or
for improving weight gain in a subject or for improving feed
efficiency in a subject or for modulating (e.g. improving) the
immune response of the subject or for promoting the growth of
beneficial bacteria in the gastrointestinal tract of a subject or
for reducing populations of pathogenic bacteria in the
gastrointestinal tract of a subject, or for reducing nutrient
excretion in manure, which method comprising administering to a
subject a direct fed microbial in combination with a protease, a
xylanase, an amylase and a phytase.
[0010] A yet further aspect of the present invention is use of a
direct fed microbial in combination with a protease, a xylanase, an
amylase and a phytase for improving the performance of a subject or
for improving digestibility of a raw material in a feed (e.g.
nutrient digestibility, such as amino acid digestibility) or for
improving nitrogen retention) or for avoiding the negative effects
of necrotic enteritis or for improving feed conversion ratio (FCR)
or for improving weight gain in a subject or for improving feed
efficiency in a subject or for modulating (e.g. improving) the
immune response of the subject or for promoting the growth of
beneficial bacteria in the gastrointestinal tract of a subject or
for reducing populations of pathogenic bacteria in the
gastrointestinal tract of a subject, or for reducing nutrient
excretion in manure.
[0011] In a further aspect of the present invention there is
provided a kit comprising a direct fed microbial, a protease, a
xylanase, an amylase, a phytase (and optionally at least one
vitamin and/or optionally at least one mineral) and instructions
for administration.
[0012] In another aspect the present invention provides a method of
preparing a feed additive composition, comprising admixing a direct
fed microbial with a protease, a xylanase, an amylase and a phytase
and (optionally) packaging.
[0013] In a yet further aspect the present invention provides feed
or feedstuff comprising a feed additive composition comprising (or
consisting essentially of or consisting of) a direct fed microbial
in combination with a protease, a xylanase, an amylase and a
phytase.
[0014] A premix comprising a feed additive composition comprising
(or consisting essentially of or consisting of) a direct fed
microbial in combination with a protease, a xylanase, an amylase
and a phytase, and at least one mineral and/or at least one
vitamin.
[0015] In another aspect, the present invention provides a method
of preparing a feedstuff comprising admixing a feed component with
a feed additive composition comprising (or consisting essentially
of or consisting of) a direct fed microbial in combination with a
protease, a xylanase, an amylase and a phytase.
[0016] In a further aspect, the present invention relates to a feed
additive composition for preventing and/or treating coccidiosis
and/or necrotic enteritis in a subject.
[0017] The present invention yet further provides a method of
preventing and/or treating necrotic enteritis and/or coccidiosis
wherein an effective amount of a feed additive composition
according to the present invention is administered to a
subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows that a combination of DFM (Enviva Pro.RTM.
available from Danisco A/S) with a combination of a xylanase (e.g.
an endo-xylanase from Trichoderma xylanase), an amylase (e.g. a
Bacillus licheniformis alpha-amylase), a protease (e.g. Bacillus
subtilis protease) and a phytase (e.g. 500 FTU/kg of Phyzyme XP (an
E. coli phytase) available from Danisco A/S) significantly improved
(reduced) necrotic enteritis lesion scores in the gut of the
animals compared with the challenged control. In some embodiments
the xylanase, amylase and protease may formulated together in
AxtraXAP.RTM. [containing 2000 XU/kg feed of xylanase; 200 AU/kg
feed of amylase and 4000 PU/kg feed of protease] also available
from Danisco A/S).
[0019] FIG. 2 shows that a combination of (Enviva Pro.RTM.
available from Danisco A/S) with a combination of a xylanase (e.g.
an endo-xylanase from Trichoderma xylanase), an amylase (e.g. a
Bacillus licheniformis alpha-amylase), a protease (e.g. Bacillus
subtilis protease) and a phytase (e.g. 500 FTU/kg of Phyzyme XP (an
E. coli phytase) available from Danisco A/S) significantly improved
Body weight gain (BW gain) in broiler chickens challenged with
Clostridium perfringens compared with the challenged control--even
resulting in a BW gain which was improved over a negative control
(i.e. an unchallenged control). This was significantly better than
any other combinations of enzymes such as either amylase and
phytase or protease and phytase, and significantly better than DFM
applied on the challenged control. In some embodiments the
xylanase, amylase and protease may formulated together in
AxtraXAP.RTM. [containing 2000 XU/kg feed of xylanase; 200 AU/kg
feed of amylase and 4000 PU/kg feed of protease] also available
from Danisco A/S). Pooled SEM=28.6
[0020] FIG. 3 shows a combination of (Enviva Pro.RTM. available
from Danisco A/S) with a combination of a xylanase (e.g. an
endo-xylanase from Trichoderma xylanase), an amylase (e.g. a
Bacillus licheniformis alpha-amylase), a protease (e.g. Bacillus
subtilis protease) and a phytase (e.g. 500 FTU/kg of Phyzyme XP (an
E. coli phytase) available from Danisco A/S) significantly improved
feed conversion ratio (FCR) (g feed intake/g BW gain) in broiler
chickens challenged with Clostridium perfringens to the level of
unchallenged birds. This was significantly better than other
combinations of enzymes with the DFM such as either amylase and
phytase or protease and phytase. In some embodiments the xylanase,
amylase and protease may formulated together in AxtraXAP.RTM.
[containing 2000 XU/kg feed of xylanase; 200 AU/kg feed of amylase
and 4000 PU/kg feed of protease] also available from Danisco
A/S).
[0021] FIG. 4 shows relative mRNA expression of IFN-g used as
marker of inflammation in the intestine, and shows that a
combination of DFM (Enviva Pro.RTM.) with a combination of
xylanase, amylase, protease and phytase (Avizyme 1502.RTM.
available from Danisco A/S+500 FTU/kg of Phyzyme XP (an E. coli
phytase) increased IFN-g expression at 11 days and reduced it at 20
days.
[0022] FIG. 5 shows apparent ileal digestible energy (mCal/kg) and
shows that a combination of DFM (Enviva Pro.RTM.) with a xylanase,
amylase, protease and phytase (two different enzyme mixes were used
the first was Avizyme 1502.RTM. available from Danisco A/S+500
FTU/kg of Phyzyme XP (an E. coli phytase); and the second was
AxtraXAP [containing 2000 XU/kg feed of xylanase; 200 AU/kg feed of
amylase and 4000 PU/kg feed of protease] also available from
Danisco A/S+500 FTU/kg of Phyzyme XP (an E. coli phytase)
significantly improved energy digestibility effects.
[0023] FIG. 6 shows amino acid digestibility significantly improved
with a combination of DFM (Enviva Pro.RTM.) with a xylanase,
amylase, protease and phytase. The improvement of digestibility of
the undigested fractions of amino acid at the ileal level with a
combination of DMF with xylanase, amylase, protease and phytase was
greater than the improvement of DFM alone or the combination of
xylanase, amylase, protease and phytase without DFM.
[0024] FIG. 7 shows energy digestibility improved with a
combination of DFM (Enviva Pro.RTM.) with a xylanase, amylase,
protease and phytase.
[0025] FIG. 8 shows nitrogen-corrected apparent metabolizable
energy AMEn of dietary treatments fed to 17 to 21-d-old broiler
chickens.
[0026] FIG. 9 shows that a combination of DFM (Enviva Pro.RTM.)
with a xylanase, amylase, protease and phytase (two different
enzyme mixes were used the first was Avizyme 1502.RTM. available
from Danisco A/S+500 FTU/kg of Phyzyme XP (an E. coli phytase); and
the second was AxtraXAP also available from Danisco A/S+500 FTU/kg
of Phyzyme XP (an E. coli phytase) significantly improved nitrogen
retention.
[0027] FIG. 10 shows that a combination of DFM (Enviva Pro.RTM.)
with a xylanase, amylase, protease and phytase (Avizyme 1502.RTM.
available from Danisco A/S+Phyzyme XP (an E. coli phytase))
significantly reduces the mRNA abundance of MUC-2 in the ileal
mucosal scrapings at day 14 treated with an overdosed coccidian
vaccine at hatch, compared to the challenged and unchallenged
control treatments.
[0028] FIG. 11 shows the amino acid sequence (SEQ ID No. 1) of a
pepsin resistant alpha amylase from Bacillus licheniformis.
[0029] FIG. 12 shows the nucleotide sequence (SEQ ID No. 2) of a
pepsin resistant alpha amylase from Bacillus licheniformis.
[0030] FIG. 13 shows the amino acid sequence (SEQ ID No. 3) of a
pepsin resistant alpha amylase from Trichoderma reesei.
[0031] FIG. 14 shows the nucleotide sequence (SEQ ID No. 4) of a
pepsin resistant alpha amylase from Trichoderma reesei.
[0032] FIG. 15 shows feed conversion ratio of broiler chickens at
48 d of age.
[0033] FIG. 16 shows a heat map of expression profiles of genes of
interest for all treatments for jejunum at 23 days of age.
[0034] Unchallenged control=Unchallenged Control+phytase
[0035] CC=Challenged Control+phytase
[0036] CC+Amylase=Challenged Control+phytase+amylase
[0037] CC+XAP=Challenged
Control+phytase+xylanase+amylase+protease
[0038] CC+EP=Challenged Control+phytase+Enviva Pro
[0039] CC+EP+Amylase=Challenged Control+phytase+amylase+Enviva
Pro
[0040] CC+EP+XAP=Challenged
Control+phytase+xylanase+amylase+protease+Enviva Pro.
[0041] FIG. 17 shows a heat map of expression profile of chicken
alpha amylase for all treatments in pancreas at 23 days of age.
[0042] Unchallenged control=Unchallenged Control+phytase
[0043] CC=Challenged Control+phytase
[0044] CC+Amylase=Challenged Control+phytase+amylase
[0045] CC+XAP=Challenged
Control+phytase+xylanase+amylase+protease
[0046] CC+EP=Challenged Control+phytase+Enviva Pro
[0047] CC+EP+Amylase=Challenged Control+phytase+amylase+Enviva
Pro
[0048] CC+EP+XAP=Challenged
Control+phytase+xylanase+amylase+protease+Enviva Pro.
[0049] FIG. 18 shows apparent metabolizable energy corrected by
nitrogen retention (AME.sub.n) of 21 d old broiler chickens. Effect
of DFM; P<0.001; Effect of Enzyme; P<0.001; Effect of
DFM.times.Enzyme; P=0.27; Pooled SEM=32 kcal.
[0050] FIG. 19 shows feed conversion ratio (FCR) of broiler
chickens in a necrotic enteritis challenge model (Pooled SEM:
0.015).
[0051] FIG. 20 shows relative proportion of Lactobacillus spp. at
21 d in jejunum in broiler chickens, ChSq<0.0001.
DETAILED DESCRIPTION OF THE INVENTION
[0052] Preferably each of the enzymes used in the present invention
are exogenous to the DFM. In other words the enzymes are preferably
added to or admixed with the DFM.
[0053] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR
BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale
& Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper
Perennial, N.Y. (1991) provide one of skill with a general
dictionary of many of the terms used in this disclosure.
[0054] This disclosure is not limited by the exemplary methods and
materials disclosed herein, and any methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of embodiments of this disclosure. Numeric ranges are
inclusive of the numbers defining the range. Unless otherwise
indicated, any nucleic acid sequences are written left to right in
5' to 3' orientation; amino acid sequences are written left to
right in amino to carboxy orientation, respectively.
[0055] The headings provided herein are not limitations of the
various aspects or embodiments of this disclosure which can be had
by reference to the specification as a whole. Accordingly, the
terms defined immediately below are more fully defined by reference
to the specification as a whole.
[0056] Amino acids are referred to herein using the name of the
amino acid, the three letter abbreviation or the single letter
abbreviation.
[0057] The term "protein", as used herein, includes proteins,
polypeptides, and peptides.
[0058] As used herein, the term "amino acid sequence" is synonymous
with the term "polypeptide" and/or the term "protein". In some
instances, the term "amino acid sequence" is synonymous with the
term "peptide". In some instances, the term "amino acid sequence"
is synonymous with the term "enzyme".
[0059] The terms "protein" and "polypeptide" are used
interchangeably herein. In the present disclosure and claims, the
conventional one-letter and three-letter codes for amino acid
residues may be used. The 3-letter code for amino acids as defined
in conformity with the IUPACIUB Joint Commission on Biochemical
Nomenclature (JCBN). It is also understood that a polypeptide may
be coded for by more than one nucleotide sequence due to the
degeneracy of the genetic code.
[0060] Other definitions of terms may appear throughout the
specification. Before the exemplary embodiments are described in
more detail, it is to understand that this disclosure is not
limited to particular embodiments described, as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting, since the scope of the
present disclosure will be limited only by the appended claims.
[0061] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within this disclosure. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within this disclosure, subject to any specifically excluded limit
in the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in this disclosure.
[0062] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "an enzyme" includes a plurality of such
candidate agents and reference to "the feed" includes reference to
one or more feeds and equivalents thereof known to those skilled in
the art, and so forth.
[0063] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
such publications constitute prior art to the claims appended
hereto.
[0064] The enzymes for use in the present invention can be produced
either by solid or submerged culture, including batch, fed-batch
and continuous-flow processes. Culturing is accomplished in a
growth medium comprising an aqueous mineral salts medium, organic
growth factors, the carbon and energy source material, molecular
oxygen, and, of course, a starting inoculum of one or more
particular microorganism species to be employed.
Direct Fed Microbial (DFM)
[0065] The term "microbial" herein is used interchangeably with
"microorganism".
[0066] Preferably the DFM comprises a viable microorganism.
Preferably the DFM comprises a viable bacterium or a viable yeast
or a viable fungi.
[0067] In one preferred embodiment the DFM comprises a viable
bacteria.
[0068] The term "viable microorganism" means a microorganism which
is metabolically active or able to differentiate.
[0069] In one embodiment the DFM may be a spore forming bacterium
and hence the term DFM may be comprised of or contain spores, e.g.
bacterial spores. Therefore in one embodiment the term "viable
microorganism" as used herein may include microbial spores, such as
endospores or conidia.
[0070] In another embodiment the DFM in the feed additive
composition according to the present invention is not comprised of
or does not contain microbial spores, e.g. endospores or
conidia.
[0071] The microorganism may be a naturally occurring microorganism
or it may be a transformed microorganism. The microorganism may
also be a combination of suitable microorganisms.
[0072] In some aspects, the DFM according to the present invention
may be one or more of the following: a bacterium, a yeast or a
fungi.
[0073] Preferably the DFM according to the present invention is a
probiotic microorganism.
[0074] In the present invention, the term direct fed microbial
(DFM) encompasses direct fed bacteria, direct fed yeast, direct fed
yeast and combinations thereof.
[0075] Preferably the DFM is a direct fed bacterium.
[0076] Preferably the DFM is a combination comprising two or more
bacteria, e.g. three or more or four or more; or the DFM is a
combination comprising two or more bacterial strains, e.g. three or
more or four or more.
[0077] Preferably the bacterium or bacteria is or are isolated.
[0078] Suitably the DFM may comprise a bacterium from one or more
of the following genera: Lactobacillus, Lactococcus, Streptococcus,
Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium,
Propionibacterium, Bifidobacterium, Clostridium and Megasphaera and
combinations thereof.
[0079] In one embodiment the DFM may be selected from the following
Bacillus spp: Bacillus subtilis, Bacillus cereus, Bacillus
licheniformis and Bacillus amyloliquefaciens.
[0080] In one embodiment the DFM may be a combination comprising
two or more Bacillus strains.
[0081] In one embodiment the DFM may be a combination of two or
more the Bacillus subtilis strains 3A-P4 (PTA-6506); 15A-P4
(PTA-6507); 22C-P1 (PTA-6508); 2084 (NRRL B-500130); LSSA01
(NRRL-B-50104); BS27 (NRRL B-50105); BS 18 (NRRL B-50633); and BS
278 (NRRL B-50634).
[0082] Strains 3A-P4 (PTA-6506), 15A-P4 (PTA-6507) and 22C-P1
(PTA-6508) are publically available from American Type Culture
Collection (ATCC).
[0083] Strains 2084 (NRRL B-500130); LSSA01 (NRRL-B-50104); BS27
(NRRL B-50105) are publically available from the Agricultural
Research Service Culture Collection (NRRL). Strain Bacillus
subtilis LSSA01 is sometimes referred to as B. subtilis 8.
[0084] These strains are taught in U.S. Pat. No. 7,754,469 B2.
[0085] Bacillus subtilis BS 18 and Bacillus subtilis BS 278 were
deposited by Andy Madisen of W227 N752 Westmound Dr. Waukesha, Wis.
53186, USA or Danisco USA Inc. of W227 N752 Westmound Dr. Waukesha,
Wis. 53186, USA under the Budapest Treaty at the Agricultural
Research Service Culture Collection (NRRL) at 1815 North University
Street, Peoria, Ill. 61604, United States of America, under deposit
numbers NRRL B-50633 and NRRL B-50634, respectively on 9 Jan.
2012.
[0086] Andy Madisen of W227 N752 Westmound Dr. Waukesha, Wis.
53186, USA and Danisco USA Inc. of W227 N752 Westmound Dr.
Waukesha, Wis. 53186, USA authorise Danisco A/S of Langebrogade 1,
PO Box 17, DK-1001, Copenhagen K, Denmark to refer to these
deposited biological materials in this patent application and have
given unreserved and irrevocable consent to the deposited material
being made available to the public.
[0087] In some embodiments the DFM may be a combination comprising
the Bacillus subtilis strains as detailed in the table below:
TABLE-US-00001 B. subtilis strain Bs Bs Bs Bs Bs Bs Bs 2084 8
(LSSAO1) 3A-P4 15A-P4 278 18 22C-P1 DFM X X X X Combi- X X X nation
X X X comprises X X X X X X X X X X X X X X X X X X
[0088] In one embodiment the DFM may be selected from the following
Lactococcus spp: Lactococcus cremoris and Lactococcus lactis and
combinations thereof.
[0089] In one embodiment the DFM may be selected from the following
Lactobacillus spp: Lactobacillus buchneri, Lactobacillus
acidophilus, Lactobacillus casei, Lactobacillus kefiri,
Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus
helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus,
Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus
bulgaricus, Lactobacillus sakei, Lactobacillus reuteri,
Lactobacillus fermentum, Lactobacillus farciminis, Lactobacillus
lactis, Lactobacillus delbreuckii, Lactobacillus plantarum,
Lactobacillus paraplantarum, Lactobacillus farciminis,
Lactobacillus rhamnosus, Lactobacillus crispatus, Lactobacillus
gasseri, Lactobacillus johnsonii and Lactobacillus jensenii, and
combinations of any thereof.
[0090] In one embodiment the DFM may be selected from the following
Bifidobacteria spp: Bifidobacterium lactis, Bifidobacterium
bifidium, Bifidobacterium longum, Bifidobacterium animalis,
Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium
catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium
adolescentis, and Bifidobacterium angulatum, and combinations of
any thereof.
[0091] Suitably the DFM may comprise a bacterium from one or more
of the following species: Bacillus subtilis, Bacillus
licheniformis, Bacillus amyloliquefaciens, Enterococcus,
Enterococcus spp, and Pediococcus spp, Lactobacillus spp,
Bifidobacterium spp, Lactobacillus acidophilus, Pediococsus
acidilactici, Lactococcus lactis, Bifidobacterium bifidum, Bacillus
subtilis, Propionibacterium thoenii, Lactobacillus farciminis,
Lactobacillus rhamnosus, Megasphaera elsdenii, Clostridium
butyricum, Bifidobacterium animalis ssp. animalis, Lactobacillus
reuteri, Bacillus cereus, Lactobacillus salivarius ssp. Salivarius,
Propionibacteria sp and combinations thereof.
[0092] The direct fed bacterium used in the present invention may
be of the same type (genus, species and strain) or may comprise a
mixture of genera, species and/or strains.
[0093] Suitably the DFM according to the present invention may be
one or more of the products or the microorganisms contained in
those products as in the Table below:
TABLE-US-00002 Symbiotic Product Name Company Microorganism(s)
ingredients Enviva Pro .RTM.. Danisco A/S Bacillus subtilis strain
2084 (formerly known as Accession No. NRRl B-50013, Avicorr .RTM.)
Bacillus subtilis strain LSSAO1 Accession No. NRRL B- 50104 and
Bacillus subtilis strain 15A-P4 ATCC Accession No. PTA-6507
Calsporin .RTM. Calpis - Japan Bacillus subtilis Strain C3102
Clostat .RTM. Kemin Industries Bacillus subtilis Strain PB6 Inc.
Cylactin .RTM. DSM Enterococcus NCIMB 10415 (SF68) Gallipro .RTM.
& Chr. Hansen A/S Bacillus subtilis Strain C3102 GalliproMax
.RTM. Gallipro .RTM.Tect .RTM. Chr. Hansen A/S Bacillus
licheniformis Poultry star .RTM. Biomin, Inc Enterococcus and
Pediococcus Fructo- oligosaccharides Protexin .RTM. Protexin Int
Lactobacillus, Bifidobacterium and another Proflora .RTM. Alpharma
Inc. Bacillus subtilis strain QST 713 .beta.-Mos .beta.-mannan
oligosaccharides and .beta.-glucans Ecobiol .RTM. & Norel S.A.
Bacillus amyloliquefaciens Ecobiol .RTM. Plus CECT-5940 Fortiflora
.RTM. Enterococcus faecium SF68 BioPlus2B .RTM. DSM Bacillus
subtilis and Bacillus licheniformis Lactiferm .RTM. Chr. Hansen
Lactic acid bacteria 7 Enterococcus faecium CSI .RTM. Danisco A/S
Bacillus strain Yea-Sacc .RTM. Alltech Saccharomyces cerevisiae
Biomin IMB52 .RTM. Biomin Enterococcus Biomin C5 .RTM. Biomin
Pediococcus acidilactici, Enterococcus, Bifidobacterium animalis
ssp. animalis, Lactobacillus reuteri Lactobacillus salivarius ssp.
salivarius Biacton .RTM. ChemVet Lactobacillus farciminis Oralin
E1707 .RTM. Chevita GmBH Enterococcus Probios-pioneer Chr Hansen
Enterococcus (2 strains) PDFM .RTM. Lactococcus lactis DSM 11037
Sorbiflore .RTM. Danisco Animal Lactobacillus rhamnosus and
Nutrition Lactobacillus farciminis Animavit .RTM. KRKA Bacillus
subtilis Bonvital .RTM. Lactosan GmbH Enterococcus Levucell SB 20
.RTM. Lallemand Saccharomyces cerevisiae Levucell SC 0 &
Lallemand Saccharomyces cerevisiae SC10 .RTM. ME Bactocell
Lallemand Pediococcus acidilacti ActiSaf .RTM. Le Saffre
Saccharomyces cerevisiae (formerly BioSaf .RTM.) Actisaf .RTM. SC47
Le Saffre Saccharomyces cerevisiae NCYC Sc47 Miya-Gold .RTM.
Miyarisan Clostridium butyricum Pharma Fecinor and Norel S.A
Enterococcus Fecinor Plus .RTM. InteSwine .RTM. ntegro Gidave
Saccharomyces cerevisiae Ticaret AS NCYC R-625 represented by R M
Associates Ltd BioSprint .RTM. ProSol SpA Saccharomyces cerevisia
Provita .RTM. Provita Enterococcus and Lactobacillus rhamnosus
PepSoyGen-C .RTM. Regal BV Bacillus subtilis and (Nutraferma)
Aspergillus oryzae Toyocerin .RTM. Rubinum Bacillus cereus
TOYOCERIN .RTM. Rubinum Bacillus cereus var. toyoi NCIMB 40112/CNCM
I-1012
[0094] In one embodiment suitably the DFM may be Enviva Pro.RTM..
Enviva Pro.RTM. is commercially available from Danisco A/S and is a
combination of Bacillus strain 2084 Accession No. NRR1 B-50013,
Bacillus strain LSSAO1 Accession No. NRRL B-50104 and Bacillus
strain 15A-P4 ATCC Accession No. PTA-6507 (as taught in U.S. Pat.
No. 7,754,469 B--incorporated herein by reference).
[0095] Suitably, the DFM may comprise a yeast from the genera:
Saccharomyces spp.
[0096] Preferably the DFM to be used in accordance with the present
invention is a microorganism which is generally recognised as safe
and, which is preferably GRAS approved.
[0097] A skilled person will readily be aware of specific species
and or strains of microorganisms from within the genera described
herein which are used in the food and/or agricultural industries
and which are generally considered suitable for animal
consumption.
[0098] Preferably, the DFM used in accordance with the present
invention is one which is suitable for animal consumption.
[0099] Advantageously, where the product is a feed or feed additive
composition, the viable DFM should remain effective through the
normal "sell-by" or "expiration" date of the product during which
the feed or feed additive composition is offered for sale by the
retailer. The desired lengths of time and normal shelf life will
vary from feedstuff to feedstuff and those of ordinary skill in the
art will recognise that shelf-life times will vary upon the type of
feedstuff, the size of the feedstuff, storage temperatures,
processing conditions, packaging material and packaging
equipment.
[0100] In some embodiments it is important that the DFM is tolerant
to heat, i.e. is thermotolerant. This is particularly the case
where the feed is pelleted. Therefore in one embodiment the DFM may
be a thermotolerant microorganism, such as a thermotolerant
bacteria,_including for example Bacillus spp.
[0101] In some embodiments it may be preferable that the DFM is a
spore producing bacteria, such as Bacilli, e.g. Bacillus spp.
Bacilli are able to from stable endospores when conditions for
growth are unfavorable and are very resistant to heat, pH, moisture
and disinfectants.
[0102] In one embodiment suitably the DFM may decrease or prevent
intestinal establishment of pathogenic microorganism (such as
Clostridium perfringens and/or E. coli and/or Salmonella spp and/or
Campylobacter spp.).
[0103] The DFM according to the present invention may be any
suitable DFM. In one embodiment the following assay "DFM ASSAY" may
be used to determine the suitability of a microorganism to be a
DFM. For the avoidance of doubt in one embodiment a DFM selected as
an inhibitory strain (or an antipathogen DFM) in accordance with
the "DFM ASSAY" taught herein is a suitable DFM for use in
accordance with the present invention, i.e. in the feed additive
composition according to the present invention.
DFM Assay:
[0104] Tubes were seeded each with a representative pathogen from a
representative cluster.
[0105] Supernatant from a potential DFM grown aerobically or
anaerobically was added to the seeded tubes and incubated.
[0106] After incubation, the optical density (OD) of the control
and supernatant treated tubes was measured for each pathogen.
[0107] Colonies of (potential DFM) strains that produced a lowered
OD compared with the control were classified as an inhibitory
strain (or an antipathogen DFM).
[0108] The DFM assay as used herein is explained in more detail in
US2009/0280090--incorporated herein by reference.
[0109] Preferably the representative pathogen used in assay is one
(or more) of the following: Clostridium, such as Clostridium
perfringens and/or Clostridium difficile, and/or E. coli and/or
Salmonella spp and/or Campylobacter spp. In one preferred
embodiment the assay is conducted with one or more of Clostridium
perfringens and/or Clostridium difficile and/or E. coli, preferably
Clostridium perfringens and/or Clostridium difficile, more
preferably Clostridium perfringens.
[0110] In one embodiment the DFM of the present invention is
preferably an antipathogen.
[0111] The term "antipathogen" as used herein means the DFM
counters an effect (negative effect) of a pathogen.
[0112] In one embodiment to determine if a DFM is an antipathogenic
DFM the above mentioned DFM assay may be used. A DFM is considered
to be an antipathogen or antipathogenic DFM if it is classed as an
inhibitory strain in the above mentioned "DFM assay", for example
when the pathogen is Clostridium perfringens.
[0113] In one embodiment the antipathogen DFM may be one or more of
the following bacteria:
Bacillus subtilis strain 2084 Accession No. NRRL B-50013, Bacillus
subtilis strain LSSAO1 Accession No. NRRL B-50104, Bacillus
subtilis strain 15A-P4 ATCC Accession No. PTA-6507, Bacillus
subtilis strain 3A-P4 ATCC Accession No. PTA-6506, and Bacillus
subtilis strain BS27 ATCC Accession No. NRRL B-50105.
[0114] For the avoidance of doubt these strains are available and
are referred to in U.S. Pat. No. 7,754,459 B.
[0115] In one embodiment the DFM used in accordance with the
present invention is not Lactobacillus gasseri BNR 17 Strain Acc
No. KCTC 10902BP as taught in WO2008/016214.
[0116] Preferably the DFM is not an inactivated microorganism.
[0117] In one embodiment the DFM as used here is a composition
comprising one or more DFM microorganisms as described herein. The
composition may additionally comprise the enzymes of the present
invention. The composition can be fed to an animal as a direct-fed
microbial (DFM). One or more carrier(s) or other ingredients can be
added to the DFM. The DFM may be presented in various physical
forms, for example, as a top dress, as a water soluble concentrate
for use as a liquid drench or to be added to a milk replacer,
gelatin capsule, or gels. In one embodiment of the top dress form,
freeze-dried fermentation product is added to a carrier, such as
whey, maltodextrin, sucrose, dextrose, limestone (calcium
carbonate), rice hulls, yeast culture, dried starch, and/or sodium
silico aluminate. In one embodiment of the water soluble
concentrate for a liquid drench or milk replacer supplement,
freeze-dried fermentation product is added to a water soluble
carrier, such as whey, maltodextrin, sucrose, dextrose, dried
starch, sodium silico aluminate, and a liquid is added to form the
drench or the supplement is added to milk or a milk replacer. In
one embodiment of the gelatin capsule form, freeze-dried
fermentation product is added to a carrier, such as whey,
maltodextrin, sugar, limestone (calcium carbonate), rice hulls,
yeast culture dried starch, and/or sodium silico aluminate. In one
embodiment, the bacteria and carrier are enclosed in a degradable
gelatin capsule. In one embodiment of the gels form, freeze-dried
fermentation product is added to a carrier, such as vegetable oil,
sucrose, silicon dioxide, polysorbate 80, propylene glycol,
butylated hydroxyanisole, citric acid, ethoxyquin, and/or
artificial coloring to form the gel.
[0118] The DFM(s) may optionally be admixed with a dry formulation
of additives including but not limited to growth substrates,
enzymes, sugars, carbohydrates, extracts and growth promoting
micro-ingredients. The sugars could include the following: lactose;
maltose; dextrose; maltodextrin; glucose; fructose; mannose;
tagatose; sorbose; raffinose; and galactose. The sugars range from
50-95%, either individually or in combination. The extracts could
include yeast or dried yeast fermentation solubles ranging from
5-50%. The growth substrates could include: trypticase, ranging
from 5-25%; sodium lactate, ranging from 5-30%; and, Tween 80,
ranging from 1-5%. The carbohydrates could include mannitol,
sorbitol, adonitol and arabitol. The carbohydrates range from 5-50%
individually or in combination. The micro-ingredients could include
the following: calcium carbonate, ranging from 0.5-5.0%; calcium
chloride, ranging from 0.5-5.0%; dipotassium phosphate, ranging
from 0.5-5.0%; calcium phosphate, ranging from 0.5-5.0%; manganese
proteinate, ranging from 0.25-1.00%; and, manganese, ranging from
0.25-1.0%.
[0119] To prepare DFMs described herein, the culture(s) and
carrier(s) (where used) can be added to a ribbon or paddle mixer
and mixed for about 15 minutes, although the timing can be
increased or decreased. The components are blended such that a
uniform mixture of the cultures and carriers result. The final
product is preferably a dry, flowable powder. The DFM(s) or
composition comprising same can then be added to animal feed or a
feed premix, added to an animal's water, or administered in other
ways known in the art (preferably simultaneously with the enzymes
of the present invention). A feed for an animal can be supplemented
with one or more DFM(s) described herein or with a composition
described herein.
[0120] By "a mixture of at least two strains," is meant a mixture
of two, three, four, five, six or even more strains. In some
embodiments of a mixture of strains, the proportions can vary from
1% to 99%. Other embodiments of a mixture of strains are from 25%
to 75%. Additional embodiments of a mixture of strains are
approximately 50% for each strain. When a mixture comprises more
than two strains, the strains can be present in substantially equal
proportions or in different proportions in the mixture.
[0121] The DFM may be dosed appropriately.
[0122] Suitably dosages of DFM in the feed may be between about
1.times.10.sup.3 CFU/g feed to about 1.times.10.sup.9 CFU/g feed,
suitably between about 1.times.10.sup.4 CFU/g feed to about
1.times.10.sup.8 CFU/g feed, suitably between about
7.5.times.10.sup.4 CFU/g feed to about 1.times.10.sup.7 CFU/g
feed.
[0123] In one embodiment the DFM is dosed in the feedstuff at more
than about 1.times.10.sup.3 CFU/g feed, suitably more than about
1.times.10.sup.4 CFU/g feed, suitably more than about
7.5.times.10.sup.4 CFU/g feed.
[0124] Suitably dosages of DFM in the feed additive composition may
be between about 1.times.10.sup.5 CFU/g composition to about
1.times.10.sup.13 CFU/g composition, suitably between about
1.times.10.sup.6 CFU/g composition to about 1.times.10.sup.12 CFU/g
composition, suitably between about 3.75.times.10.sup.7 CFU/g
composition to about 1.times.10.sup.11 CFU/g composition.
[0125] In one embodiment the DFM is dosed in the feed additive
composition at more than about 1.times.10.sup.5 CFU/g composition,
suitably more than about 1.times.10.sup.6 CFU/g composition,
suitably more than about 3.75.times.10.sup.7 CFU/g composition.
[0126] In one embodiment the DFM is dosed in the feed additive
composition at more than about 2.times.10.sup.5 CFU/g composition,
suitably more than about 2.times.10.sup.6 CFU/g composition,
suitably more than about 3.75.times.10.sup.7 CFU/g composition.
[0127] As used herein the term "CFU" means colony forming units and
is a measure of viable cells in which a colony represents an
aggregate of cells derived from a single progenitor cell.
Xylanase
[0128] Xylanase is the name given to a class of enzymes which
degrade the linear polysaccharide beta-1,4-xylan into xylose, thus
breaking down hemicellulose, one of the major components of plant
cell walls.
[0129] The xylanase for use in the present invention may be any
commercially available xylanase.
[0130] Suitably the xylanase may be an endo-1,4-.beta.-d-xylanase
(classified as E.C. 3.2.1.8) or a 1,4,0-xylosidase (classified as
E.C. 3.2.1.37).
[0131] In one embodiment preferably the xylanase in an
endoxylanase, e.g. an endo-1,4-.beta.-d-xylanase. The
classification for an endo-1,4-.beta.-d-xylanase is E.C.
3.2.1.8.
[0132] In one embodiment the present invention relates to a DFM in
combination with an endoxylanase, e.g. an
endo-1,4-.beta.-d-xylanase, and another enzyme.
[0133] All E.C. enzyme classifications referred to here relate to
the classifications provided in Enzyme
Nomenclature--Recommendations (1992) of the nomenclature committee
of the International Union of Biochemistry and Molecular
Biology--ISBN 0-12-226164-3.
[0134] Suitably, the xylanase for use in the present invention may
be a xylanase from Bacillus, Trichoderma, Thermomyces, Aspergillus
and Penicillium.
[0135] In one embodiment the xylanase may be the xylanase in Axtra
XAP.RTM. or Avizyme 1502.RTM., both commercially available products
from Danisco A/S.
[0136] In one preferred embodiment the xylanase for use in the
present invention may be one or more of the xylanases in one or
more of the commercial products below:
TABLE-US-00003 Commercial Name .RTM. Company Xylanase type Xylanase
source Allzyme PT Alltech endo-1,4-.beta.-xylanase Aspergillus
Niger Amylofeed Andres endo-1,4-.beta.-xylanase Aspergillus Niger
(phoenicis) Pintaluba S.A Avemix 02 CS Aveve
endo-1,4-.beta.-xylanase Trichoderma reesei AveMix XG 10 Aveve, NL
endo-1,4-.beta.-xylanase Trichoderma reesei Avizyme 1100 Danisco
endo-1,4-.beta.-xylanase Trichoderma longibrachiatum Avizyme 1110
Danisco endo-1,4-.beta.-xylanase Trichoderma longibrachiatum
Avizyme 1202 Danisco endo-1,4-.beta.-xylanase Trichoderma
longibrachiatum Avizyme 1210 Danisco endo-1,4-.beta.-xylanase
Trichoderma longibrachiatum Avizyme 1302 Danisco
endo-1,4-.beta.-xylanase Trichoderma longibrachiatum Avizyme 1500
Danisco endo-1,4-.beta.-xylanase Trichoderma longibrachiatum
Avizyme 1505 Danisco endo-1,4-.beta.-xylanase Trichoderma
longibrachiatum Avizyme SX Danisco endo-1,4-.beta.-xylanase
Trichoderma longibrachiatum Belfeed MP100 Beldem
endo-1,4-.beta.-xylanase Bacillus subtilis Biofeed Plus DSM
endo-1,4-.beta.-xylanase Humicola insolens Danisco Glycosidase
Danisco Animal endo-1,4-.beta.-xylanase Trichoderma reesei (TPT/L)
Nutrition Danisco Xylanase Danisco endo-1,4-.beta.-xylanase
Trichoderma reesei Econase XT ABVista endo-1,4-.beta.-xylanase
Trichoderma reesei Endofeed .RTM. DC Andres
endo-1,4-.beta.-xylanase Aspergillus Niger Pintaluba S.A. Feedlyve
AXL Lyven endo-1,4-.beta.-xylanase Trichoderma longibrachiatum
Grindazym GP Danisco endo-1,4-.beta.-xylanase Aspergillus Niger
Grindazym GV Danisco endo-1,4-.beta.-xylanase Aspergillus Niger
Hostazym X Huvepharma endo-1,4-.beta.-xylanase Trichoderma
longibrachiatum Kemzyme Plus Dry Kemin endo-1,4-.beta.-xylanase
Trichoderma viride Kemzyme Plus Liquid Kemin
endo-1,4-.beta.-xylanase Trichoderma viride Kemzyme W dry Kemin
endo-1,4-.beta.-xylanase Trichoderma viride Kemzyme W liquid Kemin
endo-1,4-.beta.-xylanase Trichoderma viride Natugrain BASF
endo-1,4-.beta.-xylanase Trichoderma longibrachiatum Natugrain TS
Plus BASF endo-1,4-.beta.-xylanase Aspergillus Niger Natugrain
Wheat BASF endo-1,4-.beta.-xylanase Aspergillus Niger Natugrain
.RTM. TS/L BASF endo-1,4-.beta.-xylanase Aspergillus Niger Natuzyme
Bioproton endo-1,4-.beta.-xylanase Trichoderma longibrachiatum/
Trichoderma reesei Porzyme 8100 Danisco endo-1,4-.beta.-xylanase
Trichoderma longibrachiatum Porzyme 8300 Danisco
endo-1,4-.beta.-xylanase Trichoderma longibrachiatum Porzyme 9102
Danisco endo-1,4-.beta.-xylanase Trichoderma longibrachiatum
Porzyme 9310/ Danisco endo-1,4-.beta.-xylanase Trichoderma
longibrachiatum Avizyme 1310 Porzyme tp100 Danisco
endo-1,4-.beta.-xylanase Trichoderma longibrachiatum Ronozyme AX
DSM endo-1,4-.beta.-xylanase Thermomyces lanuginosus gene expressed
in Aspergillus oryzae Ronozyme WX DSM/Novozymes
endo-1,4-.beta.-xylanase Thermomyces lanuginosus gene expressed in
Aspergillus oryzae Rovabio Excel Adisseo endo-1,4-.beta.-xylanase
Penicillium funiculosum Roxazyme G2 DSM/Novozymes
endo-1,4-.beta.-xylanase Trichoderma longibrachiatum Safizym X Le
Saffre endo-1,4-.beta.-xylanase Trichoderma longibrachiatum
Xylanase Lyven endo-1,4-.beta.-xylanase Trichoderma
longibrachiatum
[0137] Preferably, the xylanase is present in the feedstuff in
range of about 500 XU/kg to about 16,000 XU/kg feed, more
preferably about 750 XU/kg feed to about 8000 XU/kg feed, and even
more preferably about 1000 XU/kg feed to about 4000 XU/kg feed
[0138] In one embodiment the xylanase is present in the feedstuff
at more than about 500 XU/kg feed, suitably more than about 600
XU/kg feed, suitably more than about 700 XU/kg feed, suitably more
than about 800 XU/kg feed, suitably more than about 900 XU/kg feed,
suitably more than about 1000 XU/kg feed.
[0139] In one embodiment the xylanase is present in the feedstuff
at less than about 16,000 XU/kg feed, suitably less than about 8000
XU/kg feed, suitably less than about 7000 XU/kg feed, suitably less
than about 6000 XU/kg feed, suitably less than about 5000 XU/kg
feed, suitably less than about 4000 XU/kg feed.
[0140] Preferably, the xylanase is present in the feed additive
composition in range of about 100 XU/g to about 320,000 XU/g
composition, more preferably about 300 XU/g composition to about
160,000 XU/g composition, and even more preferably about 500 XU/g
composition to about 50,000 XU/g composition, and even more
preferably about 500 XU/g composition to about 40,000 XU/g
composition.
[0141] In one embodiment the xylanase is present in the feed
additive composition at more than about 100 XU/g composition,
suitably more than about 200 XU/g composition, suitably more than
about 300 XU/g composition, suitably more than about 400 XU/g
composition, suitably more than about 500 XU/g composition.
[0142] In one embodiment the xylanase is present in the feed
additive composition at less than about 320,000 XU/g composition,
suitably less than about 160,000 XU/g composition, suitably less
than about 50,000 XU/g composition, suitably less than about 40,000
XU/g composition, suitably less than about 30000 XU/g
composition.
[0143] It will be understood that one xylanase unit (XU) is the
amount of enzyme that releases 0.5 .mu.mol of reducing sugar
equivalents (as xylose by the Dinitrosalicylic acid (DNS)
assay-reducing sugar method) from a oat-spelt-xylan substrate per
min at pH 5.3 and 50.degree. C. (Bailey, M. J. Biely, P. and
Poutanen, K., Journal of Biotechnology, Volume 23, (3), May 1992,
257-270).
[0144] In one embodiment suitably the enzyme is classified using
the E.C. classification above, and the E.C. classification
designates an enzyme having that activity when tested in the assay
taught herein for determining 1 XU.
Amylase
[0145] Amylase is the name given to a class of enzymes capable of
hydrolysing starch to shorter-chain oligosaccharides such as
maltose. The glucose moiety can then be more easily transferred
from maltose to a monoglyceride or glycosylmonoglyceride than from
the original starch molecule.
[0146] The term amylase includes .alpha.-amylases (E.C. 3.2.1.1),
G4-forming amylases (E.C. 3.2.1.60), .beta.-amylases (E.C. 3.2.1.2)
and 7-amylases (E.C. 3.2.1.3).
[0147] In one embodiment preferably the amylase is an
.alpha.-amylase. .alpha.-Amylases are classified as (E.C.
3.2.1.1).
[0148] These can include amylases of bacterial or fungal origin,
chemically modified or protein engineered mutants are included.
[0149] In one embodiment preferably the amylase may be an amylase,
e.g. an .alpha.-amylase, from Bacillus licheniformis and/or an
amylase, e.g. an .alpha.-amylase, from Bacillus
amyloliquefaciens.
[0150] In one embodiment the .alpha.-amylase may be the
.alpha.-amylase in Axtra XAP.RTM. or Avizyme 1502.RTM., both
commercially available products from Danisco A/S.
[0151] In another embodiment the amylase may be a pepsin resistant
.alpha.-amylase, such as a pepsin resistant Trichoderma (such as
Trichoderma reesei) alpha amylase. A suitably pepsin resistant
.alpha.-amylase is taught in UK application number 1011513.7 (which
is incorporated herein by reference) and PCT/IB2011/053018 (which
is incorporated herein by reference).
[0152] In one embodiment the amylase may be a pepsin resistant
.alpha.-amylase comprising or consisting of an amino acid sequence:
[0153] i) as set forth in SEQ ID No. 1 or SEQ ID No. 3; [0154] ii)
as set forth in SEQ ID No. 1 or SEQ ID No. 3 except for one or
several amino acid additions/insertions, deletions or
substitutions; [0155] iii) having at least 85% (preferably, at
least 90%, 95%, 97%, 98% or 99%) identity to SEQ ID No. 1 or at
least 70% (preferably, at least 75%, 80%, 85%, 90%, 95%, 97%, 98%
or 99%) identity to SEQ ID No. 3; [0156] iv) which is produced by
expression of a nucleotide sequence comprising the sequence of SEQ
ID No. 2 or SEQ ID No. 4; [0157] v) which is produced by expression
of a nucleotide sequence which differs from SEQ ID No. 2 or SEQ ID
No. 4 due to the degeneracy of the genetic code; [0158] vi) which
is produced by expression of a nucleotide sequence which differs
from SEQ ID No. 2 or SEQ ID No. 4 by one or several nucleotide
additions/insertions, deletions or substitutions; or [0159] vii)
which is produced by expression of a nucleotide sequence which has
at least 70% (preferably, at least 75%, 80%, 85%, 90%, 95%, 97%,
98% or 99%) identity to SEQ ID No. 2 or SEQ ID No. 4.
[0160] The pepsin resistant alpha amylase may also be encoded by a
nucleotide sequence which hybridises to SEQ ID No. 2 or SEQ ID No.
4 under stringent or highly stringent conditions.
[0161] In one preferred embodiment the amylase for use in the
present invention may be one or more of the amylases in one or more
of the commercial products below:
TABLE-US-00004 Commercial product .RTM. Company Amylase type
Amylase source Amylofeed Andres alpha amylase Aspergillus oryzae
Pintaluba S.A Avizyme 1500 Danisco alpha amylase Bacillus
amyloliquefaciens Avizyme 1505 Danisco alpha amylase Bacillus
amyloliquefaciens Kemzyme Plus Dry Kemin alpha-amylase Bacillus
amyloliquefaciens Kemzyme Plus Liquid Kemin alpha-amylase Bacillus
amyloliquefaciens Kemzyme W dry Kemin alpha-amylase Bacillus
amyloliquefaciens Kemzyme W liquid Kemin alpha-amylase Bacillus
amyloliquefaciens Natuzyme Bioproton alpha-amylase Trichoderma
longibrachiatum/ Trichoderma reesei Porzyme 8100 Danisco
alpha-amylase Bacillus amyloliquefaciens Porzyme tp100 Danisco
alpha-amylase Bacillus amyloliquefaciens Ronozyme A DSM/Novozymes
alpha-amylase Bacillus amyloliquefaciens Ronozyme AX DSM
alpha-amylase Bacillus amyloliquefaciens Ronozyme .RTM. RumiStar
DSM/Novozymes alpha-amylase Bacillus stearothermophilus (L/CT)
expressed in Bacillus licheniformis
[0162] In one embodiment the amylase may be a maltogenic
alpha-amylase from Bacillus (see EP 120 693). This amylase is
commercially available under the trade name Novamyl (Novo Nordisk
A/S, Denmark). Novamyl is described in detail in International
Patent Publication WO 91/104669.
[0163] Preferably, the amylase is present in the feedstuff in range
of about 50 AU/kg to about 10,000 AU/kg feed, more preferably about
70 AU/kg feed to about 7500 AU/kg feed, more preferably about 70
AU/kg feed to about 5000 AU/kg feed and even more preferably about
100 AU/kg feed to about 2000 AU/kg feed.
[0164] In one embodiment the amylase is present in the feedstuff at
more than about 50 AU/kg feed, suitably more than about 60 AU/kg
feed, suitably more than about 70 AU/kg feed, suitably more than
about 80 AU/kg feed, suitably more than about 90 AU/kg feed,
suitably more than about 100 AU/kg feed.
[0165] In one embodiment the amylase is present in the feedstuff at
less than about 10,000 AU/kg feed, suitably less than about 8000
AU/kg feed, suitably less than about 7000 AU/kg feed, suitably less
than about 5000 AU/kg feed, suitably less than about 4000 AU/kg
feed, suitably less than about 3000 AU/kg feed, suitably less than
about 2000 AU/kg feed.
[0166] Preferably, the amylase is present in the feed additive
composition in range of about 10 AU/kg to about 200,000 AU/g
composition, more preferably about 30 AU/g composition to about
100,000 AU/g composition, and even more preferably about 40 AU/g
composition to about 50,000 AU/g composition, and even more
preferably about 50 AU/g composition to about 20,000 AU/g
composition.
[0167] In one embodiment the amylase is present in the feed
additive composition at more than about 10 AU/g composition,
suitably more than about 20 AU/g composition, suitably more than
about 30 AU/g composition, suitably more than about 40 AU/g
composition, suitably more than about 50 AU/g composition.
[0168] In one embodiment the amylase is present in the feed
additive composition at less than about 200,000 AU/g composition,
suitably less than about 100,000 AU/g composition, suitably less
than about 50,000 AU/g composition, suitably less than about 40,000
AU/g composition, suitably less than about 30000 AU/g composition,
suitably less than about 20000 AU/g composition.
[0169] It will be understood that one amylase unit (AU) is the
amount of enzyme that releases 1 mmol of glucosidic linkages from a
water insoluble cross-linked starch polymer substrate per min at pH
6.5 and 37.degree. C. (this may be referred to herein as the assay
for determining 1 AU).
[0170] 1 TAU (.alpha.-amylase activity) is the amount of enzyme
required to release (in the presence of excess .alpha.-glucosidase)
0.20 mmol of glucosidic linkages (expressed as p-nitrophenol
equivalents) from a maltoheptaoside substrate per minute at pH 8.0
and 40.degree. C. This may be referred to herein as the assay for
determining 1 TAU unit.
[0171] In one embodiment suitably the enzyme is classified using
the E.C. classification above, and the E.C. classification
designates an enzyme having that activity when tested in the assay
taught herein for determining 1 AU.
Protease
[0172] The term protease as used herein is synonymous with
peptidase or proteinase.
[0173] The protease for use in the present invention may be a
subtilisin (E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or
an alkaline serine protease (E.C. 3.4.21.x) or a keratinase (E.C.
3.4.x.x).
[0174] Preferably the protease in accordance with the present
invention is a subtilisin.
[0175] Suitable proteases include those of animal, vegetable or
microbial origin. Chemically modified or protein engineered mutants
are also suitable. The protease may be a serine protease or a
metalloprotease, e.g., an alkaline microbial protease or a
trypsin-like protease. Examples of alkaline proteases are
subtilisins, especially those derived from Bacillus sp., e.g.,
subtilisin Novo, subtilisin Carlsberg, subtilisin 309 (see, e.g.,
U.S. Pat. No. 6,287,841), subtilisin 147, and subtilisin 168 (see,
e.g., WO 89/06279). Examples of trypsin-like proteases are trypsin
(e.g., of porcine or bovine origin), and Fusarium proteases (see,
e.g., WO 89/06270 and WO 94/25583). Examples of useful proteases
also include but are not limited to the variants described in WO
92/19729 and WO 98/20115.
[0176] In one preferred embodiment the protease for use in the
present invention may be one or more of the proteases in one or
more of the commercial products below:
TABLE-US-00005 Commercial product .RTM. Company Protease type
Protease source Avizyme 1100 Danisco A/S Subtilisin Bacillus
subtilis Avizyme 1202 Danisco A/S Subtilisin Bacillus subtilis
Avizyme 1302 Danisco A/S Subtilisin Bacillus subtilis Avizyme 1500
Danisco A/S Subtilisin Bacillus subtilis Avizyme 1505 Danisco A/S
Subtilisin Bacillus subtilis Kemzyme Plus Dry Kemin Bacillolysin
Bacillus amyloliquefaciens Kemzyme W dry Kemin Bacillolysin
Bacillus amyloliquefaciens Natuzyme Bioproton Protease Trichoderma
longibrachiatum/ Trichoderma reesei Porzyme 8300 Danisco Subtilisin
Bacillus subtilis Ronozyme ProAct DSM/Novozymes Alkaline serine
Nocardiopsis prasina protease gene expressed in Bacillus
licheniformis Versazyme/Cibenza Novus Keratinase Bacillus
licheniformis DP100
[0177] In one embodiment the protease may be a protease from B.
subtilis.
[0178] In one embodiment the protease may be a Nocardiopsis
protease available from Novozymes A/S.
[0179] Preferably, the protease is present in the feedstuff in
range of about 1000 U/kg to about 20,000 PU/kg feed, more
preferably about 1500 PU/kg feed to about 10000 PU/kg feed, more
preferably about 2000 PU/kg feed to about 6000 PU/kg feed.
[0180] In one embodiment the protease is present in the feedstuff
at more than about 1000 PU/kg feed, suitably more than about 1500
PU/kg feed, suitably more than about 2000 PU/kg feed.
[0181] In one embodiment the protease is present in the feedstuff
at less than about 20,000 PU/kg feed, suitably less than about
10000 PU/kg feed, suitably less than about 7000 PU/kg feed,
suitably less than about 6000 PU/kg feed.
[0182] Preferably, the protease is present in the feed additive
composition in range of about 200 PU/g to about 400,000 PU/g
composition, more preferably about 300 PU/g composition to about
200,000 PU/g composition, and even more preferably about 5000 PU/g
composition to about 100,000 PU/g composition, and even more
preferably about 700 PU/g composition to about 70,000 PU/g
composition, and even more preferably about 1000 PU/g composition
to about 60,000 PU/g composition.
[0183] In one embodiment the protease is present in the feed
additive composition at more than about 200 PU/g composition,
suitably more than about 300 PU/g composition, suitably more than
about 400 PU/g composition, suitably more than about 500 PU/g
composition, suitably more than about 750 PU/g composition,
suitably more than about 1000 PU/g composition.
[0184] In one embodiment the protease is present in the feed
additive composition at less than about 400,000 PU/g composition,
suitably less than about 200,000 PU/g composition, suitably less
than about 100,000 PU/g composition, suitably less than about
80,000 PU/g composition, suitably less than about 70000 PU/g
composition, suitably less than about 60000 PU/g composition.
[0185] It will be understood that one protease unit (PU) is the
amount of enzyme that liberates from the substrate (0.6% casein
solution) one microgram of phenolic compound (expressed as tyrosine
equivalents) in one minute at pH 7.5 (40 mM Na.sub.2PO.sub.4/lactic
acid buffer) and 40.degree. C. This may be referred to as the assay
for determining 1 PU.
[0186] In one embodiment suitably the enzyme is classified using
the E.C. classification above, and the E.C. classification
designates an enzyme having that activity when tested in the assay
taught herein for determining 1 PU.
Phytase
[0187] The phytase for use in the present invention may be
classified a 6-phytase (classified as E.C. 3.1.3.26) or a 3-phytase
(classified as E.C. 3.1.3.8).
[0188] In one embodiment the phytase may be a 6-phytase (E.C.
3.1.3.26).
[0189] In one preferred embodiment the phytase for use in the
present invention may be one or more of the phytases in one or more
of the commercial products below:
TABLE-US-00006 Commercial product .RTM. Company Phytase type
Phytase source Finase ABVista 3-phytase Trichoderma reesei Finase
EC ABVista 6-phytase E. coli gene expressed in Trichoderma reesei
Natuphos BASF 3-phytase Aspergillus Niger Natuzyme Bioproton
phytase (type Trichoderma longibrachiatum/ not specified)
Trichoderma reesei OPTIPHOS .RTM. Huvepharma AD 6-phytase E. coli
gene expressed in Pichia pastoris Phytase sp1002 DSM 3-phytase A
consensus gene expressed in Hansenula polymorpha Phyzyme XP Danisco
6-phytase E. coli gene expressed in Schizosaccahomyces pombe
Quantum 2500D, 5000L ABVista 6-phytase E. coli gene expressed in
Pichia pastoris or Trichoderma Ronozyme Hi-Phos DSM/Novozymes
6-phytase Citrobacter braakii (M/L) gene expressed in Aspergillus
oryzae Ronozyme NP DSM/Novozymes 6-phytase Peniphora lycii gene
expressed in Aspergillus oryzae Ronozyme P DSM/Novozymes 6-phytase
Peniphora lycii gene expressed in Aspergillus oryzae Rovabio PHY
Adisseo 3-phytase Penicillium funiculosum
[0190] The term consensus gene as used herein means that the DNA
vector used to transform the organism contains a synthetic phytase
gene based on a consensus sequence, a URA gene from the
non-pathogenic yeast Saccharomyces cerevisiae and the origin of
replication of the Escherichia coli plasmid pBR322.
[0191] In one embodiment the phytase is a Citrobacter phytase
derived from e.g. Citrobacter freundii, preferably C. freundii
NCIMB 41247 and variants thereof e.g. as disclosed in WO2006/038062
(incorporated herein by reference) and WO2006/038128 (incorporated
herein by reference), Citrobacter braakii YH-15 as disclosed in WO
2004/085638, Citrobacter braakii ATCC 51113 as disclosed in
WO2006/037328 (incorporated herein by reference), as well as
variants thereof e.g. as disclosed in WO2007/112739 (incorporated
herein by reference) and WO2011/117396 (incorporated herein by
reference), Citrobacter amalonaticus, preferably Citrobacter
amalonaticus ATCC 25405 or Citrobacter amalonaticus ATCC 25407 as
disclosed in WO2006037327 (incorporated herein by reference),
Citrobacter gillenii, preferably Citrobacter gillenii DSM 13694 as
disclosed in WO2006037327 (incorporated herein by reference), or
Citrobacter intermedius, Citrobacter koseri, Citrobacter murliniae,
Citrobacter rodentium, Citrobacter sedlakii, Citrobacter werkmanii,
Citrobacter youngae, Citrobacter species polypeptides or variants
thereof.
[0192] In one embodiment the phytase may be a phytase from
Citrobacter, e.g. from Citrobacter freundii, such as the phytase
enzyme(s) taught in WO2006/038128, which reference is incorporated
herein by reference.
[0193] In preferred embodiments, the phytase is preferably E. coli
phytase marketed under the name Phyzyme XP.TM. by Danisco A/S.
[0194] Alternatively the phytase may be a Buttiauxella phytase,
e.g. a Buttiauxella agrestis phytase, for example, the phytase
enzymes taught in WO 2006/043178, WO 2008/097619, WO2009/129489,
WO2008/092901, PCT/U52009/41011 or PCTAB2010/051804, all of which
are incorporated herein by reference.
[0195] In one embodiment the phytase may be a phytase from Hafnia,
e.g. from Hafnia alvei, such as the phytase enzyme(s) taught in
US2008263688, which reference is incorporated herein by
reference.
[0196] In one embodiment the phytase may be a phytase from
Aspergillus, e.g. from Apergillus orzyae.
[0197] In one embodiment the phytase may be a phytase from
Penicillium, e.g. from Penicillium funiculosum.
[0198] Preferably, the phytase is present in the feedstuff in range
of about 200 FTU/kg to about 1000 FTU/kg feed, more preferably
about 300 FTU/kg feed to about 750 FTU/kg feed, more preferably
about 400 FTU/kg feed to about 500 FTU/kg feed.
[0199] In one embodiment the phytase is present in the feedstuff at
more than about 200 FTU/kg feed, suitably more than about 300
FTU/kg feed, suitably more than about 400 FTU/kg feed.
[0200] In one embodiment the phytase is present in the feedstuff at
less than about 1000 FTU/kg feed, suitably less than about 750
FTU/kg feed.
[0201] Preferably, the phytase is present in the feed additive
composition in range of about 40 FTU/g to about 40,000 FTU/g
composition, more preferably about 80 FTU/g composition to about
20,000 FTU/g composition, and even more preferably about 100 FTU/g
composition to about 10,000 FTU/g composition, and even more
preferably about 200 FTU/g composition to about 10,000 FTU/g
composition.
[0202] In one embodiment the phytase is present in the feed
additive composition at more than about 40 FTU/g composition,
suitably more than about 60 FTU/g composition, suitably more than
about 100 FTU/g composition, suitably more than about 150 FTU/g
composition, suitably more than about 200 FTU/g composition.
[0203] In one embodiment the phytase is present in the feed
additive composition at less than about 40,000 FTU/g composition,
suitably less than about 20,000 FTU/g composition, suitably less
than about 15,000 FTU/g composition, suitably less than about
10,000 FTU/g composition.
[0204] It will be understood that as used herein 1 FTU (phytase
unit) is defined as the amount of enzyme required to release 1
.mu.mol of inorganic orthophosphate from a substrate in one minute
under the reaction conditions defined in the ISO 2009 phytase
assay--A standard assay for determining phytase activity and 1 FTU
can be found at International Standard ISO/DIS 30024: 1-17,
2009.
[0205] In one embodiment suitably the enzyme is classified using
the E.C. classification above, and the E.C. classification
designates an enzyme having that activity when tested in the assay
taught herein for determining 1 FTU.
Advantages
[0206] The interaction of DFMs with enzymes is complicated and
without wishing to be bound by theory, it is very surprising that
we can see an improvement in the subject's resistance to necrotic
enteritis, e.g. that we see a reduction in lesion scores for
instance. Prior to the present invention the combination of DFMs
and enzymes (e.g. as taught herein) had not been taught for this
specific purpose.
[0207] One advantage of the present invention is that the feed
additive composition according to the present invention can avoid
the negative effects of necrotic enteritis or can be used for
improving the subject's resistance to necrotic enteritis.
[0208] Without wishing to be bound by theory, phytase catalyses the
sequential hydrolysis of phytate, a principal storage form of
phosphorus in cereals and legumes, to less phosphorylated
myo-inositol derivatives with concomitant release of inorganic
phosphate. Hydrolysis of phytate causes a reduction of endogenous
losses of amino acids to the intestinal lumen. A reduction of
endogenous amino acid losses in the intestine reduces the
availability of nitrogen for bacterial growth, which helps the
activity of DFMs on inhibition of C. perfringens and other
pathogenic bacteria.
[0209] Without wishing to be bound in theory proteases cause
non-specific hydrolysis of dietary protein yielding a variety of
polypeptides in the intestinal lumen. Animals finalise protein
hydrolysis and absorb such amino acids. However, in the case of
enteric pathogenic challenges, pathogenic bacteria may take
advantage of higher peptide availability in the lumen of jejunum
and ileum. DFMs inhibit the growth of entero-pathogens by for
example competing for N sources, as well as by direct
inhibition.
[0210] In addition, xylanase degrades the linear polysaccharide
beta-1,4-xylan into xylose. Without wishing to be bound by theory,
the inventors herein have shown that the increased energy
digestibility with the combination of DFMs and enzymes is not
explained by starch, fat or protein, therefore it must be explained
by non-starch polysaccharides.
[0211] Amylase activity hydrolyses alpha-bonds of large
alpha-linked polysaccharides such as starch yielding dextrins and
oligosaccharides, which are mainly absorbed in the small intestine
after hydrolysis to maltose and glucose in the gut wall.
Surprisingly, rapid starch hydrolysis in the foregut and greater
absorption of glucose in the duodenum deprives pathogenic bacteria
from an important energy source (glucose) in the jejunum and ileum,
which improves the DFM activity because of a competitive advantage
against pathogens that cannot use pentoses as efficiently.
[0212] In combination the four enzymes and DFMs surprisingly
provide a significant improvement on the pathogen reduction and/or
resistance to necrotic enteritis compared with other DFM and enzyme
combinations and/or DFMs alone and/or enzyme(s) alone.
[0213] The specific combination of DFMs and the enzymes taught
herein may advantageously lead to reduced mucin secretion. Without
wishing to be bound by theory this reduced mucin secretion may
result in a reduction of endogenous amino acid losses, and/or may
be responsible for improved performance.
[0214] The specific combination of DFMs and the enzymes taught
herein may advantageously reduce inflammation in the ileum. This
can be seen by the downregulation of IFR-g expression in the ileum.
The inventors have shown that modulation of immune response may
improve performance.
Formulation of the DFM with the Enzymes
[0215] The DFM and the enzymes may be formulated in any suitable
way to ensure that the formulation comprises viable DFMs and active
enzymes.
[0216] In one embodiment the DFM and enzymes may be formulated as a
liquid, a dry powder or a granule.
[0217] The dry powder or granules may be prepared by means known to
those skilled in the art, such as, in top-spray fluid bed coater,
in a buttom spray Wurster or by drum granulation (e.g. High sheer
granulation), extrusion, pan coating or in a microingredients
mixer.
[0218] For some embodiments the DFM and/or the enzyme(s) may be
coated, for example encapsulated. Suitably the DFM and enzymes may
be formulated within the same coating or encapsulated within the
same capsule. Alternatively one or two or three or four of the
enzymes may be formulated within the same coating or encapsulated
within the same capsule and the DFM could be formulated in a
coating separate to the one or more or all of the enzymes. In some
embodiments, such as where the DFM is capable of producing
endospores, the DFM may be provided without any coating. In such
circumstances, the DFM endospores may be simply admixed with one or
two or three or four enzymes. In the latter case, the enzymes may
be coated, e.g. encapsulated, for instance one or more or all of
the enzymes may be coated, e.g. encapsulated. The enzymes may be
encapsulated as mixtures (i.e. comprising one or more, two or more,
three or more or all) of enzymes or they may be encapsulated
separately, e.g. as single enzymes. In one preferred embodiment all
four enzymes may be coated, e.g. encapsulated, together.
[0219] In one embodiment the coating protects the enzymes from heat
and may be considered a thermoprotectant.
[0220] In one embodiment the feed additive composition is
formulated to a dry powder or granules as described in
WO2007/044968 (referred to as TPT granules) or WO1997/016076 or
WO1992/012645 (each of which is incorporated herein by
reference).
[0221] In one embodiment the feed additive composition may be
formulated to a granule for feed compositions comprising: a core;
an active agent; and at least one coating, the active agent of the
granule retaining at least 50% activity, at least 60% activity, at
least 70% activity, at least 80% activity after conditions selected
from one or more of a) a feed pelleting process, b) steam-heated
feed pretreatment process, c) storage, d) storage as an ingredient
in an unpelleted mixture, and e) storage as an ingredient in a feed
base mix or a feed premix comprising at least one compound selected
from trace minerals, organic acids, reducing sugars, vitamins,
choline chloride, and compounds which result in an acidic or a
basic feed base mix or feed premix.
[0222] With regard to the granule at least one coating may comprise
a moisture hydrating material that constitutes at least 55% w/w of
the granule; and/or at least one coating may comprise two coatings.
The two coatings may be a moisture hydrating coating and a moisture
barrier coating. In some embodiments, the moisture hydrating
coating may be between 25% and 60% w/w of the granule and the
moisture barrier coating may be between 2% and 15% w/w of the
granule. The moisture hydrating coating may be selected from
inorganic salts, sucrose, starch, and maltodextrin and the moisture
barrier coating may be selected from polymers, gums, whey and
starch.
[0223] The granule may be produced using a feed pelleting process
and the feed pretreatment process may be conducted between
70.degree. C. and 95.degree. C. for up to several minutes, such as
between 85.degree. C. and 95.degree. C.
[0224] In one embodiment the feed additive composition may be
formulated to a granule for animal feed comprising: a core; an
active agent, the active agent of the granule retaining at least
80% activity after storage and after a steam-heated pelleting
process where the granule is an ingredient; a moisture barrier
coating; and a moisture hydrating coating that is at least 25% w/w
of the granule, the granule having a water activity of less than
0.5 prior to the steam-heated pelleting process.
[0225] The granule may have a moisture barrier coating selected
from polymers and gums and the moisture hydrating material may be
an inorganic salt. The moisture hydrating coating may be between
25% and 45% w/w of the granule and the moisture barrier coating may
be between 2% and 10% w/w of the granule.
[0226] The granule may be produced using a steam-heated pelleting
process which may be conducted between 85.degree. C. and 95.degree.
C., for up to several minutes.
[0227] In some embodiments the DFM (e.g. DFM endospores for
example) may be diluted using a diluent, such as starch powder,
lime stone or the like.
[0228] In one embodiment, the composition is in a liquid
formulation suitable for consumption preferably such liquid
consumption contains one or more of the following: a buffer, salt,
sorbitol and/or glycerol.
[0229] In another embodiment the feed additive composition may be
formulated by applying, e.g. spraying, the enzyme(s) onto a carrier
substrate, such as ground wheat for example.
[0230] In one embodiment the feed additive composition according to
the present invention may be formulated as a premix. By way of
example only the premix may comprise one or more feed components,
such as one or more minerals and/or one or more vitamins.
[0231] In one embodiment the DFM and/or enzymes for use in the
present invention are formulated with at least one physiologically
acceptable carrier selected from at least one of maltodextrin,
limestone (calcium carbonate), cyclodextrin, wheat or a wheat
component, sucrose, starch, Na.sub.2SO.sub.4, Talc, PVA, sorbitol,
benzoate, sorbiate, glycerol, sucrose, propylene glycol,
1,3-propane diol, glucose, parabens, sodium chloride, citrate,
acetate, phosphate, calcium, metabisulfite, formate and mixtures
thereof.
Packaging
[0232] In one embodiment the feed additive composition and/or
premix and/or feed or feedstuff according to the present invention
is packaged.
[0233] In one preferred embodiment the feed additive composition
and/or premix and/or feed or feedstuff is packaged in a bag, such
as a paper bag.
[0234] In an alternative embodiment the feed additive composition
and/or premix and/or feed or feedstuff may be sealed in a
container. Any suitable container may be used.
Feed
[0235] The feed additive composition of the present invention may
be used as--or in the preparation of--a feed.
[0236] The term "feed" is used synonymously herein with
"feedstuff".
[0237] The feed may be in the form of a solution or as a
solid--depending on the use and/or the mode of application and/or
the mode of administration.
[0238] When used as--or in the preparation of--a feed--such as
functional feed--the composition of the present invention may be
used in conjunction with one or more of: a nutritionally acceptable
carrier, a nutritionally acceptable diluent, a nutritionally
acceptable excipient, a nutritionally acceptable adjuvant, a
nutritionally active ingredient.
[0239] In a preferred embodiment the feed additive composition of
the present invention is admixed with a feed component to form a
feedstuff.
[0240] The term "feed component" as used herein means all or part
of the feedstuff. Part of the feedstuff may mean one constituent of
the feedstuff or more than one constituent of the feedstuff, e.g. 2
or 3 or 4. In one embodiment the term "feed component" encompasses
a premix or premix constituents.
[0241] Preferably the feed may be a fodder, or a premix thereof, a
compound feed, or a premix thereof. In one embodiment the feed
additive composition according to the present invention may be
admixed with a compound feed, a compound feed component or to a
premix of a compound feed or to a fodder, a fodder component, or a
premix of a fodder.
[0242] The term fodder as used herein means any food which is
provided to an animal (rather than the animal having to forage for
it themselves). Fodder encompasses plants that have been cut.
[0243] The term fodder includes hay, straw, silage, compressed and
pelleted feeds, oils and mixed rations, and also sprouted grains
and legumes.
[0244] Fodder may be obtained from one or more of the plants
selected from: alfalfa (lucerne), barley, birdsfoot trefoil,
brassicas, Chau moellier, kale, rapeseed (canola), rutabaga
(swede), turnip, clover, alsike clover, red clover, subterranean
clover, white clover, grass, false oat grass, fescue, Bermuda
grass, brome, heath grass, meadow grasses (from naturally mixed
grassland swards, orchard grass, rye grass, Timothy-grass, corn
(maize), millet, oats, sorghum, soybeans, trees (pollard tree
shoots for tree-hay), wheat, and legumes.
[0245] The term "compound feed" means a commercial feed in the form
of a meal, a pellet, nuts, cake or a crumble. Compound feeds may be
blended from various raw materials and additives. These blends are
formulated according to the specific requirements of the target
animal.
[0246] Compound feeds can be complete feeds that provide all the
daily required nutrients, concentrates that provide a part of the
ration (protein, energy) or supplements that only provide
additional micronutrients, such as minerals and vitamins.
[0247] The main ingredients used in compound feed are the feed
grains, which include corn, soybeans, sorghum, oats, and
barley.
[0248] Suitably a premix as referred to herein may be a composition
composed of microingredients such as vitamins, minerals, chemical
preservatives, antibiotics, fermentation products, and other
essential ingredients. Premixes are usually compositions suitable
for blending into commercial rations.
[0249] Any feedstuff of the present invention may comprise one or
more feed materials selected from the group comprising a) cereals,
such as small grains (e.g., wheat, barley, rye, oats and
combinations thereof) and/or large grains such as maize or sorghum;
b) by products from cereals, such as corn gluten meal, Distillers
Dried Grain Solubles (DDGS), wheat bran, wheat middlings, wheat
shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus
pulp; c) protein obtained from sources such as soya, sunflower,
peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried
plasma protein, meat and bone meal, potato protein, whey, copra,
sesame; d) oils and fats obtained from vegetable and animal
sources; e) minerals and vitamins.
[0250] A feedstuff of the present invention may contain at least
30%, at least 40%, at least 50% or at least 60% by weight corn and
soybean meal or corn and full fat soy, or wheat meal or sunflower
meal.
[0251] In addition or in the alternative, a feedstuff of the
present invention may comprise at least one high fibre feed
material and/or at least one by-product of the at least one high
fibre feed material to provide a high fibre feedstuff. Examples of
high fibre feed materials include: wheat, barley, rye, oats, by
products from cereals, such as corn gluten meal, Distillers Dried
Grain Solubles (DDGS), wheat bran, wheat middlings, wheat shorts,
rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp.
Some protein sources may also be regarded as high fibre: protein
obtained from sources such as sunflower, lupin, fava beans and
cotton.
[0252] In the present invention the feed may be one or more of the
following: a compound feed and premix, including pellets, nuts or
(cattle) cake; a crop or crop residue: corn, soybeans, sorghum,
oats, barley, corn stover, copra, straw, chaff, sugar beet waste;
fish meal; freshly cut grass and other forage plants; meat and bone
meal; molasses; oil cake and press cake; oligosaccharides;
conserved forage plants: hay and silage; seaweed; seeds and grains,
either whole or prepared by crushing, milling etc.; sprouted grains
and legumes; yeast extract.
[0253] The term feed in the present invention also encompasses in
some embodiments pet food. A pet food is plant or animal material
intended for consumption by pets, such as dog food or cat food. Pet
food, such as dog and cat food, may be either in a dry form, such
as kibble for dogs, or wet canned form. Cat food may contain the
amino acid taurine.
[0254] The term feed in the present invention also encompasses in
some embodiments fish food. A fish food normally contains macro
nutrients, trace elements and vitamins necessary to keep captive
fish in good health. Fish food may be in the form of a flake,
pellet or tablet. Pelleted forms, some of which sink rapidly, are
often used for larger fish or bottom feeding species. Some fish
foods also contain additives, such as beta carotene or sex
hormones, to artificially enhance the color of ornamental fish.
[0255] The term feed in the present invention also encompasses in
some embodiment bird food. Bird food includes food that is used
both in birdfeeders and to feed pet birds. Typically bird food
comprises of a variety of seeds, but may also encompass suet (beef
or mutton fat).
[0256] As used herein the term "contacted" refers to the indirect
or direct application of the composition of the present invention
to the product (e.g. the feed). Examples of the application methods
which may be used, include, but are not limited to, treating the
product in a material comprising the feed additive composition,
direct application by mixing the feed additive composition with the
product, spraying the feed additive composition onto the product
surface or dipping the product into a preparation of the feed
additive composition.
[0257] In one embodiment the feed additive composition of the
present invention is preferably admixed with the product (e.g.
feedstuff). Alternatively, the feed additive composition may be
included in the emulsion or raw ingredients of a feedstuff.
[0258] For some applications, it is important that the composition
is made available on or to the surface of a product to be
affected/treated. This allows the composition to impart one or more
of the following favourable characteristics: performance
benefits.
[0259] The feed additive compositions of the present invention may
be applied to intersperse, coat and/or impregnate a product (e.g.
feedstuff or raw ingredients of a feedstuff) with a controlled
amount of DFM and enzymes.
[0260] The DFM and enzymes may be used simultaneously (e.g. when
they are in admixture together or even when they are delivered by
different routes) or sequentially (e.g. they may be delivered by
different routes). In one embodiment preferably the DFM and enzymes
are applied simultaneously. Preferably the DFM and enzymes are
admixed prior to being delivered to a feedstuff or to a raw
ingredient of a feedstuff.
[0261] The DFM in feed additive compositions according to the
present invention--can be added in suitable concentrations--such as
for example in concentrations in the final feed product which offer
a daily dose of between about 2.times.10.sup.5 CFU to about
2.times.10.sup.11 CFU, suitably between about 2.times.10.sup.6 to
about 1.times.10.sup.10, suitably between about 3.75.times.10.sup.7
CFU to about 1.times.10.sup.10 CFU.
[0262] Preferably, the feed additive composition of the present
invention will be thermally stable to heat treatment up to about
70.degree. C.; up to about 85.degree. C.; or up to about 95.degree.
C. The heat treatment may be performed for up to about 1 minute; up
to about 5 minutes; up to about 10 minutes; up to about 30 minutes;
up to about 60 minutes. The term thermally stable means that at
least about 75% of the enzyme components and/or DFM that were
present/active in the additive before heating to the specified
temperature are still present/active after it cools to room
temperature. Preferably, at least about 80% of the enzyme
components and/or DFM that were present and active in the additive
before heating to the specified temperature are still present and
active after it cools to room temperature.
[0263] In a particularly preferred embodiment the feed additive
composition is homogenized to produce a powder.
[0264] In an alternative preferred embodiment, the feed additive
composition is formulated to granules as described in WO2007/044968
(referred to as TPT granules) incorporated herein by reference.
[0265] In another preferred embodiment when the feed additive
composition is formulated into granules the granules comprise a
hydrated barrier salt coated over the protein core. The advantage
of such salt coating is improved thermo-tolerance, improved storage
stability and protection against other feed additives otherwise
having adverse effect on the enzyme and/or DFM.
[0266] Preferably, the salt used for the salt coating has a water
activity greater than 0.25 or constant humidity greater than 60% at
20.degree. C.
[0267] Preferably, the salt coating comprises a
Na.sub.2SO.sub.4.
[0268] The method of preparing a feed additive composition may also
comprise the further step of pelleting the powder. The powder may
be mixed with other components known in the art. The powder, or
mixture comprising the powder, may be forced through a die and the
resulting strands are cut into suitable pellets of variable
length.
[0269] Optionally, the pelleting step may include a steam
treatment, or conditioning stage, prior to formation of the
pellets. The mixture comprising the powder may be placed in a
conditioner, e.g. a mixer with steam injection. The mixture is
heated in the conditioner up to a specified temperature, such as
from 60-100.degree. C., typical temperatures would be 70.degree.
C., 80.degree. C., 85.degree. C., 90.degree. C. or 95.degree. C.
The residence time can be variable from seconds to minutes and even
hours. Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1
minutes 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes
and 1 hour.
[0270] It will be understood that the feed additive composition of
the present invention is suitable for addition to any appropriate
feed material.
[0271] As used herein, the term feed material refers to the basic
feed material to be consumed by an animal. It will be further
understood that this may comprise, for example, at least one or
more unprocessed grains, and/or processed plant and/or animal
material such as soybean meal or bone meal.
[0272] As used herein, the term "feedstuff" refers to a feed
material to which one or more feed additive compositions have been
added.
[0273] It will be understood by the skilled person that different
animals require different feedstuffs, and even the same animal may
require different feedstuffs, depending upon the purpose for which
the animal is reared.
[0274] Preferably, the feedstuff may comprise feed materials
comprising maize or corn, wheat, barley, triticale, rye, rice,
tapioca, sorghum, and/or any of the by-products, as well as protein
rich components like soybean mean, rape seed meal, canola meal,
cotton seed meal, sunflower seed mean, animal-by-product meals and
mixtures thereof. More preferably, the feedstuff may comprise
animal fats and/or vegetable oils.
[0275] Optionally, the feedstuff may also contain additional
minerals such as, for example, calcium and/or additional
vitamins.
[0276] Preferably, the feedstuff is a corn soybean meal mix.
[0277] In one embodiment, preferably the feed is not pet food.
[0278] In another aspect there is provided a method for producing a
feedstuff. Feedstuff is typically produced in feed mills in which
raw materials are first ground to a suitable particle size and then
mixed with appropriate additives. The feedstuff may then be
produced as a mash or pellets; the later typically involves a
method by which the temperature is raised to a target level and
then the feed is passed through a die to produce pellets of a
particular size. The pellets are allowed to cool. Subsequently
liquid additives such as fat and enzyme may be added. Production of
feedstuff may also involve an additional step that includes
extrusion or expansion prior to pelleting--in particular by
suitable techniques that may include at least the use of steam.
[0279] The feedstuff may be a feedstuff for a monogastric animal,
such as poultry (for example, broiler, layer, broiler breeders,
turkey, duck, geese, water fowl), swine (all age categories), a pet
(for example dogs, cats) or fish, preferably the feedstuff is for
poultry.
[0280] In one embodiment the feedstuff is not for a layer.
[0281] By way of example only a feedstuff for chickens, e.g.
broiler chickens may be comprises of one or more of the ingredients
listed in the table below, for example in the % ages given in the
table below:
TABLE-US-00007 Ingredients Starter (%) Finisher (%) Maize 46.2 46.7
Wheat Middlings 6.7 10.0 Maize DDGS 7.0 7.0 Soyabean Meal 48% CP
32.8 26.2 An/Veg Fat blend 3.0 5.8 L-Lysine HCl 0.3 0.3
DL-methionine 0.3 0.3 L-threonine 0.1 0.1 Salt 0.3 0.4 Limestone
1.1 1.1 Dicalcium Phosphate 1.2 1.2 Poultry Vitamins and Micro- 0.3
0.3 minerals
[0282] By way of example only the diet specification for chickens,
such as broiler chickens, may be as set out in the Table below:
TABLE-US-00008 Diet specification Crude Protein (%) 23.00 20.40
Metabolizable Energy Poultry 2950 3100 (kcal/kg) Calcium (%) 0.85
0.85 Available Phosphorus (%) 0.38 0.38 Sodium (%) 0.18 0.19 Dig.
Lysine (%) 1.21 1.07 Dig. Methionine (%) 0.62 0.57 Dig. Methionine
+ Cysteine (%) 0.86 0.78 Dig. Threonine (%) 0.76 0.68
[0283] By way of example only a feedstuff laying hens may be
comprises of one or more of the ingredients listed in the table
below, for example in the % ages given in the table below:
TABLE-US-00009 Ingredient Laying phase (%) Maize 10.0 Wheat 53.6
Maize DDGS 5.0 Soybean Meal 48% CP 14.9 Wheat Middlings 3.0 Soybean
Oil 1.8 L-Lysine HCl 0.2 DL-methionine 0.2 L-threonine 0.1 Salt 0.3
Dicalcium Phosphate 1.6 Limestone 8.9 Poultry Vitamins and Micro-
0.6 minerals
[0284] By way of example only the diet specification for laying
hens may be as set out in the Table below:
TABLE-US-00010 Diet specification Crude Protein (%) 16.10
Metabolizable Energy Poultry 2700 (kcal/kg) Lysine (%) 0.85
Methionine (%) 0.42 Methionine + Cysteine (%) 0.71 Threonine (%)
0.60 Calcium (%) 3.85 Available Phosphorus (%) 0.42 Sodium (%)
0.16
[0285] By way of example only a feedstuff for turkeys may be
comprises of one or more of the ingredients listed in the table
below, for example in the % ages given in the table below:
TABLE-US-00011 Phase 1 Phase 2 Phase 3 Phase 4 Ingredient (%) (%)
(%) (%) Wheat 33.6 42.3 52.4 61.6 Maize DDGS 7.0 7.0 7.0 7.0
Soyabean Meal 48% CP 44.6 36.6 27.2 19.2 Rapeseed Meal 4.0 4.0 4.0
4.0 Soyabean Oil 4.4 4.2 3.9 3.6 L-Lysine HCl 0.5 0.5 0.4 0.4
DL-methionine 0.4 0.4 0.3 0.2 L-threonine 0.2 0.2 0.1 0.1 Salt 0.3
0.3 0.3 0.3 Limestone 1.0 1.1 1.1 1.0 Dicalcium Phosphate 3.5 3.0
2.7 2.0 Poultry Vitamins and 0.4 0.4 0.4 0.4 Micro-minerals
[0286] By way of example only the diet specification for turkeys
may be as set out in the Table below:
TABLE-US-00012 Diet specification Crude Protein (%) 29.35 26.37
22.93 20.00 Metabolizable Energy Poultry 2.850 2.900 2.950 3.001
(kcal/kg) Calcium (%) 1.43 1.33 1.22 1.02 Available Phosphorus (%)
0.80 0.71 0.65 0.53 Sodium (%) 0.16 0.17 0.17 0.17 Dig. Lysine (%)
1.77 1.53 1.27 1.04 Dig. Methionine (%) 0.79 0.71 0.62 0.48 Dig.
Methionine + Cysteine (%) 1.12 1.02 0.90 0.74 Dig. Threonine (%)
1.03 0.89 0.73 0.59
[0287] By way of example only a feedstuff for piglets may be
comprises of one or more of the ingredients listed in the table
below, for example in the % ages given in the table below:
TABLE-US-00013 Ingredient Phase 1 (%) Phase 2 (%) Maize 20.0 7.0
Wheat 25.9 46.6 Rye 4.0 10.0 Wheat middlings 4.0 4.0 Maize DDGS 6.0
8.0 Soyabean Meal 48% CP 25.7 19.9 Dried Whey 10.0 0.0 Soyabean Oil
1.0 0.7 L-Lysine HCl 0.4 0.5 DL-methionine 0.2 0.2 L-threonine 0.1
0.2 L-tryptophan 0.03 0.04 Limestone 0.6 0.7 Dicalcium Phosphate
1.6 1.6 Swine Vitamins and Micro- 0.2 0.2 minerals Salt 0.2 0.4
[0288] By way of example only the diet specification for piglets
may be as set out in the Table below:
TABLE-US-00014 Diet specification Crude Protein (%) 21.50 20.00
Swine Digestible Energy 3380 3320 (kcal/kg) Swine Net Energy
(kcal/kg) 2270 2230 Calcium (%) 0.80 0.75 Digestible Phosphorus (%)
0.40 0.35 Sodium (%) 0.20 0.20 Dig. Lysine (%) 1.23 1.14 Dig.
Methionine (%) 0.49 0.44 Dig. Methionine + Cysteine (%) 0.74 0.68
Dig. Threonine (%) 0.80 0.74
[0289] By way of example only a feedstuff for grower/finisher pigs
may be comprises of one or more of the ingredients listed in the
table below, for example in the % ages given in the table
below:
TABLE-US-00015 Ingredient Grower/Finisher (%) Maize 27.5 Soyabean
Meal 48% CP 15.4 Maize DDGS 20.0 Wheat bran 11.1 Rice bran 12.0
Canola seed meal 10.0 Limestone 1.6 Dicalcium phosphate 0.01 Salt
0.4 Swine Vitamins and Micro-minerals 0.3 Lysine-HCl 0.2 Vegetable
oil 0.5
[0290] By way of example only the diet specification for
grower/finisher pigs may be as set out in the Table below:
TABLE-US-00016 Diet specification Crude Protein (%) 22.60 Swine
Metabolizable Energy 3030 (kcal/kg) Calcium (%) 0.75 Available
Phosphorus (%) 0.29 Digestible Lysine (%) 1.01 Dig. Methionine +
Cysteine (%) 0.73 Digestible Threonine (%) 0.66
Forms
[0291] The feed additive composition of the present invention and
other components and/or the feedstuff comprising same may be used
in any suitable form.
[0292] The feed additive composition of the present invention may
be used in the form of solid or liquid preparations or alternatives
thereof. Examples of solid preparations include powders, pastes,
boluses, capsules, pellets, tablets, dusts, and granules which may
be wettable, spray-dried or freeze-dried. Examples of liquid
preparations include, but are not limited to, aqueous, organic or
aqueous-organic solutions, suspensions and emulsions.
[0293] In some applications, DFM or feed additive compositions of
the present invention may be mixed with feed or administered in the
drinking water. In one embodiment the dosage range for inclusion
into water is about 1.times.10.sup.3 CFU/animal/day to about
1.times.10.sup.10 CFU/animal/day, and more preferably about
1.times.10.sup.7 CFU/animal/day.
[0294] Suitable examples of forms include one or more of: powders,
pastes, boluses, pellets, tablets, pills, capsules, ovules,
solutions or suspensions, which may contain flavouring or colouring
agents, for immediate-, delayed-, modified-, sustained-, pulsed- or
controlled-release applications.
[0295] By way of example, if the composition of the present
invention is used in a solid, e.g. pelleted form, it may also
contain one or more of: excipients such as microcrystalline
cellulose, lactose, sodium citrate, calcium carbonate, dibasic
calcium phosphate and glycine; disintegrants such as starch
(preferably corn, potato or tapioca starch), sodium starch
glycollate, croscarmellose sodium and certain complex silicates;
granulation binders such as polyvinylpyrrolidone,
hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),
sucrose, gelatin and acacia; lubricating agents such as magnesium
stearate, stearic acid, glyceryl behenate and talc may be
included.
[0296] Examples of nutritionally acceptable carriers for use in
preparing the forms include, for example, water, salt solutions,
alcohol, silicone, waxes, petroleum jelly, vegetable oils,
polyethylene glycols, propylene glycol, liposomes, sugars, gelatin,
lactose, amylose, magnesium stearate, talc, surfactants, silicic
acid, viscous paraffin, perfume oil, fatty acid monoglycerides and
diglycerides, petroethral fatty acid esters,
hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.
[0297] Preferred excipients for the forms include lactose, starch,
a cellulose, milk sugar or high molecular weight polyethylene
glycols.
[0298] For aqueous suspensions and/or elixirs, the composition of
the present invention may be combined with various sweetening or
flavouring agents, colouring matter or dyes, with emulsifying
and/or suspending agents and with diluents such as water, propylene
glycol and glycerin, and combinations thereof.
[0299] Non-hydroscopic whey is often used as a carrier for DFMs
(particularly bacterial DFMs) and is a good medium to initiate
growth.
[0300] Bacterial DFM containing pastes may be formulated with
vegetable oil and inert gelling ingredients.
[0301] Fungal products may be formulated with grain by-products as
carriers.
[0302] In one embodiment preferably the feed additive composition
according to the present invention is not in the form of a
microparticle system, such as the microparticle system taught in
WO2005/123034.
Dosing
[0303] The DFM and/or feed additive composition according to the
present invention may be designed for one-time dosing or may be
designed for feeding on a daily basis.
[0304] The optimum amount of the composition (and each component
therein) to be used in the combination of the present invention
will depend on the product to be treated and/or the method of
contacting the product with the composition and/or the intended use
for the same.
[0305] The amount of DFM and enzymes used in the compositions
should be a sufficient amount to be effective and to remain
sufficiently effective in improving the performance of the animal
fed feed products containing said composition. This length of time
for effectiveness should extend up to at least the time of
utilisation of the product (e.g. feed additive composition or feed
containing same).
[0306] The ratio of DFM to each enzyme in the feed can be in the
ranges given below:
DFM:phytase (CFU/FTU): In range from 5.0.times.10.sup.2 CFU DFM:1
FTU enzyme to 5.0.times.10.sup.9 CFU:1 FTU enzyme; preferably in
the range from 7.5.times.10.sup.4 CFU DFM:1 FTU enzyme to
2.5.times.10.sup.7 CFU:1 FTU enzyme. DFM: xylanase (CFU/XU): In
range from 6.25.times.10.sup.1 CFU DFM:1 XU enzyme to
2.0.times.10.sup.9 CFU:1 XU enzyme; preferably in the range from
1.88.times.10.sup.4 CFU DFM:1 XU enzyme to 1.0.times.10.sup.7 CFU:1
XU enzyme. DFM:amylase (CFU/AU): In range from 1.0.times.10.sup.2
CFU DFM:1 AU enzyme to 2.0.times.10.sup.10 CFU:1 AU enzyme;
preferably in the range from 3.7.times.10.sup.4 CFU DFM:1 AU enzyme
to 1.0.times.10.sup.8 CFU:1 AU enzyme. DFM:protease (CFU/PU): In
range from 5.0.times.10.sup.1 CFU DFM:1 PU enzyme to
1.0.times.10.sup.9 CFU:1 PU enzyme; preferably in the range from
1.25.times.10.sup.4 CFU DFM:1 PU enzyme to 5.0.times.10.sup.6 CFU:1
PU enzyme.
[0307] In one embodiment preferably the feedstuff comprises the
following:
a protease at at least 4000 PU/kg of feed; a xylanase at at least
1000 XU/kg to 2000 XU/kg of feed (e.g. Avizyme at 1000 XU/kg of
feed or Axtra XAP at at least 2000 XU/kg of feed); an amylase; at
least 1800 AU/kg or 200 TAU/kg of feed (e.g. Avizyme at 1800 AU/kg
or Axtra XAP at at least 200 TAU/kg of feed); a phytase at at least
500 FTU/kg of feed; and Envivo Pro (DFM) at at least 75,000 CFU/g
to 150,000 CFU/g of feed.
[0308] In one embodiment preferably the feedstuff comprises the
following:
a protease at 4000 PU/kg of feed; a xylanase at 1000 XU/kg to 2000
XU/kg of feed (e.g. Avizyme at 1000 XU/kg of feed or Axtra XAP at
2000 XU/kg of feed); an amylase; 1800 AU/kg or 200 TAU/kg of feed
(e.g. Avizyme at 1800 AU/kg or Axtra XAP at 200 TAU/kg of feed); a
phytase at 500 FTU/kg of feed; and Envivo Pro (DFM) at 75,000 CFU/g
to 150,000 CFU/g of feed.
[0309] In one embodiment preferably the feedstuff comprises the
following:
a protease at 5000 PU/kg of feed; a xylanase at 1250 XU/kg to 2500
XU/kg of feed (e.g. Avizyme at 1000 XU/kg of feed or Axtra XAP at
2500 XU/kg of feed); an amylase; 2250 AU/kg or 250 TAU/kg of feed
(e.g. Avizyme at 1800 AU/kg or Axtra XAP at 250 TAU/kg of feed); a
phytase at 625 FTU/kg of feed; and Envivo Pro (DFM) at 75,000 CFU/g
to 150,000 CFU/g of feed.
[0310] In another embodiment the feedstuff comprises the
following:
a protease at 2000 PU/kg of feed; a xylanase at 500 XU/kg to 1000
XU/kg of feed (e.g. Avizyme at 500 XU/kg of feed or Axtra XAP at
1000 XU/kg of feed); an amylase; 900 AU/kg or 100 TAU/kg of feed
(e.g. Avizyme at 900 AU/kg or Axtra XAP at 100 TAU/kg of feed); a
phytase at 500 FTU/kg of feed; and Envivo Pro (DFM) at 37,500 CFU/g
to 75,000 CFU/g of feed.
[0311] In a preferred embodiment the feed additive composition
comprises sufficient enzyme and DFMs to dose the feedstuff as
follows:
a protease at 4000 PU/kg of feed; a xylanase at 1000 XU/kg to 2000
XU/kg of feed (e.g. Avizyme at 1000 XU/kg of feed or Axtra XAP at
2000 XU/kg of feed); an amylase; 1800 AU/kg or 200 TAU/kg of feed
(e.g. Avizyme at 1800 AU/kg or Axtra XAP at 200 TAU/kg of feed); a
phytase at 500 FTU/kg of feed; and Envivo Pro (DFM) at 75,000 CFU/g
to 150,000 CFU/g of feed.
[0312] In a preferred embodiment the feed additive composition
comprises sufficient enzyme and DFMs to dose the feedstuff as
follows:
a protease at 2000 PU/kg of feed; a xylanase at 500 XU/kg to 1000
XU/kg of feed (e.g. Avizyme at 500 XU/kg of feed or Axtra XAP at
1000 XU/kg of feed); an amylase; 900 AU/kg or 100 TAU/kg of feed
(e.g. Avizyme at 900 AU/kg or Axtra XAP at 100 TAU/kg of feed); a
phytase at 500 FTU/kg of feed; and Envivo Pro (DFM) at 37,500 CFU/g
to 75,000 CFU/g of feed. Combination with Other Components
[0313] The DFM and enzyme(s) for use in the present invention may
be used in combination with other components. Thus, the present
invention also relates to combinations. The DFM in combination with
a protease, xylanase, amylase and phytase may be referred to herein
as "the feed additive composition of the present invention".
[0314] The combination of the present invention comprises the feed
additive composition of the present invention (or one or more of
the constituents thereof) and another component which is suitable
for animal consumption and is capable of providing a medical or
physiological benefit to the consumer.
[0315] In one embodiment preferably the "another component" is not
a further enzyme or a further DFM.
[0316] The components may be prebiotics. Prebiotics are typically
non-digestible carbohydrate (oligo- or polysaccharides) or a sugar
alcohol which is not degraded or absorbed in the upper digestive
tract. Known prebiotics used in commercial products and useful in
accordance with the present invention include inulin
(fructo-oligosaccharide, or FOS) and transgalacto-oligosaccharides
(GOS or TOS). Suitable prebiotics include
palatinoseoligosaccharide, soybean oligosaccharide, alginate,
xanthan, pectin, locust bean gum (LBG), inulin, guar gum,
galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS),
non-degradable starch, lactosaccharose, lactulose, lactitol,
maltitol, maltodextrin, polydextrose (i.e. Litesse.RTM.), lactitol,
lactosucrose, soybean oligosaccharides, palatinose,
isomalto-oligosaccharides, gluco-oligosaccharides and
xylo-oligosaccharides, pectin fragments, dietary fibres,
mannan-oligosaccharides.
[0317] Dietary fibres may include non-starch polysaccharides, such
as arabinoxylans, cellulose and many other plant components, such
as resistant dextrins, inulin, lignin, waxes, chitins, pectins,
beta-glucans and oligosaccharides.
[0318] In one embodiment the present invention relates to the
combination of the feed additive composition (or one or more of the
constituents thereof) according to the present invention with a
prebiotic. In another embodiment the present invention relates to a
feed additive composition comprising (or consisting essentially of
or consisting of) a DFM in combination with a xylanase, an amylase,
a phytase, a protease and a prebiotic.
[0319] The prebiotic may be administered simultaneously with (e.g.
in admixture together with or delivered simultaneously by the same
or different routes) or sequentially to (e.g. by the same or
different routes) the feed additive composition (or constituents
thereof) according to the present invention.
[0320] Other components of the combinations of the present
invention include polydextrose, such as Litesse.RTM., and/or a
maltodextrin and/or lactitol. These other components may be
optionally added to the feed additive composition to assist the
drying process and help the survival of DFM.
[0321] Further examples of other suitable components include one or
more of: thickeners, gelling agents, emulsifiers, binders, crystal
modifiers, sweeteners (including artificial sweeteners), rheology
modifiers, stabilisers, anti-oxidants, dyes, enzymes, carriers,
vehicles, excipients, diluents, lubricating agents, flavouring
agents, colouring matter, suspending agents, disintegrants,
granulation binders etc. These other components may be natural.
These other components may be prepared by use of chemical and/or
enzymatic techniques.
[0322] In one embodiment the DFM and/or enzymes may be
encapsulated. In one embodiment the feed additive composition
and/or DFM and/or enzymes is/are formulated as a dry powder or
granule as described in WO2007/044968 (referred to as TPT
granules)--reference incorporated herein by reference.
[0323] In one preferred embodiment the DFM and/or enzymes for use
in the present invention may be used in combination with one or
more lipids.
[0324] For example, the DFM and/or enzymes for use in the present
invention may be used in combination with one or more lipid
micelles. The lipid micelle may be a simple lipid micelle or a
complex lipid micelle.
[0325] The lipid micelle may be an aggregate of orientated
molecules of amphipathic substances, such as a lipid and/or an
oil.
[0326] As used herein the term "thickener or gelling agent" refers
to a product that prevents separation by slowing or preventing the
movement of particles, either droplets of immiscible liquids, air
or insoluble solids. Thickening occurs when individual hydrated
molecules cause an increase in viscosity, slowing the separation.
Gelation occurs when the hydrated molecules link to form a
three-dimensional network that traps the particles, thereby
immobilising them.
[0327] The term "stabiliser" as used here is defined as an
ingredient or combination of ingredients that keeps a product (e.g.
a feed product) from changing over time.
[0328] The term "emulsifier" as used herein refers to an ingredient
(e.g. a feed ingredient) that prevents the separation of emulsions.
Emulsions are two immiscible substances, one present in droplet
form, contained within the other. Emulsions can consist of
oil-in-water, where the droplet or dispersed phase is oil and the
continuous phase is water; or water-in-oil, where the water becomes
the dispersed phase and the continuous phase is oil. Foams, which
are gas-in-liquid, and suspensions, which are solid-in-liquid, can
also be stabilised through the use of emulsifiers.
[0329] As used herein the term "binder" refers to an ingredient
(e.g. a feed ingredient) that binds the product together through a
physical or chemical reaction. During "gelation" for instance,
water is absorbed, providing a binding effect. However, binders can
absorb other liquids, such as oils, holding them within the
product. In the context of the present invention binders would
typically be used in solid or low-moisture products for instance
baking products: pastries, doughnuts, bread and others.
[0330] "Carriers" or "vehicles" mean materials suitable for
administration of the DFM and/or enzymes and include any such
material known in the art such as, for example, any liquid, gel,
solvent, liquid diluent, solubilizer, or the like, which is
non-toxic and which does not interact with any components of the
composition in a deleterious manner.
[0331] The present invention provides a method for preparing a feed
additive composition comprising admixing a DFM, a xylanase, a
protease, a phytase and a amylase with at least one physiologically
acceptable carrier selected from at least one of maltodextrin,
limestone (calcium carbonate), cyclodextrin, wheat or a wheat
component, sucrose, starch, Na.sub.2SO.sub.4, Talc, PVA, sorbitol,
benzoate, sorbiate, glycerol, sucrose, propylene glycol,
1,3-propane diol, glucose, parabens, sodium chloride, citrate,
acetate, phosphate, calcium, metabisulfite, formate and mixtures
thereof.
[0332] Examples of excipients include one or more of:
microcrystalline cellulose and other celluloses, lactose, sodium
citrate, calcium carbonate, dibasic calcium phosphate, glycine,
starch, milk sugar and high molecular weight polyethylene
glycols.
[0333] Examples of disintegrants include one or more of: starch
(preferably corn, potato or tapioca starch), sodium starch
glycollate, croscarmellose sodium and certain complex
silicates.
[0334] Examples of granulation binders include one or more of:
polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),
hydroxypropylcellulose (HPC), sucrose, maltose, gelatin and
acacia.
[0335] Examples of lubricating agents include one or more of:
magnesium stearate, stearic acid, glyceryl behenate and talc.
[0336] Examples of diluents include one or more of: water, ethanol,
propylene glycol and glycerin, and combinations thereof.
[0337] The other components may be used simultaneously (e.g. when
they are in admixture together or even when they are delivered by
different routes) or sequentially (e.g. they may be delivered by
different routes).
[0338] Preferably, when the feed additive composition of the
present invention is admixed with another component(s), the DFM
remains viable.
[0339] In one embodiment preferably the feed additive composition
according to the present invention does not comprise chromium or
organic chromium
[0340] In one embodiment preferably the feed additive according to
the present invention does not contain glucanase.
[0341] In one embodiment preferably the feed additive according to
the present invention does not contain sorbic acid.
Concentrates
[0342] The DFMs for use in the present invention may be in the form
of concentrates. Typically these concentrates comprise a
substantially high concentration of a DFM.
[0343] Feed additive compositions according to the present
invention may have a content of viable cells (colony forming units,
CFUs) which is in the range of at least 10.sup.4 CFU/g (suitably
including at least 10.sup.5 CFU/g, such as at least 10.sup.6 CFU/g,
e.g. at least 10.sup.7 CFU/g, at least 10.sup.8 CFU/g, e.g. at
least 10.sup.9 CFU/g) to about 10.sup.10 CFU/g (or even about
10.sup.11 CFU/g or about 10.sup.12 CFU/g).
[0344] When the DFM is in the form of a concentrate the feed
additive compositions according to the present invention may have a
content of viable cells in the range of at least 10.sup.9 CFU/g to
about 10.sup.12 CFU/g, preferably at least 10.sup.10 CFU/g to about
10.sup.12 CFU/g.
[0345] Powders, granules and liquid compositions in the form of
concentrates may be diluted with water or resuspended in water or
other suitable diluents, for example, an appropriate growth medium
such as milk or mineral or vegetable oils, to give compositions
ready for use.
[0346] The DFM or feed additive composition of the present
invention or the combinations of the present invention in the form
of concentrates may be prepared according to methods known in the
art.
[0347] In one aspect of the present invention the enzymes or feed
is contacted by a composition in a concentrated form.
[0348] The compositions of the present invention may be spray-dried
or freeze-dried by methods known in the art.
[0349] Typical processes for making particles using a spray drying
process involve a solid material which is dissolved in an
appropriate solvent (e.g. a culture of a DFM in a fermentation
medium). Alternatively, the material can be suspended or emulsified
in a non-solvent to form a suspension or emulsion. Other
ingredients (as discussed above) or components such as
anti-microbial agents, stabilising agents, dyes and agents
assisting with the drying process may optionally be added at this
stage.
[0350] The solution then is atomised to form a fine mist of
droplets. The droplets immediately enter a drying chamber where
they contact a drying gas. The solvent is evaporated from the
droplets into the drying gas to solidify the droplets, thereby
forming particles. The particles are then separated from the drying
gas and collected.
Subject
[0351] The term "subject", as used herein, means an animal that is
to be or has been administered with a feed additive composition
according to the present invention or a feedstuff comprising said
feed additive composition according to the present invention.
[0352] The term "subject", as used herein, means an animal.
Preferably, the subject is a mammal, bird, fish or crustacean
including for example livestock or a domesticated animal (e.g. a
pet).
[0353] In one embodiment the "subject" is livestock.
[0354] The term "livestock", as used herein refers to any farmed
animal. Preferably, livestock is one or more of cows or bulls
(including calves), poultry, pigs (including piglets), poultry
(including broilers, chickens and turkeys), birds, fish (including
freshwater fish, such as salmon, cod, trout and carp, e.g. koi
carp, and marine fish, such as sea bass), crustaceans (such as
shrimps, mussels and scallops), horses (including race horses),
sheep (including lambs).
[0355] In one embodiment the term livestock and/or poultry and/or
chickens does not include egg layers.
[0356] In another embodiment the "subject" is a domesticated animal
or pet or an animal maintained in a zoological environment.
[0357] The term "domesticated animal or pet or animal maintained in
a zoological environment" as used herein refers to any relevant
animal including canines (e.g. dogs), felines (e.g. cats), rodents
(e.g. guinea pigs, rats, mice), birds, fish (including freshwater
fish and marine fish), and horses.
[0358] In one embodiment the subject may be challenged by an
enteric pathogen.
[0359] By way of example a subject may have one or more enteric
pathogens present in its gut or digestive tract. For example a
subject may have one or more enteric pathogens in its gut or
digestive tract at a level which: [0360] i) results in loss of
performance of the animal and/or [0361] ii) is at clinically
relevant levels; or [0362] iii) is at sub-clinical levels.
[0363] The enteric pathogen may be Clostridium perfringens for
example.
Performance
[0364] As used herein, "animal performance" may be determined by
the feed efficiency and/or weight gain of the animal and/or by the
feed conversion ratio and/or by the digestibility of a nutrient in
a feed (e.g. amino acid digestibility) and/or digestible energy or
metabolizable energy in a feed and/or by nitrogen retention and/or
by animals ability to avoid the negative effects of necrotic
enteritis and/or by the immune response of the subject.
[0365] Preferably "animal performance" is determined by feed
efficiency and/or weight gain of the animal and/or by the feed
conversion ratio.
[0366] By "improved animal performance" it is meant that there is
increased feed efficiency, and/or increased weight gain and/or
reduced feed conversion ratio and/or improved digestibility of
nutrients or energy in a feed and/or by improved nitrogen retention
and/or by improved ability to avoid the negative effects of
necrotic enteritis and/or by an improved immune response in the
subject resulting from the use of feed additive composition of the
present invention in feed in comparison to feed which does not
comprise said feed additive composition.
[0367] Preferably, by "improved animal performance" it is meant
that there is increased feed efficiency and/or increased weight
gain and/or reduced feed conversion ratio.
[0368] As used herein, the term "feed efficiency" refers to the
amount of weight gain in an animal that occurs when the animal is
fed ad-libitum or a specified amount of food during a period of
time.
[0369] By "increased feed efficiency" it is meant that the use of a
feed additive composition according the present invention in feed
results in an increased weight gain per unit of feed intake
compared with an animal fed without said feed additive composition
being present.
Feed Conversion Ratio (FCR)
[0370] As used herein, the term "feed conversion ratio" refers to
the amount of feed fed to an animal to increase the weight of the
animal by a specified amount.
[0371] An improved feed conversion ratio means a lower feed
conversion ratio.
[0372] By "lower feed conversion ratio" or "improved feed
conversion ratio" it is meant that the use of a feed additive
composition in feed results in a lower amount of feed being
required to be fed to an animal to increase the weight of the
animal by a specified amount compared to the amount of feed
required to increase the weight of the animal by the same amount
when the feed does not comprise said feed additive composition.
Nutrient Digestibility
[0373] Nutrient digestibility as used herein means the fraction of
a nutrient that disappears from the gastro-intestinal tract or a
specified segment of the gastro-intestinal tract, e.g. the small
intestine. Nutrient digestibility may be measured as the difference
between what is administered to the subject and what comes out in
the faeces of the subject, or between what is administered to the
subject and what remains in the digesta on a specified segment of
the gastro intestinal tract, e.g. the ileum.
[0374] Nutrient digestibility as used herein may be measured by the
difference between the intake of a nutrient and the excreted
nutrient by means of the total collection of excreta during a
period of time; or with the use of an inert marker that is not
absorbed by the animal, and allows the researcher calculating the
amount of nutrient that disappeared in the entire gastro-intestinal
tract or a segment of the gastro-intestinal tract. Such an inert
marker may be titanium dioxide, chromic oxide or acid insoluble
ash. Digestibility may be expressed as a percentage of the nutrient
in the feed, or as mass units of digestible nutrient per mass units
of nutrient in the feed.
[0375] Nutrient digestibility as used herein encompasses starch
digestibility, fat digestibility, protein digestibility, and amino
acid digestibility.
[0376] Energy digestibility as used herein means the gross energy
of the feed consumed minus the gross energy of the faeces or the
gross energy of the feed consumed minus the gross energy of the
remaining digesta on a specified segment of the gastro-intestinal
tract of the animal, e.g. the ileum. Metabolizable energy as used
herein refers to apparent metabolizable energy and means the gross
energy of the feed consumed minus the gross energy contained in the
faeces, urine, and gaseous products of digestion. Energy
digestibility and metabolizable energy may be measured as the
difference between the intake of gross energy and the gross energy
excreted in the faeces or the digesta present in specified segment
of the gastro-intestinal tract using the same methods to measure
the digestibility of nutrients, with appropriate corrections for
nitrogen excretion to calculate metabolizable energy of feed.
Nitrogen Retention
[0377] Nitrogen retention as used herein means as subject's ability
to retain nitrogen from the diet as body mass. A negative nitrogen
balance occurs when the excretion of nitrogen exceeds the daily
intake and is often seen when the muscle is being lost. A positive
nitrogen balance is often associated with muscle growth,
particularly in growing animals.
[0378] Nitrogen retention may be measured as the difference between
the intake of nitrogen and the excreted nitrogen by means of the
total collection of excreta and urine during a period of time. It
is understood that excreted nitrogen includes undigested protein
from the feed, endogenous proteinaceous secretions, microbial
protein, and urinary nitrogen.
Survival
[0379] The term survival as used herein means the number of subject
remaining alive. The term "improved survival" may be another way of
saying "reduced mortality".
Carcass Yield and Meat Yield
[0380] The term carcass yield as used herein means the amount of
carcass as a proportion of the live body weight, after a commercial
or experimental process of slaughter. The term carcass means the
body of an animal that has been slaughtered for food, with the
head, entrails, part of the limbs, and feathers or skin removed.
The term meat yield as used herein means the amount of edible meat
as a proportion of the live body weight, or the amount of a
specified meat cut as a proportion of the live body weight.
Weight Gain
[0381] The present invention further provides a method of
increasing weight gain in a subject, e.g. poultry or swine,
comprising feeding said subject a feedstuff comprising a feed
additive composition according to the present invention.
[0382] An "increased weight gain" refers to an animal having
increased body weight on being fed feed comprising a feed additive
composition compared with an animal being fed a feed without said
feed additive composition being present.
Necrotic Enteritis
[0383] Necrotic enteritis is an acute or chronic enterotoxemia seen
in chickens, turkeys and ducks worldwide, caused by Clostridium
perfringens. Necrotic enteritis is often characterised by a
fibrino-necrotic enteritis, usually of the mid-small intestine.
Mortality may be 5-50%, usually around 10%. Infection occurs by
faecal-oral transmission. Spores of the causative organism are
highly resistant. Predisposing factors include
coccidiosis/coccidiasis, diet (high protein), in ducks possibly
heavy strains, high viscosity diets (often associated with high rye
and wheat inclusions in the diet), contaminated feed and/or water,
other debilitating diseases.
[0384] The present invention relates to increasing the subject's
resistance to necrotic enteritis. In other words, the present
invention relates to avoiding or reducing the negative effect of
necrotic enteritis.
[0385] The term "resistance to" as used herein may encompasses the
term "tolerance of". Therefore in one embodiment the subject may
not be resistant to necrotic enteritis but the subject may be able
to tolerate the necrotic enteritis, i.e. without negative effects
on performance of the subject.
[0386] In one embodiment the present invention relates to a feed
additive composition according to the present invention for
treating or preventing necrotic enteritis in a subject. Typically
the subject will be one which has been or will be challenged with
Clostridium perfringens and/or Eimeria species. Such challenge may
come from the environment, or the application of live
microorganisms in the feed or drinking water, e.g. when live
coccidia vaccines are used.
[0387] In another embodiment the present invention relates to a
feed additive composition for preventing and/or treating
coccidiosis and/or necrotic enteritis in a subject.
[0388] The present invention yet further provides a method of
preventing and/or treating necrotic enteritis and/or coccidiosis
wherein an effective amount of a feed additive composition
according to the present invention is administered to a
subject.
Immune Response
[0389] Immune response as used herein means one of the multiple
ways in which DFMs modulate the immune system of animals, including
increased antibody production, up-regulation of cell mediated
immunity, up-regulation of pro-inflammatory cytokines, and
augmented toll-like receptor signalling. It is understood that
immuno-stimulation of the gastro intestinal tract by DFMs may be
advantageous to protect the host against disease, and that
immuno-suppression of the gastro intestinal tract may be
advantageous to the host because less nutrients and energy are used
to support the immune function.
[0390] Preferably the immune response is a cellular immune
response.
[0391] Preferably immune response is measure by looking at immune
markers.
Pathogenic Bacteria
[0392] The term pathogenic bacteria as used herein means for
example toxigenic clostridia species, e.g. Clostridium perfringens
and/or E. coli and/or Salmonella spp and/or Campylobacter spp. In
one embodiment the pathogenic bacteria may be Avian pathogenic E.
coli species.
[0393] The present invention may reduce populations of pathogenic
bacteria in the gastrointestinal tract of a subject.
Nutrient Excretion
[0394] In one embodiment the present invention relates to reducing
nutrient excretion in manure. This has positive effects on reducing
environmental hazards. For example, in a preferred embodiment the
present invention relates to reducing nitrogen and/or phosphorus
content in the subject's manure. This, therefore, reduces the
amount of nitrogen and/or phosphorus in the environment, which can
be beneficial.
Probiotic
[0395] For some applications, it is believed that the DFM in the
composition of the present invention can exert a probiotic culture
effect. It is also within the scope of the present invention to add
to the composition of the present invention further probiotic
and/or prebiotics.
[0396] Here, a prebiotic is:
[0397] "a non-digestible food ingredient that beneficially affects
the host by selectively stimulating the growth and/or the activity
of one or a limited number of beneficial bacteria".
[0398] The term "probiotic culture" as used herein defines live
microorganisms (including bacteria or yeasts for example) which,
when for example ingested or locally applied in sufficient numbers,
beneficially affects the host organism, i.e. by conferring one or
more demonstrable health benefits on the host organism. Probiotics
may improve the microbial balance in one or more mucosal surfaces.
For example, the mucosal surface may be the intestine, the urinary
tract, the respiratory tract or the skin. The term "probiotic" as
used herein also encompasses live microorganisms that can stimulate
the beneficial branches of the immune system and at the same time
decrease the inflammatory reactions in a mucosal surface, for
example the gut.
[0399] Whilst there are no lower or upper limits for probiotic
intake, it has been suggested that at least 10.sup.6-10.sup.12,
preferably at least 10.sup.6-10.sup.10, preferably
10.sup.8-10.sup.9, cfu as a daily dose will be effective to achieve
the beneficial health effects in a subject.
Isolated
[0400] In one aspect, suitably the enzyme or DFM used in the
present invention may be in an isolated form. The term "isolated"
means that the enzyme or DFM is at least substantially free from at
least one other component with which the enzyme or DFM is naturally
associated in nature and as found in nature. The enzyme or DFM of
the present invention may be provided in a form that is
substantially free of one or more contaminants with which the
substance might otherwise be associated. Thus, for example it may
be substantially free of one or more potentially contaminating
polypeptides and/or nucleic acid molecules.
Purified
[0401] In one aspect, preferably the enzyme and/or DFM according to
the present invention is in a purified form. The term "purified"
means that the enzyme and/or DFM is present at a high level. The
enzyme and/or DFM is desirably the predominant component present in
a composition. Preferably, it is present at a level of at least
about 90%, or at least about 95% or at least about 98%, said level
being determined on a dry weight/dry weight basis with respect to
the total composition under consideration.
[0402] It is envisaged within the scope of the present invention
that the embodiments of the invention can be combined such that
combinations of any of the features described herein are included
within the scope of the present invention. In particular, it is
envisaged within the scope of the present invention that any of the
therapeutic effects of the bacteria may be exhibited
concomitantly.
Nucleotide Sequence
[0403] The scope of the present invention encompasses nucleotide
sequences encoding proteins having the specific properties as
defined herein.
[0404] The term "nucleotide sequence" as used herein refers to an
oligonucleotide sequence or polynucleotide sequence, and variant,
homologues, fragments and derivatives thereof (such as portions
thereof). The nucleotide sequence may be of genomic or synthetic or
recombinant origin, which may be double-stranded or single-stranded
whether representing the sense or anti-sense strand.
[0405] The term "nucleotide sequence" in relation to the present
invention includes genomic DNA, cDNA, synthetic DNA, and RNA.
Preferably it means DNA, more preferably cDNA sequence coding for
the present invention.
[0406] In a preferred embodiment, the nucleotide sequence when
relating to and when encompassed by the per se scope of the present
invention does not include the native nucleotide sequence according
to the present invention when in its natural environment and when
it is linked to its naturally associated sequence(s) that is/are
also in its/their natural environment. For ease of reference, we
shall call this preferred embodiment the "non-native nucleotide
sequence". In this regard, the term "native nucleotide sequence"
means an entire nucleotide sequence that is in its native
environment and when operatively linked to an entire promoter with
which it is naturally associated, which promoter is also in its
native environment. However, the amino acid sequence encompassed by
the scope of the present invention can be isolated and/or purified
post expression of a nucleotide sequence in its native organism.
Preferably, however, the amino acid sequence encompassed by scope
of the present invention may be expressed by a nucleotide sequence
in its native organism but wherein the nucleotide sequence is not
under the control of the promoter with which it is naturally
associated within that organism.
[0407] Typically, the nucleotide sequence encompassed by the scope
of the present invention is prepared using recombinant DNA
techniques (i.e. recombinant DNA). However, in an alternative
embodiment of the invention, the nucleotide sequence could be
synthesised, in whole or in part, using chemical methods well known
in the art (see Caruthers M H et al., (1980) Nuc Acids Res Symp Ser
215-23 and Horn T et al., (1980) Nuc Acids Res Symp Ser
225-232).
Preparation of the Nucleotide Sequence
[0408] A nucleotide sequence encoding either a protein which has
the specific properties as defined herein or a protein which is
suitable for modification may be identified and/or isolated and/or
purified from any cell or organism producing said protein. Various
methods are well known within the art for the identification and/or
isolation and/or purification of nucleotide sequences. By way of
example, PCR amplification techniques to prepare more of a sequence
may be used once a suitable sequence has been identified and/or
isolated and/or purified.
[0409] By way of further example, a genomic DNA and/or cDNA library
may be constructed using chromosomal DNA or messenger RNA from the
organism producing the enzyme. If the amino acid sequence of the
enzyme is known, labelled oligonucleotide probes may be synthesised
and used to identify enzyme-encoding clones from the genomic
library prepared from the organism. Alternatively, a labelled
oligonucleotide probe containing sequences homologous to another
known enzyme gene could be used to identify enzyme-encoding clones.
In the latter case, hybridisation and washing conditions of lower
stringency are used. Alternatively, enzyme-encoding clones could be
identified by inserting fragments of genomic DNA into an expression
vector, such as a plasmid, transforming enzyme-negative bacteria
with the resulting genomic DNA library, and then plating the
transformed bacteria onto agar plates containing a substrate for
enzyme (i.e. maltose), thereby allowing clones expressing the
enzyme to be identified.
[0410] In a yet further alternative, the nucleotide sequence
encoding the enzyme may be prepared synthetically by established
standard methods, e.g. the phosphoroamidite method described by
Beucage S. L. et al., (1981) Tetrahedron Letters 22, p 1859-1869,
or the method described by Matthes et al., (1984) EMBO J. 3, p
801-805. In the phosphoroamidite method, oligonucleotides are
synthesised, e.g. in an automatic DNA synthesiser, purified,
annealed, ligated and cloned in appropriate vectors.
[0411] The nucleotide sequence may be of mixed genomic and
synthetic origin, mixed synthetic and cDNA origin, or mixed genomic
and cDNA origin, prepared by ligating fragments of synthetic,
genomic or cDNA origin (as appropriate) in accordance with standard
techniques. Each ligated fragment corresponds to various parts of
the entire nucleotide sequence. The DNA sequence may also be
prepared by polymerase chain reaction (PCR) using specific primers,
for instance as described in U.S. Pat. No. 4,683,202 or in Saiki R
K et al., (Science (1988) 239, pp 487-491).
Amino Acid Sequences
[0412] The scope of the present invention also encompasses amino
acid sequences of enzymes having the specific properties as defined
herein.
[0413] As used herein, the term "amino acid sequence" is synonymous
with the term "polypeptide" and/or the term "protein". In some
instances, the term "amino acid sequence" is synonymous with the
term "peptide". In some instances, the term "amino acid sequence"
is synonymous with the term "enzyme".
[0414] The amino acid sequence may be prepared/isolated from a
suitable source, or it may be made synthetically or it may be
prepared by use of recombinant DNA techniques.
[0415] The protein encompassed in the present invention may be used
in conjunction with other proteins, particularly enzymes. Thus the
present invention also covers a combination of proteins wherein the
combination comprises the protein/enzyme of the present invention
and another protein/enzyme, which may be another protein/enzyme
according to the present invention.
[0416] Preferably the amino acid sequence when relating to and when
encompassed by the per se scope of the present invention is not a
native enzyme. In this regard, the term "native enzyme" means an
entire enzyme that is in its native environment and when it has
been expressed by its native nucleotide sequence.
Sequence Identity or Sequence Homology
[0417] The present invention also encompasses the use of sequences
having a degree of sequence identity or sequence homology with
amino acid sequence(s) of a polypeptide having the specific
properties defined herein or of any nucleotide sequence encoding
such a polypeptide (hereinafter referred to as a "homologous
sequence(s)"). Here, the term "homologue" means an entity having a
certain homology with the subject amino acid sequences and the
subject nucleotide sequences. Here, the term "homology" can be
equated with "identity".
[0418] The homologous amino acid sequence and/or nucleotide
sequence should provide and/or encode a polypeptide which retains
the functional activity and/or enhances the activity of the
enzyme.
[0419] In the present context, a homologous sequence is taken to
include an amino acid sequence which may be at least 75, 85 or 90%
identical, preferably at least 95 or 98% identical to the subject
sequence. Typically, the homologues will comprise the same active
sites etc. as the subject amino acid sequence. Although homology
can also be considered in terms of similarity (i.e. amino acid
residues having similar chemical properties/functions), in the
context of the present invention it is preferred to express
homology in terms of sequence identity.
[0420] In the present context, a homologous sequence is taken to
include a nucleotide sequence which may be at least 75, 85 or 90%
identical, preferably at least 95 or 98% identical to a nucleotide
sequence encoding a polypeptide of the present invention (the
subject sequence). Typically, the homologues will comprise the same
sequences that code for the active sites etc. as the subject
sequence. Although homology can also be considered in terms of
similarity (i.e. amino acid residues having similar chemical
properties/functions), in the context of the present invention it
is preferred to express homology in terms of sequence identity.
[0421] Homology comparisons can be conducted by eye, or more
usually, with the aid of readily available sequence comparison
programs. These commercially available computer programs can
calculate % homology between two or more sequences.
[0422] % homology may be calculated over contiguous sequences, i.e.
one sequence is aligned with the other sequence and each amino acid
in one sequence is directly compared with the corresponding amino
acid in the other sequence, one residue at a time. This is called
an "ungapped" alignment. Typically, such ungapped alignments are
performed only over a relatively short number of residues.
[0423] Although this is a very simple and consistent method, it
fails to take into consideration that, for example, in an otherwise
identical pair of sequences, one insertion or deletion will cause
the following amino acid residues to be put out of alignment, thus
potentially resulting in a large reduction in % homology when a
global alignment is performed. Consequently, most sequence
comparison methods are designed to produce optimal alignments that
take into consideration possible insertions and deletions without
penalising unduly the overall homology score. This is achieved by
inserting "gaps" in the sequence alignment to try to maximise local
homology.
[0424] However, these more complex methods assign "gap penalties"
to each gap that occurs in the alignment so that, for the same
number of identical amino acids, a sequence alignment with as few
gaps as possible--reflecting higher relatedness between the two
compared sequences--will achieve a higher score than one with many
gaps. "Affine gap costs" are typically used that charge a
relatively high cost for the existence of a gap and a smaller
penalty for each subsequent residue in the gap. This is the most
commonly used gap scoring system. High gap penalties will of course
produce optimised alignments with fewer gaps. Most alignment
programs allow the gap penalties to be modified. However, it is
preferred to use the default values when using such software for
sequence comparisons.
[0425] Calculation of maximum % homology therefore firstly requires
the production of an optimal alignment, taking into consideration
gap penalties. A suitable computer program for carrying out such an
alignment is the Vector NTI (Invitrogen Corp.). Examples of
software that can perform sequence comparisons include, but are not
limited to, the BLAST package (see Ausubel et al 1999 Short
Protocols in Molecular Biology, 4th Ed--Chapter 18), BLAST 2 (see
FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999
177(1): 187-8 and tatiana@ncbi.nlm.nih.gov), FASTA (Altschul et al
1990 J. Mol. Biol. 403-410) and AlignX for example. At least BLAST,
BLAST 2 and FASTA are available for offline and online searching
(see Ausubel et al 1999, pages 7-58 to 7-60).
[0426] Although the final % homology can be measured in terms of
identity, the alignment process itself is typically not based on an
all-or-nothing pair comparison. Instead, a scaled similarity score
matrix is generally used that assigns scores to each pairwise
comparison based on chemical similarity or evolutionary distance.
An example of such a matrix commonly used is the BLOSUM62
matrix--the default matrix for the BLAST suite of programs. Vector
NTI programs generally use either the public default values or a
custom symbol comparison table if supplied (see user manual for
further details). For some applications, it is preferred to use the
default values for the Vector NTI package.
[0427] Alternatively, percentage homologies may be calculated using
the multiple alignment feature in Vector NTI (Invitrogen Corp.),
based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp
P M (1988), Gene 73(1), 237-244).
[0428] Once the software has produced an optimal alignment, it is
possible to calculate % homology, preferably % sequence identity.
The software typically does this as part of the sequence comparison
and generates a numerical result.
[0429] Should Gap Penalties be used when determining sequence
identity, then preferably the following parameters are used for
pairwise alignment:
TABLE-US-00017 FOR BLAST GAP OPEN 0 GAP EXTENSION 0 FOR CLUSTAL DNA
PROTEIN WORD 2 1 K triple SIZE GAP 15 10 PENALTY GAP 6.66 0.1
EXTENSION
[0430] In one embodiment, CLUSTAL may be used with the gap penalty
and gap extension set as defined above.
[0431] Suitably, the degree of identity with regard to a nucleotide
sequence is determined over at least 20 contiguous nucleotides,
preferably over at least 30 contiguous nucleotides, preferably over
at least 40 contiguous nucleotides, preferably over at least 50
contiguous nucleotides, preferably over at least 60 contiguous
nucleotides, preferably over at least 100 contiguous
nucleotides.
[0432] Suitably, the degree of identity with regard to a nucleotide
sequence may be determined over the whole sequence.
Hybridisation
[0433] The present invention also encompasses sequences that are
complementary to the nucleic acid sequences of the present
invention or sequences that are capable of hybridising either to
the sequences of the present invention or to sequences that are
complementary thereto.
[0434] The term "hybridisation" as used herein shall include "the
process by which a strand of nucleic acid joins with a
complementary strand through base pairing" as well as the process
of amplification as carried out in polymerase chain reaction (PCR)
technologies.
[0435] The present invention also encompasses the use of nucleotide
sequences that are capable of hybridising to the sequences that are
complementary to the sequences presented herein, or any derivative,
fragment or derivative thereof.
[0436] The term "variant" also encompasses sequences that are
complementary to sequences that are capable of hybridising to the
nucleotide sequences presented herein.
[0437] Preferably, complementary sequences are those capable of
hybridising under stringent conditions (e.g. 50.degree. C. and
0.2.times.SSC {1.times.SSC=0.15 M NaCl, 0.015 M Na.sub.3citrate pH
7.0}) to the nucleotide sequences presented herein.
[0438] More preferably, complementary sequences are those that are
capable of hybridising under high stringency conditions (e.g.
65.degree. C. and 0.1.times.SSC {1.times.SSC=0.15 M NaCl, 0.015 M
Na.sub.3citrate pH 7.0}) to the nucleotide sequences presented
herein.
[0439] In a more preferred aspect, the present invention covers
nucleotide sequences that can hybridise to the nucleotide sequence
of the present invention, or the complement thereof, under high
stringent conditions (e.g. 65.degree. C. and 0.1.times.SSC).
EXAMPLES
Example 1
Materials and Methods
[0440] Three thousand six hundred one-day-old Cobb male chicks were
purchased from a commercial hatchery. At study initiation, fifty
males were allocated to each treatment pen by blocks. The study
consisted of the following treatments (Table 1):
TABLE-US-00018 TABLE 1 Experimental design of Example 1.
Clostridium perfringens Additional Treatment Challenge
Phytase.sup.1 enzyme.sup.2 DFM.sup.3 1 No 500 FTU/kg None None 2
Yes 500 FTU/kg None None 3 Yes 500 FTU/kg Amylase None (200 u/kg) 4
Yes 500 FTU/kg Protease None (5000 u/kg) 5 Yes 500 FTU/kg
Xylanase.sup.4 None (2000 u/kg) Amylase.sup.4 (200 u/kg)
Protease.sup.4 (5000 u/kg) 6 Yes 500 FTU/kg None Enviva Pro (7.5
.times. 10.sup.4 CFU/g) 7 Yes 500 FTU/kg Amylase Enviva Pro (200
u/kg) (7.5 .times. 10.sup.4 CFU/g) 8 Yes 500 FTU/kg Protease Enviva
Pro (5000 u/kg) (7.5 .times. 10.sup.4 CFU/g) 9 Yes 500 FTU/kg
Xylanase.sup.4 Enviva Pro (2000 u/kg) (7.5 .times. 10.sup.4 CFU/g)
Amylase.sup.4 (200 u/kg) Protease.sup.4 (5000 u/kg) .sup.1Phytase
from E. coli. .sup.2Amylase from Bacillus licheniformis, xylanase
from Trichoderma reesei, protease from Bacillus subtilis.
.sup.3Enviva Pro .RTM. is combination of Bacillus subtilis strains
Bs2084, LSSAO1 and 15AP4, provided by Danisco A/S. .sup.4Axtra XAP
.RTM. provided by Danisco A/S.
[0441] Bird weights by pen were recorded at study initiation, 23 d,
35 d, and termination (42 d). The pen was the unit of measure.
Broiler diets were fed as crumbles (starter) or pellets (grower and
finisher). Diets met or exceeded NRC standards (Table 2). The mixer
was flushed to prevent cross contamination of diets. All treatment
feeds were mixed using a Davis S-20 mixer and pelleted using a
California Pellet Mill (cold pellet temperature 65-70 C). Samples
were collected from each treatment diet from the beginning, middle,
and end of each batch and blended together to confirm enzyme
activities and Enviva Pro presence in feed.
TABLE-US-00019 TABLE 2 Experimental diet composition of Example 1.
Ingredient (%) Starter Grower Finisher Maize 53.62 57.87 59.82
Maize DDGS 10.00 10.00 10.00 Soybean Meal 49% CP 26.93 23.97 21.36
Ampro 55 5.00 5.00 5.00 Soy oil 2.07 0.91 1.74 Lysine 0.24 0.24
0.24 DL-methionine 0.21 0.19 0.18 L-threonine 0.01 0.01 0.01 Salt
0.30 0.34 0.35 Limestone 1.04 1.07 0.94 Dicalcium phosphate 0.26
0.11 0.02 Vitamin and trace mineral premix 0.33 0.33 0.33
Calculated Nutrient Composition (%) CP 22.60 21.50 20.39 Energy,
kcal/kg 3060 3025 3100 Digestible lysine 1.36 1.26 1.21 Digestible
methionine 0.58 0.61 0.53 Digestible threonine 0.83 0.83 0.80
[0442] Birds received feed ad-libitum appropriate to the treatment
from day 0 to 42. Enzymes and Enviva Pro were provided by Danisco
in the appropriate mixtures and levels for all experimental
treatments. All diets contained 500 FTU of E. coli phytase in the
background. The pens were arranged within the facility to prevent
direct contact in order to avoid contamination. A change from
starter to grower occurred on day 23. Grower diet was replaced with
the finisher diet on day 35. At each feed change, feeders were
removed from pens by block, weighed back, emptied, and refilled
with the appropriate treatment diet. On the final day of the study
feed was weighed. Pens were checked daily for mortality. When a
bird was culled or found dead, the date and removal weight (kg)
were recorded. A gross necropsy was performed on all dead or culled
birds to determine the sex and probable cause of death. Signs of
Necrotic Enteritis were noted.
[0443] All pens had approximately 4 inches of built up litter with
a coating of fresh pine shavings. All birds were spray vaccinated
prior to placement into pens with a commercial coccidiosis vaccine
(Coccivac-B). On days 20, 21, and 22 all birds, except Treatment 1,
were dosed with a broth culture of C. perfringens. A field isolate
of C. perfringens known to cause NE and originating from a
commercial broiler operation was utilized as the challenge
organism. Fresh inoculum was used each day. The titration levels
were approximately 1.0.times.10.sup.8-9. Each pen received the same
amount of inoculum. The inoculum was administered by mixing into
the feed found in the base of the tube feeder. On day 23, five
birds from each pen were selected, euthanized, group weighed, and
examined for the degree of presence of Necrotic Enteritis lesions.
The scoring was based on a 0 to 3 score, with 0 being normal and 3
being the most severe (0=none, 1=mild, 2=moderate, 3=marked/severe;
Hofacre et al., 2003 J. Appl. Poult. Res. 12:60-64). No concomitant
drug therapy was used during the study.
[0444] Means were separated using pair wise t-tests. Significant
differences were considered at P<0.05. Pens were used as the
experimental unit.
Results
[0445] FIG. 1 shows the necrotic enteritis lesion scores of broiler
chickens in a necrotic enteritis challenge model, based on a 0 to 3
score system. Pooled SEM=0.15
[0446] The challenged control treatment increased lesion scores
compared to the unchallenged control treatment. Addition of DFMs
with a combination of a xylanase, amylase, protease and phytase
reduced lesion scores compared to all other treatments. Addition of
DFMs in combination with the enzymes reduced lesion scores compared
DFMs alone or enzymes by themselves.
[0447] FIG. 2 shows the body weight gain of broiler chickens in a
necrotic enteritis challenge model. Pooled SEM=28.6
[0448] FIG. 2 shows that a combination of the DFM (Enviva Pro.RTM.)
with a combination of a xylanase, an amylase, a protease and a
phytase significantly improved body weight gain (BW gain) in
broiler chickens challenged with Clostridium perfringens compared
with the challenged control--even resulting in BW gain which was
improved over a negative control (i.e. an unchallenged control).
This was significantly better than any other treatments.
[0449] FIG. 3 shows the feed conversion ratio of broiler chickens
in a necrotic enteritis challenge model. Pooled SEM=0.016
[0450] The combination of Enviva Pro (DFM) with a xylanase,
amylase, protease and phytase significantly improved (reduced) FCR
(g BW gain/g feed intake) of broilers from hatch to 42 d compared
to the challenged control, and enzymes by themselves and the other
treatments.
Example 2
Materials and Methods
[0451] Cobb 500 male broiler chicks were obtained from a commercial
hatchery. A total of 26 chicks were randomly assigned to one of 8
replicate pens per treatment. Floor pens (16 ft.sup.2/pen) were
located in a curtain-sided house containing controlled heating,
circulating fans, heat lamps and fresh wood shavings. Birds were
exposed to fluorescent lighting in a 24 h light cycle for the first
four days and then 16 light:8 hour dark cycle for the remainder of
the experiment. Feed was provided in bell feeders and water
supplied via nipple drinkers ad libitum. A 5.times. dose of
Coccivac-B (Intervet) was administered manually with a syringe into
the oral cavity of chicks at one day of age.
TABLE-US-00020 TABLE 3 Experimental design of Example 2.
Coccidiosis Additional Treatment vaccine Phytase.sup.1 enzyme.sup.2
DFM.sup.3 1 5X 500 FTU/kg None None 2 5X 500 FTU/kg None Enviva Pro
(7.5 .times. 10.sup.4 CFU/g) 3 5X 500 FTU/kg Xylanase.sup.4 None
(1000 u/kg) Amylase.sup.4 (1800 u/kg) Protease.sup.4 (5000 u/kg) 4
5X 500 FTU/kg Xylanase.sup.4 Enviva Pro (1000 u/kg) (7.5 .times.
10.sup.4 CFU/g) Amylase.sup.4 (1800 u/kg) Protease.sup.4 (5000
u/kg) .sup.1Phytase from E. coli. .sup.2Amylase from Bacillus
amyloliquefaciens, xylanase from Trichoderma reesei, protease from
Bacillus subtilis. .sup.3Enviva Pro .RTM. is combination of
Bacillus subtilis strains Bs2084, LSSAO1 and 15AP4, provided by
Danisco A/S. .sup.4Avizyme 1505XAP .RTM. provided by Danisco
A/S.
[0452] Chicks were fed diets with or without either Enviva Pro or
xylanase, amylase, and protease (Avizyme 1502; Table 3). Enzymes
and Enviva Pro were provided by Danisco in the appropriate mixtures
and levels for all experimental treatments. All diets contained 500
FTU of E. coli phytase. The pens were arranged within the facility
to prevent direct contact in order to avoid contamination.
[0453] All diets were corn-soybean meal-DDGS based diets. Starter
diets were provided during the study (d1-20). Diets were pelleted
(65-70.degree. C.) and crumbled. Samples were collected from each
treatment diet from the beginning, middle, and end of each batch
and blended together to confirm enzyme activities and Enviva Pro
presence in feed.
TABLE-US-00021 TABLE 4 Experimental diet composition of Example 2.
Starter Grower Finisher Ingredient (%) (0-20 d) (20-38 d) (38-48 d)
Maize 50.60 52.3 57.40 Wheat Middilings 1.33 1.03 1.32 Maize DDGS
7.00 7.00 7.00 Soybean Meal 34.60 33.50 28.60 Vegetable fat 2.50
2.50 2.50 Limestone 1.41 1.38 1.09 MD-Phosphate 1.20 1.00 0.84 DL-
methionine 0.31 0.27 0.27 Salt 0.46 0.46 0.46 L-Lysine 0.29 0.23
0.28 Vitamin and Trace Mineral Premix 1.50 1.50 1.50 Calculated
Nutrient Composition (%) ME poultry, kcal/kg 2950 3000 3040 CP 23.0
22.5 20.4 Calcium 0.85 0.81 0.75 Av. Phosphorus 0.38 0.35 0.32 TSAA
0.98 0.94 0.89 Lysine 1.36 1.29 1.20 Methionine 0.62 0.59 0.56
[0454] Body weights and feeder weights were recorded on day 1, 11,
20, 38 and 48 for calculation of feed intake, body weight gain and
feed conversion. Mortality and culls were monitored on a daily
basis and used to adjust for feed consumption and gain. One bird
from six replicate pens was euthanized by cervical dislocation for
collection of mucosal scrapings on days 11 and 20. Mucosal
scrapings were collected from the ileum (Meckel's diverticulum to
the ileo-cecal junction). The ileum was excised and cut along its
length to expose the lumen and then flushed quickly and gently with
PBS to remove digesta. The edge of a microscope slide was used to
remove the mucosal layer by scraping along the length of the
excised tissue section. The mucosal layer was immediately freeze
clamped between aluminium plates in liquid N to preserve RNA
integrity and stored in individual whirl-pack bags. Frozen tissue
samples were stored in liquid N during sampling and at -80 C prior
to analysis. Total RNA from mucosal scraping was isolated using the
Trizol reagent (Invitrogen) using a mechanical homogenizer for
tissue disruption. Total RNA (0.5 .mu.g) was reverse transcribed to
complementary DNA using iScript (Bio-Rad) according to the
manufacturer's recommendations. The mRNA abundance of secreted
inflammatory cytokine genes (interleukin-10, interferon-.gamma. and
interleukin-17) was assessed using chicken-specific primers.
Additionally, TATA-BP, HPRT-1 and .beta.-actin mRNA abundance was
measured for data normalization using geNorm software. The
fold-change in mRNA abundance in gene expression was determined
using the modified delta-delta Ct equation as described by Rudrappa
and Humphrey (2007) J. Nutr. 137: 427-432 and log transformed for
data analysis.
[0455] Means were separated using pair wise t-tests. Significant
differences were considered at P<0.05. Birds were used as the
experimental unit for mRNA data.
Results
[0456] FIG. 4 shows mRNA abundance of interferon-gamma gene in
ileal mucosal scrapings of broiler chickens.
Age 11 d: Pooled SEM=0.1
Age 20 d: Pooled SEM=0.6
[0457] The combination of Enviva Pro and xylanase, amylase,
protease+phytase upregulated IFR-g expression in the ileum of
11-d-old-broilers that received 5 times a live coccidiosis vaccine
at hatch compared to the negative control, Enviva Pro+phytase, and
xylanase, amylase, protease+phytase. At 21 d, Enviva Pro+phytase,
and the combination of Enviva Pro and xylanase, amylase,
protease+phytase down regulated IFR-g expression in the ileum
compared to the negative control. These data suggest that
modulation of immune response may be one of the mechanisms of
improved performance of DFMs in combination with the 4 enzymes in
broilers.
[0458] FIG. 15 shows feed conversion ratio of broiler chickens at
48 d of age. Age 48 d: Pooled SEM=0.041
Example 3
Materials and Methods
[0459] One digestibility trial with broiler chickens was conducted
to determine the effects of dietary enzymes and DFMs treatments on
nutrient utilisation. The cages were housed in environmentally
controlled rooms. The birds received 20-hour fluorescent
illumination and, allowed free access to the diets and water. On
day 1, a broiler live coccidiosis vaccine was given to all chicks
via drinking water. Paper was provided on cage wire-floor for the
first three days to enable recycling of Eimeria Oocystes. The study
consisted of the following treatments (Table 5).
TABLE-US-00022 TABLE 5 Experimental design of Example 3. Treatment
Phytase.sup.1 Additional enzyme.sup.2 DFM.sup.3 1 500 FTU/kg None
None 2 500 FTU/kg Xylanase.sup.4 (1000 u/kg) None Amylase 1.sup.4
(1800 u/kg) Protease.sup.4 (5000 u/kg) 3 500 FTU/kg Xylanase (2000
u/kg) None Amylase 2 (200 u/kg) 4 500 FTU/kg Xylanase.sup.5 (2000
u/kg) None Amylase 2.sup.5 (200 u/kg) Protease.sup.5 (5000 u/kg) 5
500 FTU/kg None Enviva Pro (7.5 .times. 10.sup.4 CFU/g) 6 500
FTU/kg Xylanase.sup.4 (1000 u/kg) Enviva Pro Amylase 1.sup.4 (1800
u/kg) (7.5 .times. 10.sup.4 CFU/g) Protease.sup.4 (5000 u/kg) 7 500
FTU/kg Xylanase (2000 u/kg) Enviva Pro Amylase 2 (200 u/kg) (7.5
.times. 10.sup.4 CFU/g) 8 500 FTU/kg Xylanase.sup.5 (2000 u/kg)
Enviva Pro Amylase 2.sup.5 (200 u/kg) (7.5 .times. 10.sup.4 CFU/g)
Protease.sup.5 (5000 u/kg) .sup.1Phytase from E. coli.
.sup.2Amylase 1 from Bacillus amyloliquefaciens, amylase 2 from
Bacillus licheniformis, xylanase from Trichoderma reesei, protease
from Bacillus subtilis. .sup.3Enviva Pro .RTM. is combination of
Bacillus subtilis strains Bs2084, LSSAO1 and 15AP4, provided by
Danisco A/S. .sup.4Avizyme 1505 .RTM. provided by Danisco A/S.
.sup.5Axtra XAP .RTM. provided by Danisco A/S.
[0460] A total of 192 birds were individually weighed and assigned
on the basis of body weight to 48 cages (4 birds/cage). The 8
dietary treatments were then randomly assigned to six cages each.
Birds received starter feed ad-libitum appropriate to the treatment
from 0 to 21 days. Enzymes and Enviva Pro were provided by Danisco
in the appropriate mixtures and levels for all experimental
treatments. All diets contained 500 FTU of E. coli phytase. The
pens were arranged within the facility to prevent direct contact in
order to avoid contamination. Birds were fed starter diets (Table
6) in mash form throughout the experiment.
TABLE-US-00023 TABLE 6 Experimental diet composition of Example 3.
Ingredient (%) Starter Maize 46.22 Wheat middlings 6.73 Maize DDGS
7.00 Soybean Meal 48% CP 32.81 Maize starch/enzyme/DFM premix 0.30
Animal/vegetable fat blend (50:50) 3.00 L-Lysine.cndot.HCl 0.27
DL-methionine 0.30 L-threonine 0.11 Titanium dioxide 0.30 Salt 0.34
Limestone 1.12 Dicalcium phosphate 1.20 Vitamin and trace mineral
premix 0.30 Calculated Nutrient Composition (%) CP 23.00 ME,
kcal/kg 2950 Calcium 0.85 Available phosphorus 0.38 Sodium 0.18
Digestible lysine 1.21 Digestible methionine 0.62 Digestible TSAA
0.86 Digestible threonine 0.76
[0461] On day 21, four birds per cage were euthanized by
intracardial injection of sodium pentobarbitone and contents of the
lower ileum were expressed by gentle flushing with distilled water.
Digesta from birds within a cage were pooled, resulting in six
samples per dietary treatment. The digesta samples were frozen
immediately after collection, lyophilised and processed. Digesta
samples and diets were analysed for Ti, DM, GE, starch, fat, N and
amino acids, excluding tryptophan, as per standard procedures.
Calculation of ileal digestibility coefficients was performed as
reported by Ravindran et al. (2005), based on the concentration of
indigestible Ti. The energy contribution of starch, fat and protein
to ileal digestible energy was calculated based on mean gross
energy of starch (4.2 kcal/g), fat (9.4 kcal/g), or protein (5.5
kcal/kg). The improvement of digestible amino acids in response to
enzymes and DFMs was expressed in relation to the amount of
non-digested amino acids at the ileal level; the slope of that
linear function was used as an indicator of the effects of the
additives on amino acid digestibility.
[0462] Means were separated using pair wise t-tests. Significant
differences were considered at P<0.05. Cages were used as the
experimental unit.
Results
[0463] FIG. 5 shows apparent ileal digestible energy of broiler
chickens at 21 d of age. Pooled SEM=0.027
[0464] The addition of Enviva Pro (a DFM) in combination with an
amylase, xylanase, protease and phytase exhibited commercially
relevant increments of ileal digestible energy compared with the
enzymes by themselves and the negative controls. These data
indicates that DFMs improved the effects of these exogenous enzymes
on the energy digestibility of poultry diets. For the avoidance of
doubt Amylase 2 is through use of the amylase in AxtraXAP and
Amylase 1 is through use of the amylase in Avizyme 1502.
[0465] FIG. 6 show increments of ileal amino acid digestibility for
three dietary treatments versus the control treatment as function
of ileal undigested amino acids in the control treatment using
21-d-old broiler chickens.
[0466] The figure presents the improvement on ileal amino acid
digestibility of dietary treatments with respect to the undigested
fraction of amino acids in the ileum of broilers in the control
treatment. Each point within a treatment represents one of the
measured amino acids. The addition of Enviva Pro on top of
xylanase, amylase 2, protease+phytase increased the ileal
digestibility of amino acids (+11.3%) compared to Enviva
Pro+Phytase (+3.6%) and xylanase, amylase 2, protease+phytase by
themselves (i.e. without DFM) (+4.7%). These data indicates that
DFMs improved the efficacy of these exogenous enzymes to increase
amino acid digestibility of poultry diets.
[0467] FIG. 7 shows the improvement of ileal digestible energy with
respect to the control treatment using 21-d-old broiler
chickens.
[0468] The figure presents the increment of ileal digestible energy
of each dietary treatment compared a negative control treatment
with phytase. Additionally, the calculated contributions of energy
from starch, fat or protein are presented. Addition of Enviva Pro
in combination with xylanase, amylase 2, protease+phytase increased
the ileal digestible energy compared to the Enviva Pro+phytase
treatment and the xylanase, amylase 2, protease+phytase by
themselves treatment. Addition of Enviva Pro in combination with
xylanase, amylase 1, protease+phytase produced commercially
important increments on ileal digestible energy versus the enzymes
by themselves. These data indicate an improved ability of the 4
enzymes to increase the ileal digestible energy of broiler diets in
the presence of DFMs.
Example 4
Materials and Methods
[0469] One digestibility trial with broiler chickens was conducted
to determine the effects of dietary enzymes and DFMs treatments on
nutrient utilisation. The cages were housed in environmentally
controlled rooms. The birds received 20-hour fluorescent
illumination and, allowed free access to the diets and water. On
day 1, a broiler live coccidiosis vaccine was given to all chicks
via drinking water. Paper was provided on cage wire-floor for the
first three days to enable recycling of Eimeria Oocystes. The study
consisted of the following treatments (Table 7)
TABLE-US-00024 TABLE 7 Experimental design of Example 4. Treatment
Phytase.sup.1 Additional enzyme.sup.2 DFM.sup.3 1 None None None 2
500 FTU/kg Xylanase.sup.4 (1000 u/kg) None Amylase 1.sup.4 (1800
u/kg) Protease.sup.4 (5000 u/kg) 3 500 FTU/kg Xylanase.sup.5 (2000
u/kg) None Amylase 2.sup.5 (200 u/kg) Protease.sup.5 (5000 u/kg) 4
None None Enviva Pro (7.5 .times. 10.sup.4 CFU/g) 5 500 FTU/kg
Xylanase.sup.4 (1000 u/kg) Enviva Pro Amylase 1.sup.4 (1800 u/kg)
(7.5 .times. 10.sup.4 CFU/g) Protease.sup.4 (5000 u/kg) 6 500
FTU/kg Xylanase.sup.5 (2000 u/kg) Enviva Pro Amylase 2.sup.5 (200
u/kg) (7.5 .times. 10.sup.4 CFU/g) Protease.sup.5 (5000 u/kg)
.sup.1Phytase from E. coli. .sup.2Amylase 1 from Bacillus
amyloliquefaciens, amylase 2 from Bacillus licheniformis, xylanase
from Trichoderma reesei, protease from Bacillus subtilis.
.sup.3Enviva Pro .RTM. is combination of Bacillus subtilis strains
Bs2084, LSSAO1 and 15AP4, provided by Danisco A/S. .sup.4Avizyme
1505 .RTM. provided by Danisco A/S. .sup.5Axtra XAP .RTM. provided
by Danisco A/S.
[0470] A total of 144 birds were individually weighed and assigned
on the basis of body weight to 36 cages (4 birds/cage). The 6
dietary treatments were then randomly assigned to six cages each.
Birds received starter feed ad-libitum appropriate to the treatment
from 0 to 21 days. Enzymes and Enviva Pro were provided by Danisco
in the appropriate mixtures and levels for all experimental
treatments. The pens were arranged within the facility to prevent
direct contact in order to avoid contamination. Birds were fed
starter diets (Table 6) in mash form throughout the experiment.
TABLE-US-00025 TABLE 8 Experimental diet composition of Example 4.
Ingredient (%) Starter Maize 46.22 Wheat middlings 6.73 Maize DDGS
7.00 Soybean Meal 48% CP 32.81 Maize starch/enzyme/DFM premix 0.30
Animal/vegetable fat blend (50:50) 3.00 L-Lysine.cndot.HCl 0.27
DL-methionine 0.30 L-threonine 0.11 Titanium dioxide 0.30 Salt 0.34
Limestone 1.12 Dicalcium phosphate 1.20 Vitamin and trace mineral
premix 0.30 Calculated Nutrient Composition (%) CP 23.00 ME,
kcal/kg 2950 Calcium 0.85 Available phosphorus 0.38 Sodium 0.18
Digestible lysine 1.21 Digestible methionine 0.62 Digestible TSAA
0.86 Digestible threonine 0.76
[0471] Feed intake and total excreta output were measured
quantitatively per cage over four consecutive days (from day 17 to
20) for the determination of nitrogen-corrected apparent
metabolizable energy (AMEn) and Nitrogen retention. Daily excreta
collections were pooled within a cage, mixed in a blender and
sub-sampled. Each sub sample was lyophilized, ground to pass
through a 0.5 mm sieve and stored in airtight plastic containers at
-4 C pending analysis. Processed samples were analysed for DM, GE
and N, using standard procedures.
[0472] Means were separated using pair wise t-tests. Significant
differences were considered at P<0.05. Cages were used as the
experimental unit.
Results
[0473] FIG. 8 shows nitrogen-corrected apparent metabolizable
energy AMEn of dietary treatments fed to 17 to 21-d-old broiler
chickens. Pooled SEM=0.015
[0474] Addition of Enviva Pro in combinations with xylanase,
amylase, protease+phytase increased the AMEn of diets in response
to enzymes compared to the negative control diet. In particular,
addition of Enviva Pro in combination with xylanase, amylase 2,
protease+phytase increased the AMEn of diets in response to enzymes
compared to diets with only Enviva Pro.
[0475] FIG. 9 shows nitrogen retention of 17 to 21-d-old broiler
chickens. Pooled SEM=0.006
[0476] Addition of Enviva Pro in combination with xylanase,
amylase, protease+phytase increased the nitrogen retention of
broiler chickens in response to enzymes compared to the negative
control diet. In particular, addition of Enviva Pro on top of
xylanase, amylase 2, protease+phytase increased the nitrogen
retention of broilers in response to enzymes compared to broilers
fed diets with Enviva Pro only.
Example 5
Materials and Methods
[0477] Ross 308 male broiler chicks were obtained from a commercial
hatchery. A total of 10 chicks were randomly assigned to one of 6
replicate cages per treatment. Birds were exposed to fluorescent
lighting in a 24 h light cycle for the first four days and then 16
light:8 hour dark cycle for the remainder of the experiment. Feed
and water were supplied ad libitum. The experimental design
consisted of the following treatments.
TABLE-US-00026 TABLE 9 Experimental design of Example 5.
Coccidiosis Coccidio Additional Treatment vaccine stat
Phytase.sup.1 enzyme.sup.2 DFM.sup.3 1 None None 500 FTU/kg None
None 2 5X None 500 FTU/kg None None 3 5X Salinomycin 500 FTU/kg
None None 4 5X None 500 FTU/kg None Enviva Pro (7.5 .times.
10.sup.4 CFU/g) 5 5X None 500 FTU/kg Xylanase.sup.4 (1000 u/kg)
None Amylase.sup.4 (1800 u/kg) Protease.sup.4 (5000 u/kg) 6 5X None
500 FTU/kg Xylanase.sup.4 (1000 u/kg) Enviva Pro Amylase.sup.4
(1800 u/kg) (7.5 .times. 10.sup.4 CFU/g) Protease.sup.4 (5000 u/kg)
.sup.1Phytase from E. coli. .sup.2Amylase from Bacillus
amyloliquefaciens, xylanase from Trichoderma reesei, protease from
Bacillus subtilis. .sup.3Enviva Pro .RTM. is combination of
Bacillus subtilis strains Bs2084, LSSAO1 and 15AP4, provided by
Danisco A/S. .sup.4Avizyme 1505 .RTM. provided by Danisco A/S.
[0478] In treatments 2 to 6, an overdosed (recommended dose
.times.5) coccidiosis vaccine (B, Intervet) was administered
manually with a syringe into the oral cavity of chicks at one day
of age. In treatment 2, Salinomycin (Bio-cox) was used at the
approved level (60 g/MT) as a coccidiostat. The pens were arranged
within the facility to prevent direct contact in order to avoid
cross contamination with Eimeria oocysts and DFMs. Enzymes and
Enviva Pro were provided by Danisco A/S in the appropriate mixtures
and levels for all experimental treatments. All diets contained 500
FTU of E. coli phytase in the background.
TABLE-US-00027 TABLE 10 Experimental diet composition of Example 5.
Ingredient (%) Starter Maize 53.18 Maize DDGS 10.00 Soyabean Meal
48% CP 32.05 Soyabean Oil 1.07 L-Lysine HCl 0.31 DL-methionine 0.31
L-threonine 0.12 Salt 0.33 Limestone 1.14 Dicalcium Phosphate 1.19
Vitamin and Trace Mineral Premix 0.30 Calculated Nutrient
Composition (%) CP 23.00 ME, kcal/kg 2950 Calcium 0.85 Available
phosphorus 0.38 Sodium 0.18 Digestible lysine 1.21 Digestible
methionine 0.63 Digestible TSAA 0.86 Digestible threonine 0.76
[0479] A total of 2 birds per replicate cage were euthanized at 14
d of age for collection of mucosal scrapings from mid-ileum. Ileums
were flushed with distilled water and cut open with a pair of
scissors. Opened sections were laid flat on a clean glass plate.
Mucosa was carefully scraped from the mid region of ileum with the
long edge of a glass slide. Each sample was stored in 2 ml of RNA
later (Ambion) and frozen in a -80 C freezer. Samples were thawed
on ice. Total RNA was isolated with Trizol reagent according to
standard protocols. Integrity of RNA was determined on an agarose
gel. RNA was reverse transcribed with the MMLV reverse
transcriptase. Expression of mucin (MUC2) was determined by real
time PCR on a Biorad real-time MyIQ machine.
[0480] Means were separated using pair wise t-tests. Significant
differences were considered at P<0.05. Birds were used as the
experimental unit for mRNA data.
Results
[0481] FIG. 10 shows mRNA abundance of MUC2 gene in ileal mucosal
scrapings of broiler chickens at 14 d of age. Pooled SEM=0.14
[0482] Addition of Enviva Pro in combination with xylanase,
amylase, protease+phytase down regulated the expression of MUC 2 in
the ileum of broilers challenged with a 5.times. dose of a live
coccidiosis vaccine compared to the challenged control. These data
suggest that a reduction of endogenous amino acid losses due to
reduced mucin secretion may be responsible for improved performance
of broilers receiving combinations of DFMs and the 4 enzymes.
Example 6
Materials and Methods
[0483] Tissue samples were taken from broiler chicks from the trial
presented in Example 1 at 23 days of age. Treatment specifications
are presented in Table 1. The jejunum, pancreas and liver were
removed from 2 birds from every pen and the mucosa pooled resulting
in eight samples per treatment. The samples were rinsed in buffer
solution (PBS) immersed in a tissue storage reagent (RNAlater)
according to manufacturer's protocol and stored at -80.degree. C.
Total RNA was isolated from each tissue sample using a single step
phenol-chloroform extraction method as described by Chomczynski and
Saachi (1987; Anal. Biochem. 162:156-9). Concentration of the RNA
was determined by measuring the absorbance at 260 nm (Nanodrop) and
monitored for integrity by gel electrophoresis on 1.2% agarose
gels. Only RNA of sufficient purity and having a ratio of
absorption at 260 nm vs. 280 nm greater than 1.87 were considered
for use.
[0484] Microarrays were manufactured using 70 base pair
oligo-nucleotides (Opereon Biotechnologies Inc) according to the
protocol described by Druyan et al. (2008; Poult. Sci. 87:2418-29).
The experimental design of the array was a complete interwoven loop
design as described by Garosi et al. (2005; Br. J. Nutr. 93:425-32)
which each sample is compared directly with the others in a
multiple pair wise fashion allowing all treatments to be compared.
The samples were labelled according to the method described by
Druyan et al. (2008; Poult. Sci. 87:2418-29) in that that half the
samples would be labelled with Cy3 and half with Cy5 which are
fluorescent dyes of cyanine. Hybridisation was carried out using
the Pronto Plus! Microarray Hybridisation Kit prior to the addition
of Cy3 and Cy5 labelled cDNA probes and covered with a clean glass
coverslip (Lifterslip) and left to hybridise for 16 hours. The
microarrays were then scanned on a Scan Array Gx PLUS Microarray
Scanner set to 65% laser power to acquire images.
[0485] Total RNA from individual samples was reversed transcribed
to produce cDNA which was then used as a template for the qPCR
amplifications as described by Druyan et al. (2008; Poult. Sci.
87:2418-29). Thermocycling parameters were optimised for each gene
and each gene was amplified independently in duplicate within a
single instrument run.
[0486] Data files were generated from the scanned images of the
microarrays but extracting the intensity raw data for each slide
and dye combination using ScanAlyze Softare. Intensity data files
were then analysed using JMP Genomics including and initial log 2
transformation. Data normalisation was performed using
locally-weighted regression and smoothing first within array and
across all arrays. The resulting normalised log 2 intensities were
analysed using a mixed model ANOVA.
[0487] Mean intensities were compared using a threshold of
significance based on Bongerroni correction of P=0.05. For the
complete array, including all replicates, a mean by grid intensity
was calculated for each gene using the 3 side by side probes,
resulting in a total of four replicated means, one from each grid,
per gene. Data for the Ct ratio from the samples in duplicate
(sample gene Ct:Sample GAPDH Ct) depending on treatment were
subjected to one way ANOVA.
Results
[0488] Expression data was collected using the microarray platform
and a "heat map" produced to visualise the data for the jejunum
(FIG. 16) and pancreas (FIG. 17). Relative expression levels of six
genes of interest were converted to visual cues based on the scale
seen in FIG. 16. Lowly expressed genes are marked with a minus sign
("-"), and highly expressed genes are marked with a plus sign
("+"); whereas a greater gray intensity depicts a greater
difference from the mean expression level of the treatments. The
genes that were measured and their purported functions are seen in
Table 11. Real-time PCR was used to validate the gene expression
shown in the heat map for sucrase-isomaltase (SI) and amylase 2A
(AMY2a) and were highly correlated to the array data.
TABLE-US-00028 TABLE 11 Purported function of genes measured. Gene
Identity Function PEPT1 Oligo-peptide transporter 1 Nutrient
transport GCK Glucokinase Initial step in glucose metabolism SI
Sucrase isomaltase Glucose metabolism ZO1 Tight Junction protein 1
Tight junction formation, intestinal integrity CD3d T- cell antigen
CD3 T-cell marker AMY2A Amylase 2A Starch and sucrose
metabolism
[0489] FIG. 16 shows a heat map of expression profiles of genes of
interest for all treatments for jejunum at 23 days of age.
[0490] FIG. 17 shows a heat map of expression profile of chicken
alpha amylase for all treatments in pancreas at 23 days of age.
[0491] In FIGS. 16 and 17 the key is as follows:
Unchallenged control=Unchallenged Control+phytase CC=Challenged
Control+phytase CC+Amylase=Challenged Control+phytase+amylase
CC+XAP=Challenged Control+phytase+xylanase+amylase+protease
CC+EP=Challenged Control+phytase+Enviva Pro
CC+EP+Amylase=Challenged Control+phytase+amylase+Enviva Pro
CC+EP+XAP=Challenged
Control+phytase+xylanase+amylase+protease+Enviva Pro
[0492] The expression of oligo-peptide transport 1 (PEPT1) was
increased by xylanase+amylase+protease+phytase, and this was
increased further when in combination with Enviva Pro. PEPT1 is
part of a peptide transport system and is responsible for the
uptake of a wide range of di- and tri-peptides.
[0493] The expression of Glucokinase (GCK) was down-regulated by
the challenged control but the combination of amylase+phytase or
xylanase+amylase+protease+phytase with Enviva Pro produced an
up-regulation similar to the unchallenged control. The extent of
the up-regulation was greater than when
xylanase+amylase+protease+phytase were used with Enviva Pro.
[0494] A similar pattern was also seen with sucrase iso-maltase
(SI) where the combination of Enviva Pro with amylase+phytase or
xylanase+amylase+protease+phytase produced a greater up-regulation
than both the challenged and unchallenged control. GCK is a key
enzyme in glucose metabolism and SI is responsible for hydrolysis
of sucrose and iso-maltose, and so has an important role in the
digestion and absorption of carbohydrates in animals.
[0495] Tight Junction protein 1 (ZO1) was most highly expressed in
the challenged control. A reduction was seen with the enzyme
treatments but a greater down-regulation in expression was seen
when Enviva Pro was used and particularly so when in combination
with xylanase+amylase+protease+phytase which produced a similar
level of down-regulation as the non-challenged control. ZO1 is a
protein that is on the cytoplasmic face of tight junctions, there
are various roles for this protein ranging from signal transduction
for tight junction assembly to stability of the tight junctions
themselves.
[0496] The T-cell antigen CD3 (CD3D) was highly expressed in the
challenged control. The enzyme alone treatments did reduce
expression somewhat but it was significantly down-regulated when in
combination with Enviva Pro. The combination of
xylanase+amylase+protease+phytase produced the largest
down-regulation of the enzyme treatments, and, when in combination
with Enviva Pro, produced an even larger down-regulation close to
that seen for the unchallenged control. CD3D is a surface molecule
found on T cells and plays an important role in signal transduction
during T-cell receptor engagement and is part of the T-cell
receptor/CD3 complex.
[0497] The alpha amylase (AMY2A) was highly expressed in the
unchallenged and challenged controls but the addition of
amylase+phytase or xylanase+amylase+protease+phytase resulted in
reduced expression, which was further reduced when Enviva Pro was
used in combination, particularly for
xylanase+amylase+protease+phytase. Chicken alpha amylase is mainly
produced in the pancreas and has a major role in starch
digestion.
Discussion
[0498] The increase in expression of the peptide transporter
oligopeptide transporter 1 (PEPT1) when
xylanase+amylase+protease+phytase were given, particularly in
combination with Enviva Pro, suggests increased availability of
peptides and thus an increased requirement of peptide transporters,
which indicates a synergistic effect of enzymes and DFMs to
increase the adsorption of peptides for the animal which allows for
greater growth. Animal performance results of Example 1 support
this conclusion. The increase in expression of glucokinase and
sucrase isomerase with the combination of amylase+phytase, or
xylanase+amylase+protease+phytase, and Enviva Pro suggests that
there was increased absorption of glucose, and increased
availability of sucrose and isomaltose in the brush border, which
indicates a positive interaction between the enzyme and DFMs to
increase carbohydrate absorption in the small intestine and thus
increase energy availability from the diet. The decrease of
glucokinase expression for the challenged control suggests that the
Clostridium perfringens challenge caused damage to the mucosa and
that addition of Enviva Pro and xylanase+amylase+protease+phytase
alleviated this.
[0499] The effect of Enviva Pro on reducing the expression of Tight
junction protein 1 indicates lower requirement for protein turn
over in the intestine, which may be related to a high intestinal
integrity. The increased expression in the challenged control,
however, suggests that turnover/requirement of the protein was high
due to failing intestinal integrity possibly due to the coccidia
and Clostridium perfringens infections. The enzymes alone did have
some effect on ameliorating this but the additive effect seen with
Enviva Pro suggests a greater benefit from the combination. This
indicates that Enviva Pro acts to increase intestinal integrity and
thus benefit the health of the animal. Increased intestinal
integrity, and thus absorptive capacity, may be one of the
mechanisms by which the effectiveness of exogenous enzymes is
increased when a DFM is present.
[0500] The increased expression of T cell antigen CD3 d in the
challenged control indicates increased cell-mediated immune
response due to the challenge. In these conditions, birds will be
undergoing sub-optimal performance because the immune response will
demand energy that could be used for growth, and because some birds
will experience a systemic disease response. The increased
expression of this immunological marker was markedly reversed when
Enviva Pro was used alone or in combination with enzymes. Down
regulation of immune response in the intestine may be one of the
mechanisms by which the effectiveness of exogenous enzymes in
nutrient absorption and performance is increased when a DFM is
present.
[0501] The down-regulation of alpha amylase (AMY2A) production that
was seen with the combination of amylase+phytase, or
xylanase+amylase+protease+phytase suggests that the chicken is
reducing its production of endogenous amylase as a response to the
exogenous enzymes supplied. The additive effect seen with Enviva
Pro and xylanase+amylase+protease+phytase suggest that the DFM is
working synergistically with the exogenous enzymes to allow the
bird to utilise the energy that it would have spent producing
enzymes for digestion of starch in the diet.
[0502] The net effect of a down-regulated immune response and
higher intestinal integrity, and a better nutrient digestion and
absorption with the combination of enzymes and DFMs, clearly
determines enhanced production performance of broiler chickens.
Example 7
Materials and Methods
[0503] A digestibility trial with broiler chickens was conducted to
determine the effects of dietary enzymes and DFM treatments on
energy utilisation. A total of 288 day-old, male Ross 308 chicks
were obtained from a commercial hatchery and brooded in raised wire
battery pens until day 14. Birds were vaccinated with a live
coccidia vaccine at hatch (Coccivac-B). Chicks were fed a
corn-SBM-DDGS based starter diet. Chicks were provided experimental
diets from day 14 until day 21. The feed and water were provided
ad-libitum throughout the 21 day period. Six chicks were housed per
pen in battery pens located within an environmentally controlled
room, where they received supplemental heat starting at 35.degree.
C. on day-of-age and decreasing 2.degree. C. weekly. Light was
provided at 23L:1D. On day 15, chicks were individually weighed,
sorted, wing banded and randomly allocated to the experimental
units using a completely randomized design. Each treatment
consisted of 8 pens per treatment. The study consisted of the
following treatments (Table 12).
TABLE-US-00029 TABLE 12 Experimental design of Example 7. Treatment
Phytase.sup.1 Additional enzyme.sup.2 DFM.sup.3, 4 1 None None None
2 500 Xylanase (2000 u/kg) None FTU/kg Amylase (200 u/kg) Protease
(5000 u/kg) 3 None None Enviva Pro (1.5 .times. 10.sup.5 FTU/g) 4
500 Xylanase (2000 u/kg) Enviva Pro FTU/kg Amylase (200 u/kg) (1.5
.times. 10.sup.5 FTU/g) Protease (5000 u/kg) 5 None None GalliPro
Tect (8 .times. 10.sup.5 FTU/g) 6 500 Xylanase (2000 u/kg) GalliPro
Tect FTU/kg Amylase (200 u/kg) (8 .times. 10.sup.5 FTU/g) Protease
(5000 u/kg) .sup.1Phytase from Buttiauxella. .sup.2Amylase from
Bacillus licheniformis, xylanase from Trichoderma reesei, protease
from Bacillus subtilis. .sup.3Enviva Pro .RTM. is a combination of
Bacillus subtilis strains Bs2084, LSSAO1 and 15AP4, provided by
Danisco A/S. .sup.4GalliPro Tect is a DFM comprised by one strain
of Bacillus licheniformis (DSM17236).
[0504] Enzymes and DFMs were sourced and provided by Danisco in the
appropriate mixtures and levels for all experimental treatments.
The pens were arranged within the facility to prevent direct
contact in order to avoid cross contamination. Birds were fed
starter diets (Table 13) in mash form throughout the experimental
period.
TABLE-US-00030 TABLE 13 Experimental diet composition of Example 7.
Ingredient (%) Starter Corn 52.94 Corn-DDGS 12.00 Soybean meal 48%
29.38 Animal/Vegetable Fat Blend 1.08 Salt 0.40 DL Methionine 0.22
Bio-Lys 0.44 Limestone 1.30 Dicalcium Phosphate 1.27 Choline
chloride 60 0.10 Vit/Min Premix 0.63 TiO.sub.2 0.25 Calculated
Nutrient Composition (%) CP 22.25 ME, kcal/kg 2925 Calcium 0.90
Available phosphorus 0.38 Sodium 0.18 Digestible lysine 1.20
Digestible methionine 0.52 Digestible TSAA 0.85 Digestible
threonine 0.75
[0505] Clean excreta trays were put in place for the last 2 days
and excreta samples were collected by pen on day 21. The collected
excreta samples were frozen at -20.degree. C. before they were oven
dried at 65.degree. C. for 3 days to determine the dry matter (AOAC
International, 2005; method 934.01). The feed samples were also
corrected to the dry matter basis be measuring 5.0 g of each sample
and drying them in an oven at 100.degree. C. for 24 hrs. The
excreta samples were then ground through a 1-mm screen while the
feed samples were ground 0.5-mm screen. Excreta samples and diets
were analysed for Ti, DM, GE, and N, as per standard procedures.
Apparent metabolizable energy (AME) calculation was based on the
concentration of the indigestible marker (Ti) and the gross energy
of diets and excreta. Appropriate corrections were made for
differences in moisture content. N-corrected AME (AMEn) was
determined for zero nitrogen retention by multiplication with 8.22
kcal per gram of nitrogen retained in the body (Hill and Anderson,
1958; J. Nutr. 64:587-603).
[0506] Means were separated using pair wise t-tests. Significant
differences were considered at P<0.05. Cages were used as the
experimental unit.
Results
[0507] FIG. 18 shows apparent metabolizable energy corrected by
nitrogen retention (AME.sub.n) of 21 d old broiler chickens. Effect
of DFM; P<0.001; Effect of Enzyme; P<0.001; Effect of
DFM.times.Enzyme; P=0.27; Pooled SEM=32 kcal.
[0508] Addition of Xylanase, amylase, protease, and phytase, in
combination with Enviva Pro or GalliPro Tect resulted in
improvements of AME.sub.n versus the control treatment, that were
significantly greater compared to the enzymes or the DFMs by
themselves. The AME.sub.n increments due to the combination of
xylanase, amylase, protease, phytase, and Enviva Pro (235 kcal/kg)
or GalliPro Tect (215 kcal/kg) were greater than the addition of
the enzymes and the DFM effects when applied separately (152
kcal/kg for Enviva Pro, or 120 kcal/kg for GalliPro Tect), compared
to the negative control treatment.
Example 8
Materials and Methods
[0509] One thousand and four hundred one-day-old Cobb male chicks
were purchased from a commercial hatchery. At study initiation,
fifty males were allocated to one of seven pens per treatment by
blocks. The study consisted of the following treatments (Table
1):
TABLE-US-00031 TABLE 1 Experimental design of Example 8.
Clostridium perfringens Additional Treatment Challenge
Phytase.sup.1 enzyme.sup.2 DFM.sup.3 1 No 500 None None FTU/kg 2
Yes 500 None None FTU/kg 3 Yes 500 None Enviva Pro FTU/kg (7.5
.times. 10.sup.4 FTU/g) 4 Yes 500 Xylanase.sup.4 Enviva Pro FTU/kg
(2000 u/kg) (7.5 .times. 10.sup.4 FTU/g) Amylase.sup.4 (200 u/kg)
Protease.sup.4 (5000 u/kg) .sup.1Phytase from E. coli.
.sup.2Amylase from Bacillus licheniformis, xylanase from
Trichoderma reesei, protease from Bacillus subtilis. .sup.3Enviva
Pro .RTM. is combination of Bacillus subtilis strains Bs2084,
LSSAO1 and 15AP4, provided by Danisco A/S. .sup.4Axtra XAP .RTM.
provided by Danisco A/S.
[0510] Bird weights by pen were recorded at study initiation, 21 d
and termination (42 d). The pen was the unit of measure. Broiler
diets were fed as crumbles (starter) or pellets (grower and
finisher). Diets met or exceeded NRC standards (Table 2). The mixer
was flushed to prevent cross contamination of diets. All treatment
feeds were mixed using a Davis S-20 mixer and pelleted using a
California Pellet Mill (cold pellet temperature 65-70 C). Samples
were collected from each treatment diet from the beginning, middle,
and end of each batch and blended together to confirm enzyme
activities and Enviva Pro presence in feed.
TABLE-US-00032 TABLE 2 Experimental diet composition of Example 8.
Ingredient (%) Starter Grower Finisher Maize 50.959 59.6156 62.7488
Maize DDGS 12 12 12 Soybean Meal 49% CP 30.7176 22.5873 19.4
Choline Chloride 0.06 0.06 0.06 Soy oil 3.0693 2.7035 2.84841
Lysine 0.21 0.2426 0.244 DL-methionine 0.1723 0.1566 0.1341
L-threonine 0.0387 0.0551 0.0564 Salt 0.4668 0.4692 0.47 Limestone
1.4467 1.4501 1.33389 Dicalcium phosphate 0.7346 0.5349 0.571
Vitamin and trace 0.125 0.125 0.125 mineral premix Calculated
Nutrient Composition (%) CP 22.642 19.45 19.45 Energy, mcal/kg
12.761 12.012 12.012 Digestible lysine 1.327 1.124778 1.124778
Digestible methionine 0.53142 0.475425 0.475425 Digestible
threonine 0.89401 0.78494 0.78494
[0511] Birds received feed ad-libitum appropriate to the treatment
from day 0 to 42. Enzymes and Enviva Pro were provided by Danisco
in the appropriate mixtures and levels for all experimental
treatments. All diets contained 500 FTU of E. coli phytase in the
background. The pens were arranged within the facility to prevent
direct contact in order to avoid contamination.
[0512] A change from starter to grower occurred on day 21. Grower
diet was replaced with the finisher diet on day 35. At each feed
change, feeders were removed from pens by block, weighed back,
emptied, and refilled with the appropriate treatment diet. On the
final day of the study, feed was weighed. Pens were checked daily
for mortality. When a bird was culled or found dead, the date and
removal weight (kg) were recorded. A gross necropsy was performed
on all dead or culled birds to determine the sex and probable cause
of death. Signs of Necrotic Enteritis were noted.
[0513] All pens had approximately 4 inches of built up litter with
a coating of fresh pine shavings. All birds were spray vaccinated
prior to placement into pens with a commercial coccidiosis vaccine
(Coccivac-B). On days 18, 19, and 20 all birds, except Treatment 1,
were dosed with a broth culture of C. perfringens. A field isolate
of C. perfringens known to cause Necrotic Enteritis and originating
from a commercial broiler operation was utilized as the challenge
organism. Fresh inoculum was used each day. The titration levels
were approximately 1.0.times.10.sup.8-9. Each pen received the same
amount of inoculum. The inoculum was administered by mixing into
the feed found in the base of the tube feeder.
Sample Collection
[0514] On day 21, a total of 8 birds per treatment (1-2 birds per
pen) were euthanised and the total gastrointestinal tract from
below the gizzard to the ileal-cecal junction was collected from
each bird and sent overnight on ice to the laboratory. The samples
were further dissected in the laboratory to obtain a 20 cm portion
of the jejunum surrounding the Meckle's diverticulum; the remainder
of the intestinal tract was discarded. The sections were rinsed
with 0.1% peptone to remove the intestinal contents and opened
longitudinally to expose the epithelial lining. The sections were
masticated in 99 ml of 0.1% peptone at 7.0 strokes/s for 60 s to
release mucosa-associated bacterial cells. Bacteria were harvested
from the masticated solution by centrifugation at 12,000.times.g
for 10 minutes. The resultant bacterial pellet was resuspended in
10 ml of MRS broth+10% glycerol, flash-frozen in liquid nitrogen,
and stored at -20.degree. C. until further analysis.
DNA Isolation
[0515] Genomic DNA was isolated from all samples by phenol
chloroform extraction and purified using Roche Applied Science High
Pure PCR Template Purification Kit (Roche Diagnostics Corp.,
Indianapolis, Ind.). Samples were randomly pooled in pairs at the
DNA level after extraction, for a total of four samples per
treatment.
Pyrosequencing
[0516] Bacterial tag-encoded FLX amplicon pyrosequencing was
performed as described by Dowd (Dowd et al. 2008; BMC Microbiol. 8,
125). An equivalent amount of DNA isolated from the intestinal
mucosa from each bird was analyzed in pooled samples containing DNA
from two birds. The V1-V3 region of the 16S rRNA gene was amplified
in each sample using the primers 28 F (5'-GAGTTTGATCNTGGCTCAG) and
519R (5'-GTNTTACNGCGGCKGCTG). Following sequencing, raw data was
screened and trimmed based on quality. Sequences were sorted by
individual samples based on barcode sequences. Barcode tags were
removed and non-bacteria ribosomal sequences were removed. The
bacterial community composition was determined using BlastN
comparison to a quality controlled and manually curated database
derived from NCBI. The relative abundance of each bacterial ID was
determined for each sample. Data was compiled at each taxonomic
level using NCBI nomenclature.
Statistical Analysis
[0517] For performance data means were separated using pair wise
t-tests. Significant differences were considered at P<0.05. Pens
were used as the experimental unit.
[0518] Genus level identifications were used for the analysis of
the pyrosequencing data. The relative abundance of each genus was
calculated and used for the analysis. The results were analysed
using a categorical model analysis and then a Chi-square
probability calculated using JMP 8.0.2 (SAS institute, Cary, N.C.),
where each sample representing two birds was considered an
experimental unit.
Results:
[0519] FIG. 19 shows feed conversion ratio (FCR) of broiler
chickens in a necrotic enteritis challenge model (Pooled SEM:
0.015).
[0520] The combination of Enviva Pro with xylanase, amylase,
protease+phytase reduced FCR (g BW gain/g feed intake) compared to
the challenged control treatment and the use of Enviva Pro and
phytase alone. Feed conversion ratio was reduced by the combination
to the level of the unchallenged control+phytase.
[0521] FIG. 20 shows relative abundance of Lactobacillus spp. at 21
d in the jejunal mucosa of broiler chickens, ChSq<0.0001.
[0522] FIG. 20 shows the relative abundance of Lactobacillus spp.
in comparison to other species in the jejunal mucosa of broilers at
21 days in a necrotic enteritis challenge model. The proportion of
Lactobacilli was reduced in the challenged control in comparison to
the unchallenged control. The combination of Enviva Pro, xylanase,
amylase, protease+phytase increases the proportion of Lactobacilli
more so than Enviva Pro and phytase alone and the challenged
control.
[0523] Lactobacilli are widely used as probiotics for both human
and animal use (Patterson and Burkeholder 2003; Poult Sci 82 (4)
627-31) and have been documented to improve gut health to a level
that could be comparable to antibiotic growth promoters (Awad et
al. 2009 Poult Sci 88 (1) 49-56). Thus by increasing the proportion
Lactobacilli in the gut microbiota, the combination of Enviva Pro,
xylanase, amylase, protease+phytase can improve gut health and
positively impact feed efficiency.
[0524] All publications mentioned in the above specification are
herein incorporated by reference. Various modifications and
variations of the described methods and system of the present
invention will be apparent to those skilled in the art without
departing from the scope and spirit of the present invention.
Although the present invention has been described in connection
with specific preferred embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
described modes for carrying out the invention which are obvious to
those skilled in biochemistry and biotechnology or related fields
are intended to be within the scope of the following claims.
Sequence CWU 1
1
61483PRTBacillus licheniformis 1Ala Asn Leu Asn Gly Thr Leu Met Gln
Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15 Asn Asp Gly Gln His Trp Lys
Arg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30 Ala Glu His Gly Ile
Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gln
Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly
Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 65 70
75 80 Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile
Asn 85 90 95 Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala
Asp Ala Thr 100 105 110 Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala
Asp Arg Asn Arg Val 115 120 125 Ile Ser Gly Glu His Leu Ile Lys Ala
Trp Thr His Phe His Phe Pro 130 135 140 Gly Arg Gly Ser Thr Tyr Ser
Asp Phe Lys Trp His Trp Tyr His Phe 145 150 155 160 Asp Gly Thr Asp
Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170 175 Phe Gln
Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190
Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195
200 205 Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu
Gln 210 215 220 Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys
Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val Asn His Val Arg Glu
Lys Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp Gln
Asn Asp Leu Gly Ala Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr Asn
Phe Asn His Ser Val Phe Asp Val Pro Leu 275 280 285 His Tyr Gln Phe
His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met 290 295 300 Arg Lys
Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser 305 310 315
320 Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335 Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe
Ile Leu 340 345 350 Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly
Asp Met Tyr Gly 355 360 365 Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro
Ala Leu Lys His Lys Ile 370 375 380 Glu Pro Ile Leu Lys Ala Arg Lys
Gln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400 Asp Tyr Phe Asp His
His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser Val
Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly
Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435 440
445 Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser
450 455 460 Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser
Ile Tyr 465 470 475 480 Val Gln Arg 21452DNABacillus licheniformis
2gcaaatctta atgggacgct gatgcagtat tttgaatggt acatgcccaa tgacggccaa
60cattggaagc gtttgcaaaa cgactcggca tatttggctg aacacggtat tactgccgtc
120tggattcccc cggcatataa gggaacgagc caagcggatg tgggctacgg
tgcttacgac 180ctttatgatt taggggagtt tcatcaaaaa gggacggttc
ggacaaagta cggcacaaaa 240ggagagctgc aatctgcgat caaaagtctt
cattcccgcg acattaacgt ttacggggat 300gtggtcatca accacaaagg
cggcgctgat gcgaccgaag atgtaaccgc ggttgaagtc 360gatcccgctg
accgcaaccg cgtaatttca ggagaacacc taattaaagc ctggacacat
420tttcattttc cggggcgcgg cagcacatac agcgatttta aatggcattg
gtaccatttt 480gacggaaccg attgggacga gtcccgaaag ctgaaccgca
tctataagtt tcaaggaaag 540gcttgggatt gggaagtttc caatgaaaac
ggcaactatg attatttgat gtatgccgac 600atcgattatg accatcctga
tgtcgcagca gaaattaaga gatggggcac ttggtatgcc 660aatgaactgc
aattggacgg tttccgtctt gatgctgtca aacacattaa attttctttt
720ttgcgggatt gggttaatca tgtcagggaa aaaacgggga aggaaatgtt
tacggtagct 780gaatattggc agaatgactt gggcgcgctg gaaaactatt
tgaacaaaac aaattttaat 840cattcagtgt ttgacgtgcc gcttcattat
cagttccatg ctgcatcgac acagggaggc 900ggctatgata tgaggaaatt
gctgaacggt acggtcgttt ccaagcatcc gttgaaatcg 960gttacatttg
tcgataacca tgatacacag ccggggcaat cgcttgagtc gactgtccaa
1020acatggttta agccgcttgc ttacgctttt attctcacaa gggaatctgg
ataccctcag 1080gttttctacg gggatatgta cgggacgaaa ggagactccc
agcgcgaaat tcctgccttg 1140aaacacaaaa ttgaaccgat cttaaaagcg
agaaaacagt atgcgtacgg agcacagcat 1200gattatttcg accaccatga
cattgtcggc tggacaaggg aaggcgacag ctcggttgca 1260aattcaggtt
tggcggcatt aataacagac ggacccggtg gggcaaagcg aatgtatgtc
1320ggccggcaaa acgccggtga gacatggcat gacattaccg gaaaccgttc
ggagccggtt 1380gtcatcaatt cggaaggctg gggagagttt cacgtaaacg
gcgggtcggt ttcaatttat 1440gttcaaagat ga 14523463PRTTrichoderma
reesei 3Met Lys Leu Arg Tyr Ala Leu Pro Leu Leu Leu Gln Leu Ser Leu
Pro 1 5 10 15 Val Leu Ser Ala Asp Thr Ala Ala Trp Arg Ser Arg Thr
Ile Tyr Phe 20 25 30 Ala Leu Thr Asp Arg Ile Ala Arg Gly Ser Gly
Asp Thr Gly Gly Ser 35 40 45 Ala Cys Gly Asn Leu Gly Asp Tyr Cys
Gly Gly Thr Phe Gln Gly Leu 50 55 60 Glu Ser Lys Leu Asp Tyr Ile
Lys Gly Met Gly Phe Asp Ala Ile Trp 65 70 75 80 Ile Thr Pro Val Val
Thr Ser Asp Asp Gly Gly Tyr His Gly Tyr Trp 85 90 95 Ala Glu Asp
Ile Asp Ser Ile Asn Ser His Tyr Gly Ser Ala Asp Asp 100 105 110 Leu
Lys Ser Leu Val Asn Ala Ala His Ser Lys Gly Phe Tyr Met Met 115 120
125 Val Asp Val Val Ala Asn His Met Gly Tyr Ala Asn Ile Ser Asp Asp
130 135 140 Ser Pro Ser Pro Leu Asn Gln Ala Ser Ser Tyr His Pro Glu
Cys Asp 145 150 155 160 Ile Asp Tyr Asn Asn Gln Thr Ser Val Glu Asn
Cys Trp Ile Ser Gly 165 170 175 Leu Pro Asp Leu Asn Thr Gln Ser Ser
Thr Ile Arg Ser Leu Tyr Gln 180 185 190 Asp Trp Val Ser Asn Leu Val
Ser Thr Tyr Gly Phe Asp Gly Val Arg 195 200 205 Ile Asp Thr Val Lys
His Val Glu Gln Asp Tyr Trp Pro Gly Phe Val 210 215 220 Asn Ala Thr
Gly Val Tyr Cys Ile Gly Glu Val Phe Asp Gly Asp Pro 225 230 235 240
Asn Tyr Leu Leu Pro Tyr Ala Ser Leu Met Pro Gly Leu Leu Asn Tyr 245
250 255 Ala Ile Tyr Tyr Pro Met Thr Arg Phe Phe Leu Gln Gln Gly Ser
Ser 260 265 270 Gln Asp Met Val Asn Met His Asp Gln Ile Gly Ser Met
Phe Pro Asp 275 280 285 Pro Thr Ala Leu Gly Thr Phe Val Asp Asn His
Asp Asn Pro Arg Phe 290 295 300 Leu Ser Ile Lys Asn Asp Thr Ala Leu
Leu Lys Asn Ala Leu Thr Tyr 305 310 315 320 Thr Ile Leu Ser Arg Gly
Ile Pro Ile Val Tyr Tyr Gly Thr Glu Gln 325 330 335 Ala Phe Ser Gly
Gly Asn Asp Pro Ala Asn Arg Glu Asp Leu Trp Arg 340 345 350 Ser Gly
Phe Asn Ala Gln Ser Asp Met Tyr Asp Ala Ile Ser Lys Leu 355 360 365
Thr Tyr Ala Lys His Ala Val Gly Gly Leu Ala Asp Asn Asp His Lys 370
375 380 His Leu Tyr Val Ala Asp Thr Ala Tyr Ala Phe Ser Arg Ala Gly
Gly 385 390 395 400 Asn Met Val Ala Leu Thr Thr Asn Ser Gly Ser Gly
Ser Ser Ala Gln 405 410 415 His Cys Phe Gly Thr Gln Val Pro Asn Gly
Arg Trp Gln Asn Val Phe 420 425 430 Asp Glu Gly Asn Gly Pro Thr Tyr
Ser Ala Asp Gly Asn Gly Gln Leu 435 440 445 Cys Leu Asn Val Ser Asn
Gly Gln Pro Ile Val Leu Leu Ser Ser 450 455 460 41392DNATrichoderma
reesei 4atgaagctcc ggtacgctct cccgctgctc ttgcagctct ctttgccggt
cctctccgca 60gacaccgccg cctggaggtc ccgcaccatc tactttgccc tgacagaccg
catcgctcgt 120ggaagcggtg acacgggggg cagtgcgtgt gggaacctgg
gggactactg cggtggcacg 180ttccagggct tggagagcaa gttggactac
atcaagggca tgggattcga tgccatctgg 240atcacacctg ttgtgacgag
tgatgatggg ggctaccatg gctattgggc ggaggacatc 300gactccatca
actctcatta tggctctgcg gacgatctca agagtctcgt caacgccgcg
360catagcaagg gcttctatat gatggtggac gtcgtggcca accacatggg
ctacgccaat 420atctctgacg atagtccctc tccactgaac caggcctcgt
cgtatcaccc cgagtgtgat 480atcgactaca acaaccaaac cagcgtcgag
aactgctgga tcagcggcct cccggatctc 540aacacgcaga gctcaaccat
ccgcagcctc taccaggact gggtctccaa cctcgtgtcc 600acgtacggct
tcgacggcgt ccgcatcgac accgtcaagc acgtcgagca agactactgg
660cccggcttcg tcaacgccac cggcgtctac tgcatcggcg aggtctttga
cggagaccca 720aactacctgc tgccctacgc cagcctcatg ccgggcctgc
tcaactacgc catctactac 780cccatgacgc gcttcttcct ccagcagggc
tcctcgcagg acatggtcaa catgcacgac 840cagatcggca gcatgttccc
cgacccgacc gcgctcggca cctttgtcga caaccacgac 900aacccgcgct
tcctgagcat caagaacgac acggccctgc tcaagaacgc gctgacgtac
960accatcctct cgcgcggcat ccccatcgtc tactacggca ccgagcaggc
cttctcgggc 1020ggcaacgacc cggccaacag ggaggacctc tggcgcagcg
gcttcaacgc ccagtccgac 1080atgtacgacg ccatctccaa gctcacctac
gccaagcacg ccgtcggcgg cctcgccgac 1140aacgaccaca agcacctgta
cgtcgccgac acggcctacg ccttcagccg cgccggcggc 1200aacatggtgg
ccctgaccac caacagcggc agcgggagct cggcccagca ctgcttcggc
1260acgcaggtgc ccaacggccg ctggcagaat gtctttgacg agggcaatgg
gccgacgtat 1320tccgccgacg gcaacggcca gctttgcttg aatgtgtcca
acggtcagcc cattgtcttg 1380ctgtcttcgt ga 1392519DNAArtificial
SequencePrimer 28F 5gagtttgatc ntggctcag 19618DNAArtificial
SequencePrimer 519R 6gtnttacngc ggckgctg 18
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