U.S. patent application number 14/575916 was filed with the patent office on 2015-04-16 for bacterial composition.
This patent application is currently assigned to MICROBIOS, INC.. The applicant listed for this patent is MicroBios, Inc.. Invention is credited to Joseph F. Flint, Matthew Ryan Garner.
Application Number | 20150104418 14/575916 |
Document ID | / |
Family ID | 52809860 |
Filed Date | 2015-04-16 |
United States Patent
Application |
20150104418 |
Kind Code |
A1 |
Flint; Joseph F. ; et
al. |
April 16, 2015 |
BACTERIAL COMPOSITION
Abstract
A bacterial composition that inhibits E. coli O157:H7 growth by
as much as 93% and Salmonella growth by as much as 97%, together
with commensurate inhibition rates against the Big-Six Escherichia
coli strains referred to as the non-O157 STECs that include E. coli
O121:H19; E. coli O45:H2; E. coli O103:H11; E. coli O145, E. coli
O26:H11; and E. coli O111. The composition is constituted of
various combinations of the following four unique
pathogen-inhibiting bacteria: (1) Lactobacillus animalis strain
MB101; (2) Lactobacillus animalis strain MB102; (3) Enterococcus
faecium strain MB505; and (4) Pediococcus acidilactici strain
MB902. Each of the discovered bacteria is deposited with the
American Type Culture Collection (ATCC) and respectively has an
individual ATCC Accession Number.
Inventors: |
Flint; Joseph F.; (Ithaca,
NY) ; Garner; Matthew Ryan; (Amarillo, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MicroBios, Inc. |
Houston |
TX |
US |
|
|
Assignee: |
MICROBIOS, INC.
Houston
TX
|
Family ID: |
52809860 |
Appl. No.: |
14/575916 |
Filed: |
December 18, 2014 |
Current U.S.
Class: |
424/93.3 ;
424/93.45 |
Current CPC
Class: |
A23K 50/10 20160501;
A61K 35/744 20130101; A61K 35/744 20130101; A23K 50/75 20160501;
A23K 50/20 20160501; A61K 2300/00 20130101; A61K 2300/00 20130101;
A23K 10/18 20160501; A61K 35/747 20130101; A23Y 2220/07 20130101;
A61K 35/742 20130101; A61K 35/747 20130101; A23K 50/30 20160501;
A61K 2300/00 20130101; A23Y 2280/15 20130101; A61K 35/742
20130101 |
Class at
Publication: |
424/93.3 ;
424/93.45 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A23K 1/00 20060101 A23K001/00 |
Claims
1-26. (canceled)
27. A method for preparing a probiotic composition comprising:
mixing into a combination at least two bacteria selected from the
group consisting of: Lactobacillus animalis strain MB101 having
ATCC Accession Number PTA-121710; Lactobacillus animalis strain
MB102 having ATCC Accession Number PTA-121711; Enterococcus faecium
strain MB505 having ATCC Accession Number PTA-121709; and
Pediococcus acidilactici strain MB902 having ATCC Accession Number
PTA-121712; and packaging the combination.
28. The method recited in claim 27 comprising mixing: Lactobacillus
animalis strain MB101 having ATCC Accession Number PTA-121710; and
Lactobacillus animalis strain MB102 having ATCC Accession Number
PTA-121711.
29. The method recited in claim 27 comprising mixing: Lactobacillus
animalis strain MB101 having ATCC Accession Number PTA-121710;
Lactobacillus animalis strain MB102 having ATCC Accession Number
PTA-121711; and Enterococcus faecium strain MB505 having ATCC
Accession Number PTA-121709.
30. The method recited in claim 27 comprising mixing: Lactobacillus
animalis strain MB101 having ATCC Accession Number PTA-121710;
Lactobacillus animalis strain MB102 having ATCC Accession Number
PTA-121711; Enterococcus faecium strain MB505 having ATCC Accession
Number PTA-121709; and Pediococcus acidilactici strain MB902 having
ATCC Accession Number PTA-121712.
31. The method recited in claim 27 comprising mixing: Lactobacillus
animalis strain MB101 having ATCC Accession Number PTA-121710; and
Enterococcus faecium strain MB505 having ATCC Accession Number
PTA-121709.
32. The method recited in claim 27 comprising mixing: Lactobacillus
animalis strain MB101 having ATCC Accession Number PTA-121710;
Enterococcus faecium strain MB505 having ATCC Accession Number
PTA-121709; and Pediococcus acidilactici strain MB902 having ATCC
Accession Number PTA-121712.
33. The method recited in claim 27 comprising mixing: Lactobacillus
animalis strain MB101 having ATCC Accession Number PTA-121710; and
Pediococcus acidilactici strain MB902 having ATCC Accession Number
PTA-121712.
34. The method recited in claim 27 comprising mixing: Lactobacillus
animalis strain MB101 having ATCC Accession Number PTA-121710;
Lactobacillus animalis strain MB102 having ATCC Accession Number
PTA-121711; and Pediococcus acidilactici strain MB902 having ATCC
Accession Number PTA-121712.
35. The method recited in claim 27 comprising mixing: Lactobacillus
animalis strain MB102 having ATCC Accession Number PTA-121711; and
Enterococcus faecium strain MB505 having ATCC Accession Number
PTA-121709.
36. The method recited in claim 27 comprising mixing: Lactobacillus
animalis strain MB102 having ATCC Accession Number PTA-121711;
Enterococcus faecium strain MB505 having ATCC Accession Number
PTA-121709; and Pediococcus acidilactici strain MB902 having ATCC
Accession Number PTA-121712.
37. The method recited in claim 27 comprising mixing: Lactobacillus
animalis strain MB102 having ATCC Accession Number PTA-121711; and
Pediococcus acidilactici strain MB902 having ATCC Accession Number
PTA-121712.
38. The method recited in claim 27 comprising mixing: Enterococcus
faecium strain MB505 having ATCC Accession Number PTA-121709; and
Pediococcus acidilactici strain MB902 having ATCC Accession Number
PTA-121712.
39. The method recited in claim 27 wherein at least one strain is a
pathogen growth inhibitor.
40. The method recited in claim 27 wherein at least one strain is
an E. coli O157:H7 growth inhibitor.
41. The method recited in claim 27 wherein at least one strain is a
growth inhibitor to the non-O157 STEC, Big-Six Escherichia coli
strains including E. coli O121:H19; E. coli O45:H2; E. coli
O103:H11; E. coli O145, E. coli O26:H11; and E. coli O111.
42. The method recited in claim 27 wherein at least one strain is a
Salmonella growth inhibitor.
43. The method recited in claim 27 wherein at least one strain is a
Salmonella typhimurium growth inhibitor.
44. The method recited in claim 27 wherein at least one strain is a
Salmonella enteriditis growth inhibitor.
45. The method recited in claim 27 wherein each strain is a
pathogen growth inhibitor.
46. The method recited in claim 27 wherein each strain is an E.
coli O157:H7 growth inhibitor.
47. The method recited in claim 27 wherein each strain is a growth
inhibitor to the non-O157 STEC, Big-Six Escherichia coli strains
including E. coli O121:H19; E. coli O45:H2; E. coli O103:H11; E.
coli O145, E. coli O26:H11; and E. coli O111.
48. The method recited in claim 27 wherein each strain is a
Salmonella growth inhibitor.
49. The method recited in claim 27 wherein each strain is a
Salmonella typhimurium growth inhibitor.
50. The method recited in claim 27 wherein at least one strain is a
Salmonella enteriditis growth inhibitor.
51. The method recited in claim 27 further comprises mixing in a
carrier and thereby establishing a direct fed bacterial animal feed
additive.
52. A method for preparing a probiotic composition comprising:
obtaining at least one bacteria selected from the group consisting
of: Lactobacillus animalis strain MB102 having ATCC Accession
Number PTA-121711; Enterococcus faecium strain MB505 having ATCC
Accession Number PTA-121709; and Pediococcus acidilactici strain
MB902 having ATCC Accession Number PTA-121712; and packaging the
composition.
Description
FIELD
[0001] The present disclosure relates generally to compositions and
methods for manufacture and use of pathogen inhibiting
bacteria.
BACKGROUND
[0002] Certain bacteria have been recognized for their capability
to inhibit the growth of certain pathogenic bacteria, and have
therefore been utilized as additives in products for which
pathogenic inhibition is advantageous. At least one example is
animal feed to which bacteria are sometimes added and that have
been found to improve animal efficiency and health. Such bacterial
feed additives are frequently referred to as probiotics, as well as
Direct Fed Microbials, or DFMs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0003] Implementations of the present technology will now be
described, by way of example only, with reference to the attached
figures, wherein:
[0004] FIG. 1 is a diagram representing pathogen inhibition in a
ruminant animal by the administration of the bacterial compositions
disclosed herein; and
[0005] FIGS. 2 and 3 are tables detailing discovered pathogen
inhibition efficiencies of the bacteria and bacterial compositions
disclosed herein.
DETAILED DESCRIPTION
[0006] One of the larger economic burdens facing cattle owners is
the high cost of rearing and/or replacing animals to maintain or
increase herd size. A major factor contributing to the high cost of
cattle replacement is the prevalence of diarrheal disease, known as
scours, in livestock. Scours causes greater than 60% of all deaths
associated with pre-weaned calves, and accounts for 6.2% of total
calf losses. The prevalence of scours can vary dramatically (4.3%
to 52.4%) depending on herd, diet, season, or "outbreak"
occurrences. Estimates of scouring rates within a herd are
difficult to obtain, though they are believed to between 15% and
35%. Nonetheless, it is agreed that diarrheal events comprise the
largest health challenge to pre-weaned calves.
[0007] Moreover, diarrhea (scours) remains the predominant cause of
mortality among calves. There are multiple causes of scours
including malabsorption and improper nutrition; however, infections
by bacteria, viruses, and protozoa are the primary etiological
agents. It is important to consider that scours in calves may be
due to a number of concurrent gastrointestinal insults by numerous
pathogens. Susceptibility to acute undifferentiated diarrhea can be
largely determined by the quantity, quality, and administration
time of colostrum.
[0008] The costs associated with scours are difficult to estimate;
however, mortality alone represents a large expense, since, at
birth, a heifer has an estimated value of $400-$600. Scours does
not always result in death, but costs associated with treatment
(e.g. electrolytes, antibiotics, veterinary services and associated
labor) can be significant. In addition, animal sickness and death
can negatively impact the morale of farm laborers and must be taken
into consideration, though the financial costs of this cannot be
readily quantified.
[0009] Serum immunoglobulin obtained from colostrum can offer some
limited protection to calves from bacterial and viral infections.
However, this protective effect begins to diminish <96 hours
after birth, which could explain the high onset of viral scours 5-7
days following birth. Prophylactic antibiotics and vaccines
administered to calves are frequent measures used to prevent scours
in calves. While antibiotic administration can be effective against
bacterial infections, antibiotics are ineffective against viruses
and protozoa and, in fact, they can promote the development of
viral or protozoal scours by diminishing the normal protective
flora. Moreover, the use of antibiotics is disfavored in many
settings and can otherwise compromise the health and/or value of
the animal. Vaccinations can also confer protection against scours;
however, the full protective immune response does not occur until
after a few weeks of administration. Despite some advances in
prevention and treatment, the incidence of scours can vary wildly
between cattle herds.
[0010] A major factor contributing to the onset of scours in calves
is the practice of removing calves from their mother cows
immediately after birth, and transporting them to facilities away
from adult animals. The gastrointestinal tracts of mammals,
including calves, are sterile at birth, but rapidly become
colonized by microflora located near the mother's vagina and anus.
Other bacteria begin to establish themselves when the neonate comes
into contact with new objects (feed, dirt, gates, fences, handlers,
etc.). Prior to the current practice of removing a calf from its
mother, protective microflora would become established in the calf
due to contact with the mother via licking, nursing, and grooming.
Thus, one possible avenue to reduce the incidence and severity of
scours includes manipulating the bacterial flora of a calves'
digestive tract.
[0011] It has long been known that a number of beneficial bacteria
colonize the intestinal tracts of mammals and can promote the
well-being of the host. It has also been recognized for many years
that the consumption of exogenous bacteria, often referred to as
probiotics, can elicit beneficial effects upon a host. In humans,
these probiotic bacteria have been shown to reduce the severity and
duration of rotaviral-induced diarrhea, alleviate lactose
intolerance, and enhance gastrointestinal immune function.
Traditionally, food sources such as yogurt have been considered
probiotic-carriers providing these health-promoting benefits. It is
believed that the consumption of foods rich in probiotic bacteria,
including lactic acid bacteria and bifidobacteria, leads to
colonization of the human gastrointestinal tract of humans.
[0012] The consumption of probiotics by animals used in food
production can improve their health and feed utilization
efficiencies, as well as decrease certain pathogen loads within the
animal and pathogen sheading outside, from the animal. Probiotics
work by competitive exclusion in which live bacterial cultures act
antagonistically on specific organisms to cause a decrease in the
numbers of that organism. Mechanisms of competitive exclusion
include production of antibacterial agents (bacteriocins) and
metabolites (organic acids and hydrogen peroxide), competition for
nutrients, and competition for adhesion sites on the gut epithelial
surface. Lactic acid producing bacteria are generally considered as
food grade organisms and there are many potential applications of
protective cultures in various foods. A number of different factors
have been identified that contribute to the antibacterial activity
of lactic acid producing bacteria. These bacteria produce different
antibacterials, such as lactic acid, acetic acid, hydrogen
peroxide, carbon dioxide and bacteriocins, which can inhibit
pathogenic microorganisms.
[0013] A majority of bacteriocins produced by bacteria are
lantibiotics or small hydrophobic heat stable peptides. Nisin, a
lantibiotic is effective at inhibition of Gram-positive bacteria
such as Bacillus and Clostridium. However, Nisin has demonstrated
no effectiveness against Gram-negative bacteria. Among the small
hydrophobic heat stable peptides, pediocins are frequently
encountered and possess the ability to inhibit Listeria
monocytogenes.
[0014] Lactobacillus genus includes the most prevalently
administered probiotic bacteria. Lactobacillus is a genus of more
than 25 species of gram-positive, catalase-negative,
non-sporulating, rod-shaped organisms. Lactobacillus species
ferment carbohydrates to form lactic acid. Lactobacillus species
are generally anaerobic, non-motile, and do not reduce nitrate.
Lactobacillus species are often used in the manufacture of food
products including dairy products and other fermented foods.
Lactobacillus species inhabit various locations including the
gastrointestinal tracts of animals and intact and rotting plant
material. Lactobacillus strains appear to be present in the
gastrointestinal tract of approximately 70% of humans that consume
a Western-style diet. The number of Lactobacillus cells in neonates
is approximately 105 colony forming units (CFU) per gram CFU/g of
feces. The amount in infants of one month and older is higher,
ranging from 106 to 108 CFU/g of feces.
[0015] Lactic acid and products containing lactic acid enhance
gains in the starting period of feedlot cattle (first 28 days) and
reduce liver abscesses when administered during the transition from
a primarily roughage diet of grass to a feedlot diet including more
grains. Certain strains of Lactobacillus acidophilus have been
isolated which restore and stabilize the internal bacterial balance
of animals. Some strains demonstrate a greater propensity to adhere
to the epithelial cells of some animals which would increase their
ability to survive, initiate and maintain a population within an
animal intestine. Thus, the primary mode of action as previously
understood relative to Lactobacillus acidophilus occurs
post-ruminally.
[0016] The most common method used today to control pathogenic
populations in livestock is through the use of antibacterial
compounds. While these are effective for short-term treatments,
prolonged application of antibacterial compounds leads to the
evolution of antibiotic resistance in the pathogenic organisms. The
widespread occurrence of antibiotic resistant microorganisms is
well known; two examples are methicillin resistant Staphylococcus
aureus (MRSA) and vancomycin resistant enterococci (VRE). Bacteria
are remarkably adaptable to deleterious environments with their
abilities to rapidly reproduce and modify their genetic content.
Thus, it is inevitable that after prolonged application of any
method that disrupts or kills bacteria, a population that is
recalcitrant to its effects will eventually arise. It is not
uncommon now in the veterinary environment that doctors often
resort to using multiple antibiotics concurrently or in succession
to eradicate pathogenic organisms.
[0017] As with antibiotics, bacteria can also become resistant to
other biological treatments. For example, bacteriophages are able
to reduce pathogen populations, but inevitably, a fraction of the
targeted bacterial population is not affected. This small
sub-population then rapidly reproduces and attains sizable
population numbers.
[0018] Similar circumstances have been seen with the application of
probiotic bacteria that are meant to inhibit or reduce the numbers
of pathogenic bacteria within a gastrointestinal system. Some
researchers have commented that significantly better animal
performance and pathogen reductions were seen in treated animals
early in their experiments, but the beneficial effects were no
longer statistically different after prolonged application of the
probiotic product. It is possible that the target populations were
initially affected, but prolonged usage of the probiotic product
led to the selection of bacterial populations that were not
influenced by the application of the product. According to this
disclosure, the adaptation of pathogens to probiotic treatment can
be avoided with the inclusion of multiple strains of bacteria.
[0019] There are numerous advantages provided by the inclusion of
multiple strains of bacteria in a bacterial composition such as
that which is disclosed herein. These advantages, whether working
independently or concurrently, support the superiority of the
presently disclosed bacterial composition and the enhanced benefits
for an animal to which it is administered.
[0020] Different bacteria strains utilize certain nutrients more
efficiently than others. The ability to use available nutrients in
a gut environment is necessary for the bacteria to produce
antibacterial compounds or to beneficially affect the host GI
system. However, the nutrient availability is constantly changing
because of animal behavior, different foods consumed, antibiotic
use, energy requirements, or health of the animal. These
fluctuations allow different bacteria to proliferate while other
bacterial populations diminish.
[0021] The use of different bacterial strains also produces
different bacterial metabolites. Different metabolites have
different effects upon pathogenic populations. Lactic acid is a
powerful antibacterial agent against some pathogens, while
propionic acid is more effective against other populations. It
should also be considered that just as metabolites produced from
cells in bacterial products affect GI populations, endogenous
microorganisms produce chemicals that can be inhibitory to some
bacteria strains. The present inclusion of different strains in a
direct fed feed supplement increases the likelihood that the
product will have a positive effect.
[0022] Additionally, the production of bacteriocins influences
bacterial populations. There is a large diversity of bacteriocins
that target specific bacteria populations. Thus, a bacterial
product that contains multiple strains will produce multiple
bacteriocins and target different groups of pathogenic populations.
Conversely, the intestinal tract contains a large diversity of
bacteriocin producing bacteria. While some of the produced
bacteriocins can affect one of the included strains, it is unlikely
to affect all of the included microorganisms.
[0023] Another benefit of the presently disclosed multi-strain
bacterial composition when utilized as a direct fed feed additive
is its ability to target more than one pathogen population for
inhibition. Bacterial pathogens are very diverse and require
different methods to reduce or eliminate their populations. Thus, a
product containing different pathogen inhibiting bacteria that are
able to effect different pathogenic populations will result in an
overall healthier animal and herd.
[0024] Different microorganisms positively influence the
gastrointestinal system through different mechanisms. Therefore,
including bacteria that work through different modes will result in
a superior product. One strain may reduce pathogen populations,
while another has an immunostimulative effect, while another
produces micronutrients essential for the host. Interestingly,
multiple strains can also provide synergistic effects upon the host
or pathogen inhibition abilities. One strain alone may not be able
to reduce certain populations, but the combination of two or more
different strains working through different mechanisms can reduce
pathogen populations.
[0025] Additionally, the use of multiple beneficial microorganisms
can help overcome bacteriophages that infect and kill bacteria.
Bacteriophages are very common in gastrointestinal systems and have
profound effects upon the bacterial community. Bacteriophages
require specific sites on a cell to bind and infect. Thus, by
including multiple microorganisms in a product, the greater the
likelihood that at least some populations from the product will
evade bacteriophage attack and elicit beneficial effects upon the
bacterial community and host.
[0026] Certain examples in the present disclosure concern a method
of inhibiting or reducing a population of pathogenic bacteria in,
on and/or outside the animal. In one aspect, the disclosed
compositions reduce a population of pathogenic bacteria in the
gastrointestinal tract of the animal.
[0027] Other examples concern the inhibition or reduction of a
population of pathogenic bacteria by providing to the animal a
composition containing the multiple probiotic bacteria described
herein.
[0028] In the examples of the present disclosure wherein an
administration of pathogen inhibiting bacteria is contemplated, the
number of bacteria (concentration) per administration can be any
amount capable of providing some inhibition or reduction of a
population of pathogenic bacteria. In specific embodiments, the
number of bacteria per administration is between 1.times.10.sup.3
and 1.times.10.sup.9 bacteria. Preferably, the number of bacteria
in an administration is approximately 1.times.10.sup.6
bacteria.
[0029] The administrations can be timed such that a series of
administrations of the composition or its separate components is
separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 17, 19, 20, 21, 22 or 23 hours or 1, 2, 3, 4, 5, or 6 days or
1, 2 or 3 weeks or 1 month or some duration in between. In some
specific embodiments, the administrations are daily.
[0030] Among others, administration of the bacterial composition to
the animal can be oral, nasal, topical, rectal, and via injection.
In certain embodiments, the administrations are oral
administrations. In embodiments wherein the administrations are
oral administrations, the composition comprising probiotic bacteria
can be mixed with animal feed or mixed with animal drinking water.
In such embodiments, the composition or compositions can be
formulated as a liquid formulation for administration, or as a
freeze dried formulation, or as a gel formulation or as a spore
formulation.
[0031] Additional steps in inhibiting or reducing the population of
pathogenic bacteria in an animal include assessing the presence of
pathogenic bacteria in the gastrointestinal tract of the animal
between administrations. In more specific embodiments, the animal
is assessed for the presence of pathogenic bacteria, for strains of
pathogenic bacteria, species of pathogenic bacteria and number
(concentration) of pathogenic bacteria present. In specific
embodiments, this assessment is done by examining the feces of the
animal.
[0032] In the context of the present disclosure, the terms
"substantially" and "about" are defined to be essentially
conforming to the particular quantity, concentration, dimension,
shape or other thing that "substantially" or "about" modifies, such
that the so described characteristic need not be exact, but within
reasonable tolerances. The terms "comprising," "including" and
"having" (and variants thereof) are used interchangeably in this
disclosure. The terms "comprising," "including" and "having" mean
to include, but not necessarily be limited to the things so
described.
[0033] It should be understood that this disclosure is not limited
to particular compositions or biological systems, which can, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting.
[0034] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the disclosure belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
disclosure, the preferred methods and materials are now
described.
[0035] In this specification and the claims that follow, reference
will be made to a number of terms which can be considered to have
the following meanings: "inhibit" and/or "reduce" or other forms of
the words and their synonyms, such as "reducing" or "reduction,"
refer to slowing the growth of the so-referenced pathogen and/or
lowering its incidence. It is understood that this is typically in
relation to some standard or expected value, in other words it is
relative, but that it is not always necessary for the standard or
relative value to be referred to. For example, "reduces the
population of bacteria" in certain instances refer to lowering the
amount of bacteria relative to a standard or a control. "Inhibit"
and "inhibition" refer to slowing or deterring pathogen growth,
including pathogenic bacterial growth that would have otherwise
occurred except for the provision of the characterized
deterrent.
[0036] By "treat" or other forms of the word, such as "treated" or
"treatment," means to administer a composition or to perform a
method in order to reduce, prevent, inhibit, break-down, or
eliminate a particular characteristic or event.
[0037] The term "viable cell" means a microorganism, and in
particular, bacteria that are alive and capable of regeneration
and/or propagation, while in a vegetative, frozen, preserved, or
reconstituted state.
[0038] The term "viable cell yield" or "viable cell concentration"
refers to the number of viable cells in a liquid culture,
concentrated, or preserved state per a unit of measure, such as
liter, milliliter, kilogram, gram or milligram.
[0039] The term "cell preservation" refers to a process that takes
a vegetative cell and preserves it in a metabolically inert state
that retains viability over time. As used herein, the term
"product" refers to a bacterial composition that can be blended
with other components and contains specified concentration of
viable cells that can be sold and used.
[0040] As used herein, the terms "microorganism" or "microbe"
refers to an organism of microscopic size. The definition of
microorganism herein includes bacteria, Archaea, single-celled
Eukaryotes (protozoa, fungi, and ciliates), and viral agents
(viruses). The term "microbial" is used herein to describe
processes or compositions of microorganisms, thus a
"microbial-based product" is a composition that includes
microorganisms, cellular components of the microorganisms, and/or
metabolites produced by the microorganisms. Microorganisms can
exist in various states and occur in vegetative, dormant, or spore
states. Microorganisms can also occur as either motile or
non-motile, and may be found as planktonic cells (unattached),
substrate affixed cells, cells within colonies, or cells within a
biofilm.
[0041] The term "bacteria" refers to one-celled organisms that are
Prokaryotes in that their genetic material, or DNA, is not enclosed
in a nucleus.
[0042] The term "prebiotic" refers to food ingredients that are not
readily digestible by endogenous host enzymes and confer beneficial
effects on an organism that consumes them by selectively
stimulating the growth and/or activity of a limited range of
beneficial microorganisms that are associated with the intestinal
tract.
[0043] The term "probiotic" refers to one or more live
microorganisms that confer beneficial effects on a host organism.
Benefits derived from the establishment of probiotic microorganisms
within the digestive tract include reduction of pathogen load,
improved bacterial fermentation patterns, improved nutrient
absorption, improved immune function, aided digestion and relief of
symptoms of irritable bowel disease and colitis.
[0044] The term "synbiotic" refers to a composition that contains
both probiotics and prebiotics. Synbiotic compositions are those in
which the prebiotic compound selectively favors the probiotic
microorganism.
[0045] The term "gastrointestinal tract" refers to the complete
system of organs and regions that are involved with ingestion,
digestion, and excretion of food and liquids. This system generally
consists of, but not limited to, the mouth, esophagus, stomach and
or rumen, intestines (both small and large), cecum (plural ceca),
fermentation sacs, and the anus.
[0046] The term "pathogen" refers to any microorganism that
produces a harmful effect and/or disease state in a human or animal
host.
[0047] The term "fermentation" refers to a metabolic process
performed by an organism that converts one substrate to another in
which the cell is able to obtain cellular energy, such as when an
organism utilizes glucose and converts it to lactic acid or
propionic acid. Many of the end-substrates formed in fermentation
processes are volatile fatty acids.
[0048] The term "volatile fatty acids" (VFAs) refers to short-chain
fatty acids containing six or fewer carbon atoms and at least one
carboxyl group. Some examples of VFAs include, but are not limited
to: lactic acid, acetic acid, propionic acid, butyric acid,
isobutyric acid, valeric acid, and isovaleric acid, which are
products of bacterial fermentation within the digestive tracts of
animals. Volatile fatty acids can be absorbed through the
intestines of animals and used as an energy or carbon source.
Bacteria produce VFAs based on available substrates and also rely
upon VFAs for energy and carbon sources.
[0049] The term "lactic acid" refers to a byproduct of glucose
fermentation resulting in a three-carbon acid with the chemical
formula C.sub.3H.sub.6O.sub.3. This includes, but is not limited
to, lactic acid derived from specific strains of bacteria or lactic
acid derived from other types of organisms. Lactic acid can be
microbialstatic, microbialcidal, bacteriostatic, bacteriocidal or
bacteriolytic; these concepts are known to skilled persons. "Lactic
acid producing" refers to any organism that generates lactic
acid.
[0050] The term "bacteriocin(s)" refers to (poly) peptides and
proteins that inhibit one or more bacterial species. This includes,
but is not limited to, (poly) peptides or proteins that were
derived from specific strains of bacteria or (poly) peptides that
are derived from other types of organisms.
[0051] The bacteriocin can be microbialstatic, microbialcidal,
bacteriostatic, bacteriocidal, or bacteriolytic; these concepts are
known to skilled persons. For the treatment of produce and other
food products the bacteriocin is preferably microbialcidal or
bacteriocidal. "Bacteriocin producing" in certain instances refer
to any organism that generates bacteriocins.
[0052] As used herein, "hydrogen peroxide" refers to a byproduct of
oxygen metabolism that has the chemical formula H.sub.2O.sub.2.
This includes, but is not limited to, hydrogen peroxide derived
from specific strains of bacteria or hydrogen peroxide derived from
other types of organisms. Hydrogen peroxide can be microbialstatic,
microbialcidal, bacteriostatic, bacteriocidal or bacteriolytic;
these concepts are known to skilled persons. "Hydrogen
peroxide-producing" refers to any organism that generates hydrogen
peroxide.
[0053] As used herein, the term "synergistic" refers to a property
wherein the combined result of two effects is greater than would be
expected if the two effects were added together. The term
"synergistically" is used to describe a synergistic effect.
[0054] As used herein, the phrase "foregut fermentor" refers to an
animal having an anatomical compartment in the alimentary canal
that is positioned anterior to the stomach that is used for
bacterial fermentation and digestion of ingested materials. Ruminal
fermentors are considered foregut-fermenting organisms.
[0055] As used herein, the phrase "ruminal fermentor" or "rumen
fermenting" refers to an animal having a large, multi-compartmented
section of the digestive tract, called a rumen, which is positioned
between the esophagus and the anus. Rumen are very complex
ecosystems that support bacterial fermentation of cellulose, plant
matter, and other ingested materials. Ruminal-fermentors may also
be termed "cranial fermentors" or "ruminants". Some examples of
rumen-fermenting organisms include cattle, sheep, goats, camels,
llama, bison, buffalo, deer, wildebeest and antelope.
[0056] As used herein, the phrase "hindgut fermentor" refers to an
animal having a complex large intestine that may or may not include
specialized fermentation chambers that can include a cecum or blind
sac, that is positioned posterior to the stomach in the alimentary
canal. Cecal fermentors and intestinal fermentors are both
considered hindgut-fermenting organisms.
[0057] As used herein, the phrase "cecal fermentor" refers to an
animal having a complex large intestine that includes a cecum or a
blind sac along the digestive tract. The cecum of a cecal fermentor
forms a distinct chamber, which is the primary site of bacterial
fermentation of cellulose, plant matter, or other ingesta. A
cecal-fermentor may also be referred to as "caudal fermentor".
Cecal-fermentors include horses, elephants, rabbits, mice, rats,
guinea pigs and the like.
[0058] As used herein, the term "intestinal fermentor" refers to an
animal that does not primarily rely upon bacterial fermentation of
ingesta in a rumen or large cecum. In the digestive tracts of
intestinal fermentors, bacterial fermentation occurs primarily
within the large intestine or colon. Intestinal fermentors include
chickens, pigs, humans and the like.
[0059] As used herein, the term "monogastric" refers to an animal
having a single, simple (single chambered) stomach. Typically,
cecal fermentors and intestinal-fermentors are monogastric animals.
Some examples of monogastric animals include horses, chickens,
pigs, humans and the like.
[0060] As used herein, the term "polygastric" refers to an animal
having a multiple, complex (multi-chambered) stomachs. Ruminal
fermentors are polygastric animals.
[0061] As used herein, the phrase "pre-gastric fermentation" refers
to bacterial fermentation that occurs before the food reaches a
`true` stomach, which is generally the site of gastric acid and
digestive enzyme secretion. Ruminants are pre-gastric
fermentors.
[0062] As used herein, the phrase "post-gastric fermentation"
refers to bacterial fermentation that occurs after food passes
through a stomach, which is generally the site of gastric acid and
digestive enzyme secretion. Hindgut fermentors, including cecal
fermentors and intestinal fermentors, utilize post-gastric
fermentation.
[0063] As used herein, the term "herbivore" refers to an animal
that exclusively consumes plant material.
[0064] As used herein, the term "omnivore" refers to an animal that
consumes both plant and animal material.
[0065] As used herein, the term "carnivore" refers to an animal
that exclusively consumes animal material.
[0066] As used herein, "digesta" refers to food or any other
material that enters the alimentary canal and undergoes, completely
or partially, through the process of being digested or broken down
into smaller components.
[0067] The present specification discloses a novel composition
variously comprised of multiple bacteria strains. The composition
can be added to products to inhibit pathogen growth and/or reduce
pathogen presence in downstream production processes, product
storage and/or product utilization. Among other uses, the
composition can be utilized in washes, dips and the like for
reducing pathogen load and inhibiting pathogen growth; for
instance, in and on food products. Surfaces such as countertops,
refrigerators and food preparation surfaces can also be treated,
with special benefits being provided to porous surfaces into which
the composition can be absorbed.
[0068] The presently disclosed compositions are particularly
advantageous when ingested as a probiotic supplement or utilized as
a direct fed bacterial feed additive that provides beneficial
effects to all types of animals, including amphibians, birds, fish,
invertebrates, reptiles and mammals, including fermentors, cecal
fermentors and intestinal fermentors. In one embodiment, the
probiotic formulation supplemented with prebiotic compounds is fed
to ruminal fermentors to reduce scours events and improve animal
health. Ruminal fermentors that can benefit from the present
disclosure include but are not limited to: cattle, sheep, goats,
camels, llama, bison, buffalo, deer, wildebeest, antelope, and any
other pre-gastric fermentor.
[0069] In another embodiment, the probiotic formulation
supplemented with prebiotic compounds is fed to cecal fermentors to
reduce scours events and improve animal health. Cecal fermentors
that can benefit from this disclosure include but are not limited
to: horses, ponies, elephants, rabbits, hamsters, rats, hyraxes,
guinea pigs, and any other post-gastric fermentor that using the
cecum as the primary location of fermentative digestion. In another
embodiment, the probiotic formulation supplemented with prebiotic
compounds is fed to intestinal fermentors to reduce scours events
and improve animal health. Intestinal fermentors that can benefit
from said disclosure include but are not limited to: humans, pigs,
chickens, and other post-gastric fermentor using the large
intestine as the primary location of fermentative digestion. In
each case, the composition is packaged in a format that ensures
survival of both the probiotic and prebiotic components into the
gastrointestinal system of the animal.
[0070] In one aspect, the present disclosure provides a novel
composition that reduces animal mortality and morbidity and reduces
pathogen load in the animal, as well as pathogen sheading in its
feces. Administration of the bacteria-based compositions to the
animal also can improve animal health and/or productivity by way of
increased feed efficiency. It also provides a composition that will
be used once, periodically or on a continual basis to reduce the
incidence and severity of scours and/or improve animal health. The
composition is durable and easy to apply to animal feed or other
easily ingestible materials.
[0071] The novel compositions disclosed comprise specially selected
bacteria, at least some of which can produce lactic acid which
inhibits the growth of pathogenic organisms during digestive
fermentation. The composition can comprise a mixture of lactic acid
producing bacteria. In some embodiments, the composition is mixed
with colostrum or milk replacers. In other embodiments the mixture
is applied to milk or water administered to calves. Animals can be
treated with a combination of viable microorganisms with prebiotic
compounds to improve animal efficiency and/or health. Additive, or
more preferably super-additive or more preferably synergistic
effects can be achieved with the administration of two or more
bacteria species and/or strains. Animals can be treated once,
multiple times, or therapeutically on a daily basis.
[0072] In one aspect, various compositional examples of this
disclosure include bacterial combinations that are pathogen
inhibitive in that the combination inhibits pathogen growth when
added thereto. The specially selected bacteria can be of different
species (for example, L. animalis versus Enterococcus faecium), or
they may be of the same species (for example, L. animalis and L.
animalis) but different strains (for example, MB101 and MB102)
within the same species (L. animalis). That is to say, the
composition may contain multiple species and/or multiple strains.
For example, two, three, four, five, six, and so on different
bacterial strains can be included. The use of multiple types of the
specially selected bacterial strains lead to a superiorly reliable
product for maintaining or improving animal health or inhibiting,
decreasing or eliminating the presence of pathogenic bacteria. As
an example, each of the bacteria in the composition can have
superior survival characteristics under various conditions likely
to be encountered during storage, transport and/or administration.
In that way it is better assured that viable bacteria will reach
the target (animal, object and the like), regardless of whether one
or more abusive conditions (such as overheating) have been
encountered after manufacture.
[0073] More specifically, among others, four different pathogen
inhibiting bacteria strains have been discovered and are variously
included in the instantly disclosed bacterial compositions. Each
strain was deposited on Nov. 7, 2014 with the American Type Culture
Collection (ATCC) and the corresponding certificates issued on Nov.
25, 2014 carrying the title of Budapest Restricted Certificate of
Deposit Budapest Treaty on the International Recognition of the
Deposit of Microorganisms for the Purpose of Patent Procedure
International Form Receipt in the Case of an Original Deposit
Issued Pursuant to Rule 7.3 and Viability Statement Issued Pursuant
to Rule 10.2. The deposits and the following ATCC designations for
the strains were certified:
TABLE-US-00001 ATCC PATENT DEPOSIT DESIGNATIONS Strain Name Genus
PTA Designation MB101 L. animalis PTA-121710 MB102 L. animalis
PTA-121711 MB505 Enterococcus faecium PTA-121709 MB902 Pediococcus
PTA-121712
[0074] In FIG. 2 of the present disclosure, specific compositional
combinations of the discovered strains are disclosed together with
their derived pathogen inhibition efficacies specified in terms of
percent pathogen growth inhibition determined using the processes
described below. The derived pathogen inhibition efficacies of the
individual strains are shown in FIG. 3.
[0075] Experiments demonstrating the ability of the strains to
inhibit the growth of pathogenic organisms were performed using the
following method. A "control" tube was prepared containing an
amount of modified MRS medium to which an amount {concentration at
1.times.10.sup.6 CFU/ml} of a single strain pathogenic bacteria {E.
coli or Salmonella, for example} was added. A "challenged" tube was
prepared that contained the same amount of the modified MRS medium
to which the same amount of the single strain pathogenic bacteria
was added as in the control tube, together with an equal total
amount {concentration at 1.times.10.sup.6 CFU/ml} of challenging
bacterium strain(s) as the pathogenic bacteria. The tubes were
placed into a water bath at 37.degree. C. and incubated for six
hours. After incubation, each tube was serially diluted and 100
microliters of its content spread onto Luria-Bertani plates to
enumerate the number of viable pathogen cells. The percent pathogen
growth inhibition was then calculated as: the difference between
the amount of viable pathogen cells in the challenged tube versus
the control tube, divided by the amount of viable pathogen cells in
the control tube, and then multiplied by 100.
[0076] Similar illustrative procedures are disclosed and described
in US Patent Publication 2011-0189132, the entirety of which is
hereby incorporated herein by reference.
[0077] New compositions that deliver pathogen growth inhibition are
variously disclosed in FIG. 2, including those that comprise at
least two of any of the following four bacteria: (1) Lactobacillus
animalis strain MB101 having ATCC Accession Number PTA-121710; (2)
Lactobacillus animalis strain MB102 having ATCC Accession Number
PTA-121711; (3) Enterococcus faecium strain MB505 having ATCC
Accession Number PTA-121709; and (4) Pediococcus acidilactici
strain MB902 having ATCC Accession Number PTA-121712.
[0078] Exemplarily, a composition is described that includes (1)
Lactobacillus animalis strain MB101 having ATCC Accession Number
PTA-121710 and (2) Lactobacillus animalis strain MB102 having ATCC
Accession Number PTA-121711. The composition demonstrated the
following pathogen inhibition percentages: 91.9% inhibition of E.
coli O157:H7; 91.1% inhibition of Salmonella typhimurium and 95.3%
inhibition of Salmonella enteriditis.
[0079] Another composition is described that includes (1)
Lactobacillus animalis strain MB101 having ATCC Accession Number
PTA-121710; (2) Lactobacillus animalis strain MB102 having ATCC
Accession Number PTA-121711; and (3) Enterococcus faecium strain
MB505 having ATCC Accession Number PTA-121709. The composition
demonstrated the following pathogen inhibition percentages: 90.9%
inhibition of E. coli O157:H7; 90.0% inhibition of Salmonella
typhimurium and 94.0% inhibition of Salmonella enteriditis.
[0080] Another composition is described that includes (1)
Lactobacillus animalis strain MB101 having ATCC Accession Number
PTA-121710; (2) Lactobacillus animalis strain MB102 having ATCC
Accession Number PTA-121711; (3) Enterococcus faecium strain MB505
having ATCC Accession Number PTA-121709; and (4) Pediococcus
acidilactici strain MB902 having ATCC Accession Number PTA-121712.
The composition demonstrated the following pathogen inhibition
percentages: 93.1% inhibition of E. coli O157:H7; 97.4% inhibition
of Salmonella typhimurium and 96.0% inhibition of Salmonella
enteriditis.
[0081] Another composition is described that includes (1)
Lactobacillus animalis strain MB101 having ATCC Accession Number
PTA-121710 and (2) Enterococcus faecium strain MB505 having ATCC
Accession Number PTA-121709. The composition demonstrated the
following pathogen inhibition percentages: 92.1% inhibition of E.
coli O157:H7; 95.8% inhibition of Salmonella typhimurium and 95.4%
inhibition of Salmonella enteriditis.
[0082] Another composition is described that includes (1)
Lactobacillus animalis strain MB101 having ATCC Accession Number
PTA-121710; (2) Enterococcus faecium strain MB505 having ATCC
Accession Number PTA-121709; and (3) Pediococcus acidilactici
strain MB902 having ATCC Accession Number PTA-121712. The
composition demonstrated the following pathogen inhibition
percentages: 85.7% inhibition of E. coli O157:H7; 87.0% inhibition
of Salmonella typhimurium and 90.5% inhibition of Salmonella
enteriditis.
[0083] Another composition is described that includes (1)
Lactobacillus animalis strain MB101 having ATCC Accession Number
PTA-121710 and (2) Pediococcus acidilactici strain MB902 having
ATCC Accession Number PTA-121712. The composition demonstrated the
following pathogen inhibition percentages: 92.7% inhibition of E.
coli O157:H7; 96.3% inhibition of Salmonella typhimurium and 95.4%
inhibition of Salmonella enteriditis.
[0084] Another composition is described that includes (1)
Lactobacillus animalis strain MB101 having ATCC Accession Number
PTA-121710; (2) Lactobacillus animalis strain MB102 having ATCC
Accession Number PTA-121711; and (3) Pediococcus acidilactici
strain MB902 having ATCC Accession Number PTA-121712. The
composition demonstrated the following pathogen inhibition
percentages: 90.8% inhibition of E. coli O157:H7; 89.7% inhibition
of Salmonella typhimurium and 94.0% inhibition of Salmonella
enteriditis.
[0085] Another composition is described that includes (1)
Lactobacillus animalis strain MB102 having ATCC Accession Number
PTA-121711 and (2) Enterococcus faecium strain MB505 having ATCC
Accession Number PTA-121709. The composition demonstrated the
following pathogen inhibition percentages: 86.3% inhibition of E.
coli O157:H7; 87.1% inhibition of Salmonella typhimurium and 90.8%
inhibition of Salmonella enteriditis.
[0086] Another composition is described that includes (1)
Lactobacillus animalis strain MB102 having ATCC Accession Number
PTA-121711; (3) Enterococcus faecium strain MB505 having ATCC
Accession Number PTA-121709; and (4) Pediococcus acidilactici
strain MB902 having ATCC Accession Number PTA-121712. The
composition demonstrated the following pathogen inhibition
percentages: 91.6% inhibition of E. coli O157:H7; 90.7% inhibition
of Salmonella typhimurium and 94.8% inhibition of Salmonella
enteriditis.
[0087] Another composition is described that includes (1)
Lactobacillus animalis strain MB102 having ATCC Accession Number
PTA-121711 and (2) Pediococcus acidilactici strain MB902 having
ATCC Accession Number PTA-121712. The composition demonstrated the
following pathogen inhibition percentages: 84.4% inhibition of E.
coli O157:H7; 82.7% inhibition of Salmonella typhimurium and 87.9%
inhibition of Salmonella enteriditis.
[0088] Another composition is described that includes (1)
Enterococcus faecium strain MB505 having ATCC Accession Number
PTA-121709 and (2) Pediococcus acidilactici strain MB902 having
ATCC Accession Number PTA-121712. The composition demonstrated the
following pathogen inhibition percentages: 86.5% inhibition of E.
coli O157:H7; 87.3% inhibition of Salmonella typhimurium and 91.7%
inhibition of Salmonella enteriditis.
[0089] As shown in FIG. 3, Lactobacillus animalis strain MB101
having ATCC Accession Number PTA-121710 alone demonstrated the
following pathogen inhibition percentages: 93.9% inhibition of E.
coli O157:H7; 97.9% inhibition of Salmonella typhimurium; 97.2%
inhibition of Salmonella enteriditis; 96.3% inhibition of E. coli
O121:H19; 94.4% inhibition of E. coli O45:H2; 92.8.% inhibition of
E. coli O103:H11; 93.0% inhibition of E. coli O145, 91.9%
inhibition of E. coli O26:H11; and 85.0% inhibition of E. coli
O111.
[0090] Individually, Lactobacillus animalis strain MB102 having
ATCC Accession Number PTA-121711 demonstrated the following
pathogen inhibition percentages: 90.1% inhibition of E. coli
O157:H7; 87.8% inhibition of Salmonella typhimurium; 93.9%
inhibition of Salmonella enteriditis; 91.2% inhibition of E. coli
O121:H19; 90.8% inhibition of E. coli O45:H2; 94.7.% inhibition of
E. coli O103:H11; 87.4% inhibition of E. coli O145, 89.3%
inhibition of E. coli O26:H11; and 85.9% inhibition of E. coli
O111.
[0091] Individually, Enterococcus faecium strain MB505 having ATCC
Accession Number PTA-121709 demonstrated the following pathogen
inhibition percentages: 88.2% inhibition of E. coli O157:H7; 87.8%
inhibition of Salmonella typhimurium; 92.3% inhibition of
Salmonella enteriditis; 86.6% inhibition of E. coli O121:H19; 70.1%
inhibition of E. coli O45:H2; 88.2.% inhibition of E. coli
O103:H11; 87.5% inhibition of E. coli O145, 86.9% inhibition of E.
coli O26:H11; and 89.8% inhibition of E. coli O111.
[0092] Individually, Pediococcus acidilactici strain MB902 having
ATCC Accession Number PTA-121712 demonstrated the following
pathogen inhibition percentages: 85.2% inhibition of E. coli
O157:H7; 68.3% inhibition of Salmonella typhimurium; 87.5%
inhibition of Salmonella enteriditis; 88.2% inhibition of E. coli
O121:H19; 88.6% inhibition of E. coli O45:H2; 91.5.% inhibition of
E. coli O103:H11; 86.0% inhibition of E. coli O145, 87.3%
inhibition of E. coli O26:H11; and 86.4% inhibition of E. coli
O111.
[0093] Each of (1) Lactobacillus animalis strain MB101 having ATCC
Accession Number PTA-121710; (2) Lactobacillus animalis strain
MB102 having ATCC Accession Number PTA-121711; (3) Enterococcus
faecium strain MB505 having ATCC Accession Number PTA-121709; and
(4) Pediococcus acidilactici strain MB902 having ATCC Accession
Number PTA-121712 inhibits the growth of E. coli O157:H7,
Salmonella typhimurium; Salmonella enteriditis; and the Big-Six
Escherichia coli strains (referred to as the non-O157 STECs) that
include E. coli O121:H19; E. coli O45:H2; E. coli O103:H11; E. coli
O145, E. coli O26:H11; and E. coli O111, albeit some more
effectively than others.
[0094] As an example, and as disclosed above, the constituent
bacteria are provided in equal amounts and sum to the same total
amount of inhibiting bacteria as pathogenic bacteria that are
initially mixed together, before incubation, and inhibition is
measured.
[0095] Certain aspects of the disclosure contemplate a carrier
formulation for the bacterial combinations. The carrier may be any
number of different percentages (weight per weight, weight per
volume, or volume per volume) of the final product. The carrier can
comprise any amount of about 99.9%, about 95%, about 90%, about
80%, about 70%, about 60% about 50%, about 40%, about 30% and so
on. The remaining composition can also include other carriers such
as lactose, glucose, sucrose, salt, cellulose and the like. In
specific aspects of the disclosure, the carrier may be 50% or more
of the total product.
[0096] The carrier and composition can also have defined
properties, such as solubility/insolubility in water or
solubility/insolubility in fat and the like.
[0097] Other chemicals or materials principally used for the
reduction or absorption of moisture may also be included. These may
include, but are not limited to: calcium stearate, sodium
aluminosilicate, silica, calcium carbonate, zeolite, bicarbonates,
sodium sulfate, silicon dioxide, or ascorbic acid.
[0098] Other chemicals or materials principally used for the
reduction or absorption of oxygen may also be included. These may
include, but are not limited to, iron oxides, ascorbic acid, sodium
sulfide, and silica materials.
[0099] The bacteria mixed with the carrier can be stored in a pouch
or bag fabricated from various materials, a bottle fabricated from
a variety of materials, a capsule, a box, or other storage
container. The composition may also be applied onto a variety of
foods including, but not limited to, meats, vegetables, fruits,
processed foods, and others for the purpose of pathogen inhibition.
The composition can also be packaged as a probiotic supplement for
human consumption, particularly when capsulized or made into
tablets.
[0100] Preservation methods for the bacteria can include a process
of freezing, freeze-drying and/or spray-drying. In certain aspects,
the preserved bacteria contain a viable cell concentration of
1.times.10.sup.8 to 5.times.10.sup.12 cfu/g. Still further, in
certain aspects the concentrations range from 5.times.10.sup.10
cfu/g to 5.times.10.sup.13 cfu/g of bacteria.
[0101] In certain instances, a bacterial formulation for
administration to a subject or a surface or other target can
include a preservation matrix, which contains and preserves the
bacterial culture. Such a matrix may include a biologically active
binding agent, an antioxidant, a polyol, a carbohydrate and a
proteinaceous material. For example, the matrix may have a pH of
from about 5.0 to about 7.0. Such a preservation matrix may be
capable of maintaining at least about 10.sup.6 viable cells for a
period of at least about 12 months in vitro. In other examples,
such a matrix maintains at least about 10.sup.7 viable cells for a
period of at least about 12 months in vitro, and more preferably,
at least about 10.sup.8 viable cells for a period of at least about
12 months in vitro. A preservation matrix may be comprised of
ingredients to minimize the damaging effects encountered during the
preservation process and to provide functional properties. For
example when a Lactobacillus strain of the present disclosure is
added to a preservation matrix for preservation, it may be
converted from an actively growing metabolic state to a
metabolically inactive state.
[0102] In formulations of the present disclosure wherein a
preservation matrix is contemplated, a biologically acceptable
binding agent can be used to both affix the bacterial culture or
cultures to an inert carrier during a preservation process and to
provide protective effects (i.e., maintains cell viability)
throughout preservation and storage of the bacterial cells.
[0103] Antioxidants included in a preservation matrix may be
provided to retard oxidative damage to the bacterial cells during
the preservation and storage process.
[0104] Polyols (i.e., polyhydric alcohols) included in a
preservation matrix may be provided to maintain the native,
uncollapsed state of cellular proteins and membranes during the
preservation and storage process. In particular, polyols interact
with the cell membrane and provide support during the dehydration
portion of the preservation process.
[0105] Carbohydrates included in a preservation matrix may be
provided to maintain the native, uncollapsed state of cellular
proteins and membranes during the preservation and storage process.
In particular, carbohydrates provide cell wall integrity during the
dehydration portion of the preservation process.
[0106] A proteinaceous material included in a preservation matrix
may provide further protection of the bacterial cell during the
dehydration portion of the preservation process. Examples of the
proteinaceous materials include, but are not limited to skim milk
and albumin.
[0107] One example of a method of preserving bacterial cells within
a preservation matrix includes coating the cell matrix suspension
onto an inert carrier that preferably is a maltodextrin bead. The
coated beads can then be dried, preferably by a fluid bed drying
method. The coated maltodextrin beads can be stored as a powder,
placed into gelatin capsules, or pressed into tablets.
[0108] In other formulations of the disclosure, the combinations of
strains of bacteria contemplated to be cultured can be formulated
as a hard gelatin capsule.
[0109] In certain applications, the bacteria cultured with the
methods described herein may be placed in a microencapsulation
formulation. Such microencapsulation formulations may have
applicability for example in administration to subjects via oral,
nasal, rectal, vaginal or urethral routes. Spray drying is the most
commonly used microencapsulation method in the food industry as it
is economical, flexible and produces a good quality product. The
process involves the dispersion of the core material into a polymer
solution, forming an emulsion or dispersion, followed by
homogenization of the liquid, then atomization of the mixture into
the drying chamber. This leads to evaporation of the solvent
(water) and hence the formation of matrix type microcapsules.
[0110] The drying process is carried out in such a manner that a
low residual moisture content is present in the dry material. The
percentage water content is preferably from about 2 to 3% by
weight. This may be achieved by adding a post-drying step
subsequent to the spray-drying step. The drying material for this
purpose is, for example, post-dried in a fluidized bed.
[0111] Instead of the above-described physical post-drying
processes, desiccants can also be added to the dry material
obtained from the spray-drying.
[0112] The content of viable microorganisms is in the range of from
about 5.times.10.sup.5 to 1.times.10.sup.12 cfu/g of dry matter.
These preparations are also referred to as powder concentrates.
[0113] Some end uses require fewer viable microorganisms and are
therefore blended to a lower final concentration by mixing the
bacteria with larger proportions of inert carrier material.
[0114] Some bacteria can survive environmental stresses through the
formation of spores. This complex developmental process is often
initiated in response to nutrient deprivation. It allows the
bacteria to produce a dormant and highly resistant cell. Spores can
survive environmental assaults that would normally kill other
bacteria. Some stresses that endospores can withstand include
exposure to high temperatures, high UV irradiation, desiccation,
chemical damage and enzymatic destruction. The extraordinary
resistance properties of endospores make them of particular
importance because they are not readily killed by many
antibacterial treatments. Common bacteria that form spores include
species from the Bacillus and Clostridium genera. Spores formed by
these bacteria remain in their dormant state until the spores are
exposed to conditions favorable for growth. Spores of the specified
bacteria can be beneficially utilized in the disclosed compositions
because of their ability to withstand processing methods and can
have extended shelf life viabilities. Additionally, bacterial
spores can require less processing because they do not require
additional steps for preservation such as freeze drying, spray
drying, freezing and the like.
[0115] The disclosed composition can be fed to ruminal fermentors
to reduce scours events, improve animal health and animal
productivity. Ruminal fermentors that can benefit from the present
disclosure include but are not limited to: cattle, sheep, goats,
camels, llama, bison, buffalo, deer, wildebeest, antelope, and any
other pre-gastric fermentor. Alternatively, the composition can be
fed to cecal fermentors to reduce scours events, improve animal
health and animal productivity. Cecal fermentors that can benefit
from the present disclosure include but are not limited to: horses,
ponies, elephants, rabbits, hamsters, rats, hyraxes, guinea pigs,
and any other post-gastric fermentor that using the cecum as the
primary location of fermentative digestion. The composition can
also be fed to intestinal fermentors to reduce scours events,
improve animal health and animal productivity. Intestinal
fermentors that can benefit from said disclosure include but are
not limited to: humans, pigs, chickens, and other post-gastric
fermentor using the large intestine as the primary location of
fermentative digestion.
[0116] The amount of bacteria administered to the animal feed can
be any amount sufficient to achieve the desired increase in animal
efficiency and/or animal health. This amount can be anywhere from 1
to 10.sup.13 organisms per kg of animal feed. For example, amounts
of about 10.sup.4 cfu/gram feed, about 5.times.10.sup.4 cfu/gram
feed, about 10.sup.5 cfu/gram feed, about 5.times.10.sup.5 cfu/gram
feed, or ranges between 1 to 10.sup.13 organisms per kg of animal
feed can be used. In some embodiments, the dried biological
(bacteria) may be administered to an animal through a variety of
means including, but not limited to, being distributed in an
aqueous solution and subsequently being applied to animal feed,
water source, or directly fed to the animal, or through direct
application of the product onto animal feed or direct
administration or consumption by the animal.
[0117] In certain examples of the instant composition, the bacteria
and methods of the present disclosure involve two or more bacteria.
In certain examples, at least some of the bacteria are lactic
acid-producing bacteria. These compositions would be provided in a
combined amount effective to achieve the desired effect, for
example, the killing or growth inhibition of a pathogenic
microorganism. This process may involve administering different
strains or species of lactic acid producing microorganisms at the
same time. In certain embodiments the different strains or species
may be combined into a single formulation for administration. In
other embodiments, the different strains or species of lactic acid
producing microorganisms may be each in a single formulation for
administration. Still in other embodiments, some lactic acid
producing microorganism strains or species may be combined into a
single formulation and others may be combined into a different
formulation.
[0118] When more than one inhibiting strain is included in the
composition, the several bacterial components may be administered
to the animal at the same time or in a sequence sufficiently close
together to instill similar effects in the animal as when the
bacteria are administered at the same time. When serially
administered, it is contemplated that the separate formulations can
be administered within about 12-24 hours of each other and, more
preferably, within about 6-12 hours of each other. In some
situations, it may be desirable to extend the time period for
administration significantly such that days or weeks can lapse
between the respective administrations.
[0119] Various combinations may be employed, for example a
formulation containing two species of lactic acid producing
microorganisms is "A" and a second formulation containing three
species of lactic acid producing microorganisms is "B."
[0120] In such embodiments, the administration may be, for example
as such: A/B/A, B/A/B, B/B/A, A/B/B, A/B/B, B/A/A, A/B/B/B,
B/A/B/B, B/B/B/A, B/B/A/B, A/A/B/B, A/B/A/B, A/B/B/A, B/B/A/A,
B/A/B/A, B/A/A/B, A/A/A/B, B/A/A/A, A/B/A/A or A/A/B/A. It is
further contemplated that other administrations may be used with
three or more different formulations of lactic acid producing
microorganisms.
[0121] In one example, the composition is designed for continual
(once daily, for example) or periodic administration to ruminal
fermentors throughout a feeding period in order to reduce the
incidence and severity of diarrhea and/or improve overall health
and/or inhibit pathogens associated with the animal. In this
embodiment, the composition comprises a mixture of probiotic
bacteria supplemented with prebiotic substances that can be
introduced into the rumen and intestines of a ruminal
fermentor.
[0122] In another example, the composition is designed for
continual or periodic administration to cecal fermentors throughout
feeding period in order to reduce the incidence and severity of
diarrhea and/or improve overall health and/or inhibit pathogens
associated with the animal. In this embodiment, the composition
comprises a mixture of probiotic bacteria supplemented with
prebiotic substances that can be introduced into the cecum and
intestines of a cecal fermentor.
[0123] In yet another example, the novel composition is designed
for continual or periodic administration to intestinal fermentors
throughout the feeding period in order to reduce the incidence and
severity of diarrhea and/or improve overall health and/or inhibit
pathogens associated with the animal. In this embodiment, the
composition comprises a mixture of probiotic bacteria supplemented
with prebiotic substances that can be introduced into the
intestines of an intestinal fermentor.
[0124] A wide range of pathogenic bacteria can be inhibited or
eliminated through the disclosed compositions of bacteria such as
lactic acid producing probiotic bacteria. Specific examples of
infectious diseases or conditions of animals which can be caused by
pathogenic bacteria include, but are not limited to: staphylococcal
infections (caused, for example, by Staphylococcus aureus,
Staphylococcus epidermis, or Staphylococcus saprophyticus),
streptococcal infections (caused, for example, by Streptococcus
pyogenes, Streptococcus pneumoniae, or Streptococcus agalactiae),
enterococcal infections (caused, for example, by Enterococcus
faecalis) diphtheria (caused, for example, by Corynebacterium
diptheriae), anthrax (caused, for example, by Bacillus anthracis),
listeriosis (caused, for example, by Listeria monocytogenes),
gangrene (caused, for example, by Clostridium perfringens), tetanus
(caused, for example, by Clostridium tetanus), botulism (caused,
for example, by Clostridium botulinum), toxic enterocolitis
(caused, for example, by Clostridium difficile), bacterial
meningitis (caused, for example, by Neisseria meningitidis),
bacteremia (caused, for example, by Neisseria gonorrhoeae), E. coli
infections (colibacilliocis), including urinary tract infections
and intestinal infections, shigellosis (caused, for example, by
Shigella species), salmonellosis (caused, for example, by
Salmonella species), Yersinia infections (caused, for example, by
Yersinia pestis, Yersinia pseudotuberculosis, or Yersinia
enterocolitica), cholera (caused, for example, by Vibrio cholerae),
campylobacteriosis (caused, for example, by Campylobacter jejuni or
Campylobacter fetus), gastritis (caused, for example, by
Helicobacter pylori), pseudomonas infections (caused, for example,
by Pseudomonas aeruginosa or Pseudomonas mallei), Haemophilus
influenzae type B (HIB) meningitis, HIB acute epiglottitis, or HIB
cellulitis (caused, for example, by Haemophilus influenzae),
pertussis (caused, for example, by Bordetella pertussis),
mycoplasma pneumonia (caused, for example, by Mycoplasma
pneumoniae), nongonococcal urethritis (caused, for example, by
Ureaplasma urealyticum), legionellosis (caused, for example, by
Legionella pneumophila), syphillis (caused, for example, by
Treponema pallidum), leptospirosis (caused, for example, by
Leptospira interrogans), Lyme borreliosis (caused, for example, by
Borrelia burgdorferi), tuberculosis (caused, for example, by
Mycobacterium tuberculosis), leprosy (caused, for example, by
Mycobacterium leprae), actinomycosis (caused, for example, by
Actinomyces species), nocardiosis (caused, for example, by Nocardia
species), chlamydia (caused, for example, by Chlamydia psittaci,
Chlamydia trachomatis, or Chlamydia pneumoniae), Rickettsial
diseases, including spotted fever (caused, for example, by
Rickettsia ricketsii) and Rickettsial pox (caused, for example, by
Rickettsia akari), typhus (caused, for example, by Rickettsia
prowazekii), brucellosis (caused, for example, by Brucella abortus,
Brucella melitens, or Brucella suis), and tularemia (caused, for
example, by Francisella tularensis).
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