U.S. patent application number 11/012946 was filed with the patent office on 2005-07-14 for canine probiotic bifidobacteria globosum.
This patent application is currently assigned to The Procter & Gamble Company. Invention is credited to Boileau, Thomas William-Maxwell, Ceddia, Michael Anthony, Collins, John Kevin, Davenport, Gary Mitchell, Kiely, Barry Pius, O'Mahony, Liam Diarmuid, Sunvold, Gregory Dean, Tetrick, Mark Alan, Vickers, Robert Jason.
Application Number | 20050152884 11/012946 |
Document ID | / |
Family ID | 34738669 |
Filed Date | 2005-07-14 |
United States Patent
Application |
20050152884 |
Kind Code |
A1 |
Boileau, Thomas William-Maxwell ;
et al. |
July 14, 2005 |
Canine probiotic Bifidobacteria globosum
Abstract
According to the invention there is provided a strain of lactic
acid bacteria of the species Bifidobacteria globosum obtainable by
isolation from resected and washed canine gastrointestinal tract
having a probiotic activity in animals. Methods of use and
compositions comprising the Bifidobacteria globosum of the present
invention are also provided.
Inventors: |
Boileau, Thomas
William-Maxwell; (Galloway, OH) ; Ceddia, Michael
Anthony; (Newburg, IN) ; Collins, John Kevin;
(Cork, IE) ; Davenport, Gary Mitchell; (Dayton,
OH) ; Kiely, Barry Pius; (Cork, IE) ;
O'Mahony, Liam Diarmuid; (Cork, IE) ; Sunvold,
Gregory Dean; (Lewisburg, OH) ; Tetrick, Mark
Alan; (Dayton, OH) ; Vickers, Robert Jason;
(Dayton, OH) |
Correspondence
Address: |
THE PROCTER & GAMBLE COMPANY
INTELLECTUAL PROPERTY DIVISION
WINTON HILL TECHNICAL CENTER - BOX 161
6110 CENTER HILL AVENUE
CINCINNATI
OH
45224
US
|
Assignee: |
The Procter & Gamble
Company
Cincinnati
OH
|
Family ID: |
34738669 |
Appl. No.: |
11/012946 |
Filed: |
December 15, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60531588 |
Dec 19, 2003 |
|
|
|
Current U.S.
Class: |
424/93.45 ;
435/252.9 |
Current CPC
Class: |
A61P 17/00 20180101;
A61P 31/00 20180101; A23K 50/40 20160501; A61P 31/04 20180101; A23K
10/16 20160501; A23K 10/18 20160501; A23K 50/42 20160501; A61K
35/745 20130101; A61P 1/00 20180101; A61P 1/12 20180101; A61P 13/00
20180101; C12N 1/205 20210501; A23K 50/48 20160501; C12R 2001/01
20210501; A23L 33/135 20160801; A61P 37/00 20180101; A61P 37/02
20180101; C12N 1/20 20130101; A23K 50/45 20160501 |
Class at
Publication: |
424/093.45 ;
435/252.9 |
International
Class: |
A61K 045/00; C12N
001/20 |
Claims
What is claimed is:
1. A strain of Bifidobacterium globosum obtainable by isolation
from resected and washed canine gastrointestinal tract having
probiotic activity.
2. The strain according to claim 1 having the ability to survive
and colonise the gastrointestinal tract of a companion animal.
3. The strain according to claim 2 having at least 33% growth when
cultured in the presence of 0.5% bile salts.
4. The strain according to claim 2 able to maintain viability
following 1 hour at pH 2.5.
5. The strain according to claim 1 having a 16s-23s intergenic
polynucleotide sequence having at least 93% homology with the
sequence according to SEQ ID NO. 1.
6. The strain according to claim 5, wherein said strain is
Bifidobacterium globosum strain NCIMB 41198, or mutant thereof.
7. The strain according to claim 6 wherein the mutant is a natural,
genetically modified or induced mutant.
8. The strain according to claim 2 wherein the companion animal is
a dog.
9. The strain according to claim 2 wherein the companion animal is
a cat.
10. A method of maintaining or improving the health of a companion
animal comprising orally administering a strain of Bifidobacterium
globosum obtainable by isolation from resected and washed canine
gastrointestinal tract.
11. The method according to claim 10 wherein the strain is selected
for the ability to survive and colonise the gastrointestinal tract
of companion animals.
12. The method according to claim 11 wherein the strain has at
least 33% growth when cultured in the presence of 0.5% bile
salts.
13. The method according to claim 11 wherein the strain is able to
maintain viability following 1 hour at pH 2.5.
14. The method according to claim 10 wherein the strain has a
16s-23s intergenic polynucleotide sequence having at least 93%
homology with the sequence according to SEQ ID NO. 1.
15. The method according to claim 14, wherein said strain is
Bifidobacterium globosum strain NCIMB41198, or mutant or variant
thereof.
16. The method according to claim 15 wherein the mutant is a
natural, genetically modified or induced mutant.
17. The method according to claim 10 for the regulation of the
immune system of companion animals.
18. The method according to claim 17 which is a method of treating
or preventing autoimmune disease in said companion animal.
19. The method according to claim 17 which is a method of treating
or preventing inflammation in said companion animal.
20. The method according to claim 10 which is a method of
maintaining or improving the health of the skin and/or coat system
of said companion animal.
21. The method according to claim 20 which is a method of treating
or preventing atopic disease of the skin of said companion
animal.
22. The method according to claim 10 which is a method of
ameliorating or reducing the effects of aging in said companion
animal.
23. The method according to claim 10 which is a method of
preventing weight loss during and following infection in said
companion animal.
24. The method according to claim 10 which is a method of treating
or preventing gastrointestinal infection in said companion
animal.
25. The method according to claim 24 which is a method of improving
microbial ecology of said companion animal.
26. The method according to claim 25 wherein the level of
pathogenic bacteria in the faeces of said companion animal is
reduced.
27. The method according to claim 26 wherein said pathogenic
bacteria are selected from the group consisting of Clostridia,
Escherichia, Salmonella, and mixtures thereof.
28. The method according to claim 10 which is a method of treating
or preventing a urinary tract ailment in said companion animal.
29. The method according to claim 28 wherein said urinary tract
ailment comprises urinary tract infection.
30. The method according to claim 28 wherein said urinary tract
ailment comprises kidney stones.
31. The method according to claim 28 wherein said probiotic
Bifidobacteria globosum increase the degradation of oxalic acid in
vitro.
32. The method according to claim 10 wherein fiber digestion in
said companion animal is increased.
33. The method according to claim 10 wehrein stress levels in said
companion animal is decreased.
34. The method according to claim 33 wherein said stress levels are
measured by determining the level of stress hormones selected from
the group consisting of epinephrine, norepinephrine, dopamine,
cortisol, C-reactive protein and mixtures thereof.
35. The method according to claim 10 wherein the strain is fed to
an animal in any amount from 1.0E+04 to 1.0E+12 CFU/animal per
day.
36. The method according to claim 10 for the preparation of a
composition intended for maintaining or improving pet health.
37. The method according to claim 10 wherein the companion animal
is a dog.
38. The method according to claim 10 wherein the companion animal
is a cat.
39. A composition comprising a strain of Bifidobacterium globosum
obtainable by isolation from resected and washed canine
gastrointestinal tract having probiotic activity, and a
carrier.
40. The composition according to claim 39 wherein the strain is
selected for the ability to survive and colonise the
gastrointestinal tract of companion animals.
41. The composition according to claim 40 wherein the strain has at
least 33% growth when cultured in the presence of 0.5% bile
salts.
42. The composition according to claim 40 able to maintain
viability following 1 hour at pH 2.5.
43. The composition according to claim 39 wherein said strain has a
16s-23s intergenic polynucleotide sequence having at least 93%
homology with the sequence according to SEQ ID NO. 1.
44. The composition according to claim 43, wherein said strain is
Bifidobacterium globosum strain NCIMB 41198, or mutant thereof.
45. The composition according to claim 44 wherein the mutant is a
natural, genetically modified or induced mutant.
46. The composition according to claim 40 wherein the companion
animal is a dog.
47. The composition according to claim 40 wherein the companion
animal is a cat.
48. The composition according to claim 39 wherein said composition
is a companion animal food.
49. The composition according to claim 48 wherein the composition
is a cat food.
50. The composition according to claim 48 wherein the composition
is a dog food.
51. The composition according to claim 48 wherein the composition
is a wet animal food or a dry animal food.
52. The composition according to claim 51 wherein the composition
is in the form a kibble, a chew or a biscuit.
53. The composition according to claim 39 wherein said composition
is a companion animal food supplement.
54. The composition according to claim 53 wherein the composition
is in the form of a gravy.
55. The composition according to claim 53 wherein the composition
is in the form of a dairy product.
56. The composition according to claim 53 wherein the composition
is in the form of a capsule, a tablet, or a pill.
57. The composition according to claim 39 comprising at least
0.001% of from about 1.0E+04 CFU/g to about 1.0E+12 CFU/g of the
lactic acid bacteria.
58. The composition according to claim 39 wherein the
Bifidobacterium globosum bacteria are in the form of viable
cells.
59. The composition according to claim 39 wherein the lactic acid
bacteria are in the form of non-viable cells, or the constituent
active fractions thereof.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This Application claims the benefit of U.S. Provisional
Application No. 60/531,588 filed on Dec. 19, 2003.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of probiotic
micro-organisms, more specifically canine probiotic lactic acid
bacteria and methods of use.
BACKGROUND OF THE INVENTION
[0003] The defense mechanisms to protect the mammalian
gastrointestinal (GI) tract from colonisation by pathogenic
bacteria are highly complex. The GI tract of most mammals are
colonised by native microflora, and invasive pathogenic
micro-organisms. In a healthy individual, these competing
microflora are in a state of equilibrium. Modification of the
intestinal microflora equilibrium may lead to or prevent many GI
disorders, both in humans, and other mammalian species, such as
companion animals including cats, dogs and rabbits. The well being
of companion animals is closely related to their feeding and GI
health, and maintenance of the intestinal microflora equilibrium in
these animals may result in healthier pets.
[0004] The number and composition of the intestinal microflora tend
to be stable, although age and diet may modify it. Gastric acidity,
bile, intestinal peristalsis and local immunity are factors thought
to be important in the regulation of bacterial flora in the small
intestine of human beings and various other mammals. Often pet GI
disorders, including those found in canines and felines, are linked
to bacterial overgrowth and the production of enterotoxins by
pathogenic bacteria. These factors disrupt the intestinal
microflora equilibrium and can promote inflammation and aberrant
immune responses.
[0005] During the last few years, research has begun to highlight
some valuable strains of bacteria and their potential use as
probiotic agents. Probiotics are considered to be preparations of
bacteria, either viable or dead, their constituents such as
proteins or carbohydrates, or purified fractions of bacterial
ferments that promote mammalian health by preserving and promoting
the natural microflora in the GI tract, and reinforcing the normal
controls on aberrant immune responses. It is believed by some that
probiotic bacteria are more effective when derived from the
species, or a closely related species to the individual intended to
be treated. Therefore, there is a need for probiotic strains
derived from companion animals to be used for companion animals,
that are different to those derived from humans.
[0006] WO 01/90311 discloses probiotic micro-organisms isolated
from faecal samples obtained from cats and dogs having probiotic
activity. However, these bacteria were obtained from faecal
samples, and may not form part of the natural intestinal microflora
present in the upper portion of the GI tract.
[0007] Consequently, there is a need to provide strains of bacteria
obtainable by isolation from the natural intestinal microflora
present in the upper portion of the GI tract that are particularly
adapted for companion animals, and have been selected for their
probiotic properties and ability to survive processing, and to
incorporate these strains into compositions that are suitable for
their use.
SUMMARY OF THE INVENTION
[0008] According to the invention there is provided a strain of
lactic acid bacteria of the species Bifidobacteria globosum
obtainable by isolation from resected and washed canine
gastrointestinal tract having a probiotic activity in animals.
[0009] In a preferred embodiment, the lactic acid bacterial strain
is a Bifidobacteria globosum having a 16s-23s spacer region DNA
sequence having at least 93% homology to SEQ. ID NO. 1.
[0010] In a further preferred embodiment, the lactic acid bacterial
strain is Bifidobacteria globosum AHCF (NCIMB 41198).
[0011] Furthermore, the present invention is directed towards
providing uses of Bifidobacteria globosum bacteria obtainable by
isolation from resected and washed canine gastrointestinal tract
for maintaining and improving pet health, and compositions
comprising the lactic acid bacteria.
[0012] These and other features, aspects, and advantages of the
present invention will become evident to those skilled in the art
from a reading of the present disclosure.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 demonstrates the inhibition of the growth in vitro of
Salmonella typhimurium by the Bifidobacteria globosum bacteria of
the present invention according to methodology set out in example
2.
[0014] FIG. 2 demonstrates the inhibition of the growth in vitro of
Listeria monocytogenes by the Bifidobacteria globosum bacteria of
the present invention according to methodology set out in example
2.
[0015] FIG. 3 demonstrates the inhibition of the growth in vitro of
Listeria innocua by the Bifidobacteria globosum bacteria of the
present invention according to methodology set out in example
2.
[0016] FIG. 4 demonstrates the inhibition of the growth in vitro of
Escherichia coli 0157H45 by the Bifidobacteria globosum bacteria of
the present invention according to methodology set out in example
2.
[0017] FIG. 5 demonstrates the in vitro acid stability of the
Bifidobacteria globosum bacteria of the present invention according
to methodology set out in example 3.
[0018] FIG. 6 demonstrates the growth characteristics of the
Bifidobacteria globosum bacteria of the present invention in the
presence of 0.5%, 1% and 5% porcine bile salts.
[0019] FIG. 7 demonstrates the in vitro ability of the
Bifidobacteria globosum bacteria of the present invention to adhere
to HT-29 gut epithelial cells.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Sequences
[0021] SEQ. ID NO. 1-16s-23s intergenic spacer nucleotide sequence
from Bifidobacteria globosum AHCF (NCIMB 41198).
[0022] SEQ. ID NO. 2-16s-23s left PCR primer sequence for sequence
analysis.
[0023] SEQ. ID NO. 3-16s-23s right PCR primer sequence for sequence
analysis.
[0024] Bacterial Deposit Numbers
[0025] The table below indicates Bifidobacteria globosum strain
that is example of the present invention. The bacterial strain is
deposited with the National Collections of Industrial Food and
Marine Bacteria (NCIMB), Aberdeen, UK.
1 Strain Deposit Number 16 s-23 s Sequence Bifidobacteria globosum
AHCF NCIMB 41198 SEQ. ID NO. 1
[0026] All weights, measurements and concentrations herein are
measured at 25.degree. C. on the composition in its entirety,
unless otherwise specified.
[0027] Unless otherwise indicated, all percentages of compositions
referred to herein are weight percentages and all ratios are weight
ratios.
[0028] Unless otherwise indicated, all molecular weights are weight
average molecular weights.
[0029] Unless otherwise indicated, the content of all literature
sources referred to within this text are incorporated herein in
full by reference.
[0030] Except where specific examples of actual measured values are
presented, numerical values referred to herein should be considered
to be qualified by the word "about".
[0031] Within the following description, the abbreviation CFU
("colony-forming unit") designates the number of bacterial cells
revealed by microbiological counts on agar plates, as will be
commonly understood in the art.
[0032] As used herein, the term "mutants thereof" includes derived
bacterial strains having at least 93% homology, preferably at least
96% homology, more preferably 98% homology to the 16s-23s
intergenic spacer polynulceotide sequence of a referenced strain,
but otherwise comprising DNA mutations in other DNA sequences in
the bacterial genome.
[0033] As used herein, the term "DNA mutations" includes natural or
induced mutations comprising at least single base alterations
including deletions, insertions, transversions, and other DNA
modifications known to those skilled in the art, including genetic
modification introduced into a parent nucleotide or amino acid
sequence whilst maintaining at least 50% homology to the parent
sequence. Preferably, the sequence comprising the DNA mutation or
mutations has at least 60%, more preferably at least 75%, more
preferably still 85% homology with the parental sequence. As used
herein, sequence "homology" can be determined using standard
techniques known to those skilled in the art. For example, homology
may be determined using the on-line homology algorithm "BLAST"
program, publicly available at
http://www.ncbi.nlm.nih.gov/BLAST/.
[0034] As used herein "genetic modification" includes the
introduction of exogenous and/or endogenous DNA sequences into the
genome of an organism either by insertion into the genome of said
organism or by vectors including plasmid DNA or bacteriophage as
known by one skilled in the art, said DNA sequence being at least
two deoxyribonucleic acid bases in length.
[0035] As used herein, "companion animal" means a domestic animal.
Preferably, "companion animal" means a domestic canine, feline,
rabbit, ferret, horse, cow, or the like. More preferably,
"companion animal" means a domestic canine or feline.
[0036] Bifidobacteria globosum Strains
[0037] The first aspect of the present invention comprises a strain
of Bifidobacteria globosum obtainable by isolation from resected
and washed canine gastrointestinal tract having probiotic activity
in animals. Probiotics are micro-organisms, either viable or dead,
processed compositions of micro-organisms, their constituents such
as proteins or carbohydrates, or purified fractions of bacterial
ferments that beneficially affect a host. The general use of
probiotic bacteria is in the form of viable cells. However, it can
be extended to non-viable cells such as killed cultures or
compositions containing beneficial factors expressed by the
probiotic bacteria. This may include thermally killed
micro-organisms, or micro-organisms killed by exposure to altered
pH or subjected to pressure. For the purpose of the present
invention, "probiotics" is further intended to include the
metabolites generated by the micro-organisms of the present
invention during fermentation, if they are not separately
indicated. These metabolites may be released to the medium of
fermentation, or they may be stored within the micro-organism. As
used herein "probiotic" also includes bacteria, bacterial
homogenates, bacterial proteins, bacterial extracts, bacterial
ferment supernatants, and mixtures thereof, which perform
beneficial functions to the host animal when given at a therapeutic
dose.
[0038] It has been found that strains of Bifidobacteria globosum
obtainable by isolation directly from resected and washed GI tract
of mammals are adherent to the GI tract following feeding of viable
bacterial cells, and are also significantly immunomodulatory when
fed to animals in viable, non-viable or fractionated form. Without
being bound by theory, it is believed that the Bifidobacteria
globosum obtainable by isolation from resected and washed GI tract
closely associate with the gut mucosal tissues. Without further
being bound by theory, this is believed to result in the probiotic
Bifidobacteria globosum of the present invention generating
alternative host responses that result in its probiotic action. It
has been found that probiotic bacteria obtainable by isolation from
resected and washed GI tract can modulate the host's immune system
via direct interaction with the mucosal epithelium, and the host's
immune cells. This immunomodulation, in conjunction with the
traditional mechanism of action associated with probiotic bacteria,
i.e. the prevention of pathogen adherence to the gut by occlusion
and competition for nutrients, results in the Bifidobacteria
globosum of the present invention being highly efficacious as a
probiotic organism.
[0039] The Bifidobacteria globosum of the present invention,
obtainable by isolation from resected and washed canine GI tract,
have in vitro anti-microbial activity against a number of
pathogenic bacterial strains/species, as measured by zones of
inhibition or bacterial growth inhibition assays known to those
skilled in the art. Without being bound by theory, it is believed
that this in vitro anti-microbial activity is indicative of
potential probiotic activity in vivo in animals, preferably
companion animals such as canines and felines. The lactic acid
bacteria of the present invention preferably have in vitro
anti-microbial activity against Salmonella typhimurium, Listeria
monocytogenes, Listeria innocua or Eschericia coli, more preferably
a mixture of these strains, more preferably still, all of these
strains.
[0040] Without being bound by theory, it is believed that the
anti-microbial activity of the Bifidobacteria globosum bacteria of
the present invention may be the result of a number of different
actions by the Bifidobacteria globosum bacteria herein. It has
previously been suggested in the art that several strains of
bacteria isolated from faecal samples exert their probiotic effect
in the GI tract following oral consumption by preventing the
attachment of pathogenic organisms to the gut mucosa by occlusion.
This requires oral consumption of "live" or viable bacterial cells
in order for a colony of bacteria to be established in the gut.
However, it is believed that the Bifidobacteria globosum of the
present invention, obtainable by isolation from resected and washed
canine GI tract, whilst exerting some probiotic effect due to
occlusion if given in a viable form, may deliver a substantial
probiotic effect in either the viable or non-viable form due to the
production during fermentation in vitro of a substance or
substances that either inhibit the growth of or kill pathogenic
micro-organisms, and/or alter the host animal's immune competence.
This form of probiotic activity is desirable, as the bacteria of
the present invention can be given as either viable or non-viable
cultures or purified fermentation products and still deliver a
beneficial therapeutic effect to the host animal.
[0041] Preferably, the Bifidobacteria globosum bacteria of the
present invention are able to maintain viability following transit
through the GI tract. This is desirable in order for live cultures
of the bacteria to be taken orally, and for colonisation to occur
in the intestines and bowel following transit through the
oesophagus and stomach. Colonisation of the intestine and bowel by
the lactic acid bacteria of the present invention is desirable for
long-term probiotic benefits to be delivered to the host. Oral
dosing of non-viable cells or purified isolates thereof induces
temporary benefits, but as the bacteria are not viable, they are
not able to grow, and continuously deliver a probiotic effect in
situ. As a result this may require the host to be dosed regularly
in order to maintain the health benefits. In contrast, viable cells
that are able to survive gastric transit in the viable form, and
subsequently colonise by adhering to and proliferating on the gut
mucosa are able to deliver probiotic effects continuously in
situ.
[0042] Therefore, it is preferable that the lactic acid bacteria of
the present invention maintain viability after suspension in a
media having a pH of 2.5 for 1 hour. As used herein, "maintain
viability" means that at least 25% of the bacteria initially
suspended in the test media are viable using the plate count method
known to those skilled in the art. Preferably, "maintain viability"
means that at least 50% of the bacteria initially suspended are
viable. It is desirable for the lactic acid bacteria of the present
invention to maintain viability following exposure to low pH as
this mimics the exposure to gastric juices in the stomach and upper
intestine in vivo following oral consumption in animals.
[0043] Furthermore, it is preferable that the lactic acid bacteria
of the present invention have a growth of at least 33% when in the
presence of at least 0.5% porcine bile salts. Growth, as used
herein is described in further detail in example 3. More
preferably, the bacteria of the present invention have a growth of
at least 33% when in the presence of at least 1% porcine bile
salts. Without being bound by theory it is believed that the lactic
acid bacteria of the present invention, capable of growth in the
presence of at least 0.5% porcine bile salts, are able to survive
the conditions present in the intestine. This is thought to be a
result of the addition of porcine bile to the culture medium
mimicking the conditions of the intestine.
[0044] Further still, it is preferable that the Bifidobacteria
globosum bacteria of the present invention have significant
adhesion to gut epithelial cells in vitro. As used herein,
"significant adhesion" means at least 4% of the total number of
lactic acid bacteria co-incubated with the epithelial cells in
vitro adhere to the epithelial cells. More preferably, at least 6%
of bacterial cells co-incubated adhere to epithelial cells in
vitro. Without being bound by theory, it is believed that gut
epithelial cell adherence in vitro is indicative of the lactic acid
bacteria's ability to colonise the GI tract of an animal in
vivo.
[0045] The 16s-23s intergenic polynucelotide sequence is known to
those skilled in the art as the sequence of DNA in the bacterial
genome that can be used in order to identify different species and
strains of bacteria. This intergenic polynucelotide sequence can be
determined by the method detailed below in example 4.
[0046] In a preferred embodiment of the present invention, the
strain of Bifidobacteria globosum has a 16s-23s intergenic
polynucleotide sequence that has at least 93%, preferably at least
96%, more preferably at least 99% homology with the polynucleotide
sequence according to SEQ. ID NO. 1. More preferably, the strain of
lactic acid bacteria according to the present invention has a
16s-23s polynucelotide sequence according to SEQ. ID NO. 1. More
preferably still, the strain of lactic acid bacteria according to
the present invention is Bifidobacteria globosum strain NCIMB 41198
(AHCF), or a mutant thereof.
[0047] The strain of lactic acid bacteria of the genus
Bifidobacteria globosum obtainable by isolation from resected and
washed canine gastrointestinal tract can be used to deliver
probiotic benefit following oral consumption in animals, preferably
companion animals or humans. This probiotic benefit generally
maintains and improves the overall health of the animal.
Non-limiting elements of animal health and physiology that benefit,
either in therapeutically relieving the symptoms of, or disease
prevention by prophylaxis include inflammatory disorders,
immunodeficiency, inflammatory bowel disease, irritable bowel
syndrome, cancer (particularly those of the gastrointestinal and
immune systems), diarrhoeal disease, antibiotic associated
diarrhoea, appendicitis, autoimmune disorders, multiple sclerosis,
Alzheimer's disease, amyloidosis, rheumatoid arthritis, arthritis;
joint mobility, diabetes mellitus, insulin resistance, bacterial
infections, viral infections, fungal infections, periodontal
disease, urogenital disease, surgical associated trauma,
surgical-induced metastatic disease, sepsis, weight loss, weight
gain, excessive adipose tissue accumulation, anorexia, fever
control, cachexia, wound healing, ulcers, gut barrier infection,
allergy, asthma, respiratory disorders, circulatory disorders,
coronary heart disease, anaemia, disorders of the blood coagulation
system, renal disease, disorders of the central nervous system,
hepatic disease, ischaemia, nutritional disorders, osteoporosis,
endocrine disorders, and epidermal disorders. Preferred are
treatment of the gastrointestinal tract, including treatment or
prevention of diarrhoea; immune system regulation, preferably the
treatment or prevention of autoimmune disease and inflammation;
maintaining or improving the health of the skin and/or coat system,
preferably treating or preventing atopic disease of the skin;
ameliorating or reducing the effects of aging, including mental
awareness and activity levels; and preventing weight loss during
and following infection.
[0048] The treatment of the disorders disclosed above may be
measured using techniques known to those skilled in the art. For
example, inflammatory disorders including autoimmune disease and
inflammation may be detected and monitored using in vivo immune
function tests such as lymphocyte blastogenesis, natural killer
cell activity, antibody response to vaccines, delayed-type
hypersensitivity, and mixtures thereof. Such methods are briefly
described herein, but well known to those skilled in the art.
[0049] 1. Lymphocyte blastogenesis: This assay measures the
proliferative response in vitro of lymphocytes isolated from fresh
whole blood of test and control animals to various mitogens and is
a measure of overall T- and B-cell function. Briefly, peripheral
blood mononucleocytes (PBMC) are isolated from whole blood by
Ficoll-Hypaque density centrifugation methods known to those
skilled in the art. The isolated PBMCs are washed twice in RPMI
1640 cell media supplemented with HEPES, L-glutamine and
penicillin/streptomycin. The washed cells are resuspended in RPMI
1640, counted, and the cell density adjusted appropriately. The
2.times.10.sup.5 cells are exposed to a range of concentrations
(0.1.quadrature.g/ml to 100 .quadrature.g/ml) of various mitogens,
some examples of which include pokeweed mitogen (Gibco),
phytohaemagglutinin (Gibco) and conconavalin A (Sigma) in
triplicate for 72 hours at 37.degree. C. and 5% CO.sub.2 with 10%
foetal bovine serum (Sigma). At 54 hours the cells are pulsed with
1 .quadrature.Ci .sup.3H-thymidine, and the cells harvested and
scintillation counts read on a TopCount NXT at 72 hours.
[0050] 2. Natural killer cell activity: As described in U.S. Pat.
No. 6,310,090, this assay measures the in vitro effector activity
of natural killer cells isolated from fresh whole blood of test and
control animals. Natural killer cells are a component of the innate
immune function of a mammal. Canine thyroid adenocarcinoma cells
were used as target cells in assessing NK cell cytotoxic activity.
This cell line was previously shown to be susceptible to killing by
canine NK cell. Target cells were cultured in a T75 flask with 20
mL minimum essential medium (MEM; Sigma Chem. Co., St. Louis, Mo.)
supplemented with 10% fetal calf serum (FCS), 100 U/mL of
penicillin and 100 .quadrature.g/mL of streptomycin. When
confluent, target cells were trypsinized, washed 3 times and
resuspended to 5.times.10.sup.5 cells/mL in complete medium
(RPMI-1640+10% FCS+100 U/mL of penicillin+100 .quadrature.g/mL of
streptomycin). Triplicate 100 ..quadrature.L aliquots of the target
cells were pipetted into 96-well U-bottom plates (Costar,
Cambridge, Mass.) and incubated for 8 hours to allow cell
adherence. Lymphocytes (effector cells; 100 ..quadrature.L)
isolated by Ficoll-Hypaque separation (as described above) were
then added to the target cells to provide an effector/target cell
(E:T) ratio of 10:1. After 10 hours of incubation at 37.degree. C.,
20..quadrature.l of a substrate containing 5 ..quadrature.g of
3-(4,5-dimethylthiazol-2-yl- )-2,5-diphenyltetrazolium bromide
(MTT) was added. The mixture was incubated for 4 hours at
37.degree. C. after which the unmetabolized MTT was removed by
aspiration. The formazan crystals were dissolved by adding 200
.quadrature.L of 95% ethanol. Optical density was measured at 570
nm using a microplate reader. The percentage of NK cell-specific
lysis was calculated as follows:
Specific Cytotoxicity (%)=100.times.{1-[(OD of target cells and
effector cells-OD of effector cells)/(OD of target cells)]}
[0051] 3. Antibody response to vaccines: The test subjects are
given an array (up to 5) of vaccines after at least 12 weeks of
probiotic or control feeding. The vaccines may be a mixture of
novel and redundant vaccines. Non-limiting examples of vaccine
arrays that may be used include mixtures of vaccines prepared by
Fort Dodge Animal Health. Non-limiting examples of vaccines
suitable for use herein include Canine distemper, adenovirus,
coronavirus, parainfluenza, and parvovirus. The test subject's
vaccine history will determine the vaccines to be used. The
specific antibodies to the vaccines given are measured in blood for
3 weeks and the length and strength of response in control and
probiotic feeding groups compared.
[0052] 4. Delayed-type hypersensitivity: An in vivo, non-invasive
method of assessing immune system status. This test comprises an
intradermal injection of the polyclonal mitogen Phytohemmaglutinin
(PHA) in combination with sheep red blood cells a multivalent
vaccine, histamine (100 .mu.L of 0.0275 g/L Histamine Phosphate;
Greer, Lenoir, N.C.), or PBS (100 .mu.L of Phosphate Buffered
Saline, 8.5 g/L; Sigma). The immune response to the antigen is
recorded as skinfold thickness using calipers at time intervals of
0, 24, 48 and 72 hours post-injection.
[0053] An increase in skinfold thickness is indicative of a greater
hypersensitivity response that should be decreased by treatment
with the bacteria of the present invention.
[0054] Additional methods for determining the effect of the
Bifidobacteria bacteria of the present invention are described in
U.S. Pat. No. 6,133,323 and U.S. Pat. No. 6,310,090.
[0055] Furthermore, ameliorating the effects of age may be
determined using dual x-ray absorptometry or CT scan for measuring
body composition, including body fat mass, fat-free mass and bone
mineral content. Similarly, this method may be used to determine
anatomy changes such as weight loss or bone density in subjects
following infection.
[0056] The Bifidobacteria of the present invention may also be used
in a method for reducing stress levels in companion animals.
Concentrations of blood stress hormones including epinephrine,
norepinephrine, dopamine, cortisol and C-reactive protein may be
measured to determine stress levels and their reduction or
maintenance. These hormones are recognized biomarkers of stress and
can be readily measured using techniques known to those skilled in
the art.
[0057] Further still, maintenance or improvement of the health of
the skin and/or coat system of companion animals, including atopic
disease of the skin, may be measured using skin and coat
assessments conducted by two trained individuals. Examples of
criteria examined during such assessments include:
[0058] a) Shedding index: A shedding index is assigned to each test
subject by collecting hair produced during a standardized brushing
session. The hair is retained and weighed, and control and test
subjects compared.
[0059] b) Subjective skin/coat evaluations: Trained panelists
subjectively evaluate skin and coat condition by assessing
shedding, dander, shine, uniformity, softness and density.
[0060] c) Skin functional assessment: The barrier function of the
skin may be assessed by wiping the skin surface with an
acetone-soaked gauze. This technique effectively disrupts the skin
barrier by removing single cell layers and associated lipid
fractions of the stratum corneum. Barrier disruption is quantified
by measuring the increase in transepidermal water loss (TEWL) and
the degree of redness of the insulted site using methods known to
those skilled in the art. Redness (erythema) scores are obtained
using the previously described camera and lighting system. TEWL
readings and redness scores are obtained immediately before and
after disruption, and at five and 24-hour endpoints to assess the
protective and healing properties of skin.
[0061] The treatment or prevention of gastrointestinal infection,
including diarrhoea, in companion animals may be measured using
stool scores. Stools scores may be recorded daily according to the
following guidelines and control and test groups compared before
and after feeding with the bacteria according to the present
invention.
[0062] Score: 5 Extremely Dry
[0063] This stool is hard and does not stick to surfaces. Stool
will roll when pushed. No indentations are made when stool is
picked up. Stool is often defecated in groups of individual stools
instead of one complete unit. The stool maintains original shape
after collection.
[0064] Score: 4 Firm (Ideal Stool)
[0065] This stool is firm, well shaped, and cylindrical. This stool
does not break apart easily when picked up. This stool may leave
residue on surfaces and gloves. This stool is often defecated as
one unit. The stool maintains original shape after collection.
[0066] Score: 3 Soft, with Shape
[0067] This stool is soft, however there are definite shapes. This
stool will break apart easily and will definitely leave residue on
surfaces and gloves. The stool often loses original shape after
collection. This stool is often present with another score but can
comprise whole stool sample.
[0068] Score: 2 Soft, without Shape
[0069] This stool is soft and will have no cylindrical shape. The
shape often associated with a "2" is a "cow patty" shape. This
stool will lose the original shape when collected and will
definitely leave residue on surfaces and gloves. This stool score
is often present with another score but can comprise the whole
stool sample. This stool sample may spread over an area of several
inches.
[0070] Score: 1 Liquid
[0071] This stool score will always resemble liquid and there may
or may not be particulate matter present. This stool will often be
defecated in groups of piles instead of one complete unit. Mucous
is often present with this stool sample. This stool sample is very
difficult to collect and residue is always left on surfaces and
gloves. This stool sample may spread over an area of several
inches.
[0072] In addition, other observations are also recorded,
including: blood in stool; foreign object in stool; or mucous in
stool.
[0073] Furthermore, the treatment of gastrointestinal infection in
companion animals may comprise improving microbial ecology of
companion animals. Improving the microbial ecology of companion
animals preferably comprises reducing the levels of pathogenic
bacteria in the faeces of companion animals. The levels of
pathogenic bacteria present in the faeces of companion animals may
be enumerated using the standard plate count method known to those
skilled in the art. More preferably, the pathogenic bacteria are
selected from the group consisting of Clostridia, Escherichia,
Salmonella, bacteriodes and mixtures thereof. Non-limiting examples
of suitable strains of pathogenic bacteria include C. perfringens,
C. difficile, Eschericia coli, Salmonella typhimurium and mixtures
thereof.
[0074] The method of use of the bacteria of the present invention
may also include the treatment, either prophylactic or therapeutic
of the urinary tract of mammals, preferably companion animals.
Non-limiting examples of urinary tract treatment include treatment
or prevention of urinary tract infections, treatment or prevention
of kidney disease, including kidney stones, treatment or prevention
of bladder infections and the like. Without being bound by theory,
it is believed that the Bifidobacteria of the present invention are
useful in preventing these ailments as a result of their ability to
degrade oxalic acid, as demonstrated in vitro. Oxalic acid is a
by-product of urinary metabolism that can form insoluble
precipitates that result in kidney, bladder and other urinary tract
infections. By degrading oxalic acid, and therefore potentially
preventing its precipitation and build up in the urinary tract, the
bacteria of the present invention may treat and prevent infections
and other ailments of the urinary tract. Oxalic acid degradation
may be measured in vitro using the Oxalic acid test kit cat #
755699 commercially available from Boehringer
Mannheim/R-Biopharm.
[0075] The Bifidobacteria globosum of the present invention may be
used in a method for improving or maintaining the health of
companion animals comprising improving fiber digestion. Improving
fiber digestion is desirable as it promotes the growth of said
probiotic bacteria, as well as beneficial endogenous microflora,
which aid in the suppression of some potentially pathogenic
bacteria. In addition, a decrease in the amount of toxic
metabolites and detrimental enzymes that result from colonic
fermentation has been documented in humans (Tomomatsu, H. "Health
effects of oligosaccharides", (1994) Food Technol, 48, 61-65).
Fiber digestion may be determined using the method described in
Vickers et al. (2001), "Comparison of fermentation of selected
fructooligosaccharides and other fiber substrates by canine colonic
microflora", Am. J. Vet. Res. 61 (4), 609-615, with the exception
that instead of inoculating using diluted fecal samples each
experiment used pure cultures of the bacterial strains of
interest.
[0076] The method of use of the Bifidobacteria globosum bacteria of
the present invention typically involves oral consumption by the
animal. Oral consumption may take place as part of the normal
dietary intake, or as a supplement thereto. The oral consumption
typically occurs at least once a month, preferably at least once a
week, more preferably at least once per day. The Bifidobacteria
globosum bacteria of the present invention may be given to the
companion animal in a therapeutically effective amount to maintain
or improve the health of the animal, preferably a companion animal.
As used herein, the term "therapeutically effective amount" with
reference to the lactic acid bacteria, means that amount of the
bacteria sufficient to provide the desired effect or benefit to a
host animal in need of treatment, yet low enough to avoid adverse
effects such as toxicity, irritation, or allergic response,
commensurate with a reasonable benefit/risk ratio when used in the
manner of the present invention. The specific "therapeutically
effective amount" will vary with such factors as the particular
condition being treated, the physical condition of the user, the
duration of the treatment, the nature of concurrent therapy (if
any), the specific dosage form to be used, the carrier employed,
the solubility of the dose form, and the particular dosing
regimen.
[0077] Preferably, the lactic acid bacteria are given to the
companion animal at a dose of from 1.0E+04 to 1.0E+14 CFU per day,
more preferably from 1.0E+06 to 1.0E+12 CPU per day. The
composition preferably may contain at least 0.001% of from 1.0E+04
to 1.0E+12 CFU/g of the Bifidobacteria globosum obtainable by
isolation from resected and washed canine GI tract. The
Bifidobacteria globosum bacteria can be given to the animal in
either viable form, or as killed cells, or distillates, isolates or
other fractions of the fermentation products of the lactic acid
bacteria of the present invention, or any mixture thereof.
[0078] Preferably, the Bifidobacteria globosum bacteria, or a
purified or isolated fraction thereof, are used to prepare a
composition intended to maintain or improve the health of an
animal. As indicated above, the composition may be part of the
normal dietary intake, or a supplement. Where the composition
comprises part of the normal dietary intake, the composition may be
in the form of a dried animal food such as biscuits or kibbles, a
processed grain feed, a wet animal food, yogurts, gravies, chews,
treats and the like.
[0079] Such compositions may comprise further components. Other
components are beneficial for inclusion in the compositions used
herein, but are optional for purposes of the invention. For
example, food compositions are preferably nutritionally balanced.
In one embodiment, the food compositions may comprise, on a dry
matter basis, from about 20% to about 50% crude protein, preferably
from about 22% to about 40% crude protein, by weight of the food
composition. The crude protein material may comprise any material
having a protein content of at least about 15% by weight,
non-limiting examples of which include vegetable proteins such as
soybean, cotton seed, and peanut, animal proteins such as casein,
albumin, and meat tissue. Non-limiting examples of meat tissue
useful herein include fresh meat, and dried or rendered meals such
as fish meal, poultry meal, meat meal, bone meal and the like.
Other types of suitable crude protein sources include wheat gluten
or corn gluten, and proteins extracted from microbial sources such
as yeast.
[0080] Furthermore, the food compositions may comprise, on a dry
matter basis, from about 5% to about 35% fat, preferably from about
10% to about 30% fat, by weight of the food composition. Further
still, food compositions comprising the lactic acid bacteria of the
present invention may also comprise from about 4% to about 25%
total dietary fiber. The compositions may also comprise a multiple
starch source as described in WO99/51108.
[0081] The compositions of the present invention may further
comprise a source of carbohydrate. Grains or cereals such as rice,
corn, milo, sorghum, barley, alfalfa, wheat, and the like are
illustrative sources. In addition, the compositions may also
contain other materials such as dried whey and other dairy by
products.
[0082] The compositions comprising the bacteria of the present
invention may also comprise a prebiotic. "Prebiotic" includes
substances or compounds that are fermented by the intestinal flora
of the pet and hence promote the growth or development of lactic
acid bacteria in the gastro-intestinal tract of the pet at the
expense of pathogenic bacteria. The result of this fermentation is
a release of fatty acids, in particular short-chain fatty acids in
the colon. This has the effect of reducing the pH value in the
colon. Non-limiting examples of suitable prebiotics include
oligosaccharides, such as inulin and its hydrolysis products
commonly known as fructooligosaccharides, galacto-oligosaccarides,
xylo-oligosaccharides or oligo derivatives of starch. The
prebiotics may be provided in any suitable form. For example, the
prebiotic may be provided in the form of plant material which
contains the fiber. Suitable plant materials include asparagus,
artichokes, onions, wheat or chicory, or residues of these plant
materials. Alternatively, the prebiotic fiber may be provided as an
inulin extract, for example extracts from chicory are suitable.
Suitable inulin extracts may be obtained from Orafti SA of
Tirlemont 3300, Belgium under the trade mark "Raftiline". For
example, the inulin may be provided in the form of Raftiline (g) ST
which is a fine white powder which contains about 90 to about 94%
by weight of inulin, up to about 4% by weight of glucose and
fructose, and about 4 to 9% by weight of sucrose. Alternatively,
the fiber may be in the form of a fructooligosaccharide such as
obtained from Orafti SA of Tirlemont 3300, Belgium under the trade
mark "Raftilose". For example, the inulin may be provided in the
form of Raftilose (g) P95. Otherwise, the fructooligosaccharides
may be obtained by hydrolyzing inulin, by enzymatic methods, or by
using micro-organisms.
[0083] For dried pet foods a suitable process is extrusion cooking,
although baking and other suitable processes may be used. When
extrusion cooked, the dried pet food is usually provided in the
form of a kibble. If a prebiotic is used, the prebiotic may be
admixed with the other ingredients of the dried pet food prior to
processing. A suitable process is described in European patent
application No 0850569. If a probiotic micro-organism is used, the
organism is best coated onto or filled into the dried pet food. A
suitable process is described in European patent publication Number
EP 0 862 863.
[0084] For wet foods, the processes described in U.S. Pat. Nos.
4,781,939 and 5,132,137 may be used to produce simulated meat
products. Other procedures for producing chunk type products may
also be used; for example cooking in a steam oven. Alternatively,
loaf type products may be produced by emulsifying a suitable meat
material to produce a meat emulsion, adding a suitable gelling
agent, and heating the meat emulsion prior to filling into cans or
other containers. Typical wet food compositions may comprise from
about 5% to about 15% protein, from about 1% to about 10% fat, and
from about 1% to about 7% fiber. Non-limiting ingredients that may
be used in wet food compositions include chicken, turkey, beef,
whitefish, chicken broth, turkey broth, beef broth, chicken liver,
brewers rice, corn grits, fish meal, egg, beet pulp, chloride, flax
meal, lamb, beef by-products, chicken by-products and mixtures
thereof.
[0085] In another embodiment, supplement compositions such as
biscuits, chews, and other treats may comprise, on a dry matter
basis, from about 20% to about 60% protein, or from about 22% to
about 40% protein, by weight of the supplement composition. As
another example, the supplement compositions may comprise, on a dry
matter basis, from about 5% to about 35% fat, or from about 10% to
about 30% fat, by weight of the supplement composition. Food and
supplement compositions intended for use by canines or felines are
commonly known in the art.
[0086] The pet foods may contain other active agents such as long
chain fatty acids and zinc. Suitable long chain fatty acids include
alpha-linoleic acid, gamma linolenic acid, linoleic acid,
eicosapentanoic acid, and docosahexanoic acid. Fish oils are a
suitable source of eicosapentanoic acids and docosahexanoic
acid.
[0087] Borage oil, blackcurrent seed oil and evening primrose oil
are suitable sources of gamma linolenic acid. Safflower oils,
sunflower oils, corn oils and soy bean oils are suitable sources of
linoleic acid. These oils may also be used in the coating
substrates referred to above. Zinc may be provided in various
suitable forms, for example as zinc sulfate or zinc oxide. Further,
many ingredients commonly used in pet foods are sources of fatty
acids and zinc. It has been observed that the combination of
chicory, as a source of prebiotic, with a linoleic-acid rich oil,
such as soy bean oil, provides unexpected benefits, suggestive of a
synergistic effect.
[0088] Where the composition is in the form of a gravy, the
composition preferably comprises at least 10% of a broth, or stock,
non-limiting examples of which include vegetable beef, chicken or
ham stock. Typical gravy compositions may comprise from about 0.5%
to about 5% crude protein, from about 2% to about 5% crude fat, and
from about 1% to about 5% fiber.
[0089] Further non-limiting examples of supplements suitable for
use herein include powders, oil suspensions, milk-based suspensions
cheeses, and pills or capsules. Where the composition is in the
form of a pill, suitable binding agents are required to maintain
the pill in a solid, pressed form. Non-limiting examples of
suitable binding agents include the natural gums such as xanthan
gum, pectins, lecithins, alginates and others known to those
skilled in the art. Where the composition is in the form of a
capsule, the composition is preferably encapsulated using
technologies known to those skilled in the art. Non-limiting
examples of suitable encapsulation materials include polyvinyl
alcohol (PVA), polyvinylpyrrolidone (PVP), alginates, and gelatin.
Yogurt-based compositions may comprise from about 1% to about 5%
protein, from about 10% to about 20% carbohydrate, from about 1% to
about 5% fiber, from about 1% to about 5% fat and from about 50% to
about 90% liquid carrier such as milk.
EXAMPLES
[0090] The following examples are provided to illustrate the
invention and are not intended to limit the scope thereof in any
manner.
Example 1
Isolation of Bifidobacteria globosum Bacteria from Canine GI
Tracts
[0091] Canine intestinal samples were obtained from healthy dogs
presenting at the local veterinarians for owner initiated and
approved euthanasia. All animals were healthy and disease-free. The
colon, mid-colon, caecum and ileum of each dog were dissected in
order to expose the mucosa.
[0092] Supernatants were removed following agitation of the mucosal
tissue (vortexed for 1 minute) and following mechanical
homogenisation of the tissue. Each supernatant was plated on
Reinforced Clostridia Agar (RCA) or MRS plus 0.05% cysteine plus
mupirocin. These were incubated anaerobically, using the Anerocult
GasPak system, for 24 hours at 37.degree. C. Isolated colonies from
the plates were re-streaked onto either MRS or RCA and again grown
anaerobically under the same conditions. Isolated colonies were
re-streaked a further 4 times in order to purify a single strain.
Colony morphology and microscopic appearance were assessed.
Suitable isolates were tested for Gram reaction and catalase
activity. Identification of gram positive, catalase negative rods
was performed using API testing (API 50CHL, BioMerieux). Harvested
cells were washed twice with 0.05M phosphate buffer (pH 6.5) and
cysteine-HCl (500 mg/l) followed by sonication. Centrifugation
removed cellular debris. Supernatants were incubated with NaF (6
mg/ml) and Na iodoacetate (10 mg/ml) for 30 minutes at 37.degree.
C. The reaction was stopped by incubation with hydroxylamine HCl
(pH6.5) for 10 minutes at room temperature. Colour development was
monitored following the addition of HCl (4M), FeCl.sub.3.6H.sub.2O
(5% (w/v) in 0.1M HCl) and fructose-6-phosphate (Na salt).
Formation of acetyl phosphate from fructose-6-phosphate was
evidenced by the reddish colour formed by the ferric chelate of its
hydroxymate.
[0093] Fifty-eight (58) lactic acid bacterial strains were isolated
from resected and washed canine GI tract, of which six were found
to be of the genus Bifidobacteria, and one of the strain B.
globosum.
Example 2
Screening for Anti-Microbial Activity
[0094] The isolated Bifidobacteria globosum bacterial strains were
incubated anaerobically in TPY broth. 2.quadrature.1 of each
culture were spotted onto TPY agar plates and incubated
anaerobically overnight. Salmonella typhimurium, Listeria
monocytogenes, Listeria innocua and Eschericia coli 0157H45 were
pre-grown overnight and 100 .mu.l inoculated into molten agar (1%
v/v). This indicator culture was poured onto the surface of the
inoculated MRS or TPY plates. Following overnight incubation, zones
of inhibition around the probiotic colony were measured. All
experiments were performed in duplicate on three separate
occasions. In addition, incorporating the buffer 2%
betaglycerophosphate into the agar enabled assessment of the
contribution of acid production to the observed pathogen inhibition
in vitro.
[0095] The data presented in FIGS. 1, 2, 3 and 4 clearly
demonstrate that the Bifidobacteria globosum bacteria strains of
the present invention obtainable by isolation from resected and
washed canine GI tract have anti-microbial activity in vitro,
indicative of potential probiotic activity.
Example 3
In Vitro Measures of Survival and Colonisation
[0096] pH Tolerance
[0097] Bacterial cells were harvested from overnight cultures,
washed twice in phosphate buffer (pH 6.5) and resuspended in TPY
broth adjusted with 1M HCl to pH 2.5. The cells were incubated
anaerobically at 37.degree. C. and their survival measured at
intervals of 0, 30, 60, 120, 240 and 360 minutes using the plate
count method known to those skilled in the art.
[0098] FIG. 5 clearly demonstrates that five strains were resistant
to pH 2.5 over 1 hour. Table 2 summarises this data per strain.
2TABLE 2 Strain Conc. After 1 designation Starting Conc. hour
Viability (%) AHC A 1.50E+08 1.20E+08 80 AHC B 4.00E+07 5.50E+07
137 AHC C 1.10E+08 1.50E+08 136 AHC F 6.00E+08 6.00E+08 100 AHC 7
2.50E+07 4.50E+07 180
[0099] Bile Resistance
[0100] The bacterial strains were streaked onto TPY agar
supplemented with porcine bile (Sigma) at 0.5%, 1% and 5% (w/v).
Plates were incubated at 37.degree. C. under anaerobic conditions
and the growth recorded after 24 hours. Growth was compared with
control plates by an experienced observer, and the growth of
colonies described as:
[0101] Negative (0)--no growth;
[0102] +(1)--Hazy translucent growth (<33% control-plates with
0% bile);
[0103] ++(2)--Definite growth but not as good as controls (>33%
but <66%);
[0104] +++(3)--Growth equivalent to controls (>66%).
[0105] Once the growth of the colonies in the presence of bile
salts is compared with the controls, the growth descriptors are
given numerical values of 0, 1, 2 or 3 (-; +; ++, +++respectively),
and then expressed as a percentage, where 3 represents 100%.
[0106] FIG. 6 demonstrates that the Bifidobacteria of the present
invention clearly demonstrate a resistance to bile salts, being
able to grow and form colonies at a level of at least 33% when
exposed to 0.5% bile salts.
[0107] Gut Epithelial Cell Adhesion
[0108] The human epithelial cell line, HT-29, was used to assess
the adhesion properties of selected strains. Epithelial cells were
routinely cultured as a monolayer in 75 cm.sup.2 tissue culture
flasks at 37.degree. C. in a humidified atmosphere containing 5%
CO.sub.2 in Dulbecco's Minimal Essential Media (DMEM) containing
10% foetal calf serum (FCS), pen/strep, glutamine and fungizone.
For experimental purposes, the epithelial cells were seeded at a
concentration of 5.times.10.sup.5 cells/ml (3 mls total volume) per
well in 6 well culture plates (Sarstedt). Following incubation for
7 days, to allow differentiation, the epithelial monolayers were
washed with antibiotic-free medium containing 10% FCS. Bacterial
suspensions plus/in antibiotic-free DMEM were added to each well
and the cells incubated for 90 minutes at 37.degree. C. Following
incubation, the monolayers were washed three times with PBS. The
epithelial cells were lysed in deionised H.sub.2O and the number of
adherent bacteria enumerated using the plate count method known to
those skilled in the art. Adhesion was expressed as a percentage of
the number of bacteria initially plated.
[0109] As can be seen from FIG. 7, the Bifidobacteria globosum
strain deposited with the NCIMB under deposition numbers NCIMB
41198 adhere to HT-29 gut epithelial cells at levels of at least
4%.
Example 4
16s-23s Intergenic Polynucleotide Sequencing
[0110] Bifidobacteria globosum colonies were picked from an Agar
plate and resuspended in IX PCR buffer, heated at 96.degree. C. for
5 minutes, frozen at -70.degree. C. for 5-10 minutes, thawed and an
aliquot was added to a PCR eppendorf tube. PCR was performed using
the intergenic spacer (IGS) primers, IGS L:
5'-GCTGGATCACCTCCTTTC-3' and IGS R: 5'-CTGGTGCCAAGGCATCCA-3'. The
cycling conditions were 96.degree. C. for 1 min (1 cycle),
94.degree. C. for 30 sec, 53.degree. C. for 30 sec, 72.degree. C.
for 30 sec (28 cycles). The PCR reaction contained 5 .quadrature.l
of DNA, PCR buffer (Bioline, UK), 0.2 mM dNTPs (Roche, UK), 0.4
.quadrature.M IGS L and R primer (150 ng/50 .quadrature.l) (MWG
Biotech, Germany) and Bioline Taq polymerase (0.6 units). The PCR
reactions were performed on a Hybaid thermocycler. The PCR products
(8 .quadrature.l) were ran alongside a molecular weight marker
(.quadrature.X174 Hae III, Promega) on a 2% agarose EtBr stained
gel in TAE, to determine their IGS profile. Using the same primers
as above, the intergenic spacer (IGS) DNA was sequenced for the 2
canine Bifidobacteria globosum strains using methods known to those
skilled in the art.
[0111] Following sequencing, the obtained sequences for the four
deposited strains were compared with the on-line sequence database
"BLAST", available at http://www.ncbi.nlm.nih.gov/BLAST/ for
homology with other deposited bacterial 16s-23s sequences. The
closest match for AHCF was Bifidobacterium pseudolongum ATCC 25526,
having homology scores of 92%. However, the several differences
exist between these strains and between each other.
Example 5
Example Compositions
[0112] Examples 1 to 4 are examples of dried kibble compositions
comprising the probiotic Bifidobacteria globosum of the present
invention.
3 Percentage on a weight Basis Ingredient Ex. 1 Ex. 2 Ex. 3 Ex. 4
Cereal grains To 100 To 100 To 100 To 100 Poultry by-product meal
43.5 40 45 35 Poultry fat 1.28 1.02 1.16 1.35 Egg product 2.4 2.1
2.5 2.2 Chicken liver meal 1.0 1.0 1.0 1.0 Brewer's dried yeast 1.0
1.0 1.0 1.0 Monosodium phosphate 1.0 1.0 1.0 1.0 Calcium carbonate
0.8 0.8 0.8 0.8 Potassium chloride 0.6 0.6 0.6 0.6 Vitamins 0.4 0.4
0.4 0.4 Choline chloride 0.3 0.3 0.3 0.3 Minerals 0.3 0.3 0.3 0.3
DL-Methionine 0.1 0.1 0.1 0.1 Sodium Chloride 0.03 0.03 0.03 0.03
Probiotic (1 .times. 10.sup.10 cfu/g 1 0.5 0.1 0.6 NCIMB 41198 in
sunflower oil)
[0113] Examples 5 to 7 are examples of wet pet food compositions
comprising the probiotic Bifidobacteria globosum of the present
invention.
4 Percentage on a weight Basis Ingredient Ex. 5 Ex. 6 Ex. 7 Water
To 38 To 47 To 50 Poultry Liver To 25 To 20 To 15 Poultry Products
25 20 20 Brewers Rice 5 7 10 Egg Product 3 2.5 1.5 Poultry Fat 2.9
3.0 3.2 Chicken Stock 0.6 0.7 0.9 Taurine 0.1 0.1 0.1 Vitamins 0.05
0.1 0.1 Minerals 0.05 0.1 0.1 Probiotic (1 .times. 10.sup.10 cfu/g
4 5 6 NCIMB 41198)
[0114] Examples 8 to 10 are examples of yogurt supplement
compositions comprising the probiotic Bifidobacteria globosum of
the present invention.
5 Percentage on a weight Basis Ingredient Ex. 8 Ex. 9 Ex. 10 Milk
82.75 81.9 82.7 Sugar 12 12 10 Modified Starch 1.0 0.8 0.8
Prebiotic 0.25 0.3 0.5 Probiotic (1 .times. 10.sup.10 cfu/g 4 5 6
NCIMB 41198)
[0115] While particular embodiments of the present invention have
been illustrated and described, it would be obvious to those
skilled in the art that various other changes and modifications can
be made without departing from the spirit and scope of the
invention. It is therefore intended to cover in the appended claims
all such changes and modifications that are within the scope of
this invention.
Sequence CWU 1
1
3 1 556 DNA Bifidobacteria Globosum NCIMB 41198 misc_feature
(460)..(460) n is c, g, a or t. 1 acgatttgtg ggcgcacggt ggtgcgccga
tatggggatg cttccttttc ctggccgtct 60 ggccgggtgg cgtcccttgc
tggctgggaa aagggtcaag gcgcctgcgc ccgttgtggt 120 gtgggtggtg
gtggtgtggt gcatgctgtt gggttcccgg accgccaggc cccttgtcgg 180
gggtggtgtt ccgttcccgc cgtcctggcc gtgcccctgt gtggggtggg tgcctggggt
240 ggtgtggtgt ggtggtttga gaactggaga gtggacgcga gcatgaacgg
tgtgccctgt 300 ggggtgtgcc gggtgtgttc gtactgttga ttttgtcgaa
ccgttccatc cccgcctttt 360 gggttggggg tgttggattg tgttcgcgag
tgttttggta gagccgtcca cgcccgtgtg 420 ggtgtgggtg gtgtttagat
gatctgatta gttgtcgtan ggtgttccag tgcaagtggc 480 atgggcccgt
ggcccccttt tgcggggttg gtgggtttgt tgccatgggc gtatggtgga 540
atgcctgtnc accacg 556 2 18 DNA Artificial Chemically synthesized
nucleotide sequence 2 gctggatcac ctcctttc 18 3 18 DNA Artificial
Chemically synthesized nucleotide sequence 3 ctggtgccaa ggcatcca
18
* * * * *
References