U.S. patent application number 16/895641 was filed with the patent office on 2020-12-24 for activated bifidobacteria and methods of use thereof.
The applicant listed for this patent is EVOLVE BIOSYSTEMS INC.. Invention is credited to Samara FREEMAN-SHARKY, David KYLE, David MILLS.
Application Number | 20200397837 16/895641 |
Document ID | / |
Family ID | 1000005008361 |
Filed Date | 2020-12-24 |
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United States Patent
Application |
20200397837 |
Kind Code |
A1 |
KYLE; David ; et
al. |
December 24, 2020 |
ACTIVATED BIFIDOBACTERIA AND METHODS OF USE THEREOF
Abstract
Some embodiments of the invention include a composition and
method for treating dysbiosis in infants. The composition may
include a mixture of activated bifidobacteria and a complex
oligosaccharide wherein the complex oligosaccharide may be derived
from a human or non-human source.
Inventors: |
KYLE; David; (Gualala,
CA) ; MILLS; David; (Davis, CA) ;
FREEMAN-SHARKY; Samara; (Davis, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EVOLVE BIOSYSTEMS INC. |
Davis |
CA |
US |
|
|
Family ID: |
1000005008361 |
Appl. No.: |
16/895641 |
Filed: |
June 8, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15521502 |
Apr 24, 2017 |
10716816 |
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PCT/US15/57226 |
Oct 23, 2015 |
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16895641 |
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62133243 |
Mar 13, 2015 |
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62068553 |
Oct 24, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23C 9/12 20130101; A23L
29/30 20160801; A61K 31/702 20130101; A23V 2002/00 20130101; A23L
33/135 20160801; C12N 1/04 20130101; A61K 35/745 20130101; A23Y
2300/45 20130101; C12N 1/20 20130101 |
International
Class: |
A61K 35/745 20060101
A61K035/745; A23L 29/30 20060101 A23L029/30; C12N 1/04 20060101
C12N001/04; C12N 1/20 20060101 C12N001/20; A61K 31/702 20060101
A61K031/702; A23L 33/135 20060101 A23L033/135; A23C 9/12 20060101
A23C009/12 |
Claims
1-116. (canceled)
117. A method of preparing an activated Bifidobacterium, the method
comprising (a) incubating a purified strain of Bifidobacterium in a
fermenter in the presence of a mammalian milk oligosaccharide, (b)
harvesting activated Bifidobacterium cells, and (c) purifying the
activated Bifidobacterium cells obtained in (b), wherein the
activated bifidobacterium contains a transport system capable of
internalizing one or more oligosaccharides before said
oligosaccharide is hydrolyzed and is further capable of hydrolyzing
said internalized oligosaccharide, wherein said oligosaccharide has
the structure of an oligosaccharide found in a mammalian milk.
118. The method of claim 117, wherein the activated Bifidobacterium
cells express a gene coding for a sialidase, a fucosidase, a sialic
acid transporter, or a fucose transporter.
119. The method of claim 117, wherein, in the activated
Bifidobacterium cells, gene Blon_0042 is upregulated, gene
Blon_2168 is downregulated, or a combination thereof.
120. The method of claim 117, wherein any of the genes Blon_2334,
Blon_2335, Blon_2336, Blon_2337, Blon_2338, Blon_2339, Blon_2344,
Blon_2346, Blon_2347, and Blon_2331, is upregulated in the
activated Bifidobacterium cells.
121. The method of claim 117, wherein the activated Bifidobacterium
cells comprise upregulated genes selected from the group consisting
of Blon_0042, Blon_R0015, Blon_R0017, Blon_R0021, Blon_R0022, and
combinations thereof, and/or downregulated genes selected from the
group consisting of Blon_0518, Blon_0785, Blon_2167, Blon_2168 and
combinations thereof.
122. The method of claim 117, wherein the Bifidobacterium is B.
longum, B. breve, B. bifidum or B. pseudocatenulatum.
123. The method of claim 122, wherein the B. longum is B. longum
subsp. infantis.
124. The method of claim 117, wherein the mammalian milk
oligosaccharide is isolated from a mammalian milk source.
125. The method of claim 124, wherein the mammalian milk
oligosaccharide is from a human or a bovine source, optionally
wherein the bovine source is bovine colostrum.
126. The method of claim 117, wherein the mammalian milk
oligosaccharide is selected from the group consisting of
Lacto-N-Tetraose, Lacto-N-Neotetraose, Lacto-N-Fucopentaose I,
Lacto-N-Fucopentaose II, Lacto-N-Fucopentaose III,
Lacto-N-Fucopentaose V, Lacto-N-Hexaose, Para-Lacto-N-Hexaose,
Lacto-N-Neohexaose, Para-Lacto-N-Neohexaose,
Monofucosyllacto-N-Hexaose II, Isomeric Fucosylated Lacto-N-Hexaose
(1), Monofucosyllacto-N-Hexaose, Isomeric Fucosylated
Lacto-N-Hexaose (3), Isomeric Fucosylated Lacto-N-Hexaose (2),
Difucosyl-Para-Lacto-N-Neohexaose, Difucosyl-Para-Lacto-N-Hexaose,
Difucosyllacto-N-Hexaose, Lacto-N-Neoocataose,
Para-Lacto-N-Octanose, Iso-Lacto-N-Octaose, Lacto-N-Octaose,
Monofucosyllacto-Nneoocataose, Monofucosyllacto-N-Ocataose,
Difucosyllacto-N-Octaose I, Difucosyllacto-N-Octaose 11,
Difucosyllacto-N-Neoocataose 11, Difucosyllacto-N-Neoocataose 1,
Lacto-N-Decaose, Trifucosyllacto-N-Neooctaose,
Trifucosyllacto-N-Octaose, Trifucosyl-Iso-Lacto-N-Octaose,
2'-fucosyllactose, 3-fucosyllactose, difucosyllactose,
3'-sialyllactose, 6'-sialyllactose, 3'-sialyl-3-fucosyllactose,
sialyllacto-N-tetraose, and 6'-sialyllactosamine.
127. The method of claim 117, further comprising drying the
purified activated Bifidobacterium cells.
128. The method of claim 127, wherein the drying is performed by
spray drying or freeze drying, optionally wherein the freeze drying
is performed in the presence of a suitable cryoprotectant, and
optionally wherein the cryoprotectant is selected from the group
consisting of glucose, lactose, raffinose, sucrose, trehalose,
adonitol, glycerol, mannitol, methanol, polyethylene glycol,
propylene glycol, ribitol, alginate, bovine serum albumin,
carnitine, citrate, cysteine, dextran, dimethyl sulphoxide, sodium
glutamate, glycine betaine, glycogen, hypotaurine, peptone,
polyvinyl pyrrolidone, and taurine.
129. The method of claim 127, further comprising formulating the
purified activated Bifidobacterium cells into a composition in the
form of a dry powder, dry powder suspended in an oil, solution,
packet, sachet, orally disintegrating tablet, foodstuff, capsule,
lozenge, effervescent tablet, suppository, or enema, optionally
wherein the capsule or tablet has an enteric coating, and
optionally wherein the enteric coating comprises one or more of
fatty acids, waxes, shellac, plastics, plant fibers, methyl
acrylate-methacrylic acid copolymers, cellulose acetate succinate,
hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl
cellulose acetate succinate, polyvinyl acetate phthalate (PVAP),
methyl methacrylate-methacrylic acid copolymers, cellulose acetate
trimellitate, sodium alginate, and Zein.
130. The method of claim 117, further comprising formulating the
purified activated Bifidobacterium cells into a composition such
that the activated Bifidobacterium cells are at a concentration of
from 1 billion cfu/g to 500 billion cfu/g, 1 billion cfu/g to 100
billion cfu/g, or 1 billion cfu/g to 50 billion cfu/g.
131. The method of claim 127, further comprising formulating the
purified activated Bifidobacterium cells into a composition to
include a stabilizer, optionally wherein the stabilizer is a flow
agent, and optionally wherein the stabilizer is a milk protein.
132. The method of claim 117, further comprising formulating the
activated bifidobacterium composition to include an isolated
complex oligosaccharide.
133. The method of claim 132, wherein the isolated complex
oligosaccharide is isolated from a mammalian milk source selected
from human, bovine, pigs, rabbits, goats, sheep, or camel, wherein
the bovine source is bovine milk, bovine colostrum, bovine
colostrum concentrate, bovine whey permeate, or mixtures
thereof.
134. The method of claim 132, wherein the complex oligosaccharide
comprises at least one of (3Hex,4HexNac,1Fuc),
(1Gal,1GlcNAc,1NeuAc), or (1Glu,1Gal,1NeuAc (3' or 6')), and/or
wherein the complex oligosaccharide is from bovine colostrum and
comprises any of Hex(4); Hex(4) HexNAc(2); and Hex(3) HexNAc(1)
NeuAc(1) at levels greater than 1%, and/or wherein the complex
oligosaccharide comprises one of the following ratios of
constituents: 1) a ratio of Hex(2) NeuAc(1):Hex(2) HexNAc(1) less
than 5.0; 2) a ratio of Hex(2) HexNAc(1):Hex (3) HexNAc(1) of
greater than 1.0; 3) a ratio of Hex(2) HexNAc(1):Hex (3) HexNAc(2)
of greater than 2.0; 4) a ratio of Hex(3):Hex (3) HexNAc(1)
NeuAc(1) of less than 100; or 5) a ratio of Hex(2) HexNAc(1):Hex
(4) NeuAc(2) NeuGc(1) of greater than 10, optionally wherein the
complex oligosaccharide is at least 20%, at least 50%, or at least
80% of the weight of the composition.
135. The method of claim 132, wherein the complex oligosaccharide
comprises a fucosyllactose, sialyllactose, or derivatives thereof,
optionally wherein the fucosyllactose, sialyllactose, or
derivatives thereof are synthetically produced and purified
oligosaccharides of 2'-fucosyllactose, 3'-fucosyllactose,
difucosyllactose, lacto-N-fucosylpentose I, lacto-N-fucosylpentose
II, lacto-N-fucosylpentose III, lacto-N-fucosylpentose V,
3'-sialyllactose, 6'-sialyllactose, 3'-sialyl-3-fucosyllactose,
sialyllacto-N-tetraose, 6'-sialyllactosamine, or mixtures thereof,
and/or wherein the fucosyllactose and/or sialyllactose comprises
1-5%, 5-20%, or 20-50% of the total oligosaccharides, and/or
wherein the mass ratio of the complex oligosaccharide to the
fucosyllactose and/or sialyllactose is from 20:1 to 1:10, and/or
wherein the complex oligosaccharide is less than 50%
fucosylated.
136. The method of claim 117, further comprising formulating the
purified activated Bifidobacterium cells into a composition which
includes a secondary metabolite, optionally wherein the secondary
metabolite is acetate, lactate, or a combinations thereof; or a
short chain fatty acid.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to promoting health, and
more particularly, to improving the human microbiome. Further,
embodiments of this invention relate to activated bifidobacterial
compositions, methods of making activated bifidobacteria, and the
use of the compositions in order to initiate and maintain a human
gut microbiome that is highly enriched in Bifidobacterium species
in order to facilitate the development of a human infant's
gastrointestinal (GI) tract and immune function.
BACKGROUND
[0002] When a vaginally-delivered human infant is breast-fed he/she
will have a gastrointestinal microbiome that is unique in
composition and diversity compared to any other time in their life.
The GI microbiome is dominated by a single organism which can be
present at high concentrations (up to and over 70% of the total
microbiome). However, the obstetric standard of care in a typical
hospital today involves births from both Cesarean Section
(C-section) and vaginal delivery, followed by human milk or infant
formula feeding for the baby. The surgical suite and levels of
cleanliness for the mother pre- and post-op in many modern hospital
settings are such that in many cases the infant will not get seeded
with bacteria normally found in the microbiome of the vagina or
gastrointestinal tract of the mother, resulting in a dysbiosis in
the baby whether delivered by C-Section or vaginal births or fed by
mother's milk or infant formula. Furthermore, dysbiosis can also be
caused by infants losing the beneficial Bifidobacterium as a result
of illness or medical intervention (e.g., antibiotic treatment).
The dysbiosis of the infant microbiome leads to increased
gastrointestinal problems and delayed or altered immunological
programming and tolerization. The consequences of early dysbiosis
are considered to have an impact throughout the entire life of that
individual.
[0003] Human milk contains a significant quantity of complex
oligosaccharides (up to 15% of total dry mass) in a form that is
not usable as an energy source for the baby nor for most of the
microorganisms in the gut of that baby. Certain microorganisms such
as Bifidoacterium longum subsp. infantis [B. infantis or BI] have
the unique capability to consume the specific complex
oligosaccharides such as those found in human or bovine milk (U.S.
Pat. No. 8,198,872 and U.S. Pub. No. 2013/0195803, the contents of
which are incorporated herein by reference). When B. infantis comes
in contact with certain complex oligosaccharides a number of genes
are specifically induced within the bacterium whose protein
products as enzymes and binding proteins are responsible for the
uptake and internal deconstruction of those complex
oligosaccharides, and the individual sugar components are then
catabolized to provide energy for the growth and reproduction of
that organism (Sela et al, 2008, PNAS, 105(48): p. 18964-69).
SUMMARY
[0004] The instant invention provides compositions comprising
isolated complex oligosaccharide fractions from mammalian milk
sources, optionally supplemented with purified
fucosylated/sialylated oligosaccharides. The mammalian milk may be
from human or bovine sources, and including but not limited to, the
bovine source is from bovine colostrum. The fucosylated
oligosaccharide(s) may comprise synthetically produced and purified
2'-fucosyllactose, 3-fucosyllactose, difucosyllactose, or
lacto-N-fucosylpentose.
[0005] In some embodiments, the composition further comprises
bifidobacteria that internalize the complex oligosaccharides or
dietary glycans prior to their hydrolysis and metabolism. The
combination of the bifidobacteria with the complex oligosaccharide
may result in the conversion of the bifidobacteria to an activated
bifidobacteria (ABI). The bifidobacteria is preferably selected
from B. longum, B. breve, B. bifidum or B. pseudocatenulatum, and
more preferably, the B. longum is B. longum subsp. infantis.
[0006] In other embodiments, any of the compositions described
herein provide a method of improving the health of a mammal
comprising administering to a mammal a composition comprising a
complex oligosaccharide from a mammalian milk source, optionally
supplemented with a fucosylated and/or sialylated oligosaccharide,
and a bifidobacteria that internalizes the complex oligosaccharide
prior to its hydrolysis, and wherein the fucosylated
oligosaccharide can be a synthetically produced and purified form
of 2'-fucosyllactose, 3-fucosyllactose, difucosyllactose, or
lacto-N-fucosylpentose, wherein the bifidobacteria is preferably
selected from B. longum, B. breve, B. bifdum or B.
pseudocatenulatum, and more preferably, the B. longum is B. longum
subsp. infantis. The bifidobacteria is typically provided in a
daily dose of from 10 thousand to 100 billion cfu, preferably 1
billion to 50 billion, and most preferably 5 billion to 25 billion,
and the oligosaccharides are provided in a daily dose of from 1 to
20 g, preferably in a daily dose of from 1 to 10 g.
BRIEF DESCRIPTION OF DRAWINGS
[0007] FIG. 1 is a diagram depicting whole genome expression
analysis which shows differential gene expression of B. infantis
cells grown in the presence of bovine milk oligosaccharides (BMO)
or lactose.
[0008] FIG. 2 is a diagram showing a selection of the genes of the
milk oligosaccharide cluster differentially expressed in B.
infantis during growth in the presence of bovine milk
oligosaccharides (BMO) or lactose.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
I. Introduction
[0009] Human milk glycans contain a significant quantity of human
milk oligosaccharides (designated herein as "HMOs") (about 15% of
total mass) in a form that is not usable as an energy source for
the baby or for most of the microorganisms in the gut of that baby.
HMOs can be found as free oligosaccharides (dietary glycans) or
conjugated to protein or lipids. The major HMOs in milk include
lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) and
lacto-N-hexaose, which are neutral HMOs, in addition to fucosylated
oligosaccharides such as 2-fucosyllactose (2FL), 3-fucosyllactose
(3FL), and lacto-N-fucopentaoses I, II and III. Acidic HMOs include
sialyl-lacto-N-tetraose, 3' and 6' sialyllactose (6SL). HMOs are
particularly highly enriched in fucosylated oligosaccharides (Mills
et al., U.S. Pat. No. 8,197,872). Among the enzymes that produce
HMOs in the mammary gland is the enzyme encoded by the
fucosyltransferase 2 (FUT2) gene, which catalyzes the linking of
fucose residues by an .alpha.1,2-linkage to oligosaccharides found
in human milk. Fucosylated oligosaccharides are known to inhibit
the binding of pathogenic bacteria in the gut. HMOs, and in
particular the fucosylated HMOs, share common structural motifs
with glycans on the infant's intestinal epithelia known to be
receptors for pathogens. (German et al., WO 2012/009315)
[0010] HMOs are substrates for the selective growth of certain
bifidobacteria in the mammalian gut. Certain bifidobacteria such
as, but not limited to, Bifidobacterium longum subsp. infantis
possess a gene cluster dedicated to the internalization and
deconstruction of HMOs. When such bacteria interact with HMOs, this
gene cluster, including genes for transporting and catabolizing
fucosylated oligosaccharides, is upregulated. The interaction of
certain HMOs with B. longum subsp. infantis has been shown to
activate the bacterium by inducing expression of a number of genes
including, but not limited to, those in the HMO gene cluster that
encode proteins to capture and internalize the HMOs and encode
enzymes to completely catabolize the HMOs, thereby providing that
microbe with energy and substrate to grow and multiply (Underwood,
2015, Pediatric Research, 77(1-2):229-35). The products of the
upregulated genes also allow B. longum subsp. infantis to colonize
the mucosal lining of the gut and thereby impede the binding of
pathogenic microbes. (Underwood, et al., 2015, Pediatr. Res.,
77:229-235). The activated bacterium has been shown to possess
increased binding to intestinal epithethial cells. (Chichlowski et
al., 2012, J. Pediatric Gastroenteral Nur, 55:321-327). The
activated B. longum subsp. infantis is also able to produce short
chain fatty acids which facilitate the development of the infant's
mucosal lining and the immune system (Underwood et. al., 2015,
Pediatr Res, 77: 229-235). Consequently, the proliferation of ABI
in the gut of a newborn infant, triggered and uniquely enabled by
the HMOs provided in mother's milk, is of significant benefit to
the acute health and long term survival of that infant.
Consequently, ABI provides significant benefits to a newborn infant
which include, but are not limited to, a higher binding affinity to
the gut mucosa, higher colonization of the GI tract thereby
preventing growth of other bacterial clades, a higher production of
short chain fatty acids, higher consumption of complex
oligosaccharides, and a greater stimulation of the immune response
as measured by positive alterations of immune response markers,
relative to the organism in a pre-activated state (Lewis, et al.,
2015, Microbiome, 3:13; Huda, et al., 2014, Pediatrics, 134:2
e362-e372).
[0011] In the activated form, the B. infantis becomes the sole
consumer of the human milk oligosaccharides (HMO) and has been
shown to increase its relative proportion in the gut microbiota of
infant humans to levels at least 10-fold higher than its levels at
birth (prior to consumption of HMO), or in those infants
exclusively fed commercial infant formula not containing milk
oligosaccharides, and reaching levels as high as 70% of the total
microbial population of the distal colon of breast-fed babies. When
B. infantis is present in the gut of a baby, and that baby is also
provided with its mother's milk as a sole source of nutrition, the
population of B. infantis can increase to levels as high as 90% of
the total bacterial population of the gut as measured by the
microbial quantification of the stool. The ABI will remain in the
gut at high concentrations and remain activated as long as a
dietary source of the selective complex oligosaccharides (e.g., HMO
to human babies) is provided to the infant. Once the source of the
complex oligosaccharides is withdrawn from the diet (e.g., at
weaning and the introduction of solid foods), the B. infantis is no
longer activated, it can no longer successfully compete other gut
microbiota for nutrients in the gut, and its population decreases
to less than 1% of the total microbiome. B. infantis is not
normally found in the gut of a weaned infant, child, or adult in
levels of more than 1% of the total microbiome
[0012] Surprisingly, B. infantis has also been shown to grow on
oligosaccharides isolated from bovine milk (German, et al., WO
2012/009315: Ward, 2009, Open Gyceroscience, 2:9-15). The
concentration of oligosaccharides in bovine milk oligosaccharides
that are selective in supporting the growth of B. infantis
(designated as "BMO") is low in mature bovine milk compared to that
of human milk. This difference in absolute concentration of
oligosaccharides in milk may be due to an initially low level of
BMO in mature bovine milk or due to the presence of enzymes in the
milk that break down BMO into more simple sugars. Furthermore, the
structural composition of the BMO is different from that of HMO
(Aldredge et al., 2013, Glycobiology, (6):664-76; Mehra et al.,
2014, PLoS One, 9(5):e96040). For example, BMOs are higher in
sialic acid-rich components than HMOs and lower in fucose-rich
components than HMOs (Zivkovic and Barile, 2011, Advance Nutrition,
(3): 284-289).
[0013] Recently, the inventors have found that B. infantis and B.
breve, which are human-associated bacteria, are activated by bovine
colostrum oligosaccharides (BCO), such as those found in a bovine
colostrum oligosaccharide concentrate (BCOC). Colostrum is a
special fluid that comes from the breast of mammals (e.g., humans
and bovines) during the first few days after giving birth. The
composition of colostrum is significantly different from that of
the mature milk which replaces the colostrum after the first few
days of lactation. Bovine colostrum oligosaccharides (BCOs) or
bovine colostrum oligosaccharide concentrates (BCOs) have a
composition that is different from HMO and BMO (U.S. Provisional
Application No. 62/155,553, incorporated by reference herein; Tao
et al., 2009, Journal of Dairy Science, 92:2991-3001). For example,
BCO is highly enriched in sialic acid residues but is deficient in
many fucosylated oligosaccharides compared to mature milk BMO. The
inventors also discovered that there are a large number of genes in
addition to the HMO gene cluster that are either up-regulated or
down-regulated upon the interaction of B. longum subsp. infantis
with the different oligosaccharides. Some of these genes are
regulated by HMO and some by BMO, BCO, or BCOC, and some, like the
HMO cluster, are regulated by both.
II. Definitions
[0014] The term "oligosaccharide" refers to polymeric carbohydrates
that contain 3 to 20 monosaccharides covalently linked through
glycosidic bonds. In some embodiments, the oligosaccharides are
purified from human or bovine milk/whey/cheese/dairy products,
(e.g., purified away from oligosaccharide-degrading enzymes in
bovine milk/whey/cheese/dairy products).
[0015] The term "isolated," when applied to an oligosaccharide,
refers to an oligosaccharide composition that has been at least
enriched for the oligosaccharide compared to one or more other
components in the mammalian milk. In some embodiments, the
oligosaccharide(s) is purified, e.g., such that the oligosaccharide
has been separated at least in part from one or more of the other
components of milk.
[0016] The term "bifidobacteria" and its synonyms refer to a genus
of anaerobic bacteria having beneficial properties for humans.
Bifidobacteria is one of the major taxonomic groups of bacteria
that make up the gut flora, the bifidobacteria are among the
beneficial commensal bacteria that reside in the gastrointestinal
tract and have health benefits for their hosts.
[0017] The term "synthetic" composition refers to a composition
produce by a chemi-synthetic process and can be nature-identical.
For example, the composition can include ingredients that are
chemically synthesized and purified or isolated. This does not
include compositions that are naturally synthesized by mammals.
[0018] The term "residues," when applied to an oligosaccharide,
refers to monosaccharide residues of oligosaccharides joined
through glycosidic linkages, which can be hydrolyzed by enzymes or
acid to give the constituent monosaccharide units.
III. Compositions
[0019] The compositions described herein comprise a non-pathogenic
microbe and/or at least one complex oligosaccharide that induces a
change in the non-pathogenic microbe such that the complex
oligosaccharide then becomes an energy source for the microbe, and
when ingested by a mammal, the induced or activated microbe
provides a benefit to the gut of that mammal.
[0020] A. Complex Oligosaccharide
[0021] In various embodiments, the composition comprises a
plurality of oligosaccharides. The oligosaccharide composition may
be derived from human and non-human glycan sources and may exist as
free glycans or protein-bound glycans. In some embodiments, the
oligosaccharide can be a bovine or human milk oligosaccharide. In
some embodiments, the oligosaccharide composition comprises bovine
milk oligosaccharides (BMOs). Bovine oligosaccharides may comprise
oligosaccharides from mature milk, early milk, colostrum, or
concentrates thereof. In some embodiments, the oligosaccharides can
include, but are not limited to, fucose, sialic acid,
N-acetylglucosamine, and/or gluconate residues.
[0022] In various embodiments, complex milk oligosaccharides
include an oligosaccharide consisting of 3 Hex moieties, 4 HexNAc
moieties and 1 fucose (Fuc) moiety; an oligosaccharide consisting
of 4 Hex moieties, 4 HexNAc moieties, and 1 Fuc moiety; an
oligosaccharide consisting of 3 Hex moieties, 5 HexNAc moieties,
and 1 Fuc moiety; an oligosaccharide consisting of 5 Hex moieties,
4 HexNAc moieties, and 1 Fuc moiety; an oligosaccharide consisting
of 4 Hex moieties, 5 HexNAc moieties, and 1 Fuc moiety; an
oligosaccharide consisting of 3 Hex moieties, 6 HexNAc moieties,
and 1 Fuc moiety; an oligosaccharide consisting of 3 Hexose (Hex)
moieties and 6 N-acetyl hexosamine (HexNAc) moieties; an
oligosaccharide consisting of 4 Hex moieties and 3 HexNAc moieties;
an oligosaccharide consisting of 3 Hex moieties and 4 HexNAc
moieties; an oligosaccharide consisting of 6 Hex moieties and 2
HexNAc moieties; an oligosaccharide consisting of 4 Hex moieties
and 4 HexNAc moieties; an oligosaccharide consisting of 3 Hex
moieties and 5 HexNAc moieties; an oligosaccharide consisting of 5
Hex moieties and 4 HexNAc moieties; an oligosaccharide consisting
of 4 Hex moieties and 5 HexNAc moieties; and an oligosaccharide
consisting of 3 Hex moieties and 6 HexNAc moieties. Exemplary
oligosaccharides include Lacto-N-Tetraose, Lacto-N-Neotetraose,
Lacto-N-Fucopentaose I, Lacto-N-Fucopentaose II,
Lacto-N-Fucopentaose III, Lacto-N-Fucopentaose V, Lacto-N-Hexaose,
Para-Lacto-N-Hexaose, Lacto-N-Neohexaose, Para-Lacto-N-Neohexaose,
Monofucosyllacto-N-Hexaose II, Isomeric Fucosylated Lacto-N-Hexaose
(1), Monofucosyllacto-N-Hexaose, Isomeric Fucosylated
Lacto-N-Hexaose (3), Isomeric Fucosylated Lacto-N-Hexaose (2),
Difucosyl-Para-Lacto-N-Neohexaose, Difucosyl-Para-Lacto-N-Hexaose,
Difucosyllacto-N-Hexaose, Lacto-N-Neoocataose,
Para-Lacto-N-Octanose, Iso-Lacto-N-Octaose, Lacto-N-Octaose,
Monofucosyllacto-Nneoocataose, Monofucosyllacto-N-Ocataose,
Difucosyllacto-N-Octaose I, Difucosyllacto-N-Octaose II,
Difucosyllacto-N-Neoocataose II, Difucosyllacto-N-Neoocataose I,
Lacto-N-Decaose, Trifucosyllacto-N-Neooctaose,
Trifucosyllacto-N-Octaose and Trifucosyl-Iso-Lacto-N-Octaose.
[0023] In some embodiments, the oligosaccharide described herein
comprises three or more monosaccharides (i.e., at least a
trisaccharide), and can be a bovine or human milk glycan, or the
equivalent thereof that is chemically synthesized. The complex
oligosaccharide may be, but is not limited to, (3Hex,4HexNAc,1Fuc),
(1Gal,1GcNAc,1NeuAc), and/or (1Glu,1Gal,1NeuAc (3' or 6')).
[0024] In some embodiments, the oligosaccharide described herein
comprises any of Hex(4); Hex(4) HexNAc(2); and Hex(3) HexNAc(1)
NeuAc(1) at levels greater than 1%. In another embodiment, the at
least one oligosaccharide comprise one of the following ratios of
constituents: 1) a ratio of Hex(2) NeuAc(1):Hex(2) HexNAc(1) less
than 5.0; 2) a ratio of Hex(2) HexNAc(1):Hex (3) HexNAc(1) of
greater than 1.0; 3) a ratio of Hex(2) HexNAc(1):Hex (3) HexNAc(2)
of greater than 2.0; 4) a ratio of Hex(3):Hex (3) HexNAc(1)
NeuAc(1) of less than 100; and 5) a ratio of Hex(2) HexNAc(1):Hex
(4) NeuAc(2) NeuGc(1) of greater than 10.
[0025] Complex mammalian milk oligosaccharides (MMO) can be
isolated from any number of sources and using methods known to
those of skill in the art. For example, HMOs can be obtained from
human milk using methods known in the art. Human milk may be
provided by the International Milk Bank (Sparks, Nev., USA) or any
such equivalent milk bank. Human milk may be pasteurized and then
centrifugally defatted, separating it into cream (predominantly
fat) and skim (defatted product). The defatted skim milk may then
be filtered using membranes with a 5-10 kDa cut off to concentrate
a protein fraction (predominantly whey) and the permeate,
comprising the complex HMOs. The composition of this dried HMO
fraction is about 50% lactose and about 30% HMO with the remainder
of the mass primarily peptides and ash. The HMO fraction is
predominantly fucosylated. The permeate may be further passed
through a 1 kDa cut off filter to remove lactose and provide a more
enriched HMO fraction in the retentate prior to spray drying. BMOs
can be isolated similarly, using any number of sources and methods
known to those of skill in the art. For example, BMO can be
isolated using the purification protocols as disclosed in the US
Pub. No. 20130035481, the contents of which are incorporated herein
by reference.
[0026] Colostrum oligosaccharides (COs) can be isolated from
mammalian sources such as, but not limited to cows (BCO), humans
(HCO), goats (CCO), or sheep (OCO) and used in the instant
invention. Colostrum can be used as whole colostrum or processed to
selectively enrich the CO fraction. Processing steps could include,
but are not limited to, pasteurization, centrifugation,
precipitation, ultrafiltration and spray drying. In general, the
processes are selected to remove, inhibit or destroy enzymes that
degrade the COs. In some embodiments, additional processing steps
can be used to sterilize the product to eliminate any potential
bacterial or viral contamination. Such steps include, but are not
limited to, conventional pasteurization, ultrahigh temperature
(UHT) processes, gamma irradiation, freezing and thawing,
sonication, and microfluidic disruption. In other embodiments, the
lactose content of the BCO may be reduced using processes know in
the art such as, but not limited to, the treatment of the extract
with enzymes to degrade lactose or through mechanical or biological
means of selective removal of lactose. In yet other embodiments of
the invention, the liquid CO mixtures are concentrated and/or dried
by processes such as, but not limited to, spray drying, freeze
drying, fluid bed drying, tunnel drying, and drum drying.
[0027] In various embodiments, the complex oligosaccharide
comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of the dry
weight of the composition.
[0028] In alternate embodiments, the complex oligosaccharide
further comprises synthetically produced oligosaccharides
comprising fucosyllactose (SPF) and/or synthetically-produced
sialyllactose (SPS) or derivatives thereof including, but not
limited to, 2'-fucosyllactose, 3-fucosyllactose, difucosyllactose,
lacto-N-fucosylpentaose I, lacto-N-fucosylpentaose II,
lacto-N-fucosylpentaose III, lacto-N-fucosylpentaose V,
3'-sialyllactose, 6'-sialyllactose, 3'-sialyl-3-fucosyllactose,
sialyllacto-N-tetraose, and 6'-sialyllactosamine. The synthetically
produced oligosaccharides (SPO) may be derived using any of the
number of sources and methods known to those of skill in the art.
For example, SPF is produced using protocols as disclosed in the US
Pub. No. 20130035481, the contents of which are incorporated herein
by reference.
[0029] The synthetically-produced oligosaccharides (SPOs) can be
added to the biologically produced mammalian milk oligosaccharide
(MMO) and make up from at least 5% to at least 80% of the dry
weight of the composition. In some embodiments, the composition
comprises a mixture of MMO and SPF and/or SPS. In various
embodiments, the SPO is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the dry
weight of the composition. In some embodiments, the SPF is 1-50% of
the dry weight of the composition. In other embodiments, the SPO is
5-30% of the dry weight of the composition. In other embodiments,
the SPO is 10-20% of the dry weight of the composition. The MMO
comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of the
dry weight of the composition. In some embodiments, the MMO
comprises BCOs wherein the BCOs comprise at least 20% of the dry
weight of the composition. In another preferred embodiment, the
BCOs comprise at least 50% of the dry weight of the composition. In
another preferred embodiment, the BCOs comprise at least 70% of the
dry weight of the composition. In some embodiments, the mass ratio
of MMO:SPO is from 20:1 to 1:10. In some embodiment, the ratio is
from 10:1 to 1:2, and in another embodiment, the ratio is from 5:1
to 1:1. In some examples, the ratio is about 10:1, about 9:1, about
8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about
2:1, about 1:1, about 1:2 about 1:4, about 1:5, about 1:6 about
1:3, about 1:3, 10:2, about 9:2, about 8:2, about 7:2, about 6:2,
about 5:2, about 4:2 or about 3:2.
[0030] B. Non-Pathogenic Microbes
[0031] In various embodiments, the composition comprises one or
more species of non-pathogenic microbes, where one of the
non-pathogenic microbes is from a species whose genome encodes a
transport system capable of internalizing one or more complex
oligosaccharides before the oligosaccharide is internally
hydrolyzed. In various embodiments, the microbe is from the genus
Bifidobacterium. The species may be, but is not limited to, B.
longum, B. bifdum, B. breve, B. pseudocatenulatum, B. catenulatum,
or any Bifidobacterium strain that expresses a fucosidase, or any
combination of these Bifidobacterium species. In some embodiments,
one of the species is Bifidobacterium longum and in preferred
embodiments, one or both of the species is Bifidobacterium longum
subspecies infantis.
[0032] In various embodiments, the Bifidobacterium may comprise
activated Bifidobacterium (ABI). The activated Bifidobacterium is
defined herein as the state of the cells, as measured by the
up-regulation or down-regulation of genes including but not limited
to those coding for oligosaccharide binding proteins, transport
proteins, and enzymes responsible for the degradation of the
complex oligosaccharides, which provides significant benefits to a
newborn infant. Such beneficial characteristics of the ABI include,
but are not limited to, a higher binding affinity to the gut
mucosa, higher colonization of the GI tract thereby preventing
growth of other bacterial clades, a higher production of short
chain fatty acids, the ability to consume complex oligosaccharides,
and a greater stimulation of the immune response as measured by
positive alterations of immune response markers, relative to the
organism in a pre-activated state (Lewis, et al., 2015, Microbiome,
3:13; Huda, et al., 2014, Pediatrics, 134:2 e362-e372).
[0033] In various embodiments, the bifidobacteria encodes gene
clusters containing ATP-binding cassette (ABC) transporters and
glycosyl hydrolases involved in HMO utilization, typically
including one or more genes coding for a fucosidase. In some
embodiments, the bifidobacteria contains a gene coding for a
complex oligosaccharide transporter. In some embodiments, the
bifidobacteria contains a gene coding for a fucose transporter. In
some embodiments, the bifidobacteria contains a gene coding for a
fucose or sialic acid transporter. In many embodiments, the genes
encoding these components are upregulated or expressed. The genes
may be constitutively upregulated or induced.
[0034] Certain biomarkers may be induced and/or repressed as
markers to predict an activated state for bifidobacterium species,
whereby the bacteria are optimally primed for complex
oligosaccharide consumption. Suitable biomarkers identified with B.
longum subsp. infantis activation include upregulated genes and
downregulated genes. Exemplary upregulated genes include Blon_0042
(regulatory protein); Blon_R0015 (tRNA), Blon_R0017 (tRNA);
Blon_R0021 (tRNA); and Blon_R0022 (tRNA). Exemplary downregulated
genes include Blon_0518 (hypothetical protein); Blon_0785 (membrane
lipoprotein (possible transporter component)); Blon_2167
(hypothetical protein); and Blon_2168 (phage shock protein C).
Previously, these genes were not known to be associated with an
activated cell.
[0035] In some embodiments, the activated bifidobacteria comprises
gene Blon_0042, wherein gene Blon_0042 has been upregulated. The
activated bifidobacteria may comprise gene Blon_2168, wherein gene
Blon_2168 has been downregulated. In one embodiment, the activated
bifidobacteria comprises gene Blon_0042 and gene Blon_2168, wherein
gene Blon_0042 has been upregulated and gene Blon_2168 has been
downregulated. The skilled person can readily adapt quantitative
proteomic methods to determine the expressed levels of the gene
products (e.g., mRNA and protein) for these genes, to confirm
activation.
[0036] ABI is activated by being cultivated in a medium comprising
at least one oligosaccharide among the complex oligosaccharides
described above for a sufficient period of time to undergo
induction and biosynthesis of at least one metabolic enzyme. The
oligosaccharides are typically sourced from, or are identical to,
those mammalian milk oligosaccharides (MMOs) including, but not
limited to, those from human milk and bovine milk. In some
embodiments, the oligosaccharide can be a bovine or human milk
oligosaccharide. In another embodiment, the oligosaccharide is
obtained from mammalian colostrum. In some embodiments, the
oligosaccharide composition comprises bovine milk oligosaccharides
(BMOs). Bovine oligosaccharides may comprise oligosaccharides from
mature milk, early milk, colostrum, or concentrates thereof. In
some embodiments, the oligosaccharides include fucose as component
saccharide residues. In an alternative embodiment, the MMO is
supplemented with synthetically produced and purified
oligosaccharides comprising fucosylated and/or sialylated
oligosaccharides. In some embodiments of the invention, the
synthetically-produced fucosyllactose (SPF), sialyllactose (SPS) or
derivatives thereof are used to activate bifidobacteria in a way
that is more human-like than when activated by BMOs alone. In
another embodiment, the composition is used to upregulate operons
other than the HMO cluster.
[0037] Any of the compositions described herein may be prepared by
cultivating a bifidobacteria in an axenic culture (e.g., a culture
with genetic homogeneity), the culture comprising bovine milk
glycans, (e.g., concentrated from bovine colostrum) to become
"activated." In various embodiments, any of the compositions
described herein can be made by isolating bifidobacteria; purifying
the bacteria; inoculating a fermenter with the purified strains of
the bifidobacteria; and culturing the bifidobacteria in the
presence of complex bovine or human oligosaccharides; and
harvesting the cells. Fermentations for bifidobacteria may be
carried out in stirred tank fermenters of commercial volume (e.g.,
1-500 m.sup.3) which are maintained under anaerobic conditions
throughout the fermentation process. The fermentation can include
the steps of providing at least one complex oligosaccharide at any
time during the course of the fermentation in a liquid culture at a
level of at least 1 g/L, typically from about 1-50 g/L, or 2-20
g/L, or 5-10 g/L as a sole, or supplementary, carbon source to
activate the cells.
[0038] The bifidobacteria described herein may be tested for its
ability to use bovine or human milk oligosaccharides for growth. In
some embodiments, the bifidobacteria are capable of growing on
mammalian milk glycans where less than 20% of the sialic acid
content and 20% of the fucose content of the milk glycans remains
after a culture of the composition has ceased to grow. In some
embodiments, the composition is capable of growing on mammalian
milk oligosaccharide wherein less than 10% of the sialic acid
content and 10% of the fucose content of the milk glycans remains
after a culture of the composition has ceased to grow. In a
preferable embodiment the composition is capable of growing on milk
glycans wherein less than 5% of the sialic acid and 5% of the
fucose of the milk oligosaccharides remains after a culture of the
composition has ceased to grow. In a particularly preferable
embodiment, the composition is capable of growing on milk glycans
wherein less than 1% of the sialic acid and 1% of the fucose of the
milk oligosaccharides remains after a culture of the composition
has ceased to grow.
[0039] In a further embodiment, the composition can comprise a
total count of viable bacteria from about 100 thousand to 500
billion colony forming units (cfu) per gram dry weight. In another
embodiment, the total count of viable bacteria comprises 5 billion
to 100 billion cfu per gram dry weight. In another embodiment, the
total count of viable bacteria comprises 10 billion to 50 billion
cfu per gram dry. In some embodiments, the ABI concentration is
from 10 to 100 g dry weight per liter. The fermentation products
can also be concentrated by filtration or centrifugation. The ABI
can be can be dried by controlled desiccation processes such as,
but not limited to, freeze drying.
IV. Formulating Compositions
[0040] The composition comprising MMO and ABI can be prepared by
mixing the two components together. Optionally, one can combine the
harvested and/or dried activated bifidobacteria cells with a
powdered form of a complex bovine or human milk oligosaccharide.
The harvested and/or dried activated bifidobacteria cells and the
powdered form of the complex bovine or human milk oligosaccharide
can be in a single dose packet, which can contain from about 10
million to about 100 billion cfu of bacteria and, optionally, from
about 0.5 g to about 5 g of complex oligosaccharide. The complex
bovine oligosaccharide can be present in a powder composition
wherein the blend ratio of activated bifidobacteria cells to
complex oligosaccharide is 30 billion cfu per 1.5 g complex
oligosaccharide in a powder form.
[0041] Any of the compositions described herein can further
comprise a secondary metabolite. The secondary metabolite can be a
short chain fatty acid, such as acetate, lactate, or combinations
thereof. The compositions described herein can further comprise a
stabilizer, such as a flow agent. Flow agents may include starch,
silicon dioxide, tricalcium phosphate, powdered cellulose,
magnesium stearate, sodium bicarbonate, sodium ferrocyanide,
potassium ferrocyanide, calcium ferrocyanide, bone phosphate,
sodium silicate, calcium silicate, magnesium trisilicate, talcum
powder, sodium aluminosilicate, potassium aluminum silicate,
calcium aluminosilicate, bentonite, aluminum silicate, stearic
acid, and polydimethylsiloxane. The stabilizer can be a milk
protein or another suitable pharmaceutical grade or infant formula
grade diluent (e.g., lactose). The milk protein can comprise a
protein fraction of non-fat dry milk.
[0042] Any of the compositions described herein can further
comprise surface carbohydrate binding protein (e.g., solute binding
proteins). The surface carbohydrate binding proteins can allow a
more effective binding and interaction with the gut mucosa by
binding to cell surface glycosylation of the gut mucosa and or
mucous layers. This binding of surface carbohydrate can then
exclude the binding of pathogenic bacteria.
[0043] In various embodiments, any of the compositions described
herein may be dried (e.g., by spray-drying or freeze-drying), and
formulated into a unit dose medicament, such as a packet, sachet,
orally disintegrating tablet, foodstuff, capsule, lozenge,
effervescent tablet, etc. The unit dose medicament can be formed
from a variety of materials including without limitation plastic,
or paper. In some embodiments, the unit dose medicament comprises a
moisture barrier and/or oxygen barrier layer. Alternatively, the
composition may be provided in a form for anal delivery, such as a
suppository or in an enema. Preferably, the composition is packaged
in sachets made using a moisture and/or oxygen impermeable
polymer.
[0044] In various embodiments, any of the compositions described
herein may be provided in a dry powder formulation, a solution, a
suspension, or in a tablet or capsule format with or without an
enteric coating. The dry powder can be freeze-dried or spray dried.
The freeze-dried compositions are preferably frozen in the presence
of a suitable cryoprotectant. The cryoprotectant can be, for
example, glucose, lactose, raffinose, sucrose, trehalose, adonitol,
glycerol, mannitol, methanol, polyethylene glycol, propylene
glycol, ribitol, alginate, bovine serum albumin, carnitine,
citrate, cysteine, dextran, dimethyl sulphoxide, sodium glutamate,
glycine betaine, glycogen, hypotaurine, peptone, polyvinyl
pyrrolidone, or taurine. The enteric coatings include, but are not
limited to, fatty acids, waxes, shellac, plastics, plant fibers,
methyl acrylate-methacrylic acid copolymers, cellulose acetate
succinate, hydroxy propyl methyl cellulose phthalate, hydroxy
propyl methyl cellulose acetate succinate, polyvinyl acetate
phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers,
cellulose acetate trimellitate, sodium alginate, and Zein.
[0045] In some embodiments, the microbe is mixed with a
cryopreservative such as but not limited to trehalose or glycerol
under anaerobic conditions and frozen by processes such as, but not
limited to, rapid freezing (chilling with liquid nitrogen), or by a
controlled temperature reduction in a cryopreservation freezing
system. Once frozen, the microbes can be dehydrated under vacuum
using a process that best maintains the integrity of the microbe
cells. The microbe concentration in the dry powder can be from 1
million to 500 billion cfu/g. In some embodiments, the dry powder
can be from 5 billion to 100 billion cfu/g, and in a most preferred
embodiment the dry powder can be from 10 billion to 50 billion
cfu/g.
[0046] In some embodiments of the invention, the powdered microbe
is resuspended in an edible oil such as, but not limited to
triglyceride oils (e.g., vegetable oil, olive oil, and medium chain
triglycerides), diglyceride oils, monoglyceride oil, and silicone
oils.
[0047] In various embodiments, the oligosaccharide composition can
be dissolved in a polar liquid such as, but not limited to, water,
physiological saline, mammalian milk, or an infant formula, and
provided in a liquid form to the infant while the bifidobacteria
are provided separately as a powder or suspension in a carrier
liquid which may include a solution comprising the
oligosaccharide.
[0048] In various embodiments, the microbes and the oligosaccharide
composition may be provided combined or provided separately. In
some embodiments, the microbe is combined with an oligosaccharide
in a single dose packet containing from about 1 to about 100
billion cfu of microbe and from about 0.5 to about 5 g of an
oligosaccharide.
V. Use of Compositions for Improvement of Mammalian Health
[0049] In various embodiments, the compositions described herein
are delivered as a pre-activated and purified composition of
bifidobacterium to a subject in need thereof substantially
contemporaneously with delivery of compounds to the mammalian
intestine to make the intestinal environment a more favored niche
to the aforementioned purified composition of bifidobacteria, where
the compounds may comprise complex oligosaccharides described
above, synthetically produced and purified oligosaccharides, and/or
secondary metabolites produced as a result of intestinal
fermentation.
[0050] In various embodiments, the use described herein comprises
monitoring the subject's intestinal microbiome before, during
and/or after administration of the composition described herein. A
variety of monitoring techniques are known to one of ordinary skill
in the art. For example, a routine sample of the subject's feces
may be analyzed for microbes qualitatively and/or quantitatively by
standard processes well known in the art (see, e.g., Le Pare et
al., 2014, Food and Nutrition Sciences, 5: p. 71-78).
[0051] In some embodiments, the compositions described herein are
administered to a subject in need thereof in an amount and for a
duration effective to establish the population of bifidobacteria at
high levels in the gastrointestinal tract of the subject. In some
embodiments, the compositions described herein can be administered
to a subject in need thereof in an amount and for a duration
effective to maintain the population of bifidobacteria at high
levels in the gastrointestinal tract of the subject. In some
embodiments, the composition is administered daily in an effective
amount to maintain the bifidobacteria population in the gut of the
subject at greater than at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, or at least 90% of the total fecal microbiome of the
mammal.
[0052] In some embodiments, the composition comprising activated
bifidobacteria is administered to a subject in need thereof. In
other embodiments, the composition comprising complex
oligosaccharides supplemented with synthetically produced and
purified oligosaccharide is administered to a subject in need
thereof. In another embodiment, the composition comprising both
activated bifidobacteria and complex oligosaccharide is
administered to a subject in need thereof. In another embodiment,
the composition comprising bifidobacteria, and complex
oligosaccharides supplemented with synthetically produced and
purified oligosaccharide is administered to a subject in need
thereof.
[0053] In various embodiments, the bifidobacteria are administered
at a dose of from 1 billion to 100 billion cfu of bifidobacteria
and from 1 to 20 g of complex oligosaccharides per day. In some
embodiments, a dose is administered from 5 to 50 billion cfu/day.
In another embodiment, a dose is administered from 5 to 100 billion
cfu/day. In another embodiment, the dose is administered from 10 to
25 billion cfu/day. In various embodiments, the complex
oligosaccharide is administered in a dose of from 0.5 g to 5.0
g/day. In some embodiments, the dose is administered in a dose from
1.0 g to 3.0 g/day.
[0054] Typically, the composition of this invention is presented as
a single, unit dose package that is administered once per day.
However, the doses may be presented in multiple (e.g., two, three,
four, five, six, or more) sub-doses administered at appropriate
intervals throughout the day. Alternatively, they may be
administered in the same composition, or constituent components may
be administered sequentially. In some embodiments, the treatment is
maintained for a period of at least 1-week, 2-weeks, 3-weeks, or at
least 4-weeks. In other embodiments, the treatment is administered
for a period of from at least 2-months, 4-months, 6-months,
8-months, 10-months, or at least 12-months.
[0055] The subject in need thereof can be, for example, an infant
from birth to about 36 months post-conception. In additional
embodiments, the compositions described herein may be administered
to a pregnant woman in at least the third trimester of pregnancy.
The composition administered during pregnancy may include either
the bifidobacteria, the oligosaccharide, or both. In additional
embodiments, the composition described herein is administered in a
therapeutic amount to an infant born vaginally or by cesarean
section. The compositions described herein are administered to the
infant immediately after delivery and thereafter for at least the
first month to six months of the life of the infant. The
composition may be administered directly to the infant or mixed
with a liquid including, but not limited to breast milk, infant
formula, physiological saline, or water. For infants who are not
breast fed, the compositions described herein may alternatively be
administered in an infant formula and such compositions may
preferably comprise both activated B. infantis and a milk-derived
oligosaccharide. For infants born via cesarean section,
compositions comprising of activated bifidobacteria and/or complex
oligosaccharides may be administered. For infants born vaginally,
compositions comprising activated bifidobacteria and/or complex
oligosaccharides may be administered.
[0056] The above-described embodiments of the invention are
presented for purposes of illustration and not of limitation. While
these embodiments of the invention have been described with
reference to numerous specific details, one of ordinary skill in
the art will recognize that the invention can be embodied in other
specific forms without departing from the spirit of the invention.
Thus, one of ordinary skill in the art would understand that the
invention is not to be limited by the foregoing illustrative
details, but rather is to be defined by the appended claims.
EXAMPLES
[0057] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1. Preparation of Human Milk Oligosaccharide (HMO)
Compositions that can be Used Exclusively by Certain
Bifidobacteria
[0058] A concentrated mixture of HMO is obtained by a process
similar to that described by Fournell et al (US Patent Application
20150140175). Human milk is pasteurized and then centrifugally
defatted, separating it into cream (predominantly fat) and skim
milk (defatted product). The defatted skim milk is then
ultrafiltered using membranes with a 5-10 kDa cut off to
concentrate a protein fraction (predominantly whey). The permeate
from the ultrafiltration, comprising the complex HMOs, is dried by
spray drying. The composition of this dried fraction is about 50%
lactose and about 30% complex oligosaccharides (HMO) with the
remainder of the mass primarily peptides and ash. The HMO fraction
is predominantly fucosylated.
[0059] This method or similar methods can be used to obtain
compositions containing isolated complex oligosaccharides from any
mammalian milk source. For example, complex oligosaccharides can be
isolated using the purification protocols as disclosed in the US
Pub. No. 20130035481, the contents of which are incorporated herein
by reference. Such compositions are suitable for use in embodiments
of this invention.
Example 2. Preparation of Bovine Colostrum Oligosaccharide (BCO)
Compositions and Compositions Supplemented with Synthetically
Produced and Purified Fucosylated Oligosaccharides (SPF) that can
be Used Exclusively by Certain Bifidobacteria
[0060] A concentrated mixture of bovine colostrum oligosaccharide
is obtained by a process such as that described by Christiansen et
al (2010) International Dairy Journal, 20:630-636. Bovine colostrum
(preferably from the first milking) is pasteurized by heating to
145 degrees F. for 30 minutes, cooled and centrifugally defatted,
separating it into cream (predominantly fat) and skim milk
(defatted product). The defatted skim milk is then ultra-filtered
using membranes with a 5-10 kDa cut off to concentrate a protein
fraction (predominantly whey). The whey permeate is further
microfiltered using a 1 kDa cut off to remove some lactose and
concentrate the oligosaccharides in the retentate. The final
composition is spray dried to produce a dried oligosaccharide
fraction having about 40% lactose and about 40% complex
oligosaccharides (BCO) with the remainder of the mass primarily
peptides and ash. The BCO fraction is predominantly sialylated.
[0061] Synthetically produced and purified fucosylated
oligosaccharides (SPF) can be can be obtained commercially from any
of the number of sources or derived by methods known to those of
skill in the art. 100 g of purified 3-Fucosyllactose (SPF; Elicityl
S A, Crolles, F R) may be added to 1 kg of the BCO preparation and
thoroughly mixed to produce a BCO/SPF composition with a ratio of
BCO:SPF of about 4:1. The sample is analyzed and the complex
oligosaccharide component is about 20% fucosylated
oligosaccharides.
[0062] This method or similar methods can be used to obtain
compositions containing complex oligosaccharides from any mammalian
milk source supplemented with particular synthetic
oligosaccharides. Such compositions are suitable for use in
embodiments of this invention.
Example 3. Differentiation Between BCO, BCOC, BMO and HMO
Fractions
[0063] A BCOC fraction was commercially obtained as Immunel,
(Sterling Technology, USA) and analyzed using HPLC-MS methods of
Tao et al (2008) J Dairy Science, 92: 2991-3001 and compared in
Table 1 with oligosaccharide fractions from human milk (HMO),
mature bovine milk (BMO) and bovine colostrum (BCO). The four
compositions in Table 1 are significantly different from each other
and several features are immediately apparent. BCOC contains
several oligosaccharides that are not found in BMO, BCO or HMO such
as Hex(4) and Hex(3) HexNAc(1) NeuAc(1), and several
oligosaccharides that are found in BCO and BMO are not present in
BCOC such as Hex(2) HexNAc(1) NeuAc(1), Hex (3) HexNAc(2), and Hex
(4) NeuAc(2) NeuGc(1).
TABLE-US-00001 TABLE 1 Key oligosaccharides that differentiate
colostrum and BMO compositions as disclosed in Tao (2009), HMO
compositions as disclosed in Mills et al (2012) and bovine
colostrum oligosaccharide concentrate (BCOC; Immunel) of the
instant invention. Values listed are percentages of the total
sample oligosaccharides. m/z reduced BCO BMO neutral Hex HexNAc
NeuAc NeuGc Fuc Tao BCOC Tao HMO 506.1833 3 2.27 22.70 18.70 1.48
547.2198 2 1 2.62 17.10 1.59 0.01 635.2263 2 1 20.33 26.00 22.61
1.82 667.2300 4 0.10 1.40 0.01 0.01 676.2533 1 1 1 4.47 3.90 1.01
0.01 709.2621 3 1 4.15 6.90 10.00 20.90 750.2892 2 2 5.62 3.30 0.01
0.01 797.2772 3 1 4.98 3.20 23.11 0.01 838.3056 2 1 1 3.34 0.01
0.61 0.01 855.3221 3 1 1 0.01 0.01 0.01 0.70 871.3153 4 1 7.35 7.50
12.46 0.01 912.3409 3 2 4.73 0.01 2.85 0.01 942.3234 2 1 1 11.48
0.01 0.01 0.01 999.3517 3 1 1 0.01 1.30 0.01 0.01 1074.3960 4 2
0.01 4.50 0.52 0.41 1177.4000 4 1 1 2.63 0.01 0.26 0.01 1220.4540 4
2 0.01 0.01 0.01 25.20 1366.5120 4 2 2 0.01 0.01 0.01 13.50
1439.5290 5 3 0.01 0.01 0.01 2.00 1585.5870 5 3 1 0.01 0.01 0.01
5.90 1731.6450 5 3 2 0.01 0.01 0.01 7.20 1804.6610 6 4 0.01 0.01
0.01 1.40 1877.7030 5 3 3 0.01 0.01 0.01 5.00 1950.7190 6 4 1 0.01
0.01 0.01 2.90 2096.7770 6 4 2 0.01 0.01 0.01 3.60 2242.8350 6 4 3
0.01 0.01 0.01 1.80
[0064] In addition, the ratios of the various bovine
oligosaccharides to each other are also quite different among the
bovine sources as demonstrated in Table 2. The ratio of Hex(3) to
Hex(3) NeuAc( ) of BCOC is greater than 1.0, the ratio of Hex(3) to
Hex(4) HexNAc(2) of BCOC is less than 20. and the ratio of Hex(2)
HexNAc(1) to Hex(4) of BCOC is less than 20. Further, these bovine
oligosaccharide compositions are all distinguished from HMO in that
in addition to the ratio differences all bovine oligosaccharide
samples are further characterized by being less than 50%
fucosylated. In addition, about 70% of bovine early milk
oligosaccharides are sialylated in contrast to 50% in BMO, HMO, and
N-glycolylneuraminic acid, which made up 7% of the sialic acid in
the bovine early milk is completely absent in mature BMO (Tao,
2009). Even without specifying fucosylation or sialylation, there
are some ratios of complex oligosaccharides that are unique in the
BCOC of the present invention even when considering all bovine and
human oligosaccharides. Such unique BCOC oligosaccharide signatures
include; 1) a ratio of Hex(2) NeuAc(1):Hex(2) HexNAc(1) less than
5.0; 2) a ratio of Hex(2) HexNAc(1):Hex (3) HexNAc(1) of greater
than 1.0; 3) a ratio of Hex(2) HexNAc(1): Hex (3) HexNAc(2) of
greater than 2.0; 4) a ratio of Hex(3):Hex (3) HexNAc(1) NeuAc(1)
of less than 100; and 5) a ratio of Hex(2) HexNAc(1):Hex (4)
NeuAc(2) NeuGc(1) of greater than 10 (Table 2).
TABLE-US-00002 TABLE 2 Ratios of specific oligosaccharides found in
BCO, BCOC, BMO and HMO from Table 1 Oligosaccharide ratios BCO BCOC
BMO HMO Hex(2) NeuAc(1):Hex (2) HexNAc(1) 7.8 1.5 14.2 182.0 Hex(2)
HexNAc(1):Hex (3) 0.6 2.5 0.2 0.0 HexNAc(1) Hex(2) HexNAc(1):Hex
(30 0.6 1710.0 0.6 1.0 HexNAc(2) Hex(3):Hex (3) HexNAc(1) NeuAc(1)
227.0 17.5 1870.0 148.0 Hex(2) HexNAc(1):Hex(4) NeuAc(2) 1.0 1710.0
6.1 1.0 neuGc(1) Hex(3):Hex(3) NeuAc(1) 0.5 7.1 0.8 148.0
Hex(3):Hex(4) HexNAc(2) 227.0 5.0 36.0 3.6 Hex(2) HexNAc(1):Hex(4)
26.2 12.2 159.0 1.0 Fucosylated oligosaccharides (% 0.00 0.00 0.00
0.00 of total)
[0065] This example demonstrates that HMO, BMO, BCO, and BCOC have
distinctly different compositions. However, all four of these
mixtures are able to activate B. infantis.
Example 4. Preparation of an Activated Bifidobacteria (ABI)
Composition that can Exclusively Use Certain Complex
Oligosaccharides
[0066] Bifidobacteria longum subsp. infantis (alternatively B.
infantis herein) was isolated and purified from the feces of a
vaginally delivered, breast fed human infant and its identification
was confirmed by DNA analysis that reflected the presence of a gene
set that is specifically associated with this organism (Sela et
al., 2008, PNAS, 105(48): p. 18964-69). Alternatively, a strain of
B. infantis can be obtained from a commercial culture collection
such as the American Type Culture Collection (ATCC) of Washington,
D.C.
[0067] A seed culture of this organism was added to a growth medium
comprising glucose and a BCO composition, made using the process
described in Example 2, and other standard salts and vitamins in a
500 L agitated fermenter. Following 3 days of growth under
anaerobic conditions, a sample of the culture was tested for the
presence of ABI. ABI was identified by the presence of expressed
gene transcripts for fucosidase or sialidase. The fermenter was
harvested by centrifugation and the concentrated cell mass was
mixed with a cryopreservative (e.g., trehalose plus milk proteins)
and freeze dried. The final dry product was 5.5 kg of bacterial
mass with a count of 130.times.10.sup.9 cfu/g.
[0068] This example demonstrates that bifidobacteria can be
activated by culturing the bifidobacteria with a complex bovine
milk oligosaccharide. While BCO was used herein, this method or a
similar method can be used to obtain ABI by culturing with MMO from
any mammalian milk. Such ABI would be suitable for use in
embodiments of this invention.
Example 5. Bifidobacteria Grown on Complex Oligosaccharides is
Activated for Consumption of Milk Oligosaccharides
[0069] B. infantis ATCC 15697 was grown in MRS broth containing 2%
lactose or bovine milk oligosaccharides (BMO). Cells were collected
at exponential phase, RNA was purified and converted to cDNA and
sequenced on an Illumina platform. Results clearly show
differential expression during growth on BMO.
[0070] FIG. 1 depicts whole genome expression analysis. The diagram
shows principle component analysis of all expressed genes within B.
infantis. The diagram clearly shows differential expression of
cells grown on BMO versus cells grown on lactose. 577 genes are
differentially expressed suggesting growth on milk oligosaccharides
induces a different physiological state in B. infantis than
lactose.
[0071] Further analysis shows that the 40 kb milk oligosaccharide
consumption gene cluster previously identified in B. infantis is
preferentially induced during growth on BMO by comparison to growth
on lactose as shown in FIG. 2. These results clearly show B.
infantis grown on BMO is activated for consumption of milk
oligosaccharide and a range of other genes involved in colonization
and host interface in the neonate colon, including Blon_2334,
Blon_2335, Blon_2336, Blon_2337, Blon_2338, Blon_2339, Blon_2344,
Blon_2346, Blon_2347, and Blon_2331, are upregulated.
Example 6. Preparation of Therapeutic Compositions for the
Treatment of Pregnant Women
[0072] Preparation 1 is prepared by first diluting the ABI product
of Example 4 with pharmaceutical grade lactose to provide a dose of
25 billion cfu of B. longum subsp. infantis per gram. This diluted
ABI product is then packaged in 2-piece gel caps (1 g/gel cap) made
of a gastric-resistant polymer such as pectin, to provide doses of
25 billion cfu of activated B. longum subsp. infantis per capsule
in a delivery form that releases its contents in the GI tract
beyond the stomach.
[0073] Preparation 2 is prepared by blending the ABI product of
Example 4 with the BMO/SPF composition of Example 2 whereby 25
billion cfu of B. longum subsp. infantis (170 mg of the dry
product) is blended with 5 g of the BMO/SPF composition of Example
2. Preparation 2 provides a ratio of 25 billion cfu of B. longum
subsp. infantis to about 2.5 g BCO/SPF and this mixture is packaged
in sachets made using a moisture and oxygen impermeable
polymer.
Example 7. Administering Composition to Pregnant Women
[0074] The composition of Preparation 1 of Example 6 (ABI) is
produced and the product is packaged in 2-piece gel caps (1 g/gel
cap) made of a gastric-resistant polymer such as pectin, to provide
doses of 25 billion cfu of B. longum subsp. infantis per capsule. A
second preparation is made using the dry product of Preparation 2
of Example 6 and the product is packaged in sachets made using a
moisture and oxygen impermeable polymer to provide a dose of 25
billion cfu of B. longum subsp. infantis plus 2 g BMO/SPF (5 g of
the BMO/SPF composition of Example 2) per sachet.
[0075] The compositions described herein are taken orally by a
woman throughout pregnancy but at least in the third trimester of
pregnancy. For the initial treatment, a pregnant woman is provided
with two capsules of Preparation 1, as described in Example 6, on a
daily basis for the first 2 weeks of treatment. This process
establishes the population of B. longum subsp. infantis in the gut
of the woman. For subsequent weeks of treatment, the woman with is
provided with 4 sachets per day of Preparation 2 of Example 6 to
maintain the population of B. longum subsp. infantis at high levels
in her gastrointestinal tract. The 4 sachets are taken throughout
the day, one at each meal and one before going to bed. The contents
of the sachet can be mixed with milk, yogurt or pudding to aid in
oral consumption. If the levels of B. longum subsp. infantis fall
below 25% of the levels established by the end of the 2-week
pre-treatment with Preparation 1 of Example 6 as determined by
fecal microbiome analyses, the patient is returned to the
Preparation 2 treatments for an additional 2 weeks. Compositions of
this invention according to alternative embodiments may be
administered similarly. Administration should continue until the
birth of the child. The treatment leads to a much higher likelihood
that a vaginally-delivered infant will be appropriately inoculated
with the B. longum subsp. infantis from the mother.
Example 8. Use of a Composition Comprising Bifidobacteria with an
Expressed Fucosidase to Improve the Health of an Infant
[0076] The final dry product of Example 4 (Activated
Bifidobacterium longum subsp. infantis--at a bacterial count
130.times.10.sup.9 cfu/g) is blended with pharmaceutical grade
lactose to reach a bacterial count of about 25.times.10.sup.9
cfu/g, and packaged into sachets made from moisture and oxygen
resistant materials at a mass of 500 mg (12.5.times.10.sup.9 cfu)
per sachet. The contents of one sachet is provided to a newborn
infant who is exclusively receiving breast milk every day for a
period of 6 months. The package is opened and the contents emptied
into a small cup to which a few drops of breast milk is added to
make a watery paste. This is then provided to the baby either using
a blunt-tipped plastic dropper or the parent's fingertip. Best
results are obtained if the baby consumes at least 75% of the daily
composition each day. Monitoring of the infant fecal microbiota
will indicate that the level of B. infantis in the feces will
represent more than 20% of the total microbial load of the feces.
This composition should be provided to all babies whether they are
vaginally delivered, but especially if they are delivered by
Caesarian Section.
[0077] Some mothers who are nursing their babies, may be deficient
in certain complex fucosylated oligosaccharides in their milk
because of a deficiency in .alpha.1-2-fucoslyltransferase enzyme
(FUC-2) as measured by a genetic test. For mothers who are of the
genotype FUC-2, the sachet containing the ABI product of example 4
should be supplemented with a BCO composition containing
synthetically produced and purified fucosylated oligosaccharides
(SPF) at a BCO:SPF ratio of about 2:1 as described in Example 2.
These sachets are prepared to deliver of 12.5.times.10.sup.9 cfu of
ABI and 1 g BCO plus 0.5 g fucosyllactose per sachet (i.e., 100 mg
of the undiluted ABI of Example 4 plus 2.5 g of BCO of Example 2
plus 0.5 g of fucosyllactose per sachet). This composition is
provided on a daily basis for 6 months as described above for the
sachets of ABI alone.
[0078] For infants that are receiving mixed feeds (breast milk and
infant formula) or exclusively infant formula, the ABI product of
Example 4 is blended with a bovine colostrum oligosaccharide
concentrate composition of Example 2. A composition of dried bovine
colostrum oligosaccharide containing about 40% lactose and about
40% complex oligosaccharides (BCO) is prepared according to Example
2 and packaged in a moisture resistant sachet at a dose of 5
g/sachet (i.e., 2 g BCO/sachet)--the BMO sachet. Additional sachets
containing a blend of the ABI product of Example 4 resulting in the
delivery of 12.5.times.10.sup.9 cfu of ABI, and 2 g BCO per sachet
(ca. 100 mg of the undiluted ABI of Example 4 plus 5 g of BCO of
Example 2)--the BLEND sachet. The contents of one BLEND sachet is
provided to a baby on a daily basis who is otherwise receiving
mixed feeds or exclusively receiving infant formula, by opening the
sachet and dissolving its contents into the prepared liquid infant
formula and providing it to the baby in a morning feed. Twelve
hours later the process is repeated but with the BMO sachet (no
ABI) delivered with the infant formula as described for the BLEND
sachet. This daily cycle is repeated (BLEND in the morning, BMO in
the evening) for at least the first 6 months of life. Monitoring of
the infant fecal microbiota will indicate that the level of B.
infantis in the feces will represent more than 20% of the total
microbial load of the feces. This routine should be maintained for
all babies whether they are vaginally delivered, or delivered by
Caesarian Section, as long as they are not being exclusively
breast-fed. Alternatively, the infant can be provided just the
BLEND sachets twice per day providing the BLEND sachets are
prepared with only 6.times.10.sup.9 cfu of ABI per sachet.
[0079] These dietary supplements will increase the concentration of
B. longum subsp. infantis in the lower bowel of the baby to those
levels historically seen in vaginally-delivered, breast-fed babies,
and will significantly reduce the likelihood of a pathogenic
bacterial bloom that may cause colic in that baby. This
supplementation will also significantly improve the rate of
development of that baby's gastrointestinal mucosa and mucosal
immune response. Compositions of this invention according to
alternative embodiments may be administered similarly.
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