Note: Descriptions are shown in the official language in which they were submitted.
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Probiotic Compounds Derived from Lactobacillus casei Strain KE01
FIELD OF THE INVENTION
[0002] The
present invention discloses probiotic Lactobacillus casei preparations.
Specifically, anti-infective, anti-diarrhea preparations derived from a newly
characterized strain of Lactobacillus casei designated KE01 are disclosed.
Related
methods for preparing the probiotic compounds using Lactobacillus casei strain
KE01
and related methods for using the KE01 probiotic compositions are also
disclosed.
BACKGROUND OF THE INVENTION
[0003] The newly recognized probiotic Lactobacillus casei strain described
herein
has been designated KE01. Previously, this same organism had been designated
Lactobacillus casei KE99 (see for example
A.S. Naidu, X. Xie, D.A. Leumer, S. Harrison, M.J. Burrill and E.A.
Fonda. 2001. Reduction of Sulfide, Ammonia Compounds and Adhesion Properties
of
Lactobacillus casei strain KE99 In Vitro. Cum Microbiol. 44(3): 196-205
(2002).
[0004]
Lactic acid bacteria (LAB) are indigenous microflora of mammalian
gastrointestinal tract that play an important role in the host microecology
and have
been credited with an impressive list of therapeutic and prophylactic
properties.
These therapeutic and prophylactic properties include, but not limited to the
maintenance of microbial ecology of the gut, physiological, immuno-modulatory
and
antimicrobial effects. Other LAB associated attributes include enzyme release
into the
intestinal lumen that act synergistically with LAB adhesion to alleviate
symptoms of
intestinal malabsorption. Furthermore, the LAB enzymes help regulate
intestinal pH
which results in increased aromatic amino acid degradation. [Fuller, R.
Probiotic
foods ¨ current use and future developments. IFI NR 3:23-26 (1993); Mitsuoka,
T.
Taxonomy and ecology of Bifidobacteria. Bifidobacteria Microflora 3:11 (1984);
Gibson, G.R. et al., Probiotics and intestinal infections, p.10-39. In R.
Fuller (ed.),
Probiotics 2: Applications and practical aspects. Chapman and Hall, London,
U.K
(1997); Naidu AS, et al., Probiotic spectra of lactic acid bacteria (LAB).
Crit Rev Food
1
CA 02520178 2005-09-16
WO 02/058712 PCT/US01/48707
Sci Nutr 39:3-126 (1999); Naidu, A.S., Clemens, R.A. Probiotics, p.431-462. In
A.S.
Naidu (ed.), Natural Food Antimicrobial Systems. CRC Press, Boca Raton, FL
(2000)]
[0005] Lactic acid bacteria have also demonstrated the ability to
significantly
reduce sulfide and ammonia containing compounds in animal fecal waste and thus
reduce the odor and toxicity associated with animal excrements. This ex vivo
LAB
application is becoming increasingly more important as agro-businesses expand
and
as communities continue their seemingly never ending encroachment into
previously
unoccupied rural areas. For example, LAB has been demonstrated to eliminate
offensive odors and reduce hydrogen sulfide production associated with
hatchery
waste when cockerel chicks and shell waste are blended with a mixture
containing
15% carbohydrate and LAB. Moreover, LAB compositions have demonstrated
efficacy in diminishing the Escherichia coli and Salmonella content of
hatchery waste
to negligible levels. Additionally, the odor and viscosity of poultry offals
such as
broiler-processing waste is significantly reduced by L. acidophilus mediated
lactic acid
fermentation. Furthermore, preparations containing LAB have been reported to
accelerate the breakdown of hard-to-degrade carbohydrates and decrease the
ammonia production by porcine cecal bacteria. Finally, ex vivo L.casei FG1 and
L.
plantarum FG10 silage fermentation significantly reduces ammonia levels by
inhibiting
urea-splitting organisms. [Deshmukh, A.C., Patterson, P.H. Preservation of
hatchery
wastes by lactic acid fermentation. 1. Laboratory scale fermentation. Poult
Sci
76:1212-1219 (1997);.Russell, S.M. et at., Lactic acid fermentation of broiler
processing waste: physical properties and chemical analyses. Poult Sci 71:765-
770
(1992); Tibbetts, G.W. et al., Poultry offal ensiled with Lactobacillus
acidophilus for
growing and finishing swine diets. J Anim Sci 64:182-190 (1987); Sakata, T. et
al.,
Probiotic preparations dose-dependently increase net production rates of
organic
acids and decrease that of ammonia by pig cecal bacteria in batch culture. Dig
Dis Sci
44:1485-1493 (1999); Cal, Y. et al., Effect of applying lactic acid bacteria
isolated from
forage crops on fermentation characteristics, aerobic deterioration of silage.
J Dairy
Sci 82:520-526 (1999); Modler, H.W. et al., Bifidobacteria and bifidogenic
factors. Can
Inst Food Sci Tech 23:29-41 (1990)1.
[0006] However, the greatest potential for LAB to improve life quality
for man and
domestic animals lies in LAB in vivo probiotic applications. In order for LAB
to exhibit
beneficial probiotic effects in vivo, the organisms must survive for extended
time
periods in the gastrointestinal tract. Therefore, it is critical that
probiotic LAB strains
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CA 02520178 2005-09-16
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be selected that possess qualities that prevent their rapid removal by gut
contraction.
Effective probiotic bacteria must able to survive gastric conditions and
colonize the
intestine, at least temporarily, by adhering to the intestinal epithelium.
Consequently,
LAB that demonstrate an enhanced ability to adhere to mucosal surfaces, and
therefore possess improved bacterial maintenance and prolonged
gastrointestinal
tract residence times, have a competitive advantage over LAB that do not.
[Salminen,
S. et al., Clinical uses of probiotics for stabilizing the gut mucosal
barrier: successful
strains and future challenges. Antonie Van Leeuwenhoek 70:347-358 (1996);
Conway,
P. Selection criteria for probiotic microorganisms. Asia Pacific J Clin Nutr
5:10-14
(1996); Havenaar, R. et al., Selection of strains for probiotic use, p.209-
224. In R.
Fuller (ed.), Probiotics, the scientific basis. Chapman and Hall, London, U.K.
(1992)].
[0007] Lactobacillus can successfully colonize the mammalian
gastrointestinal
tract through a number of different mechanisms. For example, some bacterial
species
bind to various sub-epithelial matrix proteins and specific receptors on the
intestinal
mucosa. Other species may adhere to mammalian intestinal cells via mechanisms
that involve different combinations of carbohydrate and protein factors on the
bacteria
and host eucaryotic cell surfaces. However, regardless of the mechanism(s) of
attachment, it is the ability of LAB to successfully colonize the human
gastrointestinal
tract that provides LAB with probiotic qualities. [Greene, J.D., Klaenhammer,
T.R.
Factors involved in adherence of lactobacilli to human Caco-2 cells. Appl
Environ
Microbiol 60:4487-4494 (1994); Sarem, F. et al., Comparison of the adherence
of
three Lactobacillus strains to Caco-2 and Int-407 human intestinal cell lines.
Lett Appl
Microbiol 22:439-442 (1996); Naidu, A.S., et al., Particle agglutination
assays for rapid
detection of fibronectin, fibriogen, and collagen receptors on Staphylococcus
aureus. J
Clin Microbiol 26:1549-1554 (1988); WadstrOm, T. et at, Surface properties of
lactobacilli isolated from the small intestines of pigs. J Appl Bacteriol
62:513-520
(1987); Bernet, M.F. et al., Lactobacillus acidophilus LA 1 binds to cultured
human
intestinal cell lines and inhibits cell attachment, invasion by entero-
virulent bacteria.
Gut 35:483-489 (1994); Jin, L.Z. et al., Effect of adherent Lactobacillus spp.
on in vitro
adherence of salmonellae to the intestinal epithelial cells of chicken. J Appl
Bacteriol
81:201-206 (1996); Reid, G. et al., Influence of lactobacilli on the adhesion
of
Staphylococcus aureus and Candida albicans to fibers and epithelial cells. J
Ind
Microbiol 15:248-253 (1995)].
3
CA 02520178 2005-09-16
WO 02/058712 PCT/US91./.48707.
[0008] Generally speaking probiotic bacteria exert their beneficial
effects by
displacing invasive or toxigenic pathogenic enteric bacteria (enteric
pathogens) from
the intestinal mucosa through a competitive binding process. Enteric pathogens
such
as, but not limited to enteropathogenic Escherichia coil (EPEC),
enterotoxigeneic E.
coli (ETEC), Salmonella enteriditis, Yersina pseudotuberculosis and Listeria
monocytogenes must be able to successively colonize an animal's intestinal
tract in
order to cause disease.
[0009] The mechanisms these organisms use to effectively colonize the
intestine
are varied. For example, ETEC bearing CFA/I or CFA/II adhesive factors
specifically
adhere to the brush border of the polarized epithelial human intestinal Caco-2
cells in
culture. S. typhimurium and EPEC adhere to the brush border of differentiated
human
intestinal epithelial Caco-2 cells in culture, whereas Y. pseudotuberculosis
and L.
monocytogenes bind to the periphery of undifferentiated Caco-2 cells.
[0010] Recently, heat-killed L. acidophilus preparations have been
proven to be
effective probiotic compositions. Heat-killed L. acidophilus preparations have
been
shown to displace known enteric pathogens from the lining of a test animal's
intestinal
wall in a dose dependent manner. Consequently, enteric pathogens were unable
to
colonize the animal's gastrointestinal tract thus preventing disease. For
example, L.
acidophilus (Lacteol strain) was found to inhibit this adhesion in a dose-
dependent
manner of E. coli strain B41 (ECB41). In other experiments live and heat-
killed L.
acidophilus strain LB successfully inhibited both Caco-2 cell association and
invasion
of enteric pathogens. An in yet another study, heat-killed L. acidophilus
strain LB was
shown to completely inhibit ETEC adhesion to Caco-2 cells. [Fourniat, J. et
al., Heat-
killed Lactobacillus acidophilus inhibits adhesion of Escherichia coli B41 to
HeLa cells.
Ann Rech Vet 23:361-370 (1992); Chauviere, G. et al., Competitive exclusion of
diarrheagenic Escherichia coli (ETEC) from human enterocyte-like Caco-2 cells
by
heat-killed Lactobacillus. FEMS Microbiol Lett 70:213-217 (1992)]. Coconnier,
M.H. et
al., Inhibition of adhesion of enteroinvasive pathogens to human intestinal
Caco-2
cells by Lactobacillus acidophilus strain LB decreases bacterial invasion.
FEMS
Microbiol Lett 110:299-305 (1993)].
[0011] As previously explained, probiotic compositions are generally
defined as
microbial dietary supplements that beneficially affect the host by improving
intestinal
microbial balance. The two major genera of microorganisms commonly associated
with probiotics include Lactobacillus sp and Bifidobacteria sp. The beneficial
effects
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attributed to probiotics include increased resistance to infectious diseases,
healthier
immune systems, reduction in irritable bowel syndrome, reductions in blood
pressure,
reduced serum cholesterol, milder allergies and tumor regression. However, in
spite
of recent scientific advances and the publication of limited in vivo and in
vitro
experimental evidence supporting the efficacy of probiotic compositions, the
major
studies reporting these and other benefits have relied on antidotal evidence.
[0012]
Examples of publications depending on antidotal and limited in vitro data
include United States patent numbers (USPAN) 3,957,974, 4,210,672, 4,314,995,
4,345,032, 4,579,734, 4,871,539, 4,879,238 and 5,292,362 (the "Hata" patents).
The
Hata patents disclose various subspecies of Lactobacillus spp and report
beneficial
probiotic-like effects. However, the Hata patents do not disclose or describe
a
Lactobacillus species that demonstrates avid binding to sub-epithelial
matrices and
competitive exclusion and microbial interference with bacterial enteric
pathogens.
[0013]
United States patent number 6,060,050 (the "'050 patent") discloses a
preparation consisting of probiotic microorganisms dispensed in a starch
containing
medium used to protect the organism during storage, serves to transport the
organism
to the large bowel and provides a growth promoting substrate. However, the
'050
patent does not provided data that directly demonstrates probiotic activity
against
enteric pathogens.
[0014] United States patent number 5,965,128 (the "1 28 patent") discloses
the
treatment of enteric diseases caused by E. coli 0157:H7 (enterohemorrhagic E.
coli).
However, the probiotic compositions of the '128 patent only includes non-
pathogenic
strains of E. coli, no Lactobacillus sp are disclosed. Consequently, while the
non-
pathogen enteric organisms of the '128 patent may afford the recipient
"probiotic-like"
protection against enterohemorrhagic E. coli, other beneficial qualities
associated with
Lactobacillus sp and Bifidobacteria sp are absent.
[0015]
United States patent numbers 4,839,281, 5,032,399 and 5,709,857 (the
"(857 patent") discloses several strains of Lactobacillus acidophilus that
adhere to
intestinal mucosal cells. Moreover, the '857 patent discloses a Lactobacillus
acidophilus strain that inhibits the growth of certain enteric pathogens.
However, the
'857 patent does not disclose L. casei strains having the proven beneficial
qualities
associated with the L. acidophilus cultures of the '857 patent.
[0016]
Therefore, there remains a need for new strains of Lactobacillus sp. that
exhibit probiotic activities.
Particularly, there is a need for more strains of
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Lactobacillus sp. that have the proven capacity to reduce enteric pathogen
diseases
and increase animal vitality and health.
SUMMARY OF THE INVENTION
[0017] It is an object of the present invention to provide a new strain
of
Lactobacillus sp that has demonstrated probiotic properties.
[0018] It is another object of the present invention to provide a new
strain of
Lactobacillus spp that has anti-enteric pathogen probiotic activity.
[0019] It is yet another object of the present invention to provide a
new strain of
Lactobacillus sp. that maintain animal health and vitality.
[0020] It is still another object of the present invention to provide
dietary
supplements and pharmaceutical preparations made from Lactobacillus sp. having
demonstrated probiotic properties.
[0021] The present invention fulfills these and other objects by
providing a new
strain of Lactobacillus casei designated KE01 that possesses scientifically
proven
probiotic properties including demonstrated in vivo anti-enteric pathogen
activity.
Moreover, the present invention provides dietary supplements and
pharmaceutical
preparations composed of L. casei strain KE01 that are formulated in a sugar
complex
composed of trehalose and fructooligosaccharides that provides long term
protection
to the organism and helps maintain its proven probiotic properties.
[0022] There is a need for new probiotic formulations that can be used to
treat and
prevent enteric-pathogen infections and help maintain the health and vitality
of
humans and livestock. Recently, the Federal Food and Drug Administration (FDA)
has intensified its campaign against the over prescription and clinical abuse
of
antibiotics. The excessive use of antibiotics has increased in the number of
human
and animal pathogens that are resistant to first-line antibiotics resulting in
an increase
in infections that do not respond to conventional antimicrobial therapies.
Moreover,
the prophylactic use of antibiotics in animal feed has resulted in an alarming
increase
in livestock intestinal infections resulting in diminished herd size and
animal weight
due to nutrient malabsorption. Consequently, the number of healthy animals
suitable
for human consumption has dropped, and those that do survive long enough to
reach
market have significantly lower weights and consequently reduced meat quality.
[0023] One means of preventing the rapid spread of drug resistant
enteric
pathogens in humans and livestock is to significantly reduce antibiotic use.
However,
the spread of communicable diseases including enteric infections is inevitable
due to
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over crowding of farms and cities. Consequently, before prophylactic
antibiotic use
can be completely discontinued a suitable antimicrobial alternative must be
available.
Recent studies have indicated that the use of foodstuffs and dietary
supplements
containing specific strains of probiotic microorganisms can help prevent, and
in many
cases actually cure, enteric pathogen diseases. However, many of the probiotic
formulas currently marketed rely on organisms including Lactobacillus spp and
Bifidobacteria sp (and other genera) that have not been subjected to
scientific scrutiny
using approved methods for assessing probiotic efficacy. Consequently, too
many of
the "probiotic" formulas currently available lack proven in vivo anti-enteric
pathogen
activity. Moreover, many of the clinically effective probiotic formulations
commercially
available are not stable upon storage and therefore do not deliver effective
amounts of
viable probiotic bacteria to the user. The present inventor has tested many
commercially available preparations and found microbial viability well below
stated
concentrations and in many cases the present inventor has found that these
commercial preparations did not contain any viable bacteria.
[0024] The present inventor has developed methods for preparing and
packaging
a new strain of L. casei, designated KE01. This new strain of L. casei was
originally
from a traditional fermented yoghurt-like Asian dairy product by the present
inventor.
Subsequently, the present inventor characterized the isolate and the strain
deposited
with the American Type Culture Collection (ATCC, MD, USA) on December 21,
2001. Lactobacillus
casei strain KE01 has been given the ATCC depository number PTA-3945.
Moreover, the
present inventor has developed preparations that maintain L. casei KE01
viability such
that a clinically effective dose of viable probiotic microorganisms reaches
the host.
[0025] The present invention provides a L. casei strain (KE01) that
interferes with
bacterial adherence (microbial interference) of enteric pathogens such as, but
not
limited to enteropathogenic and enterotoxigenic E. coli, Helicobacter pylori,
Campylobacter jejuni, S. typhimurium, and S. enteritidis to a variety of
mammalian cell
types. Moreover, the Lactobacillus of the present invention can also
competitively
exclude (competitive exclusion) these, and other bacterial pathogens, from
binding to
many mammalian cells. The beneficial properties associated with the novel
Lactobacillus strain of the present invention have resulted in improved
probiotic dietary
supplements that support general human and animal health. Moreover, the
present
invention can be used to provide prophylactics, therapeutics and palliatives
(collectively referred to herein as "probiotics") for conditions such as, but
not limited to,
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51432-13
traveler's diarrhea, gastrointestinal infections, hemolytic uremic syndrome,
and
gastric ulcers.
[0026] Additional novel features and qualities of this new L.
casei strain KE01
include, but are not limited to, L. casei KE01's ability to reduce sulfide
concentrations
by a factor exceeding 300 ppm within 48 hours when exposed to a growth medium
containing approximately 2000 ppm of sulfides and the demonstration of avid
binding
to sub-epithelial matrices including BiocoatTM (Collagen type-I, Collagen type
IV,
laminin, and fibronectin), MatrigelTM and Caco-2 cell monolayer. Most
importantly, a
reconstituted, freeze-dried preparation of the L. casei of the present
invention has
been shown to effectively detach collagen-adherent E. coli.
[0027] The methods used to maintain the viability of the L.
casei of the present
invention and preserve its E. coli displacement qualities include, but are not
limited
the use of the sugar trehalose and moisture and packaging the final
compositions in
oxygen proof polymer-lined (e.g. Mylar ) foil pouches.
[0027a] In another aspect, the invention provides Lactobacillus casei
strain
KE01 having ATCC accession number PTA-3945, wherein said Lactobacillus casei
strain KE01 is derived from a substantially pure culture.
[0027b] In another aspect, the invention provides a probiotic
composition
comprising Lactobacillus casei strain KE01 as described above and one or more
inert
or active ingredient.
[0027c] In another aspect, the invention provides a probiotic
composition
comprising: powdered Lactobacillus casei strain KE01 having ATCC accession
number PTA-3945 in the amount of from approximately 1 to 5 weight percent; a
disaccharide from approximately 25 to 95 weight percent; an oligosaccharide
from
approximately 0 to 10 weight percent; and a polysaccharide from approximately
0 to
50 weight percent.
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CA 02520178 2012-10-23
51432-13
[0027d] In another aspect, the invention provides a probiotic
composition
comprising: approximately 3 weight percent of powdered Lactobacillus casei
strain
KE01 having ATCC accession number PTA-3945 having approximately 105 to 1011
CFU per gram; approximately 62 weight percent trehalose; approximately 5
weight
percent fructo-oligosaccharide; and approximately 30 weight percent malt
dextrin.
[0027e] In another aspect, the invention provides use of the probiotic
preparation as described herein, for inhibiting enteric pathogen disease of an
animal.
[0028] These and other beneficial probiotic properties of the new
strain of
Lactobacillus will be further evident by the following, non-limiting, detailed
description
of the present invention.
BRIEF DESCRIPTION OF THE FIGURES
[0029] Figure 1 depicts the genomic fingerprint of Lactobacillus
casei strain
KE01 on 1% agarose gel compared to 12 different Lactobacillus type strains
based
on Randomly Amplified Polymorphic DNA (RAPD) assay.
[0030] Figure 2 depicts the phylogenic dendogram deduced from genomic
fingerprinting and the relatedness of Lactobacillus strain KE01 with other
species of
Lactobacillus type strains.
[0031] Figure 3 depicts the attachment of Lactobacillus casei strain
KE01 to
bovine intestinal epithelia.
[0032] Figure 4 depicts the colonization formed biofiim on the bovine
intestinal
epithelial mucosa.
[0033] Figure 5 graphically depicts the enhanced gut colonization and
fecal
shedding of lactobacilli and as a result of supplementing the animal's diet
with
Lactobacillus casei strain KE01 in accordance with the teachings of the
present
invention.
8a
CA 02520178 2005-09-16
. 51432-13
[0034]
Figure 6 graphically depicts the reduction of fecal coliform counts as a
result of supplementing the animal's diet with Lactobacillus casei strain KE01
in
accordance with the teachings of the present invention.
[0035]
Figure 7 graphically depicts the reduction in ammonia products in animal
feces as a result of supplementing the animal's diet with Lactobacillus casei
strain
KE01 in accordance with the teachings of the present invention.
[0036]
Figure 8 graphically depicts the reduction in sulfide products in animal
feces as a result of supplementing the animal's diet with Lactobacillus casei
strain
KE01 in accordance with the teachings of the present invention
[0037] Figure 9
graphically depicts the increase in weight gains in animals as a
result of supplementing the animal's diet with Lactobacillus casei strain KE01
in
accordance with the teachings of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0038] One
embodiment of the present invention is a dietary supplement
comprising approximately from 105 to 1011 colony forming units of viable
lactobacilli
per gram. In another embodiment of the present invention the dietary
supplement
comprises from approximate 105 to 1011 non-viable lactobacilli per gram. The
lactobacillus of the present invention is Lactobacillus casei strain KE01
having the
American Type Culture Collection (ATCC) accession number PTA-3945. A
colony forming unit (CFU) of Lactobacillus casei KE01 equates to one bacterial
cell.
Although generally only used when referring to viable bacteria, the term
colony
forming unit, or CFU will also be defined as a single non-viable bacterial
cell when
referring to embodiments of the present invention composed of non-viable
lactobacilli
compositions.
[0039] In one
embodiment of the present invention the dietary supplement
comprises a gelatin capsule filled with a dried lactobacillus composition. In
another
embodiment the dietary supplements is in the form of a liquid preparation. In
yet
another embodiment of the present invention the dietary supplement is in the
form of
an anal or vaginal suppository. When used as a suppository it may be formed
into a
convenient bolus and may contain non-toxic lubricants, stabilizers, waxes and
the like
to ease in the administration. In another embodiment of the present invention
the
lactobacillus dietary supplement may be compounded with foods such as, but not
limited to dairy products, grains, breads, meats, fruits, vegetables, rice and
the like.
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The form the lactobacillus dietary supplement of the present invention assumes
is not
important and is non-limiting.
[0040] Throughout this specification the present invention may be
referred to as a
probiotic composition, a lactobacillus containing composition, a dietary
supplement, a
freeze-dried powder or lactobacillus composition. All of these aforementioned
terms
mean a composition, regardless of form or the presence or absence of other
ingredients, that contains viable or non-viable Lactobacillus casei strain
KE01 having
ATCC accession number PTA-3945 or its genetic equivalent as determined using
the
methods detailed herein.
[0041] In one embodiment of the present invention the lactobacilli
composition is
freeze dried using standard methods known to those having ordinary skill in
the art of
food science. In another embodiment the lactobacilli composition of the
present
invention the lactobacilli composition is air-dried. In yet another embodiment
the
lactobacilli composition is a paste. In still another embodiment the
lactobacilli
composition of the present invention is a liquid.
[0042] The lactobacilli compositions of the present invention may or may
not be
compounded with additional ingredients. For example, in one embodiment of the
present invention the lactobacilli composition is mixed with a carbohydrate
selected
from the group consisting of trehalose, glucose, sucrose, fructose, maltose
and
combinations thereof. The carbohydrate(s) may comprise from approximately 50%
to
98% of the entire composition. In another embodiment proteins such as albumin
and/or whey may or may not be added to the lactobacilli compostions of the
present
invention.
[0043] The lactobacilli compositions of the present invention may be
taken orally
as a bolus in the form of a gelatin capsule, pressed tablet, or gel cap. In
another
embodiment the lactobacilli compositions of the present invention may be taken
orally
in the form of a liquid beverage. The liquid beverage may contain other
ingredients
such as, but not limited to flavor enhancers, sweeteners, viscosity enhancers
and
other food additives. The present invention may also be taken together with
other
foods either separately or compounded therewith.
[0044] In one embodiment of the present invention an animal is provided
with a
single dose containing from approximately 105 to 1011 lactobacilli per gram of
probiotic
composition. The total amount consumed will depend on the individual needs of
the
animal and the weight and size of the animal. The preferred dosage for any
given
CA 02520178 2005-09-16
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application can be easily determined by titration. Titration is accomplished
by
preparing a series of standard weight doses each containing from approximately
105
to 1011 lactobacilli per gram. A series of doses are administered beginning at
0.5
grams and continuing up to a logical endpoint determined by the size of the
animal
and the dose form. The appropriate dose is reached when the minimal amount of
lactobacilli composition required to achieve the desired results is
administered. The
appropriate dose is also known to those skilled in the art as an "effective
amount" of
the probiotic compositions of the present invention.
[0045] For example, if it is desired to reduce the symptoms associated
with
irritable bowel syndrome in an animal, one measured dose as described above is
administered daily, escalating the dose each successive day in 0.5 grams
increments
until symptoms subside. In one embodiment of the present invention the
preferred
dose is between approximately 103 to 108 viable lactobacilli per kilogram of
body
weight (the weight of animal recipient) per day. This equates to approximately
10
billion viable Lactobacillus casei strain KE01 per day for the average healthy
adult
human. By extrapolation, it is a simple matter to calculate the approximate
dose
appropriate for any animal of any weight. It is understood that this is a non-
limiting
example that can be varied as appropriate by persons having skill in the art
of
prescribing probiotic compositions or by using the titration method provided
above.
[0046] The probiotic compositions of the present invention can be
administered .to
any animal in need of thereof including, but not limited to mammals, birds,
reptiles and
fish. Typical applications include administering the probiotic compositions of
the
present invention to humans, horses, swine (pigs), cows, sheep, dogs, cats,
rabbits,
chickens, turkeys, pheasants, quail, parakeets, parrots, and other wild and
domesticated animals.
[0047] Specifically, the probiotic compositions of the present invention
can be
used to inhibit or treat enteric pathogen-associated diseases when
administered to an
animal in need thereof using the methods described in the present
specification.
Enteric pathogen diseases include those diseases caused by pathogens such as
diarrhea, irritable bowel syndrome and intestinal hemorrhages. Examples of
enteric
pathogens associated with these diseases include, but not limited to
enteropathogenic
Escherichia coil (EPEC), enterotoxigeneic E. coil (ETEC), Salmonella
enteriditis,
Yersina pseudotuberculosis and Listeria monocytogenes. It is theorized by the
present inventor, and not offered as a limitation, that the inhibition and
treatment of the
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enteric pathogen diseases is accomplished by the probiotic composition of the
present
invention through a competitive binding process. That is, the probiotic
lactobacilli of
the present invention compete with enteric pathogens for binding sites on the
intestinal
mucosa. Because the probiotic lactobacilli of the present invention have a
higher
affinity and avidity for these binding sites than the enteric pathogens, the
probiotic
lactobacilli of the present invention displace the enteric pathogens into the
intestinal
milieu where they are harmlessly flushed from the intestines by normal
metabolic
processes. In vivo examples of this above described competitive binding and
its
efficacy in inhibiting and treating enteric pathogen diseases is provided in
detailed
examples below and in the accompanying figures.
[0048] The
following technical discussion provides detailed teachings. In Section I
one of ordinary skill in the art is taught how to isolate and identify
probiotic lactobacilli,
specifically, the probiotic Lactobacillus of the present invention,
Lactobacillus casei
strain KE01. In Section II specific examples are provided that teach how to
prepare
the probiotic compositions of the present invention and the testing that was
conducted
to scientifically validate the probiotic qualities demonstrated by the
probiotic
compositions of the present invention. It is understood that these detailed
examples
are not intended as limitations.
I. Isolation and Characterization of Lactobacillus casei strain KE01
A. Isolation of Candidate Probiotic Bacteria
[0049] The
probiotic organism of the present invention was isolated from a
traditional fermented yogurt-like Asian dairy product. The screening process
was
limited to traditional fermented yogurt-like Asian dairy products with at
least a ten-year
history of safe human consumption. Probiotic bacteria isolation was performed
using
three selective microbiological media using methods known to those of ordinary
skill in
the art of microbiology.
Lactobacilli selective media included SL medium
supplemented with 0.05% cystein, Bifidobacterium spp. were selected for using
trypticase phytone yeast extract medium containing antibiotics; and for
Streptococcus
spp. were isolated using trypticase yeast extract cystein medium.
[0050] Candidate probiotic lactobacilli were be catalase negative, glucose
homo-
fermentative, Gram-positive non-spore forming rods demonstrating low pH,
gastric
acid and bile resistance. The lactobacilli isolates' inability to grow at pH
9.0 coupled
with their ability to grow on acetate containing media served to distinguish
them from
12
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Carnobacterium spp. A total of 81 isolates were classified as candidate
probiotic
lactobacilli based on these criteria and were further characterized with
respect to the
following criteria: i) resistance to low pancreatic juice; ii) adherence
ability to sub-
epithelial matrices such as BiocoatTM Matrigel (Becton Dickinson, Bedford, MA)
and to
cultured intestinal epithelial cells (Caco-2 cell line); iii) their ability to
competitively
exclude enterohemorrhagic E.coli serotype 0157:H7 adherent to collagen
matrices;
and iv) their capacity to reduce ammonia and sulfide containing compounds.
[001] After analyzing all 81-candidate probiotic lactobacilli, two
strains were
identified having all of the above-identified characteristics. These strains
were
designated strain KE97 and strain KE99 (re-designated KE01). Finally the
growth-
multiplication rate (generation time as determined by impedance detection
using
BioMerieuxTm Bactometer System), stability of strains in continuous culture,
freeze-
drying and revival characteristics, and aroma/flavor profiles were ascertained
for each
strain.
[0052] The isolated Lactobacillus casei strain KE01 organism is maintained
in a
substantially pure culture for use in preparing probiotic compositions of the
present
invention. As used herein "substantially pure culture" refers to a
bacteriological culture
that results in only one identifiable organism when cultured on solid or semi-
solid
microbiological culture media. It is understood that extremely low levels of
cells from
other bacterial species may be present; however, these cells are either non-
viable,
non-cultivable or below the threshold of detection using classical, non-genome-
based,
microbiological techniques. The term "non-genome-based" is intended to
excluded
such methods as PCR detection or similar methods used to detect microbial DNA
or
RNA.
[0053] Moreover, it is understood that whenever the Lactobacillus casei
strain
KE01 (or may be referred to as simply KE01) powders, pastes, gels, bolus or
other
probiotic compositions are made in accordance with the teachings of the
present
invention are described herein, that the Lactobacillus casei strain KE01 was
derived
from a substantially pure culture.
B. DNA fingerprinting by Random Amplified Polymorphic DNA (RAPD) assay.
[0054] The RAPD protocol uses PCR for generating a unique fingerprint
for
bacterial identification. The analysis by PCR can be performed in a rapid and
reliable
manner. Accordingly, the RAPD assay has been used for molecular identification
and
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WO 02/058712 PCT/US01/48707
finger printing of strain KE01. A total of 12 Lactobacillus spp. type strains
from the
ATCC collection were finger printed and compared with the KE01. For the DNA
fingerprinting all the lactobacillus strains were cultivated overnight in MRS
broth
(Difco). The Lactobacillus strains analyzed for DNA fingerprint are listed in
Table 1.
Table 1
Lactobacillus spp. SOURCE
Lactobacillus strain KE01 en-N-tech, Inc., California, USA
Lactobacillus acidophilus ATCC 4356 Human [L 917; IFO 13951; NCIB 8690]
Lactobacillus amylovorus ATCC 33620 Cattle waste-corn silage
Lactobacillus brevis ATCC 14869 Human feces
Lactobacillus casei subsp. casei ATCC Cheese [IAM 12473; Orland L-323]
393
Lactobacillus casei subsp. rhamnosus [BUCSAV 227; P.A. Hansen 300; NCDO
ATCC 7469 243; NCIB 6375; NCTC 6375; NRC 488]
Lactobacillus delbrueckii subsp. lactis Swiss cheese [DSM 20072; IAM 12476;
ATCC 12315 NCDO 1438]
Lactobacillus fermentum ATCC 14931 Fermented beets [NCIB 11840]
Lactobacillus helvaticus ATCC 15009 Swiss cheese
Lactobacillus paracasei subsp. paracasei [NCDO 151; R0941
ATCC 25302
Lactobacillus pentosus ATCC 8041 [DSM 20314; NCDO 363; NCIB 8026]
-Lactobacillus plantarum ATCC 14917 Pickled cabbage [IAM 124771]
Lactobacillus reuteri ATCC 23272 Human feces
DNA Extraction Method
[0055] DNA was extracted from the lactobacilli using the Wizard Genonnic
DNA
Purification Kit (Promega, WI, USA). Briefly, 1 mL of 24-h grown MRS broth
culture of
each lactobacillus spp. was harvested by centrifugation, cells were
resuspended in 50
mM EDTA and treated with 10mg/rni. lysozyme (Sigma, MO, USA) at 37 C for 60
min.
Lactobacilli cells were pelleted by centrifugation and supernatant was
removed. The
bacterial pellets were resuspended in the nuclei lysis solution and incubated
at 80 C
for 5 minutes. Cell suspension was allowed to cool to room temperature and
RNAse
was mixed into the solution. The suspension was incubated at 37 C for 60 min.
After
incubation, protein precipitation solution was added to the mixture. Solution
was mixed
on vortex and incubated on ice for 5 min. The mixture was centrifuged for 3
min at 15K
xg, supernatant was transferred to a fresh tube and the DNA was precipitated
with
isopropyl alcohol. The DNA was centrifuged and the isopropyl alcohol was
aspirated.
The DNA pellet was washed with 70% ethanol and harvested by centrifugation.
14
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WO 02/058712 PCT/US01/48707
Ethanol was removed and the pellet was allowed to dry. The DNA was resuspended
in
tris-EDTA buffer.
PCR Amplification of extracted DNA
[0056] One microliter of the extracted DNA was used in the PCR
reactions, which
were carried out on the iCycler (Bio-Rad, CA, USA) using a single arbitrary
nucleotide
sequence according to Cocconcelli, et al. (1995). A 2x PCR solution- Premix
Taq
(Takara, Shiga, Japan) was used for each reaction. Each reaction contained a
total
volume of 50 L, 1.25 units of Takara Ex Taq DNA Polymerase, 1X Buffer, 200
1,LM
dNTP Mix (2.5 mM each). Final concentration of the primer was 4 M, and the
primer
used for the amplification was 5'- AgCAgCgTgg- 3' (Operon Technologies, Inc.,
CA,
USA). The reaction mixtures with the template DNA were cycled through the
following
temperature profile: 1 cycle of 94 C for 5 min; 40 cycles of 94 C for 1 min;
29 C for 1
min; ramp to72 C 1.5 min and held at 72 C for 1.5 min; 1 cycle of 72 C for 2
min; and
held at 4 C. [Cocconcelli, PS et al., Development of RAPD protocol for typing
of
strains of lactic acid bacteria and entercocci. Lett. Appl. Microbiol. 21:376-
379 (1995)].
Gel electrophoresis
[0057] Aliquots of each RAPD amplified reaction (10 ILL) were analyzed
by 1%
(wt/vol) agarose gel electrophoresis in Tris-borate-EDTA buffer according to
Sambrook et al. (1989). Gels were run for 2 hr at 120V without cooling. The
DNA
molecular weight marker Hyperladder I (Bioline, Randolph, MA, USA) was used as
the
standard. After electrophoresis the gel was stained with ethidium bromide (5
p,g/mL)
for 10 min, washed for 5 min and visualized and analysed on a Fluor-S
MultiImager
(BioRad, CA, USA). [Sambrook, J., Fritsch, E.F., Maniatis, T. Molecular
Cloning ¨ A
Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory Press, New York
(1989)].
[0058] The RAPD assay using a single 10-mer primer produced distinct
banding
patterns of variable intensities and numbers of amplified products on 1%
agarose gel
with DNA samples of various lactobacillus reference strains and strain KE01.
Comparison of the different species fragments on the gel to the reference
Lactobacillus spp. was noted. The banding pattern with documentation as
depicted in
Figure 1 and TABLE 2 serves to uniquely identify strain KE01 and provided a
genomic
fingerprinting library. Based on the genomic fingerprinting a dendogram was
deduced
as shown in Figure 2. Ward's Cluster Method of Phylogenic Analysis was
applied. This
CA 02520178 2005-09-16
51432-13
method minimizes the Sum of Squares of any two clusters that can be formed at
each
step, creating clusters of small sizes. Based on the phylogenic analysis,
strain KE01
showed 13% homology with Lactobacillus helvaticus ATCC15009 and 55% homology
with Lactobacillus casei ssp. rhamnosus ATCC7469.
[0059] A pure
culture of Lactobacillus casei strain KE01 was deposited with the
American Type Culture Collection, Bethesda, MD, on December 21, 2001 which was
assigned the number ATCC PTA-3945.
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PCT/U501/48707
Table 2
r- 1
P Lane ' Band 1 ..,,,,,:. Lane Band
Number Number Mol. Wt. (bp) Identification .k,µ"\
Number Number Mol. Wt. (bp) Identification
2 1 4661.359 Strain KE-01 , 9 1 3588.07
Lactobacillus paracasel
2 2 2986.3579 = ______________
' 2 1271.414 sap. paracasel
ATCC#
2 3 2457.454% =
)-. 9 3 1107.771 25302
2 4 2255.342 ==:' 9 4 1027.939
2 5 1565.537 kik:. 9 5 932.388
2 6 1354.07 'r,',.0 __ ,, 6 892.472
2 7 991287 ''.:;.; a 7 636.154
2 8 904.264 .,'',='. t ,.,... ,,,,w,,,Mt.,,A0'
,PW,.....,R....õ'm ,M.,..,,..:\ lc' M -9lN, ,;,...0k -"k,
2 9 595.721.10 ' 1 6200.546 Lactobacillus
plantarum
..
113 2 4968.224 ATCC# 14917
3 1 2457.454 Lactobacillus acidophilus .4.,: 10 3 4263.326
3 2 1231.998 ATCC# 4356 '''".10 4 1969.14
3 3 1003.944 jg 10 5 1685.552
3 4 904284 10 6 1322.462
3 5 861.774''.:\'' 10 7 1231.998
3 6 617.813 ..?^... 10 8 1027.939
10 9 223284
4 1 4721.177
Lactobacillus amylovorus =.,,,.-,-., .,.., zA..õ.;,,..,%.., ,;,..:.N :,-....,
,....,,,,,,k,,,,a,,,aro'NA., ,...z>,,,,,,, ..,,-, .., ....,s,.\\ ,t= N',..
4 2 1969.14 ATCC# 33620 #.., 11 1 2592.843 Lactobacillus
casel ssp.
, iµ rhamnosus
4 3 1894.057 = 11 2 2324.117 ATCC#7469
4 4 1725.33 it.- 11
,.: 3 1685.552
4 5 1672.497 11 4 1430.79 ________
4 6 1271.414* 11
1312.091
4 7 1203.24 A. 11 6 1212.751
4 8 1023.9 ` 11 7 1011.88
4%ng.F.rMait, "M M -^W7µ0 it^".*,' õ= --V, - 1, 1s 8 780.732
-5 1 3611.082 Lactobacillus brevls ---..k
,..µakk = . '==:n.A .;\ ;.,,,,,;õ.., -'s .-, ..., ., Nt. e .,e -.:,õ5... .=*,
, %
s 2 3259.978 ATCC# 14869 AN 12 1 9035.455 Lactobacillus
pentosus
5 3 2534.42 s, 12 2 6787.29 ATCC# 8041
5 4 2087.693
_ .12 3 2405.288
s 5 1864.831 ; 12 4 2198.014
s 6 1271.414-t =
,,,,A 12 s 1453.497
s 7 1031.994 ; 12 6 1261.443
5 8 940.583 , , 12 7 1198.513
s 9 888.576 12 8 982.651
.< .=
5 10 651.854 ,. 12 9 667.942
k,,47kZ_W,ai,A,V,i.`,.;V....i '.wt.T..''I4W,-A,
6 1 2545.999 13
2.tr.7r;."lt:UVL.r.'aliQ14.\%.,>.
=== %
k., _______________________________________ 1
ctobacIllus case!
a 2-, ,
1281.464 Lactobacillus delbrueckil .,.._% 13 2 2374.522
ssp. easel ATCC# 393
a 3 1036.084 asp. lactis ATCC# 12315''-µ,.' 13 3 2189.899
6 4 940.583 :'.; 13 4 1436.433
6 s 892.472 , 13 5 1217.534
6 6 645.528 13 6 974.089
'4'.', VW WM:a M.01.,;W:W<WW;7;ltp"Vn7647,7k*.. 13 7 876.989
7 1 7490.875 Lactobacillus fermentum ,µ',,, 13 8 839.445
7 2 2545.999 ATCC# 14931.' 13
..*, 9 681.46
7 3 1732.051:.- = 13 10 583.785
7 4 1281.464 ck:\IV, ':,-
AW.,,,..,,6,, .=*A.74.4.I..W.gUiVii,*c
7 s 1036.064 ,...-, 14 1 2324.117
Lactobacillus reuteri
7 8 953.011 ..,= , 14 2 2114.743
ATCC# 23272
7 7 900.317 =As"' 14 3 1402.903
7 5 655.041 14 4 1184.442
,µ _
,...,\ = .,.. \st,ss.,...,-,õv4. -.\.''' .. ;.,,,,,,,r.,4xtik-->,õ.
:,.ita.,-.:.,..õ-...µ.* 14 5 944.708
8 1 14117.898 Lactobacillus helveticus -,. <;: 14 6 832.131
a 2 4936.649Al
ATCC# 15009 .s 14 7 639.264
8 3 3634.241
8 4 2776.318
8 5 2394.989
a 6 2105.687
a 7 1529.442
,-
8 s 1036.064
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WO 02/058712 PCT/US01/48707
II. Examples of the Present Invention
[0060] Bulk Lactobacilli strain KE01 as used in the following Examples
was mass-
produced using standard fermentation processes as known to those having
ordinary
skill in the art of fermentation microbiology. The purified Lactobacillus
strain KE01
was then used in the preparation of various delivery systems including fine
powder,
bolus capsule, gel, and dry animal feed-mixtures as follows:
EXAMPLE-1
Preparation of the Lactobacilli Strain KE01 Compositions of the Present
Invention
Preparation of freeze-dried KE01 into a fine Powder-form
[0061] A pure culture of Lactobacillus KE01 (starter) stored frozen at -
22 C, was
revived in a fermentation broth media containing proteins, vitamins, minerals
and
carbohydrate source. A seed culture was prepared in a fermentor attached to
NBS
1500L fermentation vessel. Microbial purity was monitored at defined time
points
(through log phase and end cycle) during the fermentation process. The
microbial
mass was harvested in a sanitized separator and the slurry of cell concentrate
was
freeze-dried after mixing with carriers containing a mixture of anhydrous
dextrose and
trehalose. Spray-dried Grade-A low-heat non-fat pasteurized milk was used as a
cryo-
protectant. The freeze-dried KE01 concentrate was milled to fine powder using
a
sanitized milling equipment. The quality of KE01 powder was assured for purity
and
viability prior to use.
Blending of KE01 powder into mixtures:
[0062] All operations are performed in a temperature (74-76 F) and humidity
(30-
40%) controlled environment. KE01 powder (approximately 10" CFU/g
lactobacilli)
and other ingredients listed in the composition below were introduced into a
closed
system for blending. Serial geometric dilution is done to ensure homogenous
distribution of all components. Mixing is done in a mass flow bin tumbling
system with
customized geometry to ensure bed splitting and cross flow. Once mixed, the
powders
are maintained in the closed system until packaged into 5 lb pails. The
blended
powder is filled into suitable containers (pails, unit dose packs, etc.) with
moisture
absorbing packs or inherent vapor barrier properties, and sealed with an
airtight lid.
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[0063] It is understood to those having ordinary skill in the art that
the term
"approximately" is used to provide a reasonable element of error inherent in
weighing
and manufacturing bulk materials. As used herein "approximately" will include
an
error factor of from 0.1 to 0.5%. Furthermore, it is understood by those
having
ordinary skill in the art that the amount of materials used to formulate a
final
composition must total 100% and cannot exceed 100%. All percentages are on a
weight percentage basis. For example, and not intended as a limitation, in one
embodiment of the present invention the probiotic composition made in
accordance
with the teachings of the present invention comprise from approximately 1 to
5% of
KE01 powder having from approximately 105 to 1011 CFU of KE01 per g and from
95
to 99% inert or active ingredients selected from the group consisting of, but
not limited
to, carbohydrates, lipids, polypeptides, fatty acids, phytochemicals and
combinations
thereof.
[0064] Carbohydrates that may be used in accordance with the teachings
of the
present invention include monosaccharides, disaccharides, oligosaccharides and
polysaccharides such as, but not limited to trehalose, maltose, sucrose,
dextrose,
lactose, inulin, ribose, malt dextrin and the like. In one embodiment of the
present
invention the disaccharide is trehalose dihydrate, the oligosaccharide is
fructo-
oligosaccharide and the polysaccharide is malt dextrin.
[0065] Suitable lipids include, but are not limited to soy bean oil, olive
oil, palm
kernel oil, peanut oil, walnut oil, cannola oil and the like. Suitable
polypeptides include
whey protein, egg albumin, gelatin, milk proteins, and other animal and plant
proteins.
Finally, phytochemicals as used herein include such compounds as polyphenols,
saponins, flavanoids, monoterpenes, allyl sulfides, lycopenes, carotenoids,
polyactetylenes, silymarin, glycyrrhizin catechins and othethers.
[0066] In one embodiment of the present invention the Lactobacillus
casei strain
KE01 probiotic has the following weight percentage of ingredients:
Table 3. Exemplary Blend
KE01 fine powder Approximately 1 to 5%
Disaccharides Approximately 25 to 95%
0 ligosaccharides Approximately 0 to 10%
Polysaccharides Approximately 0 to 50%
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Preparation of KE01 into Bolus Capsules
Freeze-dried KE01 fine powder (approximately from 105 to 1011 CFU/g
lactobacilli), trehalose dihydrate (Food Grade) and Dextrose (Food Grade) or
Malt
Dextrin (Grain Processing Corp.,. Muscatine, IA)) were thoroughly blended at
3%:
67%: 30%, respectively. The blended mixture was filled in gelatin capsules.
The
quality of the KE01 bolus capsules was tested for purity and viability prior
to use.
Table 4. Exemplary Bolus Blend
KE01 fine powder 3% ___
Trehalose dihydrate (Cargill Foods, Blair, NE) 67%
Malt Dextrin (Grain Processing Corp., Muscatine, IA) 30%
Total 100%
Preparation of KE01 in a Gel form
[0067] Freeze-dried KE01 fine powder (approximately from 105 to 1011
CFU/g
lactobacilli) was thoroughly blended with fructo-oligosaccharide
(Pharmaceutical
Grade) at 2.5% concentration. A non-GMO (Genetically Modified Organism)
extruded/expelled soybean oil was used as a primary carrier. The gel is mixed
thoroughly, samples were collected from the top, middle, and bottom of the
batch
using a sterilized spatula and analyzed for purity and viability of the KE01.
The KE01
gel was filled into 50/cc tubes for use.
Table 5. Exemplary Gel Blend
KE01 fine powder 3%
Trehalose dihydrate (Cargill Foods, Blair, NE) 62%
Fructooligosaccharide (Biomatrix, Minneapolis, MN) 5%
Malt Dextrin (Grain Processing Corp., Muscatine, IA) 30%
Total 100%
EXAM P LE-2
Cell Adhesion Studies
[0068] An in vitro Radio-Adhesion Assay (RAA) was performed to measure
the
attachment of Lactobacillus strain KE01, using the Cell EnvironmentsTM (Becton
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Dickinson, Bedford, Mass.) 24-well plates containing collagen (Cn) type-I, Cn
type-IV,
laminin, or fibronectin and using the Caco-2 cell monolayers.
Preparation of KE01 lactobacilli cells
[0069] A 50 I inoculunn of overnight culture of KE01 grown under
anaerobic
conditions in Bacto0 Lactobacilli MRS broth (Difco) was re-inoculated in 10-ml
of MRS
broth containing 3H-thymidine (20 pci). KE01 cells were grown under anaerobic
conditions at 37 C to exponential phase (approximately 7-hours) to allow
optimum
uptake and incorporation of 3H-thymidine into the bacterial DNA. 3H-thymidine
labeled
KE01 were harvested by centrifugation at 7,500 x g, washed and resuspended in
phosphate buffered saline (PBS, pH 7.2). The bacterial cell density was
optically
adjusted to 107 lactobacilli/ml at 600 nm.
Radio-Adhesion Assay (RAA)
[0070] For sub-epithelial matrix interaction assays, Cell Environments
TM 24-well
plates containing collagen (Cn) type-I, Cn type-IV, laminin, or fibronectin
were used for
testing attachment of KE01. Matrix components were applied as an even,
optically
clear coating covering a total surface area of 1.75 cm2. Biocoat Matrigel
matrix, a
soluble basement membrane extracted from Engelbreth-Holm-Swarm mouse tumor,
which when reconstituted at room temperature, forms a gel, was also used in
the
interaction studies. Two milliliters of KE01 suspension (2 x 107 lactobacilli)
were added
to each well containing matrix layer and incubated at room temperature. After
one
hour, the KE01 suspension was aspirated from the wells and discarded. One
milliliter
of trypsin type 1(110 enzyme units; Sigma Chemicals, St. Louis, MO.) was added
to
each well and allowed to hydrolyze for 30 min at room temperature to release
the
matrix protein layer. The trypsin hydrolysate was aspirated into a
scintillation vial and
an additional 1 ml from each well was homogenized (ScintigestTm; Fisher
Scientific,
Fair Lawn, NJ) for 10 min at room temperature. The homogenate was aspirated
into
the corresponding scintillation vial. A volume of 10-ml scintillation cocktail
(ScintiSafe TM Gel, Fisher Scientific) was dispensed into the vial and
thoroughly mixed.
After settling and clarification of the mixture, the radioactivity was
measured using a
liquid scintillation analyzer (Tri-Garb 2100 TRO, Packard Inc.).
[0071] For eucaryotic cell interaction assays, Caco-2, a colon carcinoma
cell line,
was grown to confluence as monolayers, in 24-well tissue culture plates, for
72-h in a
CO2 incubator using Eagle's minimal essential medium (supplemented with 1% non-
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WO 02/058712 PCT/US01/48707
essential amino acids and 10% fetal calf serum). After complete monolayers
were
obtained, each plate was washed twice with phosphate buffered saline (PBS, pH
7.2).
A 2-ml volume (approximately 2 x 107 bacteria) of 3H-thymidine labeled
lactobacilli
suspension was added to each well containing approximately 105 Caco-2 cells.
The
ratio between Caco-2 cells and lactobacilli was maintained at 1:200. After a 1-
hour
incubation at 37 C, the bacterial suspension was aspirated and the wells were
washed
with PBS. Each well was treated with trypsin type 1, tissue homogenizer and
measured for radioactivity as described above. Binding was expressed as
lactobacilli
bound per cm2 area of biological surface (for sub-epithelial matrix protein
interactions)
or per cell (for Caco-2 cell interactions).
[0072] The interaction of KE01 with different biomatrix layers was also
compared
with the interactions of three ATCC reference strains, L. casei ATCC393, L.
reuteri
ATCC23272, and L. rhamnosus ATCC 7469 in Table 6. To determine significance,
the binding data was analyzed with a 4x6 factorial analysis of variance (SAS
Inc.,
Raleigh, NC).
Table 6 =
BIOMATRIX KE01 L. Casei L. reuteri L. rhamnosus
LAYER ATCC 393 ATCC23272 ATCC7469
Collagen type-I 7.4 x 10b/cm4 3.5 x 10b/cm1 3.0 x *Ince
4.7 x 10b/cmi -
Collagen type-IV 5.0 x 10b/crre 8.7 x 104/are 6.3 x 104/cm'
9.9 x 104/crre -
Laminin 4.1 x 10b/cm2 6.4 x 104/cm2 5,3 x 104/cm2
7.2 x 104/cm2 -
Fibronectin 5.3 x 10b/cm2 6.4 x 104/cnn2 9.5 x 104/cm2
1.6 x 104/cm4
Matrigeim 6.6 x 10b/crne 2.1 x 10b/cm2 1.7 x 10/cm'
2.8 x 10b/crre
Caco-2 mondayer 108/cell 40/cell 26/cell 64/cell
[0073] KE01 demonstrated avid binding to collagen type-I, collagen type-IV,
laminin and fibronectin and Matrigel Tm layers in vitro. KE01 binding to Caco-
2 cell
monolayers was approximately 54% (108 lactobacilli/cell). All of the in vitro
interactions of KE01 with sub-epithelial matrix layers and Caco-2 cell
monolayers were
significantly higher than ATCC reference strains, L. casei ATCC393 (p<0.0001),
L.
reuteri ATCC23272 (p<0.0001), and L. rhamnosus ATCC7469 (p<0.0001).
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EXAMPLE-3
Enteric Pathogen Cell Adhesion Interference Studies
[0074] The efficacy of KE01 to inhibit the adhesion of enteric pathogens
(listed in
Table 7) to biomatrices and to detach complexes of pathogen-bionnatrices.
Table 7
ENTEROPATHOGEN SOURCE
Escherichia coil ATCC43888 Enterotoxigenic isolate that does not
produce
either Shiga-like toxin (SLT)-I or SLT-II
Escherichia coil ATCC43889 Fecal isolate from a patient with hemolytic
uremic syndrome that produces SLT-I1
Escherichia coil ATCC43890 Enterotoxigenic isolate that produces SLT-I
Escherichia coli ATCC43894 Fecal isolate from outbreak of hemorrhage
colitis that produces both SLT-I and SLT-II
Escherichia coil ATCC43895 Isolate from raw hamburger implicated in
hemolytic colitis outbreak, known to produce
SLT-I and SLI-II
Enterococcus faecalis ATCC7080 Isolated from meat involved in food poisoning
Campylobacter coil ATCC33559 Isolated from pig feces
Campylobacter jejuni ATCC29428 Isolated from diarrheic stool of child.
Salmonella enteritidis ATCC4931 Isolated from human gastroenteritis
Salmonella pullorum ATCC13036 Isolated from egg
Adhesion-detachment assay
[0075] A 10-0 inoculum of overnight culture of E.coli 0157:H7 grown in
tryptic soy
broth (TSB) or the other listed pathogens grown in appropriate broth media
were re-
inoculated in 3-ml of TSB or corresponding appropriate media containing 3H-
thymidine
(20 The binding of bacterial pathogens to Biocoat plates containing
collagen
(Cn) type-1, Cn type-1V, laminin, fibronectin and matrigel surfaces was
performed as
described in Example-1. To these Biocoat -bacterial pathogen complexes, a 2-
ml
volume of unlabeled KE01 suspension (2.0 x 107 lactobacilli/ml) was added to
each
well and incubated for 1-h at room temperature. KE01 lactobacilli suspension
was
aspirated from the well. Following steps of release of contents in the wells
and
measurement of radioactivity was done according to the protocol described in
Example-2. The difference in bacterial cell numbers in wells of adherent
pathogens
with and without KE01 treatment was expressed as logio detachment' values.
23
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WO 02/058712 PCT/US01/48707
Adhesion-Inhibition Assay
[0076] Binding of unlabeled KE01 to Biocoate plates containing collagen
(Cn)
type-I, Cn type-IV, laminin, fibronectin and matrigel surfaces was performed
as
described in Example-2. To these Biocoat -KE01 complex, a 2-ml volume of 3H-
thymidine labeled E.coli or other bacterial pathogen suspension (2.0 x 107
bacteria/ml)
was added to each well and incubated for 1-h at room temperature and the
suspension was aspirated. Following steps of release of contents in the wells
and
measurement of radioactivity was done according to the protocol described in
Example-2. BiocoatIO wells that were not treated with KE01 served as adhesion
controls for E.coli or other pathogens. The reduction in attachment of
bacterial
pathogens to biomatrices that were pretreated with KE01 was expressed as
'Logic)
inhibition' values.
[0077] The results of inhibitory and detachment effects of KE01 on
E.coli 0157:H7
(strain ATCC 43895) interactions with biomatrices and Matrigelni Biocoat
surfaces are
shown in Table 8.
Table 8
BIOMATRIX E.coli bound KE01 EFFECTS ON E.coli INTERACTIONS
LAYER (bacteria/cm2) E.colVcm2 E.coli/cm2
(log10 Inhibition) (log10 Detachment) _
Collagen type-I 6.2 x 105 . 3.3 x 103 (3.3-log) 6.7 x 102 (3.9-log)
Collagen type- 2.9x 105 1.8 x 103 (2.1-log) 1.1 x 103 (2.2-log)
Fibronectin 7.6 x 105 4.4 x 103 (2.3-log) 5.8 x 103 (2.1-log)
Lanninin 1.4 x 105 9.2 x 102 (2.2-log) 3.7 x 102 (2.7-log)
Matrigelw 9.1 x 106 8.2 x 103 (3.1-log) 6.4 x 103 (3.3-log)
[0078] KE01 demonstrated avid binding to all biosurfaces tested, however,
the
interaction was significantly higher with collagen type-I and Matrigelw. KE01
caused
>3-log inhibition of E.coli 0157:H7 attachment to collagen type-I and
MatrigelTM;
whereas >2-log inhibition with collagen type-IV, fibronectin and laminin
interactions.
The efficacy of detachment of adherent E.coli by KE01 was in the similar Log
magnitudes as inhibition values.
[0079] The spectrum of efficacy of KE01 to inhibit binding and to detach
adherent
bacteria was tested with several enteric pathogens listed in Table 9.
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Table 9
ENTEROPATHOGEN BINDING STRAIN KE01 EFFECT ON BINDING
_ (Bacteria/cm2) LogiD Inhibition Logi', Detachment
Escherichia coil ATCC43888 1.9 x 106 3.5-log 3.8-log
Escherichia coil ATCC43889 9.1 x 106 2.9-log 3.1-log
Escherichia coil ATCC43890 1.0 x 107 4.2-log 4.4-log
Escherichia coil ATCC43894 8.4 x 106 3.2-log 3.2-log
Enterococcus faecalis ATCC7080 2.6 x 106 2.8-log 3.7-log
Campylobacter coil ATCC33559 7.5 x 106 3.6-log 3.5-log
Campylobacter jejuni ATCC29428 8.1 x 106 - 3.3-log 3.5-log
Salmonella enteritidis ATCC4931 3.4 x 107 4.1-log 3.9-log
Salmonella pullorum ATCC13036 9.6 x 106 3.9-log 4.0-log
[0080] KE01 effectively inhibited the interactions of enteropathogens
ranging from
2.8-log (with Enterococcus faecalis) to 4.2-log (with Salmonella enteritidis).
Comparatively, the efficacy of KE01 to detach enteropathogens was higher
ranging
from 3.1-log (with enterohemorrhagic E.coli ATCC43889) to 4.4-log (with
enterohemorrhagic E. coil ATCC43890).
EXAMPLE-4
The Efficacy of KE01 Fine Powder to Detach Adherent Enteric Pathogens
[0081] The efficacy of KE01 fine powder to detach adherent enteric
pathogens
was tested in a cellular system using Caco-2 monolayers. The Caco-2 cell
adhesion
assay for enteropathogens was performed as described for KE01 in Example 2.
The
adhesion-detachment assay was performed as described for Biomatrix layers in
Example 3. However, in the above two protocols KE01 powder was used as a 1%
solution (1-g fine powder suspended and thoroughly vortexed in 100-ml
physiological
saline, pH 7.2). Treatment with physiological saline was used as control and
the value
was subtracted as background while interpreting the result. The data is shown
in
Table 10.
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PCT/US01/48707
Table 10
ENTEROPATHOGEN UNTREATED KE01-TREATED DETACHMENT
Bacteria/well Bacteria/well Logio
(%)
(iceII) (/cell)
Escherichia coli ATCC43888 1.1 x 106 (15) 7.3 x 104 (0.97) 1.3-
log (93.5%)
Escherichia coil ATCC43889 5.1 x 106(68) 2.2 x 104(0.29) 2.3-
log (99.6%)
Escherichia coli ATCC43890 5.7 x 106(76) 8.3 x 103(0.11) 2.7-
log (99.8%)
Escherichia coli ATCC43894 4.8 x 106(64) 1.9 x 104 (0.25) 2.3-
log (99.6%)
Escherichia coli ATCC43895 6.2 x 106 (83) 3.8 x 104 (0.51) 2.2-
log (99.4%)
Enterococcus faecalis ATCC7080 1.9 x 106 (22) 5.5 x 104 (0.73) 1.6-
log (96.7%)
Campylobacter coli ATCC33559 4.3 x 106 (57) 7.1 x 104 (0.95) 1.7-
log (98.3%)
Campylobacter jejuni ATCC29428 4.1 x 106 (55) 6.5 x 104 (0.87) 1.8-
log (98.4%)
Salmonella enteritidis ATCC4931 6.6 x 106 (88) 8.3 x 103 (0.11) 2.8-
log (99.9%)
,
Salmonella pullorum ATCC13036 6.7 x 106 (89) 7.9 x 103 (0.10) 3.1-
log (99.9%)
[0082] The Caco-2 cell adhesion profiles of enteropathogens were ranging
from
1.1 x 106 (15 bacteria per Caco-2 cell with enterohennorrhagic E. coli
ATCC43888) to
6.7 x 103 (89 bacteria per Caco-2 cell with Salmonella pullorum ATCC13036).
KE01
has effectively dissociated the Caco-2 cell adherent enteropathogen complexes
with
the detachment efficacy ranging from 1.3-log (93.5%) to 3.1-log (99.9%) for
E.coli
ATCC43888 and Salmonella pullorum, respectively.
EXAMPLE-5
Demonstration of the Ability of Fresh and Freeze-dried KEO1Preparations to
Adhere and Colonize the Epithelial Mucosa of the Bovine Intestinal Tract.
Bovine intestinal adhesion assay:
[0083] Intestine samples were obtained from freshly slaughtered animals
and
transported to the laboratory in refrigerated conditions. Intestines were cut-
open and
epithelial mucosa of the lumen was gently washed to remove fecal debris. Two
different preparations of KE01 was inoculated on a 1 inch2 mucosal surface, A)
3H-
thymidine labeled KE01 described in Example 2 (0.1 ml inoculum containing
approximately 107 lactobacilli); and B) KE01 powder blend mixture described in
Example 1 (homogenously suspended in physiological saline and diluted, 0.1 ml
inoculum containing approximately 107 lactobacilli). After 2-h incubation, the
area of
inoculum was gently washed three times with physiological saline.
[0084] Sample Preparation-A, was examined for KE01 attachment. The area of
inoculum was excised, digested with tissue homogenizer, amplified with liquid
scintillation fluid and the radioactivity was measured according to the Radio-
Adhesion
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WO 02/058712 PCTfUS01/48707
assay as described in Example-2. KE01 demonstrated avid binding to bovine
intestinal
epithelial mucosa i.e. approximately 2.5 x 106 lactobacilli/inch2.
[0085] Sample Preparation-B, was evaluated for KE01 colonization by
scanning
electron microscopy (SEM). Specimens were treated with 2% 0s04 for 30-60 min
and
rinsed with water. Specimens were treated with 50%, 70% and 95% ethanol, each
for
5 min, followed by 2x10 min rinses with 100% ethanol. After critical point
drying with
liquid carbon dioxide, specimens were mounted and sputter coated with gold
palladium. The biopsy specimens were examined using a JEOL6300 scanning
electron microscope. The microscopic observations are shown in Figures 3 & 4.
EXAMPLE-6
In vivo probiotic effects of KE01 containing animal feed-supplements
administered to
piglets. Demonstration of intestinal colonization and fecal shedding of KE01;
ability of
KE01 to decrease porcine fecal odor (reduction of fecal sulfide and ammonia
content);
and KE01 contribution to weight gains of the animal.
Feed-supplement Dosage
[0086] Feed-supplement dosages were prepared in a gel-form as described
in
Example-1. Briefly, freeze-dried KE01 fine powder (approximately from 105 to
1011
CFU/g lactobacilli) was thoroughly blended with fructo-oligosaccharide (FOS)
at 2.5%
concentration. A non-GMO (Genetically Modified Organism) extruded/expelled
soybean oil was used as a primary carrier. The gel is mixed thoroughly,
samples were
collected from the top, middle, and bottom of the batch using a sterilized
spatula and
analyzed for purity and viability of the KE01. The KE01 gel was filled into
50/cc tubes
for use. Control feed dosages containing carrier alone, carrier blended with
KE01 cells
and carrier blended with KE01 cells and FOS, were also prepared.
Animal Feed-supplement Trial
[0087] The study included a total of 20 pigs (between 6-8 week old)
weighing in
the range of 40-60 lbs. These animals were divided into four Groups, each
comprising
of 5 animals, categorized as the following:
[0088] Group-1 (Control): Animals fed with 10cc of gel containing primary
carrier
only.
[0089] Group-2: Animals fed daily with 10cc of gel containing only 2.5%
FOS
blended with the primary carrier.
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WO 02/058712 PCT/US01/48707
[0090] Group-3: Animals fed daily with 10cc of gel containing about 1010
cfu/cc of
strain KE01 cells only, blended with the primary carrier.
[0091] Group-4: Animals fed daily with 10cc of gel containing a
combination of
. 101 cfu/cc of strain KE01 cells and 2.5% FOS, blended with the primary
carrier.
[0092] Animals were separated by group and contained in isolated penns and
were regularly fed with high-protein porcine diet. The four groups of animals
were fed
daily with 10cc of their respective category of dietary-supplement dosage. The
experiment was conducted for a duration of 6 weeks. Fecal analysis and body
weight
measurements were regularly performed every week throughout the feed-
supplement
trial.
Feces collection
[0093] Fecal samples (approximately 20 g per animal) were collected in
sterile
50cc polystyrene tubes. Samples were diluted at 1:1 w/v ratio in normal
saline,
thoroughly homogenized on a vortex blender, and centrifuged at 2K xg for 5
min. The
supernatant was carefully decanted and subjected to microbiological and
chemical
analysis.
[0094] Fecal samples were collected one day prior to the actual feed-
supplement
trial to obtain a baseline microbiological count (fecal coliforms and fecal
lactobacilli),
and a baseline level of fecal ammonia and fecal sulfide content.
Microbiolooical analysis of feces
[0095] The fecal solution (1:1 w/v decanted supernatant) was serially
diluted by
10-fold in normal saline. Selected dilutions were plated in duplicate on MRS
agar
(Difco) for total lactobacilli counts, and on MacConkey Agar (Difco) for total
coliforms
counts, using an Autoplate 4000 device (Spiral Biotech, Norwood, MA). The
plating
was performed in a exponential log dilution setting on the spiral autoplater.
Agar plates
were incubated at 37 C for MacConkey agar and 32 C for MRS agar for 24 to 48
hrs,
respectively. The total colony counts were estimated using an automated infra-
red Q-
count device (Spiral Biotech, Norwood, MA). Data were expressed as bacteria
per gm
of feces and the results were shown for lactobacilli counts in Figure 5 and
fecal
coliforms counts in Figure 6.
[0096] . Animals administered with feed-supplements containing KE01 and
combination of KE01 plus FOS showed about 2.5-log increase in fecal shedding
of
lactobacilli compared to control groups, indicating proliferation and
colonization of
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WO 02/058712 PCT/US01/48707
strain KE01 in the porcine gastrointestinal tract as shown in Figure 5. On the
otherhand, fecal coliforms counts decreased by about 2.5-log in KE01 and
KE01+FOS
fed animals compared to control animals indicating an effective competitive
exclusion
by strain KE01 in the porcine gut as shown in Figure 6.
Sulfide and ammonia analysis of feces
[0097] Sulfide and ammonia levels in the feces were measured with ion-
selective
electrodes using a MP230 pH/Ion meter (Mettler Toledo, Columbus, Ohio).
[0098] For standardization of a sulfide ion-selective electrode, a 75-ml
dilute
sulfide standard (10 ppm) solution was added under gentle stirring, to 25-ml
sulfide
antioxidant buffer. The electrode potential (E1) of the solution was measured
using a
DX232 silver selective electrode combined with a DX200 reference electrode.
The E2
was measured separately with 75-ml sulfide (100 ppm) solution and 25-ml
sulfide
antioxidant buffer. The differences between E1 and E2 constituted the slope
for the
sulfide electrode. For sample measurement, a 100-ml of standard (100 ppm)
silver
solution was mixed with 2-ml of bromide ISA (ionic strength adjustment) buffer
and the
Ei value was measured. The E2 value was measured by mixing a 10-ml of fecal
solution to the above solution. The difference between Ei and E2 was
considered AE.
Based on the standard slope and AE, the concentration of sulfide in the feces
was
estimated according to the concentration ratio (Q) chart using the equation, C
= Cs x Q
(C = sulfide conc. in feces; Cs = known sulfide conc. i.e. 100 ppm standard;
and Q =
concentration ratio).
[0099] For standardization of the ammonia ion-selective electrode, a 1-
ml
ammonia standard (1000 ppm) solution was added under gentle stirring, to100-ml
distilled water premixed with 2-ml ammonia pH/ISA (ionic strength adjustment)
buffer.
The electrode potential (Ei ) of the solution was measured using ammonia DX217
electrode. The solution was then spiked with 10-ml ammonia (1000 ppm) standard
and the change in the electrode potential (E2) was measured. The difference
between
E1 and E2 constituted the slope for the ammonia electrode. For sample
measurement,
a 10-ml of fecal solution was added, under gentle stirring to 90-ml distilled
water
premixed with 2-ml ammonia pH/ISA buffer and the Ei was measured. The fecal
solution was spiked with ammonia standard and the E2 was measured. The
concentration of ammonia in the feces was estimated using the equation as
above.
[0100jAnimals administered feed-supplement containing KE01 and KE01 plus FOS
demonstrated a marked reduction of ammonia by about 35 ppm/gm and of sulfide
by
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WO 02/058712 PCT/US01/48707
about 375 ppm/gm compared to control group of animals as shown in Figures 7 &
8.
Also the feces from animals fed with KE01 and KE01 plus FOS are rancid due to
lactic
acid production by the proliferating probiotic KE01 lactobacilli.
Animal Body-weioht Gain Measurements
[0101]Animals were weighed one day prior to the actual feed-supplement trial
to
obtain baseline measurements for body weight gains. The body weights of
animals
from all the four groups were measured once every week though out the 6-week
feed
trial. At the conclusion of the experiment, animals fed with KE01 showed a
body
weight gain of about 28 lbs, and the animal group that received KE01 plus FOS
showed a body weight gain of 20 lbs compared to control group of animals as
depicted in Figure 9.
III. Conclusion
[0102] In closing, it is to be understood that the embodiments of the
invention
disclosed herein are illustrative of the principles of the present invention.
Other
modifications that may be employed are within the scope of the invention.
Thus, by
way of example, but not of limitation, alternative configurations of the
present invention
may be utilized in accordance with the teachings herein. Accordingly, the
present
invention is not limited to that precisely as shown and described.
[0103]The terms "a" and "an" and "the" and similar referents used in the
context of
describing the invention (especially in the context of the following claims)
are to be
construed to cover both the singular and the plural, unless otherwise
indicated herein
or clearly contradicted by context. Recitation of ranges of values herein are
merely
intended to serve as a shorthand method of referring individually to each
separate .
value falling within the range. Unless otherwise indicated herein, each
individual value
is incorporated into the specification as if it were individually recited
herein. All
methods described herein can be performed in any suitable order unless
otherwise
indicated herein or otherwise clearly contradicted by context. The use of any
and all
examples, or exemplary language (e.g. "such as") provided herein is intended
merely
to better illuminate the invention and does not pose a limitation on the scope
of the
invention otherwise claimed. No language in the specification should be
construed as
indicating any non-claimed element essential to the practice of the invention.
CA 02520178 2014-10-20
55710-1
= [0104]Groupings of alternative elements or embodiments of the invention
disclosed
herein are not to be construed as limitations. Each group member may be
referred to
and claimed individually or in any combination with other members of the group
or
other elements found herein. It is anticipated that one or more members of a
group
may be included in, or deleted from, a group for reasons of convenience and/or
patentability. When any such inclusion or deletion occurs, the specification
is herein
deemed to contain the group as modified thus fulfilling the written
description of all
Markush groups used in the appended claims.
[0105] Preferred embodiments of this invention are described
herein, including the
best mode known to the inventors for carrying out the invention. Of course,
variations
on those preferred embodiments will become apparent to those of ordinary skill
in the
art upon reading the foregoing description. The inventor expects skilled
artisans to
employ such variations as appropriate, and the inventors intend for the
invention to be
practiced otherwise than specifically described herein. Accordingly, this
invention
includes all modifications and equivalents of the subject matter recited in
the claims
appended hereto as permitted by applicable law. Moreover, any combination of
the
above-described elements in all possible variations thereof is encompassed by
the
invention unless otherwise indicated herein or otherwise clearly contradicted
by
context.
31
