Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHODS FOR INCREASING THE SOLUBILITY OF NUTRITIONAL MAT~R1ALS USING PROBIOTIC
LACTIC ACID-PRODUCING
BACTER1A
FIELD OF THE INVENTION
S The present invention relates to the utilization of probiotic lactic acid-
producing bacteria
in a nutritional composition. More specifically, the present invention relates
to the use of
Bacillus coagulans for increasing the solubility of nutritional materials,
preferably essential
vitamins and minerals, within the gastrointestinal tract of mammals.
BACKGROUND OF THE INVENTION
1. Probiotic Microorganisms
Probiotic microorganisms are those which confer a benefit when grow in a
particular
environment, often by inhibiting the growth of other biological organisms in
the same
environment. Examples of probiotic organisms include bacteria and
bacteriophages which
possess the ability to grow within the gastrointestinal tract, at least
temporarily, to displace or
1S destroy pathogenic organisms, as well as providing other benefits to the
host. See e.g., Salminen
et al, 1996. Antonie van Leeuwenhoek 70: 347-358; Elmer et al, 1996. JAMA 275:
870-876;
Rafter, 1995. Scand J. Gastroenterol. 30: 497-502; Perdigon et al, 1995. J.
Dairy Sci. 78: 1 597-
1606; Gandi, Townsend Lett. Doctors & Patients, pp. 108-110, Jan. 1994;
Lidbeck et al. 1992.
Eur. J. Cancer Prev. l: 341-353.
The majority of previous studies on probiosis have been observational rather
than
mechanistic in nature, and thus the processes responsible for many probiotic
phenomena have yet
to be quantitatively elucidated. Some probiotics are members of the normal
colonic microflora
and are not viewed as being overtly pathogenic. However, these organisms have
occasionally
caused infections {e.g., bacteremia) in individuals who are, for example,
immunocompromised.
2S See e.g., Sussman, J. et al., 1986. Rev Infect. Dis. 8: 771-776; Hata, D.
et al., 1988. Pediatr.
Infect. Dis. 7: 669-671.
For example, the probiotic bacteria found in sour milk, has been utilized
since ancient
times (i.e., long-before the discovery of bacteria) as a therapeutic treatment
for dysentery and
related gastrointestinal diseases. More recently, probiotic preparations were
systematically
evaluated for their effect on health and longevity in the early-1900's (see
e.g., Metchinikoff, E.,
Prolongation of Life. Willaim Heinermann. London 1910), although their
utilization has been
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markedly limited since the advent of antibiotics in the 1950's to treat
pathological microbes. See
e.g.. Winberg, et al. 1993. Pediatr. ~l~ephrol. -: 509-S 14; Malin cn al. Ann-
Nutr. rl~letab. ~0: 137-
145; and U.S. Patent No. 5,176.91 I. Similarly, lactic acid-producing bacteria
(e.g.. Bacillus,
Lactobacillus and Streptococcus species) have been utilized as food additives
and there have
been some claims that they provide nutritional and/or therapeutic value. See
e.g., Gorbach.
1990. Ann. Med Z?: 37-41; Reid et ul, I990. Clin. ~Llicrobiol. Rev. 3: 335-
344.
The best known probiotics are the lactic acid-producing bacteria (i.e.,
Lactobacilli) and
Bifidobacteria, which are widely utilized in yogurts and other dairy products.
These probiotic
organisms are non-pathogenic and non-toxigenic, retain viability during
storage, and possess the
ability to survive passage through the stomach and small intestine. Since
probiotics do not
permanently colonize the host, they need to be ingested or applied regularly
for any health-
promoting properties to persist. Commercial probiotic preparations are
generally comprised of
mixtures of Lactobacilli and Bifidobacteria, although yeast such as
Saccharomyces have also
been utilized.
2. Gastrointestinal Microflora
Perhaps the best-characterized use of probiotic microorganisms is in the
maintenance of
gastrointestinal microflora. The gastrointestinal microflora has been shown to
play a number of
vital roles in maintaining gastrointestinal tract function and overall
physiological health. For
example, the growth and metabolism of the many individual bacterial species
inhabiting the
gastrointestinal tract depend primarily upon the substrates available to them,
most of which are
derived from the diet. See e.g., Gibson G.R. et al., 1995. Gastroenterology
I06: 975-982;
Christi, S.U. et al., 1992. Gut 33: 1234-1238. These finding have led to
attempts to modify the
structure and metabolic activities of the community through diet, primarily
with probiotics which
are live microbial food supplements.
While the gastrointestinal microflora presents a microbial-based barrier to
invading
organisms, pathogens often become established when the integrity of the
microbiota is impaired
through stress, illness, antibiotic treatment, changes in diet, or
physiological alterations within
the gastrointestinal tract. For example, Bifidobacteria are known to be
involved in resisting the
colonization of pathogens in the large intestine. See e.g.. Yamazaki, S. et
al., 1982.
Bifrdobacteria and Microflora l: 5~-60. Similarly. the administration of
Bifidobacteria breve to
children with gastroenteritis eradicated the causative pathogenic bacteria
(i.e., Campylobacter
jejuni) from their stools (see e. g., Tojo, M.. 1987. ,~lcta Pediatr~. Jpn.
?9: 160-167) and
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supplementation of infant formula milk with Bifidobacteria bifidum and
Streptococcus
thermophilus was found to reduce rotavirus shedding and episodes of diarrhea
in children who
were hospitalized (see e.g., Saavedra. J.M., 1994. The Lancet 3-1-l: 1046-109.
In addition, some lactic acid producing bacteria also produce bacteriocins
which are
S inhibitory metabolites which are responsible for the bacteria's anti-
microbial effects. See e.g.,
Klaenhammer, 1993. FEMS Microbiol. Rev. 12: 39-85; Barefoot et al., 1993. J.
Diary Sci. 76:
2366-2379. For example, selected Lactobacillus strains which produce
antibiotics have been
demonstrated as effective for the treatment of infections, sinusitis.
hemorrhoids, dental
inflammations, and various other inflammatory conditions. See e.g., U.S.
Patent No. 5,439,995.
Additionally, Lactobacillus reuteri has been shown to produce antibiotics
which possess anti-
microbial activity against Gram negative and Gram positive bacteria, yeast,
and various
protozoan. See e.g., U.S. Patent Nos. x,413.960 and 5,439,678. Lactobacillus
easel ssp,
rhamnosus has been utilized to preserve animal feedstuffs, particularly S.
rubidaea F 13299,
alone or combined with an anti-fungal B. subtillis (swain FB260). See U.S.
Patent No.
5,371,011.
Probiotics have also been shown to possess anti-mutagenic properties. For
example,
Gram positive and Gram negative bacteria have been demonstrated to bind
mutagenic
pyrolysates which are produced during cooking at a high temperature. Studies
performed with
lactic acid-producing bacteria has shown that these bacteria may be either
living or dead, due to
the fact that the process occurs by adsorption of mutagenic pyrolysates to the
carbohydrate
polymers present in the bacterial cell wall. See e.g., Zang, X. Bacillus et
al., 1990. J. Dairy Sci.
73: 2702-2710. Lactobacilli have also been shown to possess the ability to
degrade carcinogens
(e.g., N-nitrosamines), which may serve an important role if the process is
subsequently found to
occur at the level of the mucosal surface. See e.g., Rowland, LR. and Grasso,
P., Appl.
Microbiol. 29: 7-12. Additionally, the co-administration of lactulose and
Bifidobacteria longum
to rats injected with the carcinogen azoxymethane was demonstrated to reduce
intestinal aberrant
crypt foci, which are generally considered to be pre-neoplastic markers. See
e.g., Challa, A. et
al., 1997. Carcinogenesis 18: 5175-21. Purified cell walls of Bifidobacteria
may also possess
anti-tumorigenic activities in that the cell wall of Bifidobacteria infantis
induces the activation of
phagocytes to destroy growing tumor cells. See e.g., Sekine. K. et al., 1994.
Bifidobacteria and
Microflora 13: 65-77. Bifidobacteria probiotics have also been shown to reduce
colon
carcinogenesis induced by 1,2-dimethylhydrazine in mice when concomitantly
administered with
fructo-oligosaccharides(FOS; see e. g.. Koo and Rao. 1991. Nutrit. Rev. .il:
137-146), as well as
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inhibiting liver and mammary tumors in rats (see e.g.. Reddy and Rivenson.
1993. C'uncer Res.
.53: 3914-3918).
3. Physiological Effects of Antibiotic Administration
Antibiotics are widely used to control pathogenic microorganisms in both
humans and
animals. Unfortunately, the widespread use of anti-microbial agents,
especially broad spectrum
antibiotics, has resulted in a number of serious clinical consequences. For
example, the
indiscriminate use of these chemicals has resulted in the generation of
multiple antibiotic-
resistant pathogens. See e.g., Mitchell, P. 1998. The Lancet 3.52: 462-463;
Shannon, K., 1998.
The Lancet 352: 490-491.
In addition, antibiotics often kill beneficial, non-pathogenic microorganisms
(i. e., flora)
within the gastrointestinal tract which contribute to digestive function and
health. Accordingly,
relapse (the return of infections and their associated symptoms) and secondary
opportunistic
infections often result from the depletion of lactic acid-producing and other
beneficial flora
within the gastrointestinal tract. Most, if not all, lactic acid-producing or
probiotic bacteria are
extremely sensitive to common antibiotic compounds. During a normal course of
antibiotic
therapy, many individuals develop a number of deleterious physiological side-
effects including:
diarrhea, intestinal cramping, and sometimes constipation. These side-effects
are primarily due
to the non-selective action of antibiotics, as antibiotics do not possess the
ability to discriminate
between beneficial, non-pathogenic and pathogenic bacteria, both bacterial
types are killed by
these agents. Thus, individuals taking antibiotics offer suffer from
gastrointestinal problems as a
result of the beneficial microorganisms (i. e., intestinal flora), which
normally colonize the
gastrointestinal tract, being killed or severely attenuated. The resulting
change in the
composition of the intestinal flora can result in vitamin deficiencies when
the vitamin-producing
intestinal bacteria are killed, diarrhea and dehydration and, more seriously,
illness should a
pathogenic organism overgrow and replace the remaining beneficial
gastrointestinal bacteria.
In addition to the gastrointestinal microflora, beneficial and/or pathological
microorganisms can also inhabit the oral cavity, the genital area and the
vagina (see e.g.,
Thomason, et al, 1991. Am. J. Obstet Gynecol. 165: 1210-1217; Marsh. 1993.
Caries Res. 27:
72-76; Lehner, 1985. vaccine 3: 65-68; Hill & Embil, 1986. Can. Med. Assoc. J.
134: 321-331).
The use of anti-microbial drugs can similarly cause an imbalance in those
microorganisms and
the therapeutic use of probiotic bacteria, especially the Lactobacillus
strains, which colonize
those areas has been disclosed (see e.g., Winberg, et al.. 1993. Pediatr.
Nephrol. 7: 509-514;
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Malm, c~t al.. 1996. Ann. ~LJar. Afetab. 40: 137-l~t~, U.S. Patent No.
x.176.911). Increasing
numbers of pathogenic microorganisms have developed antibiotic resistance,
requiring the
development and use of second and third generation antibiotics. Microorganisms
that are
resistant to multiple drugs have also developed, often with multiple drug
resistance spreading
between species. leading to serious infections that cannot be controlled by
use of antibiotics.
4. Bacillus coagulans
Bacillus coagulans is a non-pathogenic gram positive spore-forming bacteria
that
produces L(+) lactic acid (dextrorotatory) in homo-fermentation. This
microorganism has been
isolated from natural sources, such as heat-treated soil samples inoculated
into nutrient medium
(see e.g., Bergey's Manual ofSystemic Bacteriology, Vol. 2, Sneath, P.H.A., et
al., eds.,
(Williams & Wilkins, Baltimore, MD, 1986)).
Purified Bacillus coagulans strains have served as a source of various enzymes
including,
but not limited to: restriction endonucleases (see U.S. Patent No. 5,200,336);
amylase (see U.S.
Patent No. 4,980,180); lactase (see U.S. Patent No. 4,323,651); and cyclo-
malto-dextrin glucano-
transferase (see U.S. Patent No. 5,102,800). Bacillus coagulans has been used
to produce lactic
acid (see U.S. Patent No. 5,079,164). In addition, a strain ofBacillus
coagulans (designated
Lactobacillus sporogenes, Sakaguti & Nakayama (ATCC 31284)) has been combined
with other
lactic acid-producing bacteria and Bacillus natto to produce a fermented food
product from
steamed soybeans (see U.S. Patent No. 4,110,477). Bacillus coagulans strains
have also been
used as animal feed additives for poultry and livestock to reduce disease and
improve feed
utilization and to, therefore, increase growth rate in the animals (see
International Patent
Application Nos. WO 9314187 and WO 9411492).
In addition, Bacillus coagulans strains and various other lactic acid-
producing bacteria
have proven to produce copious amounts of lactic acid and enzymes that have
the ability to
increase the solubility of nutritional materials, in particular vitamins and
minerals.
Accordingly, there remains an, as yet unfulfilled, need for a composition
which possesses
the ability to increase the solubility and hence, bioavailability of
nutritional materials, preferably
vitamins and minerals, within the gastrointestinal tract of animals or humans.
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SUMMARY OF THE INVENTION
The present invention discloses the discovery that, in digestion, bacterial
enzymes and
various other metabolic products of bacteria (e.g., lactic acid), play a vital
role in the solubility
and therefor the bioavailability, of vitamins and minerals, either as a
component of food or as a
result of nutritional supplementation.
The present invention discloses a probiotic composition comprising one or more
Bacillus
bacterial species or strains, combined with an effective amount of a
bifidogenic oligosaccharide,
preferably fructo-oligosaccharide(FOS), in a pharmaceutically- or
nutritionally-acceptable carrier
which is suitable for oral administration to a human or animal species,
wherein said Bacillus: (i)
is capable of growing at temperatures of about 20°C to about
65°C; (ii) produces L(+) lactic acid
(dextrorotatory); (iii) produces spores resistant to heat up to 98°C;
and (iv) exhibits probiotic
activity. In one embodiment, the probiotic activity results from the
vegetative growth of the
isolated Bacillus. Lactobacillus, Bifrdiobacterium, or Sporolactobacillus
species that produces
extracellular metabolites which function to increase the bioavailability of
nutrients though
increased solubility.
The present invention also discloses a therapeutic composition comprising one
or more
Bacillus coagulans strain in combination with a therapeutically-effective
amount of a short or
long chain bifidogenic oligosaccharide, preferably fructa-oligosaccharide
(FOS), in a
pharmaceutically- or nutritionally-acceptable earner suitable for oral
administration to the
digestive track of a mammal. In different embodiments of the aforementioned
composition, the
Bacillus coagulans strain is included in the composition in the form of spores
which are capable
of germinating following ingestion; a dried cell mass; a stabilized gel or
paste; or a stabilized
liquid suspension..
The present invention also describes a method for decreasing the number of
pathogenic
organisms that inhibit the growth of vitamin-producing organisms within the
gastrointestinal
tract, wherein a composition is administered that would, preferably, include a
complex of the
RDA vitamins, minerals and trace minerals as well as those nutrients that have
no established
RDA, but have a beneficial role in healthy human or mammal physiology. The
preferred
composition would include a minerals which are in either in the gluconate or
citrate form, due to
the established fact that these forms are more readily metabolized by lactic
acid-producing
bacteria.
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It should be understood that both the foregoing general description and the
following
detailed description are exemplary and explanatory only and are not
restrictive of the invention
as claimed.
DESCRIPTION OF THE FIGURES
FIG. I : illustrates, in tabular form, the various metabolic characteristics
of
Bacillus coagulans.
FIG. 2: illustrates, in tabular form, the vitamins, their RDA, their ranges
and
respective preferred ranges, of the therapeutic compositions of the present
invention.
FIG. 3: illustrates, in tabular form, the preferred major minerals and their
respective RDA.
FIG. 4: illustrates, in tabular form, the preferred trace minerals and their
respective
ranges, as utilized in the therapeutic composition of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The disclosures of one or more embodiments of the invention are set forth in
the
accompanying description below. Although any methods and materials similar or
equivalent to
those described herein can be used in the practice or testing of the present
invention, the
preferred methods and materials are now described. Other features, objects,
and advantages of
the invention will be apparent from the description and from the claims. In
the specification and
the appended claims, the singular forms include plural referents unless the
context clearly
dictates otherwise. Unless defined otherwise, all technical and scientific
terms used herein have
the same meaning as commonly understood by one of ordinary skill in the art to
which this
invention belongs. Unless expressly stated otherwise, the techniques employed
or contemplated
herein are standard methodologies well known to one of ordinary skill in the
art. The examples
of embodiments are for illustration purposes only. All patents and
publications cited in this
specification are incorporated by reference.
As utilized herein, the term "probiotic" refers to microorganisms (e.g.,
bacteria, yeast,
viruses, and/or fungi) which form, at a minimum, a part of the transient or
endogenous flora and,
thus, possess a beneficial prophylactic and/or therapeutic effect upon the
host organism.
Probiotics are generally known to be clinically-safe (i.e.. non-pathogenic) by
those skilled within
the art. Although not wishing to be bound by any particular mechanism, the
probiotic activity of,
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for example, the Bacillus species, is thought to result from competitive
inhibition of growth of
pathogens due to superior colonization, parasitism of undesirable
microorganisms, lactic acid
production and/or other extracellular products having anti-microbial activity,
or combinations
thereof.
As utilized herein, the term ''nutritional supplement'' refers to any type of
orally
ingestable material which can be digested and assimilated by the
gastrointestinal tract, and from
which a physiological, nutritional benefit may be derived. This material
includes liquid and solid
foodstuffs, vitamin and mineral supplements, and the like.
The present invention discloses the utilization of Bacillus, Lactobacillus,
Bifidiobacterium, or Sporolactobacillus bacterial species, preferably Bacillus
coagulans, in
nutritional compositions, in combination with a bifidogenic oligosaccharide,
preferably fructo-
oligosaccharides (FOS), as a probiotic for increasing the solubility of
essential vitamin and
mineral nutritional materials in mammals, particularly in humans. The present
invention
therefore describes various nutritional compositions, methods for using the
compositions and
systems containing the nutritional compositions.
Non-pathogenic Bacillus species of the present invention include, but are not
limited to:
Bacillus coagulans; Bacillus coagulans Hammer; Bacillus brevis subspecies
coagulans, Bacillus
subtilis, Bacillus uniflagellatus, Bacillus lateropsorus, Bacillus
laterosporus BOD, Bacillus
megaterium, Bacillus polymyxa, Bacillus licheniformis, Bacillus pumilus, and
Bacillus
sterothermophilus, and any genetic variants thereof.
Exemplary lactic acid-producing Lactobacillus species include, but are not
limited to:
Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus DDS-l,
Lactobacillus GG,
Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri,
Lactobacillus
gasserii, Lactobacillus jensenii, Lactobacillus delbruekii, Lactobacillus,
bulgaricus,
Lactobacillus salivarius and Lactobacillus sporogenes (also designated as
Bacillus coagulans),
and any genetic variants thereof.
Exemplary lactic acid-producing Sporolactobacillus species include all
Sporolactobacillus species, for example, Sporolactobacillus P44, and any
genetic variants
thereof.
Exemplary lactic acid-producing Bifidiobacterium species include, but are not
limited to:
Bifidivbacterizrm adolescentis, Bifidiobacterium animalis, Bifidiobacterium
bifidum,
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Brfrdiobacterium bijidzrs. Bifidiobacterizrm breve. BiJidiobacterium infantis.
Bifidiobacterium
infantis. Bifidiobacterium longum, and any genetic variants thereof.
1. Probiotic, Lactic Acid-Producing Bacillus Strains
By way of example, and not of limitation to any particular mechanism, the
prophylactic
and/or therapeutic effect of a lactic acid-producing bacteria of the present
invention results. in
part, from a competitive inhibition of the growth of pathogens due to: (i)
their superior
colonization abilities; (ii) parasitism of undesirable microorganisms; (iii)
the production of lactic
acid and/or other extracellular products possessing anti-microbial activity;
or (iv) various
combinations thereof. It should be noted that the aforementioned products and
activities of the
lactic acid-producing Bacillus of the present invention act synergistically to
produce the
beneficial probiotic effect disclosed herein.
A probiotic bacteria which is suitable for use in the methods and compositions
of the
present invention: (i) possesses the ability to produce lactic acid; (ii)
demonstrates beneficial
function; and (iii) is non-pathogenic. By way of example and not of
limitation, many suitable
bacteria have been identified and are described herein, although it should be
noted that the
present invention is not to be limited to currently-classified bacterial
species insofar as the
purposes and objectives as disclosed. The physiochemical results from the in
vivo production of
lactic acid is key to the effectiveness of the probiotic lactic acid-producing
bacteria of the present
invention. Lactic acid production markedly decreases the pH (i.e., increases
acidity) within the
local micro-floral environment and does not contribute to the growth of many
undesirable,
physiologically-deleterious bacteria, fungi, and viruses. Thus, the bacterial
enzymes and various
other metabolic products of the probiotic bacteria (e.g., lactic acid) serve
to inhibit the growth of
competing pathogenic bacteria, as well as concomitantly increasing the
solubility and therefore
the bioavailability, of vitamins and minerals within the gastrointestinal
tract.
Typical lactic acid-producing bacteria useful as a probiotic of this invention
are efficient
lactic acid producers which include non-pathogenic members of the Bacillus
genus which
produce bacteriocins or other compounds which inhibit the growth of pathogenic
organisms.
Exemplary lactic acid-producing, non-pathogenic Bacillus species include, but
are not limited to:
Bacillus coagulans; Bacillus coagulans Hammer; and Bacillus brevis subspecies
coagulans.
Several Bacillus species which are preferred in the practice of the present
invention,
include, but are not limited to the lactic acid-producing Bacillus coagulans
and Bacillus
laevolacticus. Various other non-lactic acid-producing Bacillus species may be
utilized in the
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present invention so long as they produce compounds which possess the ability
to inhibit
pathogenic bacterial or mycotic growth. Examples of such suitable non-lactic
acid-producing
Bacillus include, but are not limited to: Bacillus subtilis, Bacillus
uniflagellatus, Bacillus
lateropsorus, Bacillus laterosporus BOD, Bacillus megaterium, Bacillus
polymyYa, Bacillus
licheniformis, Bacillus pumilus, and Bacillus sterothermophilus.
The Bacillus species, particularly those species having the ability to form
spores (e.g.,
Bacillus coagulans), are a preferred embodiment of the present invention. The
Bacillus species
utilized in the practice of the present invention may selected from the group
comprising: Bacillus
coagulans, Bacillus subtilis, Bacillus laterosporus and Bacillus
laevolacticus, all of which have
the ability to form spores, and can colonize tissue aerobically. There are a
variety of different
Bacillus species, including, but not limited to many different strains
available through
commercial and public sources, such as the American Tissue Culture Collection
(ATCC). For
example, Bacillus coagulans strains are available as ATCC Accession Numbers
15949, 8038,
35670, 11369, 23498, 51232, 11014, 31284, 12245, 10545 and 7050. Bacillus
subtilis strains are
available as ATCC Accession Numbers 10783, 15818, 15819, 27505, 13542, 15575,
33234,
9943, 6051a, 25369, 11838, 15811, 27370, 7003, 15563, 4944, 27689, 43223,
55033, 49822,
15561, 15562, 49760, 13933, 29056, 6537, 21359, 21360, 7067, 21394, 15244,
7060, 14593,
9799, 31002, 31003, 31004, 74$0, 9858, 13407, 21554, 21555, 27328 and 31524.
Bacillus
laterosporus strains are available as ATCC Accession Numbers 6456, 6457, 30
29653, 9141,
533694, 31932 and 64, including Bacillus laterosporus BOD. Bacillus
laevolacticus strains are
available as ATCC Accession Numbers 23495; 23493, 23494, 23549 and 23492. It
should be
noted, however, that although many of the examples herein refer to the
Bacillus coagulans
species in particular, it is intended that any of the Bacillus species can be
used in the
compositions, articles of manufacture, systems and method of the present
invention.
A Bacillus species is particularly suited for the present invention due to the
properties in
common between species of the Bacillus genus, including, but not limited to,
the ability to form
spores which are relatively resistant to heat and other conditions, making
them ideal for storage
(shelf life) in product formulations, and ideal for survival and colonization
of tissues under
conditions of pH, salinity, and the like on tissues subjected to microbial
infection. For example,
probiotic Bacillus coagulans is non-pathogenic and is generally regarded as
safe (i.e., GRAS
classification) by the U.S. Federal Drug Administration (FDA) and the U.S.
Department of
Agriculture (USDA), and by those individuals skilled within the art.
Additional useful
characteristics of the Bacillus species include. but are not limited to: non-
pathogenicity, being
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aerobic, facultative and heterotrophic. thus rendering these species safe. and
able to colonize
skin. mucous membrane tissues. and various other tissues of interest.
Because Bacillus species possesses the ability to produce heat-resistant
spores. it is
particularly useful for making pharmaceutical compositions which require heat
and pressure in
their manufacture. Accordingly, formulations that include the utilization
viable Bacillus spores
in a pharmaceutically-acceptable carrier are particularly preferred for making
and using
compositions disclosed in the present invention. The growth of these various
Bacillus species to
form cell cultures, cell pastes, and spore preparations is generally well-
known within the art. It
should also be noted that the exemplary culture and preparative methods which
are described
herein for Bacillus coagulans may be readily utilized and/or modified for
growth and preparation
of the other Bacillus strains, as well as the lactic acid-producing bacteria
disclosed in the present
invention. In addition, the exemplary methods and compositions which are
described herein
using Bacillus coagulans as a probiotic for controlling, treating, or reducing
microbial infections,
may also be readily utilized with other Bacillus species.
2. Bacillus coagulans
Although, as disclosed herein, any of the aforementioned Bacillus strains may
be utilized
in the practice of the present invention, purified Bacillus coagulans is
exemplary and preferred as
a probiotic for biological control of various microbial pathogens. Because
Bacillus coagulans
forms heat-resistant spores, it is particularly useful for making
pharmaceutical compositions for
treating microbial infections.
2.1 Characteristics and Sources of Bacillus coagulans
Probiotic Bacillus coagulans has been categorized as "safe" by the U.S.
Federal Drug
Administration (FDA), the U.S. Department of Agriculture (USDA), and by those
skilled within
the art. Examples of its beneficial properties include its ability to reduce
the numbers of
pathogenic organisms that can kill vitamin producing organisms in the gut, its
production of
lactase which is a beneficial attribute for those individuals suffering from
lactose intolerance, its
production of bacteriocins that are inhibitory to those intestinal organisms
that produce
carcinogens in the colon, and its well known ability to produce L(+) lactic
acid. which functions
to both metabolize and increase the solubility, absorption, and therefore the
bioavailability of
vitamins and minerals with the gastrointestinal tract.
The Gram positive rods of Bacillus coagulans have a cell diameter of greater
than
0 pm with variable swelling of the sporangium. without parasporal crystal
production.
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WO 00/07606 PCT/US99/17671
Bacillus coagulans is a non-pathogenic, Gram positive. spore-forming bacteria
that produces
L(+) lactic acid (dextrorotatory} under homo-fermentation conditions. It has
been isolated from
natural sources, such as heat-treated soil samples inoculated into nutrient
medium (see e.g.,
Bergey's Manual ofSystemic Bacteriology, Vol. 2, Sneath, P.H.A. et ul., eds.,
Williams &
S Wilkins, Baltimore, MD, 1986). Purified Bacillus coagulans strains have
served as a source of
enzymes including endonucleases (e.g., U.S. Patent No. 5,200,336); amylase
(U.S. Patent No.
4,980,180); lactase (U.S. Patent No. 4,323,651 ) and cyclo-malto-dextrin
glucano-transferase
(U.S. Patent No. x,102,800). Bacillus coagulans has also been utilized to
produce lactic acid
(U.S. Patent No. 5,079,164). A strain of Bacillus coagulans (also referred to
as Lactobacillus
sporogenes; Sakaguti & Nakayama, ATCC No. 31284) has been combined with other
lactic acid
producing bacteria and Bacillus natto to produce a fermented food product from
steamed
soybeans (U.S. Patent No. 4,110,477). Bacillus coagulans strains have also
been used as animal
feeds additives for poultry and livestock to reduce disease and improve feed
utilization and,
therefore, to increase growth rate in the animals (International PCT Patent
Applications No. WO
9314187 and No. WO 9411492). In particular, Bacillus coagulans strains have
been used as
general nutritional materials and agents to control constipation and diarrhea
in humans and
animals.
Purified Bacillus coagulans bacteria utilized in the present invention are
available from
the American Type Culture Collection (ATCC, Rockville, MD) using the following
accession
numbers: Bacillus coagulans Hammer NRS 727 (ATCC No. I 1014); Bacillus
coagulans
Hammer strain C (ATCC No. 11369); Bacillus coagulans Hammer (ATCC No. 31284);
and
Bacillus coagulans Hammer NCA 4259 (ATCC No. 15949). Purified Bacillus
coagulans
bacteria are also available from the Deutsche Sarumlung von Mikroorganismen
and Zellkuturen
GmbH (Braunschweig, Germany) using the following accession numbers: Bacillus
coagulans
Hammer 1915 (DSM No. 2356); Bacillus coagulans Hammer 1915 (DSM No. 2383,
corresponds to ATCC No. I 1014); Bacillus coagulans Hammer (DSM No. 2384,
corresponds to
ATCC No. 11369); and Bacillus coagulans Hammer (DSM No. 2385, corresponds to
ATCC No.
15949). Bacillus coagulans bacteria can also be obtained from commercial
suppliers such as
Sabinsa Corporation (Piscataway, NJ) or K.K. Fermentation (Kyoto, Japan).
Bacillus coagulans strains and their growth requirements have been described
previously
(see e.g., Baker, D. et al, 1960. Can. J. Microbiol. 6: 557-563; Nakamura, H.
et al, 1988. Int. J.
Svst. Bacteriol. 38: 63-73. In addition. various strains of Bacillus coagulans
can also be isolated
from natural sources (e.g., heat-treated soil samples) using well-known
procedures (see e.g.,
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WO 00/07606 PCT/US99/17671
Bergey'.s ,Manual ofSvstemic Bacteriology, 6'vl. ?. p. 1 1 17. Sheath. P.H.A.
et ul.. eds.. Wiliiams
& Wilkins, Baltimore. MD. 1986).
It should be noted that Bacillus coagulans had previously been mis-
characterized as a
Lactobacillus in view of the fact that, as originally described, this
bacterium was labeled as
Lactobacillus sporogenes (See Nakamura et al. 1988. Int. J. Syst. Bacteriol.
38: 63-73).
However, initial classification was incorrect due to the fact that Bacillus
coagulans produces
spores and through metabolism excretes L(+)-lactic acid, both aspects which
provide key
features to its utility. Instead, these developmental and metabolic aspects
required that the
bacterium be classifed as a lactic acid bacillus, and therefore it was re-
designated. In addition, it
is not generally appreciated that classic Lactobacillus species are unsuitable
for colonization of
the gut due to their instability in the harsh (i.e., acidic) pH environment of
the bile, particularly
human bile. In contrast, Bacillus coagulans is able to survive and colonize
the gastrointestinal
tract in the bile environment and even grown in this low pH range. In
particular, the human bile
environment is different from the bile environment of animal models, and
heretofore there has
not been any accurate descriptions of Bacillus coagulans growth in human
gastrointestinal tract
models.
2.2 Growth of Bacillus coagulans
Bacillus coagulans is aerobic and facultative, grown typically in nutrient
broth, pH 5.7 to
6.8, containing up to 2% (wt/vol) NaCI, although neither NaCI nor KCI are an
absolute
requirement for growth. A pH value ranging from approximately pH 4 to pH 6 is
optimum for
initiation of sporulation. It is optimally grown at about 30°C to about
55°C, and the spores can
withstand pasteurization. It exhibits facultative and heterotrophic growth by
utilizing a nitrate or
sulfate source.
Bacillus coagulans can be grown in a variety of media, although it has been
found that
certain growth conditions produce a culture which yields a high level of
sporulation. For
example, sporulation is enhanced if the culture medium includes 10 mg/liter of
manganese
sulfate, yielding a ratio of spores to vegetative cells of about 80:20. In
addition, certain growth
conditions produce a bacterial spore which contains a spectrum of metabolic
enzymes
particularly suited for the present invention (i. e., the control of microbial
infections). Although
spores produced by these particular growth conditions are preferred, spores
produced by any
compatible growth conditions are suitable for producing a Bacillus coagulans
useful in the
present invention.
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(i) Culture of Vegetative Bacillus coagulans
Bacillus coagulans is aerobic and facultative, and is typically cultured at pH
~.7 to 6.8, in
a nutrient broth containing up to 2% (by wt) NaCI, although neither NaCI, nor
KCl are required
for growth. A pH of about 4.0 to about 7.5 is optimum for initiation of
sporulation (i.e., the
formation of spores). The bacteria are optimally grown at 30°C to
45°C, and the spores can
withstand pasteurization. Additionally, the bacteria exhibit facultative and
heterotrophic growth
by utilizing a nitrate or sulfate source. The various metabolic
characteristics of Bacillus
coagulans is illustrated in FIG. 1.
Bacillus coagulans can be cultured in a variety of media, although it has been
demonstrated that certain growth conditions are more efficacious at producing
a culture which
yields a high level of sporulation. For example, sporulation is demonstrated
to be enhanced if
the culture medium includes 10 mg/1 of MgSO; sulfate, yielding a ratio of
spores to vegetative
cells of approximately 80:20. In addition, certain culture conditions produce
a bacterial spore
which contains a spectrum of metabolic enzymes particularly suited for the
present invention
(i. e., production of lactic acid and enzymes for the enhanced probiotic
activity and
biodegradation). Although the spores produced by these aforementioned culture
conditions are
preferred, various other compatible culture conditions which produce viable
Bacillus coagulans
spores may be utilized in the practice of the present invention.
Suitable media for the culture of Bacillus coagulans include: PDB (potato
dextrose
broth); TSB (tryptic soy broth); and NB (nutrient broth), which are all well-
known within the
field and available from a variety of sources. In one embodiment of the
present invention, media
supplements which contain enzymatic digests of poultry and/or fish tissue, and
containing food
yeast are particularly preferred. A preferred supplement produces a media
containing at least
60% protein, and about 20% complex carbohydrates and 6% lipids. Media can be
obtained from
a variety of commercial sources, notably DIFCO (Newark, NJ); BBL
(Cockeyesville, MD);
Advanced Microbial Systems (Shakopee, MN); and Troy Biologicals (Troy, MD.
In a preferred embodiment of the present invention, a culture of Bacillus
coagulans
Hammer bacteria (ATCC No. 31284) was inoculated and grown to a cell density of
approximately 1x108 to 1x109 cells/ml in nutrient broth containing: 5.0 g
Peptone; 3.0 g Meat
Extract; 10-30 mg MnSO, and 1,000 ml distilled water, the broth was then
adjusted to pH 7Ø
The bacteria were cultured by utilization of a standard airlift fermentation
vessel at 30°C. The
range of MnSO, acceptable for sporulation was found to be 1.0 mg/1 to 1.0 g/l.
The vegetative
bacterial cells can actively reproduce up to 65°C. and the spores are
stable up to 90°C.
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Following culture, the Bacillccs coagulans Hammer bacterial cells or spores
were
collected using standard methods (e.g., filtration, centrifugation) and the
collected cells and
spores may subsequently be lyophilized, spray dried, air dried or frozen. As
described herein,
the supernatant from the cell culture can be collected and used as an
extracellular agent secreted
by Bacillus coagulans which possesses anti-microbial activity useful in a
formulation of this
invention.
A typical yield obtained from the aforementioned culture methodology is in the
range of
approximately 1 x109 to 1 x10'3 viable spores and, more typically,
approximately 1 x 10"to l .Sx 10"
cells/spores per gram prior to being dried. It should also be noted that the
Bacillus coagulans
spores, following a drying step, maintain at least 90% viability for up to 7
years when stored at
room temperature. Hence, the effective shelf life of a composition containing
Bacillus
coagulans Hammer spores at room temperature is approximately 10 years.
(its Preparation of Bacillus coagulans Spores
A culture of dried Bacillus coagulans Hammer bacteria (ATCC No. 31284) spores
may
be prepared as follows. Approximately 1 x 10' spores were inoculated into one
liter of culture
medium containing: 24 g (wt./vol.) potato dextrose broth; 10 g of an enzymatic-
digest of poultry
and fish tissue; 5 g of fructo-oligosaccharides (FOS); and 10 g MnS04. The
culture was
maintained for 72 hours under a high oxygen environment at 37°C so as
to produce a culture
having approximately 15x10'° cells/gram of culture. The culture was
then filtered to remove the
liquid culture medium and the resulting bacterial pellet was resuspended in
water and
lyophilized. The lyophilized bacteria were ground to a fine "powder" by use of
standard good
manufacturing practice (GMP) methodologies. The powder is then combined into
Formulation 1
or Formulation 4 as described in Specific Example B to form dry powder
compositions.
It should also be noted that the most preferred embodiments of the present
invention
utilizes Bacillus coagulans in spore, rather than vegetative bacterial form.
(iii Preparation of Bacillus coagulans Extracellular Products
A one liter culture of Bacillus coagulans was prepared as described in Section
2(i),
except that the fructo-oligosaccharide (FOS) was omitted. The culture was
maintained for
~ days as described, at which time FOS was added at a concentration of 5
g/liter, and the culture
was continued. Subsequently, 20 ml of carrot pulp was then added at day 7, and
the culture was
harvested when the culture became saturated (i.e.. no substantial cell
division).
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WO 00/07606 PCT/US99/17671
The culture was first autoclaved for 30 minutes at 250°F. and then
centrifuged at
4000 r.p.m. for 15 mm. The resulting supernatant was collected and filtered in
a Buchner funnel
through a 0.8 p.m filter. The filtrate was collected and further filtered
through a 0.2 ~m Nalge
vacuum filter. The resulting final filtrate was then collected (an approximate
volume of 900 ml)
to form a liquid containing an extracellular product.
3. Bifidogenic Oligosaccharides
Bifidogenic oligosaccharides, as designated herein, are a class of
carbohydrates
particularly useful for preferentially promoting the growth of a lactic acid-
producing bacteria of
the present invention. These oligosaccharides include, but are not limited to:
fructo-
oligosaccharides (FOS); gluco-oligosaccharides (GOS); other long-chain
oligosaccharide
polymers of fructose and/or glucose; and the trisaccharide - raffinose. All of
these
aforementioned carbohydrates are not readily digested by pathogenic bacteria.
Thus. the
preferential growth of lactic acid-producing bacteria is promoted by the
utilization of these
bifidogenic oligosaccharides due to the nutrient requirements of this class of
bacterium, as
1 S compared to pathogenic bacteria.
Bifidogenic oligosaccharides are long chain polymers that are utilized almost
exclusively
by the indigenous Bifrdobacteria and Lactobacillus in the intestinal tract and
can be similarly
utilized by Bacillus. In contrast, physiologically deleterious bacteria such
as Clostridium,
Staphylococcus. Salmonella and Escherichia coli cannot metabolize FOS, or
other bifidogenic
oligosaccharides, and therefor use of these bifidogenic oligosaccharides in
combination with a
lactic acid-producing bacteria of the present , preferably Bacillus coagulans,
allows these
beneficial, probiotic bacteria to grow and effectively compete with, and
eventually replace any
undesirable, pathogenic microorganisms within the gastrointestinal tract.
The use of bifidogenic oligosaccharides in the compositions of the present
invention
provides a synergistic effect thereby increasing the effectiveness of the
probiotic-containing
compositions disclosed herein. This synergy is manifested by selectively
increasing the ability
of the probiotic bacterium to grow by, for example, increasing the level of
nutrient
supplementation which preferentially selects for growth of the probiotic
bacteria over many other
bacterial species within the infected tissue.
In addition, it is readily understood that Bifrdobacteria and Lactobacillus
are also
producers of lactic acid. Bifidogenic oligosaccharides enable these
aforementioned probiotic
organisms to proliferate preferentially over the undesirable bacteria within
the gastrointestinal
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WO 00/07606 PCT/US99/17671
tract. thereby augmenting the probiotic state of the body by further enhancing
the solubility of
these nutrients (whether of food origin or as a result of nutritional
supplement augmentation).
Thus, the presence of the bifidogenic oligosaccharides in the compositions of
the present
invention allows for more effective microbial inhibition by increasing the
ability of all varieties
of probiotic bacteria to grow, and therefore provide said benefit.
The bifidogenic oligosaccharide of the present invention may be used either
alone, or in
combination with a lactic acid-producing microorganisms in a therapeutic
composition. More
specifically, due to the growth promoting activity of bifidogenic
oligosaccharides, the present
invention contemplates a composition comprising a bifidogenic oligosaccharide
present in a
concentration sufficient to promote the growth of lactic acid-producing
microorganisms. As
shown herein, these concentrations amounts can vary widely, as the probiotic
microorganisms
will respond to any metabolic amount of nutrient oligosaccharide, and
therefore the present
invention need not be so limited.
A preferred and exemplary bifidogenic oligosaccharide is fructo-
oligosaccharide (FOS),
although other carbohydrates may also be utilized, either alone or in
combination. FOS can be
obtained from a variety of natural sources, including commercial suppliers. As
a product
isolated from natural sources, the components can vary widely and still
provide the beneficial
agent, namely FOS. Typically, FOS possesses a polymer chain length of from
approximately 4
to 200 sugar units, with the longer lengths being preferred. For example, the
degree of purity can
vary widely so long as biologically-functional FOS is present in the final
formulation. Preferred
FOS formulations contain at least 50% by weight of fructo-oligosaccharides
compared to
simple(mono or disaccharide) sugars such as glucose, fructose or sucrose,
preferably at least $0%
fructo-oligosaccharides (FOS), more preferably at least 90% and most
preferably at least 95%
FOS. Sugar content and composition can be determined by any of a variety of
complex
carbohydrate analytical detection methods as is well known. Preferred sources
of FOS include,
but are not limited to: inulin; Frutafit IQTM (Imperial Suiker Unie; Sugar
Land, Texas);
NutraFlora'~ (Americal Ingredients, Inc.; Anaheim, CA); and Fruittrimfat
Replacers and
Sweeteners (Emeryville, CA). Bifidogenic oligosaccharides such as GOS, and
other long chain
oligosaccharides are also available from commercial vendors.
4. Therapeutic Compositions
The various active agents (i.e., probiotic bacteria, FOS, and the like}
comprising the
therapeutic composition of the present invention are combined with a carrier
that is
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WO 00/07606 PCT/US99/17671
physiologically compatible with the gastrointestinal tract tissue of a human
or animal to which it
is administered. More specifically, the carrier is, preferably, substantially
inactive except for
surfactant properties which are used in making a suspension of the active
ingredients. The
therapeutic compositions may also include other physiologically-active
constituents which do not
interfere with the efficacy of, for example, the Bacillus coagulans and FOS as
active agents in
said composition.
A typical therapeutic composition of the present invention will contain
(within a one
gram dosage formulation) from approximately 2x 10' to 1 x 10'° colony
forming units (CFU) of
viable bacteria or bacterial spores and approximately 10 milligrams (mg) to
one gram of fructo-
oligosaccharides (FOS).
The therapeutic compositions of the present invention may also include known
antioxidants, buffering agents, and other agents such as coloring agents,
flavorings, vitamins or
minerals. For example, a preferred therapeutic composition may also contain
one or more of the
following minerals: calcium citrate (15-350 mg); potassium gluconate (5-150
mg); magnesium
citrate (5-15 mg); and chromium picollinate (5-200 fig). In addition, a
variety of salts may be
utilized, including calcium citrate, potassium gluconate, magnesium citrate
and chromium
picollinate. Thickening agents may be added to the compositions such as
polyvinylpyrrolidone,
polyethylene glycol or carboxymethylcellulose. Preferred additional components
of a
therapeutic composition of this invention can include assorted colorings or
flavorings, vitamins,
fiber, enzymes and other nutrients. Preferred sources of fiber include any of
a variety of sources
of fiber including, but not limited to: psyllium, rice bran, oat bran, corn
bran, wheat bran, fruit
fiber and the like. Dietary or supplementary enzymes such as lactase, amylase,
glucanase,
catalase, and the like enzymes can also be included. Chemicals used in the
present compositions
can be obtained from a variety of commercial sources, including Spectrum
Quality Products, Inc
(Gardena, CA), Sigma Chemicals (St. Louis, MI), Seltzer Chemicals, Inc.,
(Carlsbad, CA) and
Jarchem Industries, Inc., (Newark, NJ).
The various active agents (e.g., probiotic bacteria, bifidogenic
oIigosaccharides, and the
like) are combined with a carrier which is physiologically compatible with the
gastrointestinal
tissue of the species to which it is administered. Carriers can be comprised
of solid-based, dry
materials for formulation into tablet, capsule or powdered form; or the
carrier can be comprised
of liquid or gel-based materials for formulations into liquid or gel forms.
The specific type of
carrier, as well as the final formulation depends. in part. upon the selected
routes) of
administration.
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The therapeutic composition of the present invention may also include a
variety of
carriers and/or binders. A preferred carrier is micro-crystalline cellulose
(MCC), added in an
amount sufficient to complete the one gram dosage total weight. Particularly
preferred
formulations for a therapeutic composition of this invention are described in
the Specific
Examples section. Carriers can be solid-based dry materials for formulations
in tablet, capsule or
powdered form, and can be liquid or gel-based materials for formulations in
liquid or get forms,
which forms depend, in part, upon the routes of administration.
Typical carriers for dry formulations include, but are not limited to:
trehalose, malto-
dextrin, rice flour, micro-crystalline cellulose (MCC) magnesium sterate,
inositol, FOS, GOS,
dextrose, sucrose, and like carriers. Where the composition is dry and
includes evaporated oils
that produce a tendency for the composition to cake (adherence of the
component spores, salts,
powders and oils), it is preferred to include dry fillers which distribute the
components and
prevent caking. Exemplary anti-caking agents include MCC, talc, diatomaceous
earth.
amorphous silica and the like, and are typically added in an amount of from
approximately 1% to
95% by weight. It should also be noted that dry formulations which are
subsequently rehydrated
(e.g., liquid formula) or given in the dry state (e.g., chewable wafers,
pellets or tablets) are
preferred to initially hydrated formulations. Dry formulations (e.g., powders)
may be added to
supplement commercially available foods (e.g., liquid formulas, strained
foods, or drinking water
supplies). Similarly, the specific type of formulation depends upon the route
of administration.
Suitable liquid or gel-based Garners include but are not limited to: water and
physiological salt solutions; urea; alcohols and derivatives (e.g., methanol,
ethanol, propanol,
butanol); glycols (e.g., ethylene glycol, propylene glycol, and the like).
Preferably, water-based
carriers possess a neutral pH value (i. e., pH 7.0). The compositions may also
include natural or
synthetic flavorings and food-quality coloring agents, all of which must be
compatible with
maintaining viability of the lactic acid-producing microorganism. Well-known
thickening agents
may also be added to the compositions such as corn starch, guar gum, xanthan
gum, and the like.
Where a liquid-based composition containing spores is provided, it is
desirable to include a spore
germination inhibitor to promote long term storage. Any spore germination
inhibitor may be
used. By way of example and not of limitation, preferred inhibitors include:
hyper-saline
carriers. methylparaben, guargum, polysorbates, preservatives, and the like.
Other suitable carriers include, but are not limited to, aqueous and
oleaginous carriers
(e.g., white petrolatum, isopropyl myristate. lanolin or lanolin alcohols.
mineral oil, sorbitan
mono-oleate. propylene glycol. cetylstearyl alcohol, together or in various
combinations):
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WO 00/07606 PCTNS99/17671
hydroxypropyl cellulose (MW = 100.000 to 1.000.000); detergents (e.g.,
polyoxyl stearate or
sodium lauryl sulfate) and mixed with water to form a gel, liquid or semi-
solid composition.
Other suitable carriers comprise water-in-oil or oil-in-water emulsions and
mixtures of
emulsifiers and emollients with solvents such as sucrose stearate, sucrose
cocoate. sucrose
distearate, mineral oil, propylene glycol, 2-ethyl-1,3 polyoxypropylene-15-
stearyl ether and
water. Emulsions containing water, glycerol stearate, glycerin, mineral oil,
synthetic spermaceti,
cetyl alcohol, butylparaben, propylparaben and methylparaben are all
commercially available.
Preservatives may also be included in the carrier and include, methylparaben,
propylparaben,
benzyl alcohol and ethylene diamine tetraacetate (EDTA) salts. Well-known
flavorings and/or
colorants may also be included in the carrier. The composition may also
include a plasticizer
such as glycerol or polyethylene glycol (MW= 800 to 20,000). It should be
noted that, generally,
the composition of the carrier can be varied so long as it does not interfere
significantly with the
pharmacological activity of the active ingredients or the viability of the
Bacillus coagulans
spores.
Preservatives may also be included within the carrier including methylparaben,
propylparaben, benzyl alcohol and ethylene diamine tetraacetate salts. Well-
known flavorings
and/or colorants may also be included within the carrier. The compositions of
the present
invention may also include a plasticizer such as glycerol or polyethylene
glycol (with a preferred
molecular weight of MW = 800 to 20,000). The composition of the carrier can be
varied so long
as it does not interfere significantly with the pharmacological activity of
the active ingredients or
the viability of the Bacillus coagulans spores.
A therapeutic composition of the present invention can be formulated to be
suitable for
oral administration in a variety of ways, for example in a liquid, a powdered
food supplement, a
paste, a gel, a solid food, a packaged food, a wafer, and the like as
described in more detail in the
Examples. Other formulations will be readily apparent to one skilled within
the art.
Awutrient supplement component of a composition of this invention can include
any of a
variety of nutritional agents, as are well known, including vitamins,
minerals, essential and non-
essential amino acids, carbohydrates, lipids, foodstuffs, dietary supplements,
and the like.
Preferred compositions comprise vitamins and/or minerals in any combination.
Vitamins for use
in a composition of this invention can include vitamins B, C, D. E, folic
acid, K, niacin, and like
vitamins. The composition can contain any or a variety of vitamins as may be
deemed useful for
a particularly application, and therefore, the vitamin content is not to be
construed as limiting.
Typical vitamins are those, for example. recommended for daily consumption and
in the
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recommended daily amount (RDA), although precise amounts can vary. FIG. ?
illustrates. in
tabular form. the vitamins, their RDA. their ranges and respective preferred
ranges, of the
therapeutic compositions of the present invention. The composition would
preferably include a
complex of the RDA vitamins. minerals and trace minerals as well as those
nutrients that have no
established RDA, but have a beneficial role in healthy human or mammal
physiology. The
preferred major minerals and their respective RDA are shown in FIG. 3. The
preferred mineral
format would include those that are in either the gluconate or citrate form
because these forms
are more readily metabolized by lactic acid bacteria. FIG. 4, illustrates, in
tabular form, the
preferred trace minerals and their respective ranges, as utilized in the
therapeutic composition of
the present invention.
In a related embodiment, the invention contemplates a composition comprising a
viable
lactic acid-producing bacteria in combination with any material to be
adsorbed, including but not
limited to nutrient supplements. foodstuffs, vitamins, minerals, medicines,
therapeutic
compositions, antibiotics, hormones, steroids, and the like compounds where it
is desirable to
insure efficient and healthy absorption of materials from the gastrointestinal
track into the blood.
The amount of material included in the composition can vary widely depending
upon the
material and the intended purpose for its absorption, such that the invention
is not to be
considered as limiting.
A number of methodologies are disclosed herein for increasing the solubility
and,
therefore, the bioavailability of vitamins and minerals in food and
nutritional formulations. The
present invention contemplates a method for increasing the bioavailability of
vitamins and
minerals using lactic acid-producing bacteria, preferably Bacillus coagulans
spores. It should be
noted however that the scope of the invention is not to be limited to any
particular member of the
Bacillus genera, but can include any member of the Bacillus, Lactobacillus,
Bifidobacterium,
Sporolactobacillus, Streptococcus, and/or Enterococcus genus. The
aforementioned method
comprises administration of a therapeutic composition of the present invention
containing the
active ingredients, to a human or animal, in various dosage regimens as
described herein to
achieve the desired nutritional result.
Administration of a nutritional composition is, preferably, to the
gastrointestinal tract by
use of a gel, suspension, aerosol spray, capsule, tablet, wafer, powder or
semi-solid formulation
(e.g., a suppository) containing a therapeutic composition of the present
invention, all formulated
using methods well-known within the art. Administration of the compositions
containing the
active ingredients effective in producing lactic acid and/or the appropriate
enzymes for
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solublizing vitamins or minerals generally consist of from one to ten unit
dosages of 10
milligrams to 10 grams per dosage of said composition for a total time period
of from one day to
one month. Unit dosages are generally given once every twelve hours, and up to
once every four
hours. Preferably, one to four dosages of the therapeutic composition is
administered per day.
Exemplar formulations are provided in the Specific Examples section, infra.
5. Specific Examples
The following examples relating to the present invention are illustrative, and
should not
be construed as expressly limiting the invention in any manner. Moreover, such
variations of the
invention, now known or later developed, which would be within the purview of
one skilled
within the art are to be considered to fall within the scope of the present
invention hereinafter
claimed.
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Formulation 1 (Combination Vitamin/ Mineral Supplement)
Bacillus coagulans x108 viable spores (approximately
3~ mg)
Calcium citrate 35 milligrams (mg)
Potassium gluconate 20 mg
Magnesium citrate 20 mg
Chromium picollinate 100 micrograms (fig)
Fructo-oligosaccharides (FOS) 250 mg
Vitamin A 10,000 International Units
(IU)
Vitamin C 120 mg
Vitamin D 400 IU
Vitamin B-1 5 mg
Vitamin B-2 8 mg
Vitamin B-6 g mg
Vitamin B-12 15 pg
Pantothenic Acid 30 mg
Folic Acid 200 mini-equivalents (meq)
Vitamin E 75 IU
The balance of the 1 gram capsule, tablet, or wafer will be microcrystaline
cellulose {MCC)
Formulation 2 (Mineral Supplements)
Bacillus coagulans 2.5x108 viable spores (approximately
17.5 mg)
Calcium citrate 35 mg
Potassium gluconate 10 mg
Magnesium citrate 10 mg
Chromium picollinate 50 p.g
Fructo-oligosaccharides (FOS) 100 mg
Micro-crystalline cellulose (MCC)827.5 mg
Formulation 3 (Vitamin Supplement)
Bacillus coagulans 2.5x108 viable spores (approximately
17.~ mg)
Vitamin A 10,000 IU
VitaminB-1 5 mg
VitaminB-2 g mg
VitaminB-6 8 mg
VitaminB-12 15 pg
Vitamin E 75 IU
Vitamin C 120 mg
Lactase 200 IU
FOS 400 mg
The balance of the I gram capsule,
tablet, or wafer will be microcrystaline
cellulose (MCC).
23
CA 02339643 2001-02-05
WO 00/07606 PCTNS99/17671
Equivalents
From the foregoing detailed description of the specific embodiments of the
present invention, it
should be readily apparent that unique, improved methodologies for the
utilization of bacterial
enzymes and various other metabolic products (e.g., lactic acid) derived from
lactic acid-
s producing bacteria, preferably Bacillus coagulans, to increase the
solubility and. therefore, the
bioavailability, of vitamins and minerals within the gastrointestinal tract of
animals and humans,
has been disclosed. Although particular embodiments have been disclosed herein
in detail, this
has been done by way of example for purposes of illustration only, and is not
intended to be
limiting with respect to the scope of the appended claims which follow. In
particular, it is
contemplated by the inventor that various substitutions, alterations, and
modifications may be
made to the invention without departing from the spirit and scope of the
invention as defined by
the claims. For example, the final form (e.g., stabilized gel, cream,
emulsification, and the like)
which is selected for the therapeutic composition of the present invention is
believed to be a
matter of routine for a person of ordinary skill in the art with knowledge of
the embodiments
described herein.
24
