U.S. patent application number 14/198739 was filed with the patent office on 2014-09-04 for human milk oligosaccharides to promote growth of beneficial bacteria.
The applicant listed for this patent is Abbott Laboratories. Invention is credited to RACHAEL BUCK, JOMAY CHOW, STEVEN R. DAVIS.
Application Number | 20140249103 14/198739 |
Document ID | / |
Family ID | 45496315 |
Filed Date | 2014-09-04 |
United States Patent
Application |
20140249103 |
Kind Code |
A1 |
BUCK; RACHAEL ; et
al. |
September 4, 2014 |
HUMAN MILK OLIGOSACCHARIDES TO PROMOTE GROWTH OF BENEFICIAL
BACTERIA
Abstract
Disclosed are nutritional compositions including human milk
oligosaccharides that can be administered to individuals including
preterm infants, infants, toddlers, and children for improving
gastrointestinal function and tolerance, as well as the growth of
beneficial bacteria. Additional suitable methods of using the
nutritional compositions including the human milk oligosaccharides
are also disclosed.
Inventors: |
BUCK; RACHAEL; (Gahanna,
OH) ; CHOW; JOMAY; (Westerville, OH) ; DAVIS;
STEVEN R.; (Columbus, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Abbott Laboratories |
Abbott Park |
IL |
US |
|
|
Family ID: |
45496315 |
Appl. No.: |
14/198739 |
Filed: |
March 6, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13335323 |
Dec 22, 2011 |
|
|
|
14198739 |
|
|
|
|
61428867 |
Dec 31, 2010 |
|
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61474691 |
Apr 12, 2011 |
|
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Current U.S.
Class: |
514/61 |
Current CPC
Class: |
A23L 33/40 20160801;
A61P 25/00 20180101; A61P 29/00 20180101; A61P 1/14 20180101; A23V
2002/00 20130101; A61K 31/702 20130101; A61P 1/00 20180101; A23V
2002/00 20130101; A23V 2200/32 20130101; A23V 2200/3202 20130101;
A23V 2200/322 20130101; A23V 2250/28 20130101 |
Class at
Publication: |
514/61 |
International
Class: |
A61K 31/702 20060101
A61K031/702; A23L 1/29 20060101 A23L001/29 |
Claims
1. A method for regulating gut motility in an infant in need
thereof, the method comprising administering a nutritional
composition comprising a human milk oligosaccharide selected from
the group consisting of 2'-fucosyllactose and 3'-fucosyllactose, to
the infant.
2. The method of claim 1, wherein the human milk oligosaccharide is
present in a concentration of from about 0.001 mg/ml to about 20
mg/ml.
3. The method of claim 1, wherein gut motility is regulated by
reducing contractions in the infant's gastrointestinal tract.
4. The method of claim 3, wherein the human milk oligosaccharide
reduces the contractions.
5. The method of claim 3, wherein the frequency of contractions is
reduced.
6. The method of claim 3, wherein the amplitude of contractions is
reduced.
7. The method of claim 3, wherein both the frequency and amplitude
of contractions are reduced.
8. The method of claim 7, wherein the frequency and amplitude of
contractions are reduced in a dose response fashion to the
concentration of the human milk oligosaccharide in the nutritional
composition.
9. The method of claim 3, wherein the infant is in need of
regulation of gut motility as a result of susceptibility to one or
more gut motility disorders.
10. The method of claim 3, wherein reducing contractions in the
infant's gastrointestinal tract includes direct stimulation of
enteric nerve cells.
11. The method of claim 10, wherein the human milk oligosaccharide
directly stimulates the enteric nerve cells.
12. A method for reducing colic in an infant in need thereof, the
method comprising administering a nutritional composition
comprising 2'-fucosyllactose to the infant.
13. The method of claim 12, wherein 2'-fucosyllactose is present in
a concentration of from about 0.001 mg/ml to about 20 mg/ml.
14. The method of claim 12, wherein the nutritional composition
further comprises lacto-N-neotetraose.
15. The method of claim 12, wherein the infant in need thereof
exhibits symptoms of colic.
16. A method for improving gastrointestinal tolerance in an infant
in need thereof, the method comprising administering a nutritional
composition comprising 2'-fucosyllactose to the infant.
17. The method of claim 16, wherein 2'-fucosyllactose is present in
a concentration of from about 0.001 mg/ml to about 20 mg/ml.
18. The method of claim 16, wherein the infant is in need of
improved gastrointestinal tolerance as a result of susceptibility
to food intolerance.
19. The method of claim 16, wherein the infant is in need of
improved gastrointestinal tolerance as a result of susceptibility
to intolerance to enteral feeding.
20. The method of claim 16, wherein the infant is in need of
improved gastrointestinal tolerance as a result of a need for
increased digestive tolerance.
21. The method of claim 20, wherein the nutritional composition
comprises a combination of fast, medium, and slowly digested
carbohydrates.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 13/335,323 filed on Dec. 22, 2011,
which claims the benefit of U.S. Provisional Application No.
61/428,867 filed on Dec. 31, 2010; and U.S. Provisional Application
No. 61/474,691 filed on Apr. 12, 2011, which disclosures are
incorporated by reference in their entirety.
FIELD OF THE DISCLOSURE
[0002] The present disclosure relates to human milk
oligosaccharides (HMOs) for improving gastrointestinal function and
tolerance in infants, toddlers, and children. More particularly,
the present disclosure relates to human milk fortifiers, preterm
and term infant formulas, and pediatric formulas comprising HMOs
that can stimulate enteric nerve cells in the gastrointestinal
tract, thereby treating and/or preventing numerous
gastrointestinal-related conditions and diseases.
BACKGROUND OF THE DISCLOSURE
[0003] During postnatal development, a newborn's intestine
experiences a process of maturation that ends with the production
of gastrointestinal epithelium that functions as a selective
barrier (i.e., gut barrier). The main function of the
gastrointestinal epithelium is the absorption of nutrients,
electrolytes and water, while preventing exposure to dietary and
microbial antigens, including food allergens. Specifically, this
barrier limits the passage of antigens to the systemic circulation,
thereby preventing infection, inflammatory reactions, and other
gastrointestinal diseases and disorders that may occur during
infancy and later in life. For very young infants, and
particularly, preterm infants, who have an immature immune system
and intestinal tract, development of suboptimal intestinal flora
may result in infection, diarrhea, allergies, and food
intolerance.
[0004] Barrier formation and maintenance has been found to be
affected by the diet. Breast milk contains components that not only
act as pathogen receptor analogues, but also activate immune
factors by infant intestinal epithelial cells and/or associated
immune cell populations to enhance development and maturation of
the infant's gastrointestinal and immune systems.
[0005] Not all infants, however, are in a position to receive human
breast milk. It would therefore be desirable to provide nutritional
compositions, and synthetic infant formulas in particular, that can
produce nutritional benefits including improved gastrointestinal
growth, development, and maturation. It would additionally be
beneficial if the nutritional compositions could enhance immunity
against microbial infections and other gastrointestinal diseases,
conditions, and disorders.
SUMMARY OF THE DISCLOSURE
[0006] The present disclosure is directed to nutritional
compositions, including synthetic infant formulas, synthetic
pediatric formulas, and synthetic child formulas including at least
one HMO alone or in combination with other components such as
prebiotic oligosaccharides and/or probiotics, for improving gut
function and immunity in an infant, toddler, child, or adult, along
with related methods of use. More particularly, the nutritional
compositions can improve growth and maturation of the gut barrier,
thereby treating and/or preventing formula intolerance or other
gastrointestinal diseases and/or disorders resulting from a loss or
dysfunction of the gut barrier.
[0007] One embodiment is directed to a method of stimulating
enteric nerve cells in the gastrointestinal tract of an individual
in need thereof. The method comprises administering to the
individual a nutritional composition comprising a neutral human
milk oligosaccharide.
[0008] Another embodiment is directed to a method of improving
cognition in an individual in need thereof. The method comprises
administering to the individual a nutritional composition
comprising a neutral human milk oligosaccharide in a concentration
of from about 0.001 mg/mL to less than 2 mg/mL.
[0009] Another embodiment is directed to a method of promoting the
growth of beneficial bacteria in an individual in need thereof. The
method comprises administering to the individual a synthetic
composition comprising 2'-fucosyllactose.
[0010] Another embodiment is directed to a method of reducing the
incidence of colic in an infant in need thereof. The method
comprises administering to the infant a synthetic infant formula
comprising 2'-fucosyllactose.
[0011] Another embodiment is directed to a method of promoting
gastrointestinal maturation in an infant in need thereof. The
method comprises administering to the infant a synthetic infant
formula comprising lacto-N-neotetraose.
[0012] Another embodiment is directed to a method of reducing the
incidence of colic in an infant in need thereof. The method
comprises administering to the infant a synthetic infant formula
comprising lacto-N-neotetraose.
[0013] Another embodiment is directed to a method of reducing the
incidence of necrotizing enterocolitis in an infant in need
thereof. The method comprises administering to the infant a
synthetic infant formula comprising lacto-N-neotetraose.
[0014] Another embodiment is directed to a synthetic pediatric
formula comprising from about 0.001 mg/mL to about 20 mg/mL of
human milk oligosaccharides and an oligosaccharide selected from
the group consisting of galactooligosaccharides,
fructooligosaccharides, inulin, and polydextrose, wherein the human
milk oligosaccharides comprise 2'-fucosyllactose in an amount of
from 0.001 mg/mL to less than 2 mg/mL.
[0015] Another embodiment is directed to a synthetic pediatric
formula comprising from about 0.001 mg/mL to about 20 mg/mL of
human milk oligosaccharides and an oligosaccharide selected from
the group consisting of galactooligosaccharides,
fructooligosaccharides, inulin, and polydextrose, wherein the human
milk oligosaccharides comprise 2'-fucosyllactose in an amount of
from greater than 2.5 mg/mL to about 20 mg/mL.
[0016] Another embodiment is directed to a synthetic pediatric
formula comprising from about 0.001 mg/mL to about 20 mg/mL of a
neutral human milk oligosaccharides and an acidic human milk
oligosaccharide, wherein the neutral human milk oligosaccharide
comprises 2'-fucosyllactose in an amount of from 0.001 mg/mL to
less than 2 mg/mL.
[0017] Another embodiment is directed to a synthetic pediatric
formula comprising from about 0.001 mg/mL to about 20 mg/mL of a
neutral human milk oligosaccharides and an acidic human milk
oligosaccharide, wherein the neutral human milk oligosaccharide
comprises 2'-fucosyllactose in an amount of from greater than 2.5
mg/mL to about 20 mg/mL.
[0018] It has been discovered that HMOs that are delivered to the
gut tissue stimulate the gut-brain-immune axis, and improve the
immune system and enteric nervous system. Specifically, it has been
found that 2'-fucosyllactose stimulates enteric nerve cells in the
gastrointestinal tract and promotes the growth of beneficial
bacteria such that gut function may be improved and
gastrointestinal issues minimized.
[0019] Additionally, it has been found that the digestive tolerance
of an infant, toddler, child, or adult can be significantly
increased by administering to the infant, toddler, child or adult a
select blend of carbohydrates including HMOs. Specifically, the
carbohydrate blend includes a combination of fast, medium, and
slowly digested carbohydrates including specific HMOs such as
lacto-N-neotetraose, 2'-fucosyllactose, 3'-sialyllactose and/or
6'-sialyllactose.
[0020] Moreover, it has been found that intestinal barrier
integrity of an infant, toddler, child, or adult can be
significantly improved by administering to the infant, toddler,
child, or adult a synbiotic composition including HMOs.
Specifically, the synbiotic combination includes a probiotic, at
least one of a galactooligosaccharide and a fructooligosaccharide
(such as a short chain fructooligosaccharide) and at least one HMO.
The synbiotic composition promotes the colonization of beneficial
intestinal bacteria (microbiota) in order to discourage the growth
of harmful bacteria.
[0021] Although the nutritional compositions and methods are
primarily discussed herein in relation to preterm infants and
infants in general, it should be understood that many of the
benefits discussed herein may be provided to toddlers, children,
and adults administered combinations of the HMOs alone, or with
other components as described herein, such as prebiotic
oligosaccharides and/or probiotics, for example. Particularly, in
some embodiments, the incidence of gastrointestinal diseases and
disorders that generally affect adults, such as Crohn's disease,
irritable bowel syndrome and the like, can be reduced with the use
of the nutritional compositions of the present disclosure including
HMOs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a graph depicting the effect of 2'FL and 3'FL on
gut motility as measured in Example 44.
[0023] FIG. 2 is a table setting forth the microbiological medium
used in the in vitro experiment of Example 45.
[0024] FIG. 3 is a table setting forth the carbohydrate components
of the oligosaccharide substrates tested in Example 45.
[0025] FIG. 4 is a graph depicting the change in pH over time for
formula fed and breast fed infants as tested in Example 45.
[0026] FIG. 5 is a graph depicting the change in pH over time as
affected by the various oligosaccharide substrates as tested in
Example 45.
[0027] FIG. 6 is a graph depicting change in acetate production
over time for formula fed and breast fed infants as tested in
Example 45.
[0028] FIG. 7 is a graph depicting change in acetate production
over time as affected by the various oligosaccharide substrates as
tested in Example 45.
[0029] FIG. 8 is a graph depicting change in propionate production
over time for formula fed and breast fed infants as tested in
Example 45.
[0030] FIG. 9 is a graph depicting change in propionate production
over time as affected by the various oligosaccharide substrates as
tested in Example 45.
[0031] FIG. 10 is a graph depicting change in butyrate production
over time for formula fed and breast fed infants as tested in
Example 45.
[0032] FIG. 11 is a graph depicting change in butyrate production
over time as affected by the various oligosaccharide substrates as
tested in Example 45.
[0033] FIG. 12 is a graph depicting change in lactic acid
production over time for formula fed and breast fed infants as
tested in Example 45.
[0034] FIG. 13 is a graph depicting change in lactic acid
production over time as affected by the various oligosaccharide
substrates as tested in Example 45.
[0035] FIG. 14 is a graph depicting change in short chain fatty
acid production over time for formula fed and breast fed infants as
tested in Example 45.
[0036] FIG. 15 is a graph depicting change in short chain fatty
acid production over time as affected by the various
oligosaccharide substrates as tested in Example 45.
[0037] FIG. 16 is a graph depicting change in Lactobacillus spp.
populations over time in formula fed and breast fed infants as
tested in Example 45.
[0038] FIG. 17 is a graph depicting change in Lactobacillus spp.
populations over time as affected by the various oligosaccharide
substrates as tested in Example 45.
[0039] FIG. 18 is a graph depicting change in Bifidobacterium spp.
populations over time in formula fed and breast fed infants as
tested in Example 45.
[0040] FIG. 19 is a graph depicting change in Bifidobacterium spp.
populations over time as affected by the various oligosaccharide
substrates as tested in Example 45.
[0041] FIG. 20 is a graph depicting change in E. coli populations
over time in formula fed and breast fed infants as tested in
Example 45.
[0042] FIG. 21 is a graph depicting change in E. coli populations
over time as affected by various oligosaccharide substrates as
tested in Example 45.
[0043] FIG. 22 is a graph depicting change in Clostridium
perfringens populations over time in formula fed and breast fed
infants as tested in Example 45.
[0044] FIG. 23 is a graph depicting change in Clostridium
perfringens populations over time by various oligosaccharide
substrates as tested in Example 45.
[0045] FIGS. 24A-24H depict growth curves of various
Bifidobacterium spp. as evaluated in Example 46.
[0046] FIGS. 25A-25H depict growth curves of various
Bifidobacterium spp. as evaluated in Example 46.
[0047] FIGS. 26A-26G depict growth curves of various
Bifidobacterium spp. as evaluated in Example 46.
[0048] FIGS. 27-28 are graphs showing HT-29 Epithelial Cell
Proliferation in the presence of LNnT.
[0049] FIGS. 29-30 are graphs showing Caco-2 Epithelial Cell
Proliferation in the presence of LNnT.
[0050] FIGS. 31-33 are graphs showing Pre-confluent HT-29
Epithelial Cell Proliferation in the presence of LNnT, 2'FL, and
6'SL.
[0051] FIGS. 34-36 are graphs showing Pre-confluent HT-29
Epithelial Cell Differentiation in the presence of LNnT, 2'FL, and
6'FL.
[0052] FIGS. 37-39 are graphs showing Confluent HT-29 Epithelial
Cell Resistance in the presence of LNnT, 2'FL, and 6'SL.
[0053] FIGS. 40-42 are graphs showing Pre-confluent Caco-2
Epithelial Cell Proliferation in the presence of LNnT, 2'FL, and
6'SL.
[0054] FIGS. 43-45 are graphs showing Post-confluent Caco-2
Epithelial Cell Differentiation in the presence of LNnT, 2'FL, and
6'FL.
[0055] FIGS. 46-48 are graphs showing Post-confluent Caco-2
Epithelial Cell Resistance in the presence of LNnT, 2'FL, and
6'SL.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0056] The nutritional compositions and methods described herein
utilize HMOs alone or in combination with at least one other
prebiotic oligosaccharide and/or a probiotic for controlling and
reducing a number of diseases, disorders and conditions related to
the gut-brain-immune system. These and other features of the
nutritional compositions and methods, as well as some of the many
optional variations and additions, are described in detail
hereafter.
[0057] The terms "retort packaging" and "retort sterilizing" are
used interchangeably herein, and unless otherwise specified, refer
to the common practice of filling a container, most typically a
metal can or other similar package, with a nutritional liquid and
then subjecting the liquid-filled package to the necessary heat
sterilization step, to form a sterilized, retort packaged,
nutritional liquid product.
[0058] The term "aseptic packaging" as used herein, unless
otherwise specified, refers to the manufacture of a packaged
product without reliance upon the above-described retort packaging
step, wherein the nutritional liquid and package are sterilized
separately prior to filling, and then are combined under sterilized
or aseptic processing conditions to form a sterilized, aseptically
packaged, nutritional liquid product.
[0059] The terms "fat" and "oil" as used herein, unless otherwise
specified, are used interchangeably to refer to lipid materials
derived or processed from plants or animals. These terms also
include synthetic lipid materials so long as such synthetic
materials are suitable for oral administration to humans.
[0060] The terms "human milk oligosaccharide" or "HMO", unless
otherwise specified, refers generally to a number of complex
carbohydrates found in human breast milk that can be in acidic or
neutral form, and to precursors thereof. Exemplary non-limiting
human milk oligosaccharides include 3'-sialyllactose,
6'-sialyllactose, 3'-fucosyllactose, 2'-fucosyllactose, and
lacto-N-neo-tetraose. Exemplary human milk oligosaccharide
precursors include sialic acid and/or fucose.
[0061] The term "shelf stable" as used herein, unless otherwise
specified, refers to a nutritional product that remains
commercially stable after being packaged and then stored at
18-24.degree. C. for at least 3 months, including from about 6
months to about 24 months, and also including from about 12 months
to about 18 months.
[0062] The terms "nutritional formulation" or "nutritional
composition" as used herein, are used interchangeably and, unless
otherwise specified, refer to synthetic formulas including
nutritional liquids, nutritional semi-liquid, nutritional solids,
nutritional semi-solids, nutritional powders, nutritional
supplements, and any other nutritional food product as known in the
art. The nutritional powders may be reconstituted to form a
nutritional liquid, all of which comprise one or more of fat,
protein and carbohydrate and are suitable for oral consumption by a
human. The terms "nutritional formulation" and "nutritional
composition" do not include human breast milk.
[0063] The term "nutritional liquid" as used herein, unless
otherwise specified, refers to nutritional compositions in
ready-to-drink liquid form, concentrated form, and nutritional
liquids made by reconstituting the nutritional powders described
herein prior to use.
[0064] The term "nutritional powder" as used herein, unless
otherwise specified, refers to nutritional compositions in flowable
or scoopable form that can be reconstituted with water or another
aqueous liquid prior to consumption and includes both spraydried
and drymixed/dryblended powders.
[0065] The term "nutritional semi-solid," as used herein, unless
otherwise specified, refers to nutritional products that are
intermediate in properties, such as rigidity, between solids and
liquids. Some semi-solids examples include puddings, gelatins, and
doughs.
[0066] The term "nutritional semi-liquid," as used herein, unless
otherwise specified, refers to nutritional products that are
intermediate in properties, such as flow properties, between
liquids and solids. Some semi-liquids examples include thick shakes
and liquid gels.
[0067] The term "infant" as used herein, unless otherwise
specified, refers to a person 12 months or younger. The term
"preterm infant" as used herein, refers to a person born prior to
36 weeks of gestation.
[0068] The term "toddler" as used herein, unless otherwise
specified, refers to a person greater than one year of age up to
three years of age.
[0069] The term "child" as used herein, unless otherwise specified,
refers to a person greater than three years of age up to twelve
years of age.
[0070] The term "newborn" as used herein, unless otherwise
specified, refers to a person from birth up to four weeks of
age.
[0071] The terms "infant formula" or "synthetic infant formula" as
used herein, unless otherwise specified, are used interchangeably
and refer to liquid, solid, semi-liquid, and semi-solid human milk
replacements or substitutes that are suitable for consumption by an
infant. The synthetic formulas include components that are of
semi-purified or purified origin. As used herein, unless otherwise
specified, the terms "semi-purified" or "purified" refer to a
material that has been prepared by purification of a natural
material or by synthesis. The terms "infant formula" or "synthetic
infant formula" do not include human breast milk.
[0072] The term "synthetic pediatric formula" as used herein,
unless otherwise specified, refers to liquid, solid, semi-liquid,
and semi-solid human milk replacements or substitutes that are
suitable for consumption by an infant or toddler up to the age of
36 months (3 years). The synthetic formulas include components that
are of semi-purified or purified origin. As used herein, unless
otherwise specified, the terms "semi-purified" or "purified" refer
to a material that has been prepared by purification of a natural
material or by synthesis. The term "synthetic pediatric formula"
does not include human breast milk.
[0073] The term "synthetic child formula" as used herein, unless
otherwise specified, refers to liquid, solid, semi-liquid, and
semi-solid human milk replacements or substitutes that are suitable
for consumption by a child up to the age of 12 years. The synthetic
formulas include components that are of semi-purified or purified
origin. As used herein, unless otherwise specified, the terms
"semi-purified" or "purified" refer to a material that has been
prepared by purification of a natural material or by synthesis. The
term "synthetic child formula" does not include human breast
milk.
[0074] The term "preterm infant formula" as used herein, unless
otherwise specified, refers to liquid and solid nutritional
products suitable for consumption by a preterm infant.
[0075] The term "human milk fortifier" as used herein, unless
otherwise specified, refers to liquid and solid nutritional
products suitable for mixing with breast milk or preterm infant
formula or infant formula for consumption by a preterm or term
infant.
[0076] The term "postbiotics" as used herein, unless otherwise
specified, refers to metabolites produced by probiotic
bacteria.
[0077] The terms "susceptible" and "at risk" as used herein, unless
otherwise specified, mean having little resistance to a certain
condition or disease, including being genetically predisposed,
having a family history of, and/or having symptoms of the condition
or disease.
[0078] The term "cognition" as used herein, unless otherwise
specified, refers to an individual's ability for learning, memory
acquisition, and memory recall.
[0079] The terms "growth of a virus" or "growth of bacteria" as
used herein, unless otherwise specified, refer to the production,
proliferation, or replication of a virus or bacteria.
[0080] All percentages, parts and ratios as used herein, are by
weight of the total composition, unless otherwise specified. All
such weights, as they pertain to listed ingredients, are based on
the active level and, therefore, do not include solvents or
by-products that may be included in commercially available
materials, unless otherwise specified.
[0081] Numerical ranges as used herein are intended to include
every number and subset of numbers within that range, whether
specifically disclosed or not. Further, these numerical ranges
should be construed as providing support for a claim directed to
any number or subset of numbers in that range. For example, a
disclosure of from 1 to 10 should be construed as supporting a
range of from 2 to 8, from 3 to 7, from 5 to 6, from 1 to 9, from
3.6 to 4.6, from 3.5 to 9.9, and so forth.
[0082] All references to singular characteristics or limitations of
the present disclosure shall include the corresponding plural
characteristic or limitation, and vice versa, unless otherwise
specified or clearly implied to the contrary by the context in
which the reference is made.
[0083] All combinations of method or process steps as used herein
can be performed in any order, unless otherwise specified or
clearly implied to the contrary by the context in which the
referenced combination is made.
[0084] The nutritional compositions and methods may comprise,
consist of, or consist essentially of the essential elements of the
compositions and methods as described herein, as well as any
additional or optional element described herein or otherwise useful
in nutritional composition applications.
Product Form
[0085] The nutritional compositions of the present disclosure may
be formulated and administered in any known or otherwise suitable
oral product form. Any solid, liquid, semi-liquid, semi-solid or
powder product form, including combinations or variations thereof,
are suitable for use herein, provided that such forms allow for
safe and effective oral delivery to the individual of the essential
ingredients and any optional ingredients, as also defined
herein.
[0086] The nutritional compositions of the present disclosure are
desirably formulated as dietary product forms, which are defined
herein as those embodiments comprising the ingredients of the
present disclosure in a product form that then contains at least
one of fat, protein, and carbohydrate, and preferably also contains
vitamins, minerals, or combinations thereof. The nutritional
compositions will comprise at least one HMO, and many times at
least two or more HMOs, desirably in combination with at least one
of protein, fat, vitamins, and minerals, to produce a nutritional
combination.
[0087] The nutritional composition may be formulated with
sufficient kinds and amounts of nutrients to provide a sole,
primary, or supplemental source of nutrition, or to provide a
specialized nutritional composition for use in individuals
afflicted with specific diseases, disorders, or conditions or with
a targeted nutritional benefit as described below.
[0088] Specific non-limiting examples of product forms suitable for
use with the HMO-containing compositions as disclosed herein
include, for example, liquid and powdered dietary supplements,
liquid and powdered human milk fortifiers, liquid and powdered
preterm infant formulas, liquid and powdered infant formulas,
liquid and powdered elemental and semi-elemental formulas, liquid
and powdered pediatric formulas, liquid and powdered toddler
formulas, liquid and powdered follow-on formulas, liquid, powdered
and solid adult nutritional formulas suitable for use with
individuals suffering from food intolerance, allergies, immune
disorders, and other gastrointestinal diseases, conditions, and/or
disorders. Other non-limiting examples of product forms suitable
for use with the HMO-containing compositions disclosed herein
include nutritional formulas suitable for use with individuals who
have been treated or are receiving antibiotic therapy or oral
rehydration solutions suitable for use with infants, children, or
adults suffering from diarrhea. Another non-limiting example
includes a supplement including one or more HMOs that might or
might not contain other nutrients. This supplement can be added to
human milk or formula, or can be provided by itself during an
enteral feeding period or used prior to an enteral feeding.
Nutritional Liquids
[0089] Nutritional liquids include both concentrated and
ready-to-feed nutritional liquids. These nutritional liquids are
most typically formulated as suspensions or emulsions, although
other liquid forms are within the scope of the present
disclosure.
[0090] Nutritional emulsions suitable for use may be aqueous
emulsions comprising proteins, fats, and carbohydrates. These
emulsions are generally flowable or drinkable liquids at from about
1.degree. C. to about 25.degree. C. and are typically in the form
of oil-in-water, water-in-oil, or complex aqueous emulsions,
although such emulsions are most typically in the form of
oil-in-water emulsions having a continuous aqueous phase and a
discontinuous oil phase.
[0091] The nutritional emulsions may be and typically are shelf
stable. The nutritional emulsions typically contain up to about 95%
by weight of water, including from about 50% to about 95%, also
including from about 60% to about 90%, and also including from
about 70% to about 85%, by weight of water. The nutritional
emulsions may have a variety of product densities, but most
typically have a density greater than about 1.03 g/mL, including
greater than about 1.04 g/mL, including greater than about 1.055
g/mL, including from about 1.06 g/mL to about 1.12 g/mL, and also
including from about 1.085 g/mL to about 1.10 g/mL.
[0092] The nutritional emulsions may have a caloric density
tailored to the nutritional needs of the ultimate user, although in
most instances the emulsions comprise generally at least 19 kcal/fl
oz (660 kcal/liter), more typically from about 20 kcal/fl oz
(675-680 kcal/liter) to about 25 kcal/fl oz (820 kcal/liter), even
more typically from about 20 kcal/fl oz (675-680 kcal/liter) to
about 24 kcal/fl oz (800-810 kcal/liter). Generally, the 22-24
kcal/fl oz formulas are more commonly used in preterm or low birth
weight infants, and the 20-21 kcal/fl oz (675-680 to 700
kcal/liter) formulas are more often used in term infants. In some
embodiments, the emulsion may have a caloric density of from about
50-100 kcal/liter to about 660 kcal/liter, including from about 150
kcal/liter to about 500 kcal/liter. In some specific embodiments,
the emulsion may have a caloric density of 25, or 50, or 75, or 100
kcal/liter.
[0093] The nutritional emulsion may have a pH ranging from about
3.5 to about 8, but are most advantageously in a range of from
about 4.5 to about 7.5, including from about 5.5 to about 7.3,
including from about 6.2 to about 7.2.
[0094] Although the serving size for the nutritional emulsion can
vary depending upon a number of variables, a typical serving size
is generally at least about 1 mL, or even at least about 2 mL, or
even at least about 5 mL, or even at least about 10 mL, or even at
least about 25 mL, including ranges from about 2 mL to about 300
mL, including from about 4 mL to about 250 mL, and including from
about 10 mL to about 240 mL.
Nutritional Solids
[0095] The nutritional solids may be in any solid form, but are
typically in the form of flowable or substantially flowable
particulate compositions, or at least particulate compositions.
Particularly suitable nutritional solid product forms include spray
dried, agglomerated and/or dryblended powder compositions. The
compositions can easily be scooped and measured with a spoon or
similar other device, and can easily be reconstituted by the
intended user with a suitable aqueous liquid, typically water, to
form a nutritional composition for immediate oral or enteral use.
In this context, "immediate" use generally means within about 48
hours, most typically within about 24 hours, preferably right after
reconstitution.
[0096] The nutritional powders may be reconstituted with water
prior to use to a caloric density tailored to the nutritional needs
of the ultimate user, although in most instances the powders are
reconstituted with water to form compositions comprising at least
19 kcal/fl oz (660 kcal/liter), more typically from about 20
kcal/fl oz (675-680 kcal/liter) to about 25 kcal/fl oz (820
kcal/liter), even more typically from about 20 kcal/fl oz (675-680
kcal/liter) to about 24 kcal/fl oz (800-810 kcal/liter). Generally,
the 22-24 kcal/fl oz formulas are more commonly used in preterm or
low birth weight infants, and the 20-21 kcal/fl oz (675-680 to 700
kcal/liter) formulas are more often used in term infants. In some
embodiments, the reconstituted powder may have a caloric density of
from about 50-100 kcal/liter to about 660 kcal/liter, including
from about 150 kcal/liter to about 500 kcal/liter. In some specific
embodiments, the emulsion may have a caloric density of 25, or 50,
or 75, or 100 kcal/liter.
Human Milk Oligosaccharides (HMOs)
[0097] The nutritional compositions of the present disclosure
include at least one HMO, and in many embodiments, a combination of
two or more HMOs. Oligosaccharides are one of the main components
of human breast milk, which contains, on average, 10 grams per
liter of neutral oligosaccharides and 1 gram per liter of acidic
oligosaccharides. The compositional structure of HMOs is very
complex and more than 200 different oligosaccharide-like structures
are known.
[0098] The HMO or HMOs may be included in the nutritional
compositions alone, or in some embodiments, in combination with
other components (e.g., prebiotic oligosaccharides, probiotics,
etc.) as described herein. In many embodiments, HMOs are included
in the nutritional compositions with multiple additional
components. The HMO or HMOs may be isolated or enriched from
milk(s) secreted by mammals including, but not limited to: human,
bovine, ovine, porcine, or caprine species. The HMOs may also be
produced via microbial fermentation, enzymatic processes, chemical
synthesis, or combinations thereof.
[0099] Suitable HMOs for use in the nutritional compositions may
include neutral oligosaccharides, acidic oligosaccharides,
n-acetylglucosylated oligosaccharides, and HMO precursors. Specific
non-limiting examples of HMOs that may be included individually or
in combination in the compositions of the present disclosure
include: sialic acid (i.e., free sialic acid, lipid-bound sialic
acid, protein-bound sialic acid); D-glucose (Glc); D-galactose
(Gal); N-acetylglucosamine (GlcNAc); L-fucose (L-Fuc); D-fucose
(D-fuc); fucosyl oligosaccharides (i.e., Lacto-N-fucopentaose I;
Lacto-N-fucopentaose II; 2'-Fucosyllactose; 3'-Fucosyllactose;
Lacto-N-fucopentaose III; Lacto-N-difucohexaose I; and
Lactodifucotetraose); non-fucosylated, non-sialylated
oligosaccharides (i.e., Lacto-N-tetraose and Lacto-N-neotetraose);
sialyl oligosaccharides (i.e., 3'-Sialyl-3-fucosyllactose;
Disialomonofucosyllacto-N-neohexaose;
Monofucosylmonosialyllacto-N-octaose (sialyl Lea);
Sialyllacto-N-fucohexaose II; Disialyllacto-N-fucopentaose II;
Monofucosyldisialyllacto-N-tetraose); and sialyl fucosyl
oligosaccharides i.e., 2'-Sialyllactose; 2-Sialyllactosamine;
3'-Sialyllactose; 3'-Sialyllactosamine; 6'-Sialyllactose;
6'-Sialyllactosamine; Sialyllacto-N-neotetraose c;
Monosialyllacto-N-hexaose; Disialyllacto-N-hexaose I;
Monosialyllacto-N-neohexaose I; Monosialyllacto-N-neohexaose II;
Disialyllacto-N-neohexaose; Disialyllacto-N-tetraose;
Disialyllacto-N-hexaose II; Sialyllacto-N-tetraose a;
Disialyllacto-N-hexaose I; and Sialyllacto-N-tetraose. Also useful
are variants in which the glucose (Glc) at the reducing end is
replaced by N-acetylglucosamine (e.g.,
2'-fucosyl-N-acetylglucosamine (2'-FLNac) is such a variant to
2'-fucosyllactose). These HMOs are described more fully in U.S.
Patent Application No. 2009/0098240, which is herein incorporated
by reference in its entirety. Other suitable examples of HMOs that
may be included in the compositions of the present disclosure
include 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), 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-neoocataose,
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,
trifucosyl-iso-lacto-N-octaose, lacto-N-difuco-hexaose II,
sialyl-lacto-N-tetraose a, sialyl-lacto-N-tetraose b,
sialyl-lacto-N-tetraose c, sialyl-fucosyl-lacto-N-tetraose I,
sialyl-fucosyl-lacto-N-tetraose II, and disialyl-lacto-N-tetraose,
and combinations thereof. Particularly suitable nutritional
compositions include at least one of the following HMOs or HMO
precursors: sialic acid (SA); 2'-Sialyllactose (2'SL);
3'-Sialyllactose (3'SL); 6'-Sialyllactose (6'SL); 2'-Fucosyllactose
(2'FL); 3'-Fucosyllactose (3'FL); and Lacto-N-neotetraose (LNnT),
and in particular, combinations of 2'FL or 3'FL with at least one
of 6'SL and 3'SL; and combinations of LNnT with at least one of
6'SL, 2'FL, and 3'FL.
[0100] Other exemplary combinations include: SA, 3'SL, 6'SL, 3'FL,
2'FL, and LNnT; 3'SL, 6'SL, 3'FL, 2'FL, and LNnT; SA, 6'SL, 3'FL,
2'FL, and LNnT; SA, 3'SL, 3'FL, 2'FL, and LNnT; SA, 3'SL, 6'SL,
2'FL, and LNnT; SA, 3'SL, 6'SL, 3'FL, and LNnT; SA, 3'SL, 6'SL,
3'FL, and 2'FL; SA and 3'SL; SA and 6'SL; SA and 2'FL; SA and LNnT;
SA, 3'SL, and 6'SL; SA, 3'SL and 3'FL; SA, 3'SL and 2'FL; SA, 3'SL
and LNnT; SA, 6'SL and 3'FL; SA, 6'SL, and 2'FL; SA, 6'SL, and
LNnT; SA, 3'FL, and 2'FL; SA, 3'FL, and LNnT; SA, 2'FL, and LNnT;
SA, 3'SL, 6'SL, and 3'FL; SA, 3'SL, 6'SL and 2'FL; SA, 3'SL, 6'SL,
and LNnT; SA, 3'SL, 3'FL, and 2'FL; SA, 3'SL, 3'FL, and LNnT; SA,
3'SL, 2'FL, and LNnT; SA, 6'SL, 3'FL, and 2'FL; SA, 6'SL, 2'FL, and
LNnT; SA, 6'SL, 3'FL, and LNnT; SA, 3'FL, 2'FL, and LNnT; SA, 6'SL,
2'FL, and LNnT; SA, 3'SL, 3'FL, 2'FL, and LNnT; SA, 6'SL, 3'FL,
2'FL, and LNnT; SA, 3'SL, 6'SL, 3'FL, and LNnT; SA, 3'SL, 3'FL,
2'FL, and LNnT; SA, 3'SL, 6'SL, 2'FL, and LNnT; 3'SL, 6'SL, 3'FL,
and 2'FL; 3'SL, 6'SL, 2'FL, and LNnT; 3'SL, 3'FL, 2'FL, and LNnT;
3'SL, 6'SL, 3'FL, and LNnT; 3'SL, 6'SL, and 3'FL; 3'SL, 3'FL, and
2'FL; 3'SL, 2'FL, and LNnT; 3'SL, 6'SL, and 2'FL; 3'SL, 6'SL, and
LNnT; 3'SL and 3'FL; 3'SL and 2'FL; 3'SL and LNnT; 6'SL and 3'FL;
6'SL and 2'FL; 6'SL and LNnT; 6'SL, 3'FL, and LNnT; 6'SL, 3'FL,
2'FL, and LNnT; 3'FL, 2'FL, and LNnT; 3'FL and LNnT; and 2'FL and
LNnT.
[0101] The HMOs are present in the nutritional compositions in
total amounts of HMO in the composition (mg of HMO per mL of
composition) of at least about 0.001 mg/mL, including at least
about 0.01 mg/mL, including from about 0.001 mg/mL to about 20
mg/mL, including from about 0.01 mg/mL to about 20 mg/mL, including
from about 0.01 mg/mL to about 15 mg/mL, including from about 0.01
mg/mL to about 10 mg/mL, including from about 0.01 mg/mL to about 5
mg/mL, and including from about 0.001 mg/mL to about 1 mg/mL of
total HMO in the nutritional composition, and including from about
0.001 mg/mL to about 0.23 mg/mL and from about 0.01 mg/mL to about
0.23 mg/mL. Typically, the amount of HMO in the nutritional
composition will depend on the specific HMO or HMOs present and the
amounts of other components in the nutritional compositions.
[0102] In one specific embodiment when the nutritional composition
is a nutritional powder, the total concentration of HMOs in the
nutritional powder is from about 0.0005% to about 5%, including
from about 0.01% to about 1% (by weight of the nutritional
powder).
[0103] In another specific embodiment, when the nutritional
composition is a ready-to-feed nutritional liquid, the total
concentration of HMOs in the ready-to-feed nutritional liquid is
from about 0.0001% to about 0.50%, including from about 0.001% to
about 0.15%, including from about 0.01% to about 0.10%, and further
including from about 0.01% to about 0.03% (by weight of the
ready-to-feed nutritional liquid).
[0104] In another specific embodiment, when the nutritional
composition is a concentrated nutritional liquid, the total
concentration of HMOs in the concentrated liquid is from about
0.0002% to about 0.60%, including from about 0.002% to about 0.30%,
including from about 0.02% to about 0.20%, and further including
from about 0.02% to about 0.06% (by weight of the concentrated
nutritional liquid).
[0105] In one specific embodiment, the nutritional composition
includes a neutral human milk oligosaccharide in an amount of from
about 0.001 mg/mL to about 20 mg/mL, including from 0.01 mg/mL to
about 20 mg/mL, including from about 0.001 mg/mL to less than 2
mg/mL, and including from about 0.01 mg/mL to less than 2
mg/mL.
[0106] In one specific embodiment of the present disclosure, a
nutritional composition includes 2'FL. The 2'FL may be the only HMO
included in the nutritional composition, or other additional HMOs
may also be included in the nutritional composition (e.g., the 2'FL
may be combined with 3'SL and/or 6'SL in some specific
embodiments). In one embodiment, the 2'FL is included in the
nutritional composition in an amount of from about 0.001 mg/mL to
about 20 mg/mL, including from about 0.001 mg/mL to about 10 mg/mL,
including from about 0.01 mg/mL to about 20 mg/mL, including from
about 0.001 mg/mL to about 1 mg/mL, including from about 0.001
mg/mL to less than 2 mg/mL, including from about 0.01 mg/mL to less
than 2 mg/mL, and also including from about 0.02 mg/mL to less than
2 mg/mL. In another embodiment, the 2'FL is included in the
nutritional composition in an amount of from about 0.001 mg/mL to
about 20 mg/mL, including from about 0.01 mg/mL to about 20 mg/mL,
including from greater than 2.5 mg/mL to 20 mg/mL, including from
greater than 2.5 mg/mL to 19.8 mg/mL, including from greater than
2.5 mg/mL to 15 mg/mL, and including from greater than 2.5 mg/mL to
10 mg/mL.
[0107] In one specific embodiment, the nutritional composition
includes 6'SL, alone or in combination with other HMOs, in an
amount of from about 0.001 mg/mL to about 20 mg/mL, including from
about 0.01 mg/mL to about 20 mg/mL, including from about 0.001
mg/mL to less than 0.25 mg/mL, and including from about 0.01 mg/mL
to less than 0.25 mg/mL. In another embodiment, the nutritional
composition includes 6'SL, alone or in combination with other HMOs,
in an amount of from about 0.001 mg/mL to about 20 mg/mL, including
from about 0.01 mg/mL to about 20 mg/mL, including from greater
than 0.4 mg/mL to about 20 mg/mL, including from greater than 0.4
mg/mL to about 15 mg/mL, and including from greater than 0.4 mg/mL
to about 10 mg/mL.
[0108] In one embodiment, when the nutritional composition includes
6'SL, the total amount of HMOs in the nutritional composition
includes at least about 88% (by total weight HMOs) 6'SL, including
from about 88% (by total weight HMOs) to about 96% (by total weight
HMOs), including from about 88% (by total weight HMOs) to about
100% (by total weight HMOs), and including about 100% (by total
weight HMOs) 6'SL.
[0109] In another embodiment, the nutritional composition includes
3'SL, alone or in combination with other HMOs, in an amount of from
about 0.001 mg/mL to about 20 mg/mL, including from about 0.01
mg/mL to about 20 mg/mL, including from about 0.01 mg/mL to less
than 0.15 mg/mL, including from greater than 0.25 mg/mL to about 20
mg/mL, including from greater than 0.25 mg/mL to about 15 mg/mL,
and including from greater than 0.25 mg/mL to about 10 mg/mL.
[0110] In one embodiment, when the nutritional composition includes
3'SL, the total amount of HMOs in the nutritional composition
includes at least about 85% (by total weight HMOs) 3'SL, including
from about 85% (by total weight HMOs) to about 88% (by total weight
HMOs), including from about 88% (by total weight HMOs) to about
100% (by total weight HMOs), and including about 100% (by total
weight HMOs) 3'SL.
[0111] In one specific embodiment, the nutritional composition
includes LNnT, alone or in combination with other HMOs, in an
amount of from about 0.001 mg/mL to about 20 mg/mL, including from
about 0.01 mg/mL to about 20 mg/mL, including from about 0.001
mg/mL to less than 0.2 mg/mL, including from about 0.01 mg/mL to
less than 0.2 mg/mL, including from greater than 0.32 mg/mL to
about 20 mg/mL, including from greater than 0.32 mg/mL to about 15
mg/mL, and including from greater than 0.32 mg/mL to about 10
mg/mL.
Additional Prebiotic Oligosaccharides
[0112] The nutritional compositions of the present disclosure may,
in addition to the HMOs described above, comprise an additional
source or sources of prebiotic oligosaccharides (the total amount
of oligosaccharides being referred to herein as an "oligosaccharide
blend" of the nutritional composition). Suitable additional sources
of prebiotic oligosaccharides for use in the nutritional
compositions include any prebiotic oligosaccharide that is suitable
for use in an oral nutritional composition and is compatible with
the essential elements and features of such compositions. In some
embodiments, the nutritional composition includes a combination of
one or more HMOs and one or more additional prebiotic
oligosaccharides such that the composition provides a synergistic
benefit to the end user, such as a synergistic benefit in improving
feeding intolerance in infants.
[0113] In some embodiments, the combinations of HMO or HMOs with
the additional prebiotic oligosaccharides to provide the
synergistic effect include HMOs and additional prebiotic
oligosaccharides that ferment at a rapid rate ("rapidly-fermenting
oligosaccharides"), oligosaccharides that ferment at a moderate
rate ("medium-fermenting oligosaccharides"), and/or
oligosaccharides that ferment at a slow rate ("slowly-fermenting
oligosaccharides"). Some preferred embodiments provide a
nutritional composition that includes at least one HMO in
combination with a rapidly-fermenting oligosaccharide, a
medium-fermenting oligosaccharide, and/or a slowly-fermenting
oligosaccharide.
[0114] Non-limiting examples of suitable additional prebiotic
oligosaccharides for use in the nutritional compositions described
herein include prebiotic oligosaccharides that have a degree of
polymerization (DP) of at least 2 monose units, which are not or
only partially digested in the intestine by the action of acids or
digestive enzymes present in the human upper digestive tract (small
intestine and stomach), but which are fermentable by the human
intestinal flora. The term "monose units" refers to units having a
closed ring structure, preferably hexose, e.g., the pyranose or
furanose forms. Particularly preferred oligosaccharides for use in
combination with the HMO or HMOs in the nutritional compositions of
the present disclosure include galactooligosaccharides (GOS),
fructooligosaccharides (FOS), short chain fructooligosaccharides,
inulin, oligofructose, polydextrose (PDX), pectin hydrolysate, and
gum fiber. In one specific embodiment, the gum fiber is gum
arabic.
[0115] The oligosaccharide blend is present in the nutritional
compositions in a total amount of at least about 1 mg/mL, including
from about 1 mg/mL to about 20 mg/mL, including from about 1 mg/mL
to about 15 mg/mL, including from about 1 mg/mL to about 10 mg/mL,
including from about 1 mg/mL to about 5 mg/mL In one embodiment,
the oligosaccharide blend is present in the nutritional composition
in a total amount of from about 1 mg/mL to about 4 mg/mL.
[0116] Typically, when used as an oligosaccharide blend, the
nutritional compositions, in addition to the HMO or HMOs, include
at least one rapidly-fermented oligosaccharide, at least one
medium-fermented oligosaccharide, and, optionally, at least one
slowly-fermented oligosaccharide to provide a nutritional
composition that is tolerated well by preterm and term infants
(i.e., reduced gassiness and/or stool frequency). Rapidly-fermented
oligosaccharides generally have a fermentation rate of greater than
4,000 .mu.g/g of dry matter/hour; medium-fermented oligosaccharides
generally have a fermentation rate of from 1,500 .mu.g/g of dry
matter/hour to 4,000 .mu.g/g of dry matter/hour; and
slowly-fermented oligosaccharides generally have a fermentation
rate of less than 1,500 .mu.g/g of dry matter/hour.
[0117] By way of specific example, rapidly-fermented
oligosaccharides include FOS, GOS (about 9,304 .mu.g/g of dry
matter/hour), LNnT (about 4,488 .mu.g/g of dry matter/hour), 2'FL
(about 4,872 .mu.g/g of dry matter/hour), and combinations thereof.
Medium-fermented oligosaccharides include 6'SL (about 1,809 .mu.g/g
of dry matter/hour), 3'SL, 2'FL, 3'FL, LNnT and combinations
thereof. Slowly-fermented oligosaccharides include longer chain
carbohydrates such as inulin (about 1,435 .mu.g/g of dry
matter/hour), gum fibers (e.g., gum arabic (about 785 .mu.g/g of
dry matter/hour)), and combinations thereof.
[0118] When used in an oligosaccharide blend, the rapidly-fermented
oligosaccharides can be included in the nutritional compositions in
amounts of from about 0.05 mg/mL to about 20 mg/mL, including from
about 0.5 mg/mL to about 15 mg/mL, including from about 0.5 mg/mL
to about 10 mg/mL, including from about 1 mg/mL to about 15 mg/mL,
including from about 1 mg/mL to about 10 mg/mL, including from
about 2 mg/mL to about 8 mg/mL, and also including from about 3
mg/mL to about 5 mg/mL. The medium-fermented oligosaccharides can
be included in the nutritional compositions in amounts of from
about 0.05 mg/mL to about 20 mg/mL, including from about 0.05 mg/mL
to about 15 mg/mL, including from about 0.05 mg/mL to about 10
mg/mL, including from about 0.05 mg/mL to about 5 mg/mL, including
from about 0.05 mg/mL to about 2.5 mg/mL, including from about 0.05
mg/mL to about 1 mg/mL, including from about 0.05 mg/mL to about
0.5 mg/mL, and including from about 0.05 mg/mL to about 0.25 mg/mL.
The slowly-fermented oligosaccharides can be included in the
nutritional compositions in amounts of from about 0.05 mg/mL to
about 20 mg/mL, including from about 0.05 mg/mL to about 15 mg/mL,
including from about 0.05 mg/mL to about 10 mg/mL, including from
about 0.05 mg/mL to about 5 mg/mL, and also including from about
0.05 mg/mL to about 2.5 mg/mL.
[0119] In one specific embodiment, the nutritional composition
includes an oligosaccharide blend including LNnT, 6'SL and inulin
in a total amount of oligosaccharide blend of from about 0.05 mg/mL
to about 20 mg/mL.
[0120] In another specific embodiment, the nutritional composition
includes an oligosaccharide blend including 2'FL, 6'SL and inulin
in a total amount of oligosaccharide blend of from about 0.05 mg/mL
to about 20 mg/mL.
[0121] Other exemplary combinations include: FOS, GOS, 2'FL, LNnT,
3'SL, and 6'SL; FOS, GOS, 2'FL, 3'SL, and 6'SL; FOS, GOS, LNnT,
3'SL, and 6'SL; FOS, 2'FL, LNnT, 3'SL, and 6'SL; GOS, 2'FL, LNnT,
3'SL, and 6'SL; FOS, GOS, 3'SL, and 6'SL; FOS, 2'FL, 3'SL, and
6'SL; FOS, LNnT, 3'SL, and 6'SL; GOS, 2'FL, 3'SL, and 6'SL; GOS,
LNnT, 3'SL, and 6'SL; 2'FL, LNnT, 3'SL, and 6'SL; FOS, 3'SL, and
6'SL; GOS, 3'SL, and 6'SL; 2'FL, 3'SL, and 6'SL; LNnT, 3'SL, and
6'SL; FOS, GOS, 2'FL, LNnT, and 3'SL; FOS, GOS, 2'FL, and 3'SL;
FOS, GOS, LNnT, and 3'SL; FOS, 2'FL, LNnT, and 3'SL; GOS, 2'FL,
LNnT, and 3'SL; FOS, GOS, and 3'SL; FOS, 2'FL, and 3'SL; FOS, LNnT,
and 3'SL; GOS, 2'FL, and 3'SL; GOS, LNnT, and 3'SL; 2'FL, LNnT, and
3'SL; FOS and 3'SL; GOS and 3'SL; 2'FL and 3'SL; LNnT and 3'SL;
FOS, GOS, 2'FL, LNnT, and 6'SL; FOS, GOS, 2'FL, and 6'SL; FOS, GOS,
LNnT, and 6'SL; FOS, 2'FL, LNnT, and 6'SL; GOS, 2'FL, LNnT, and
6'SL; FOS, GOS, and 6'SL; FOS, 2'FL, and 6'SL; FOS, LNnT, and 6'SL;
GOS, 2'FL, and 6'SL; GOS, LNnT, and 6'SL; 2'FL, LNnT, and 6'SL; FOS
and 6'SL; GOS and 6'SL; 2'FL and 6'SL; and LNnT and 6'SL.
[0122] Further exemplary combinations include: FOS, GOS, 2'FL,
LNnT, 3'SL, 6'SL, inulin, a gum, and polydextrose; FOS, GOS, 2'FL,
3'SL, 6'SL, inulin, a gum, and polydextrose; FOS, GOS, LNnT, 3'SL,
6'SL, inulin, a gum, and polydextrose; FOS, 2'FL, LNnT, 3'SL, 6'SL,
inulin, a gum, and polydextrose; GOS, 2'FL, LNnT, 3'SL, 6'SL,
inulin, a gum, and polydextrose; FOS, GOS, 3'SL, 6'SL, inulin, a
gum, and polydextrose; FOS, 2'FL, 3'SL, 6'SL, inulin, a gum, and
polydextrose; FOS, LNnT, 3'SL, 6'SL, inulin, a gum, and
polydextrose; GOS, 2'FL, 3'SL, 6'SL, inulin, a gum, and
polydextrose; GOS, LNnT, 3'SL, 6'SL, inulin, a gum, and
polydextrose; 2'FL, LNnT, 3'SL, 6'SL, inulin, a gum, and
polydextrose; FOS, 3'SL, 6'SL, inulin, a gum, and polydextrose;
GOS, 3'SL, 6'SL, inulin, a gum, and polydextrose; 2'FL, 3'SL, 6'SL,
inulin, a gum, and polydextrose; LNnT, 3'SL, 6'SL, inulin, a gum,
and polydextrose; FOS, GOS, 2'FL, LNnT, 3'SL, inulin, a gum, and
polydextrose; FOS, GOS, 2'FL, 3'SL, inulin, a gum, and
polydextrose; FOS, GOS, LNnT, 3'SL, inulin, a gum, and
polydextrose; FOS, 2'FL, LNnT, 3'SL, inulin, a gum, and
polydextrose; GOS, 2'FL, LNnT, 3'SL, inulin, a gum, and
polydextrose; FOS, GOS, 3'SL, inulin, a gum, and polydextrose; FOS,
2'FL, 3'SL, inulin, a gum, and polydextrose; FOS, LNnT, 3'SL,
inulin, a gum, and polydextrose; GOS, 2'FL, 3'SL, inulin, a gum,
and polydextrose; GOS, LNnT, 3'SL, inulin, a gum, and polydextrose;
2'FL, LNnT, 3'SL, inulin, a gum, and polydextrose; FOS, 3'SL,
inulin, a gum, and polydextrose; GOS, 3'SL, inulin, a gum, and
polydextrose; 2'FL, 3'SL, inulin, a gum, and polydextrose; LNnT,
3'SL, inulin, a gum, and polydextrose; FOS, GOS, 2'FL, LNnT, 6'SL,
inulin, a gum, and polydextrose; FOS, GOS, 2'FL, 6'SL, inulin, a
gum, and polydextrose; FOS, GOS, LNnT, 6'SL, inulin, a gum, and
polydextrose; FOS, 2'FL, LNnT, 6'SL, inulin, a gum, and
polydextrose; GOS, 2'FL, LNnT, 6'SL, inulin, a gum, and
polydextrose; FOS, GOS, 6'SL, inulin, a gum, and polydextrose; FOS,
2'FL, 6'SL, inulin, a gum, and polydextrose; FOS, LNnT, 6'SL,
inulin, a gum, and polydextrose; GOS, 2'FL, 6'SL, inulin, a gum,
and polydextrose; GOS, LNnT, 6'SL, inulin, a gum, and polydextrose;
2'FL, LNnT, 6'SL, inulin, a gum, and polydextrose; FOS, 6'SL,
inulin, a gum, and polydextrose; GOS, 6'SL, inulin, a gum, and
polydextrose; 2'FL, 6'SL, inulin, a gum, and polydextrose; LNnT,
6'SL, inulin, a gum, and polydextrose; FOS, GOS, 2'FL, LNnT, 3'SL,
6'SL, inulin, and a gum; FOS, GOS, 2'FL, 3'SL, 6'SL, inulin, and a
gum; FOS, GOS, LNnT, 3'SL, 6'SL, inulin, and a gum; FOS, 2'FL,
LNnT, 3'SL, 6'SL, inulin, and a gum; GOS, 2'FL, LNnT, 3'SL, 6'SL,
inulin, and a gum; FOS, GOS, 3'SL, 6'SL, inulin, and a gum; FOS,
2'FL, 3'SL, 6'SL, inulin, and a gum; FOS, LNnT, 3'SL, 6'SL, inulin,
and a gum; GOS, 2'FL, 3'SL, 6'SL, inulin, and a gum; GOS, LNnT,
3'SL, 6'SL, inulin, and a gum; 2'FL, LNnT, 3'SL, 6'SL, inulin, and
a gum; FOS, 3'SL, 6'SL, inulin, and a gum; GOS, 3'SL, 6'SL, inulin,
and a gum; 2'FL, 3'SL, 6'SL, inulin, and a gum; LNnT, 3'SL, 6'SL,
inulin, and a gum; FOS, GOS, 2'FL, LNnT, 3'SL, inulin, and a gum;
FOS, GOS, 2'FL, 3'SL, inulin, and a gum; FOS, GOS, LNnT, 3'SL,
inulin, and a gum; FOS, 2'FL, LNnT, 3'SL, inulin, and a gum; GOS,
2'FL, LNnT, 3'SL, inulin, and a gum; FOS, GOS, 3'SL, inulin, and a
gum; FOS, 2'FL, 3'SL, inulin, and a gum; FOS, LNnT, 3'SL, inulin,
and a gum; GOS, 2'FL, 3'SL, inulin, and a gum; GOS, LNnT, 3'SL,
inulin, and a gum; 2'FL, LNnT, 3'SL, inulin, and a gum; FOS, 3'SL,
inulin, and a gum; GOS, 3'SL, inulin, and a gum; 2'FL, 3'SL,
inulin, and a gum; LNnT, 3'SL, inulin, and a gum; FOS, GOS, 2'FL,
LNnT, 6'SL, inulin, and a gum; FOS, GOS, 2'FL, 6'SL, inulin, and a
gum; FOS, GOS, LNnT, 6'SL, inulin, and a gum; FOS, 2'FL, LNnT,
6'SL, inulin, and a gum; GOS, 2'FL, LNnT, 6'SL, inulin, and a gum;
FOS, GOS, 6'SL, inulin, and a gum; FOS, 2'FL, 6'SL, inulin, and a
gum; FOS, LNnT, 6'SL, inulin, and a gum; GOS, 2'FL, 6'SL, inulin,
and a gum; GOS, LNnT, 6'SL, inulin, and a gum; 2'FL, LNnT, 6'SL,
inulin, and a gum; FOS, 6'SL, inulin, and a gum; GOS, 6'SL, inulin,
and a gum; 2'FL, 6'SL, inulin, and a gum; LNnT, 6'SL, inulin, and a
gum; FOS, GOS, 2'FL, LNnT, 3'SL, 6'SL, inulin, and polydextrose;
FOS, GOS, 2'FL, 3'SL, 6'SL, inulin, and polydextrose; FOS, GOS,
LNnT, 3'SL, 6'SL, inulin, and polydextrose; FOS, 2'FL, LNnT, 3'SL,
6'SL, inulin, and polydextrose; GOS, 2'FL, LNnT, 3'SL, 6'SL,
inulin, and polydextrose; FOS, GOS, 3'SL, 6'SL, inulin, and
polydextrose; FOS, 2'FL, 3'SL, 6'SL, inulin, and polydextrose; FOS,
LNnT, 3'SL, 6'SL, inulin, and polydextrose; GOS, 2'FL, 3'SL, 6'SL,
inulin, and polydextrose; GOS, LNnT, 3'SL, 6'SL, inulin, and
polydextrose; 2'FL, LNnT, 3'SL, 6'SL, inulin, and polydextrose;
FOS, 3'SL, 6'SL, inulin, and polydextrose; GOS, 3'SL, 6'SL, inulin,
and polydextrose; 2'FL, 3'SL, 6'SL, inulin, and polydextrose; LNnT,
3'SL, 6'SL, inulin, and polydextrose; FOS, GOS, 2'FL, LNnT, 3'SL,
inulin, and polydextrose; FOS, GOS, 2'FL, 3'SL, inulin, and
polydextrose; FOS, GOS, LNnT, 3'SL, inulin, and polydextrose; FOS,
2'FL, LNnT, 3'SL, inulin, and polydextrose; GOS, 2'FL, LNnT, 3'SL,
inulin, and polydextrose; FOS, GOS, 3'SL, inulin, and polydextrose;
FOS, 2'FL, 3'SL, inulin, and polydextrose; FOS, LNnT, 3'SL, inulin,
and polydextrose; GOS, 2'FL, 3'SL, inulin, and polydextrose; GOS,
LNnT, 3'SL, inulin, and polydextrose; 2'FL, LNnT, 3'SL, inulin, and
polydextrose; FOS, 3'SL, inulin, and polydextrose; GOS, 3'SL,
inulin, and polydextrose; 2'FL, 3'SL, inulin, and polydextrose;
LNnT, 3'SL, inulin, and polydextrose; FOS, GOS, 2'FL, LNnT, 6'SL,
inulin, and polydextrose; FOS, GOS, 2'FL, 6'SL, inulin, and
polydextrose; FOS, GOS, LNnT, 6'SL, inulin, and polydextrose; FOS,
2'FL, LNnT, 6'SL, inulin, and polydextrose; GOS, 2'FL, LNnT, 6'SL,
inulin, and polydextrose; FOS, GOS, 6'SL, inulin, and polydextrose;
FOS, 2'FL, 6'SL, inulin, and polydextrose; FOS, LNnT, 6'SL, inulin,
and polydextrose; GOS, 2'FL, 6'SL, inulin, and polydextrose; GOS,
LNnT, 6'SL, inulin, and polydextrose; 2'FL, LNnT, 6'SL, inulin, and
polydextrose; FOS, 6'SL, inulin, and polydextrose; GOS, 6'SL,
inulin, and polydextrose; 2'FL, 6'SL, inulin, and polydextrose;
LNnT, 6'SL, inulin, and polydextrose; FOS, GOS, 2'FL, LNnT, 3'SL,
6'SL, a gum, and polydextrose; FOS, GOS, 2'FL, 3'SL, 6'SL, a gum,
and polydextrose; FOS, GOS, LNnT, 3'SL, 6'SL, a gum, and
polydextrose; FOS, 2'FL, LNnT, 3'SL, 6'SL, a gum, and polydextrose;
GOS, 2'FL, LNnT, 3'SL, 6'SL, a gum, and polydextrose; FOS, GOS,
3'SL, 6'SL, a gum, and polydextrose; FOS, 2'FL, 3'SL, 6'SL, a gum,
and polydextrose; FOS, LNnT, 3'SL, 6'SL, a gum, and polydextrose;
GOS, 2'FL, 3'SL, 6'SL, a gum, and polydextrose; GOS, LNnT, 3'SL,
6'SL, a gum, and polydextrose; 2'FL, LNnT, 3'SL, 6'SL, a gum, and
polydextrose; FOS, 3'SL, 6'SL, a gum, and polydextrose; GOS, 3'SL,
6'SL, a gum, and polydextrose; 2'FL, 3'SL, 6'SL, a gum, and
polydextrose; LNnT, 3'SL, 6'SL, a gum, and polydextrose; FOS, GOS,
2'FL, LNnT, 3'SL, a gum, and polydextrose; FOS, GOS, 2'FL, 3'SL, a
gum, and polydextrose; FOS, GOS, LNnT, 3'SL, a gum, and
polydextrose; FOS, 2'FL, LNnT, 3'SL, a gum, and polydextrose; GOS,
2'FL, LNnT, 3'SL, a gum, and polydextrose; FOS, GOS, 3'SL, a gum,
and polydextrose; FOS, 2'FL, 3'SL, a gum, and polydextrose; FOS,
LNnT, 3'SL, a gum, and polydextrose; GOS, 2'FL, 3'SL, a gum, and
polydextrose; GOS, LNnT, 3'SL, a gum, and polydextrose; 2'FL, LNnT,
3'SL, a gum, and polydextrose; FOS, 3'SL, a gum, and polydextrose;
GOS, 3'SL, a gum, and polydextrose; 2'FL, 3'SL, a gum, and
polydextrose; LNnT, 3'SL, a gum, and polydextrose; FOS, GOS, 2'FL,
LNnT, 6'SL, a gum, and polydextrose; FOS, GOS, 2'FL, 6'SL, a gum,
and polydextrose; FOS, GOS, LNnT, 6'SL, a gum, and polydextrose;
FOS, 2'FL, LNnT, 6'SL, a gum, and polydextrose; GOS, 2'FL, LNnT,
6'SL, a gum, and polydextrose; FOS, GOS, 6'SL, a gum, and
polydextrose; FOS, 2'FL, 6'SL, a gum, and polydextrose; FOS, LNnT,
6'SL, a gum, and polydextrose; GOS, 2'FL, 6'SL, a gum, and
polydextrose; GOS, LNnT, 6'SL, a gum, and polydextrose; 2'FL, LNnT,
6'SL, a gum, and polydextrose; FOS, 6'SL, a gum, and polydextrose;
GOS, 6'SL, a gum, and polydextrose; 2'FL, 6'SL, a gum, and
polydextrose; LNnT, 6'SL, a gum, and polydextrose; FOS, GOS, 2'FL,
LNnT, 3'SL, 6'SL, and inulin; FOS, GOS, 2'FL, 3'SL, 6'SL, and
inulin; FOS, GOS, LNnT, 3'SL, 6'SL, and inulin; FOS, 2'FL, LNnT,
3'SL, 6'SL, and inulin; GOS, 2'FL, LNnT, 3'SL, 6'SL, and inulin;
FOS, GOS, 3'SL, 6'SL, and inulin; FOS, 2'FL, 3'SL, 6'SL, and
inulin; FOS, LNnT, 3'SL, 6'SL, and inulin; GOS, 2'FL, 3'SL, 6'SL,
and inulin; GOS, LNnT, 3'SL, 6'SL, and inulin; 2'FL, LNnT, 3'SL,
6'SL, and inulin; FOS, 3'SL, 6'SL, and inulin; GOS, 3'SL, 6'SL, and
inulin; 2'FL, 3'SL, 6'SL, and inulin; LNnT, 3'SL, 6'SL, and inulin;
FOS, GOS, 2'FL, LNnT, 3'SL, and inulin; FOS, GOS, 2'FL, 3'SL, and
inulin; FOS, GOS, LNnT, 3'SL, and inulin; FOS, 2'FL, LNnT, 3'SL,
and inulin; GOS, 2'FL, LNnT, 3'SL, and inulin; FOS, GOS, 3'SL, and
inulin; FOS, 2'FL, 3'SL, and inulin; FOS, LNnT, 3'SL, and inulin;
GOS, 2'FL, 3'SL, and inulin; GOS, LNnT, 3'SL, and inulin; 2'FL,
LNnT, 3'SL, and inulin; FOS, 3'SL, and inulin; GOS, 3'SL, and
inulin; 2'FL, 3'SL, and inulin; LNnT, 3'SL, and inulin; FOS, GOS,
2'FL, LNnT, 6'SL, and inulin; FOS, GOS, 2'FL, 6'SL, and inulin;
FOS, GOS, LNnT, 6'SL, and inulin; FOS, 2'FL, LNnT, 6'SL, and
inulin; GOS, 2'FL, LNnT, 6'SL, and inulin; FOS, GOS, 6'SL, and
inulin; FOS, 2'FL, 6'SL, and inulin; FOS, LNnT, 6'SL, and inulin;
GOS, 2'FL, 6'SL, and inulin; GOS, LNnT, 6'SL, and inulin; 2'FL,
LNnT, 6'SL, and inulin; FOS, 6'SL, and inulin; GOS, 6'SL, and
inulin; FOS, GOS, 2'FL, LNnT, 3'SL, 6'SL, and polydextrose; FOS,
GOS, 2'FL, 3'SL, 6'SL, and polydextrose; FOS, GOS, LNnT, 3'SL,
6'SL, and polydextrose; FOS, 2'FL, LNnT, 3'SL, 6'SL, and
polydextrose; GOS, 2'FL, LNnT, 3'SL, 6'SL, and polydextrose; FOS,
GOS, 3'SL, 6'SL, and polydextrose; FOS, 2'FL, 3'SL, 6'SL, and
polydextrose; FOS, LNnT, 3'SL, 6'SL, and polydextrose; GOS, 2'FL,
3'SL, 6'SL, and polydextrose; GOS, LNnT, 3'SL, 6'SL, and
polydextrose; 2'FL, LNnT, 3'SL, 6'SL, and polydextrose; FOS, 3'SL,
6'SL, and polydextrose; GOS, 3'SL, 6'SL, and polydextrose; 2'FL,
3'SL, 6'SL, and polydextrose; LNnT, 3'SL, 6'SL, and polydextrose;
FOS, GOS, 2'FL, LNnT, 3'SL, and polydextrose; FOS, GOS, 2'FL, 3'SL,
and polydextrose; FOS, GOS, LNnT, 3'SL, and polydextrose; FOS,
2'FL, LNnT, 3'SL, and polydextrose; GOS, 2'FL, LNnT, 3'SL, and
polydextrose; FOS, GOS, 3'SL, and polydextrose; FOS, 2'FL, 3'SL,
and polydextrose; FOS, LNnT, 3'SL, and polydextrose; GOS, 2'FL,
3'SL, and polydextrose; GOS, LNnT, 3'SL, and polydextrose; 2'FL,
LNnT, 3'SL, and polydextrose; FOS, 3'SL, and polydextrose; GOS,
3'SL, and polydextrose; 2'FL, 3'SL, and polydextrose; LNnT, 3'SL,
and polydextrose; FOS, GOS, 2'FL, LNnT, 6'SL, and polydextrose;
FOS, GOS, 2'FL, 6'SL, and polydextrose; FOS, GOS, LNnT, 6'SL, and
polydextrose; FOS, 2'FL, LNnT, 6'SL, and polydextrose; GOS, 2'FL,
LNnT, 6'SL, and polydextrose; FOS, GOS, 6'SL, and polydextrose;
FOS, 2'FL, 6'SL, and polydextrose; FOS, LNnT, 6'SL, and
polydextrose; GOS, 2'FL, 6'SL, and polydextrose; GOS, LNnT, 6'SL,
and polydextrose; 2'FL, LNnT, 6'SL, and polydextrose; FOS, 6'SL,
and polydextrose; GOS, 6'SL, and polydextrose; 2'FL, 6'SL, and
polydextrose; LNnT, 6'SL, and polydextrose; FOS, GOS, 2'FL, LNnT,
3'SL, 6'SL, and a gum; FOS, GOS, 2'FL, 3'SL, 6'SL, and a gum; FOS,
GOS, LNnT, 3'SL, 6'SL, and a gum; FOS, 2'FL, LNnT, 3'SL, 6'SL, and
a gum; GOS, 2'FL, LNnT, 3'SL, 6'SL, and a gum; FOS, GOS, 3'SL,
6'SL, and a gum; FOS, 2'FL, 3'SL, 6'SL, and a gum; FOS, LNnT, 3'SL,
6'SL, and a gum; GOS, 2'FL, 3'SL, 6'SL, and a gum; GOS, LNnT, 3'SL,
6'SL, and a gum; 2'FL, LNnT, 3'SL, 6'SL, and a gum; FOS, 3'SL,
6'SL, and a gum; GOS, 3'SL, 6'SL, and a gum; 2'FL, 3'SL, 6'SL, and
a gum; LNnT, 3'SL, 6'SL, and a gum; FOS, GOS, 2'FL, LNnT, 3'SL, and
a gum; FOS, GOS, 2'FL, 3'SL, and a gum; FOS, GOS, LNnT, 3'SL, and a
gum; FOS, 2'FL, LNnT, 3'SL, and a gum; GOS, 2'FL, LNnT, 3'SL, and a
gum; FOS, GOS, 3'SL, and a gum; FOS, 2'FL, 3'SL, and a gum; FOS,
LNnT, 3'SL, and a gum; GOS, 2'FL, 3'SL, and a gum; GOS, LNnT, 3'SL,
and a gum; 2'FL, LNnT, 3'SL, and a gum; FOS, 3'SL, and a gum; GOS,
3'SL, and a gum; 2'FL, 3'SL, and a gum; LNnT, 3'SL, and a gum; FOS,
GOS, 2'FL, LNnT, 6'SL, and a gum; FOS, GOS, 2'FL, 6'SL, and a gum;
FOS, GOS, LNnT, 6'SL, and a gum; FOS, 2'FL, LNnT, 6'SL, and a gum;
GOS, 2'FL, LNnT, 6'SL, and a gum; FOS, GOS, 6'SL, and a gum; FOS,
2'FL, 6'SL, and a gum; FOS, LNnT, 6'SL, and a gum; GOS, 2'FL, 6'SL,
and a gum; GOS, LNnT, 6'SL, and a gum; 2'FL, LNnT, 6'SL, and a gum;
FOS, 6'SL, and a gum; GOS, 6'SL, and a gum; 2'FL, 6'SL, and a gum;
and LNnT, 6'SL, and a gum.
Probiotics
[0123] The nutritional compositions of the present disclosure may,
in addition to HMOs (and, optionally, other prebiotic
oligosaccharides as described above), comprise one or more
probiotics. In some embodiments, the nutritional composition
includes a combination of HMOs and probiotics such that the
composition provides a synergistic benefit to the end user in
promoting the growth of microbiota in the gastrointestinal tract of
infants.
[0124] Probiotics are live microorganisms thought to be healthy for
the host organism. Lactic acid bacteria (LAB) and bifidobacteria
are the most common types of microbes used as probiotics.
Probiotics maintain the microbial ecology of the gut and show
physiological, immuno-modulatory and antimicrobial effects, such
that the use of probiotics has been found to prevent and treat
gastrointestinal diseases and/or disorders, pathogen-induced
diarrhea and toxin-producing bacteria, urogenital infections, and
atopic diseases.
[0125] In order for microbes to exhibit beneficial probiotic
effects in vivo, the organisms should survive for extended time
periods in the gastrointestinal tract. Therefore, it is important
that probiotic strains be selected that possess qualities that
prevent their rapid removal by gut contraction. Effective probiotic
strains are able to survive gastric conditions and colonize the
intestine, at least temporarily, by adhering to the intestinal
epithelium.
[0126] Non-limiting examples of probiotic strains for use in the
nutritional compositions herein include the genus Lactobacillus
including L. acidophilus (e.g., L. acidophilus LA-5 and L.
acidophilus NCFM), L. amylovorus, L. brevis, L. bulgaricus, L.
casei spp. casei, L. casei spp. rhamnosus, L. crispatus, L.
delbrueckii ssp. lactis, L. fermentum (e.g., L. fermentum
CETC5716), L. helveticus, L. johnsonii, L. paracasei, L. pentosus,
L. plantarum, L. reuteri (e.g., L. reuteri ATCC 55730, L. reuteri
ATCC PTA-6475, and L. reuteri DSM 17938), L. sake, and L. rhamnosus
(e.g., L. rhamnosus LGG and L. rhamnosus HN001); the genus
Bifidobacterium including: B. animalis (e.g., B. animalis spp.
lactis Bb-12), B. bifidum, B. breve (e.g., B. breve M-16V), B.
infantis (e.g., B. infantis M-63, B. infantis ATCC 15697, B.
Infantis 35624, B. infantis CHCC2228, B. infantis BB-02, B.
infantis DSM20088, and B. infantis R-0033), B. longum (e.g., B.
longum BB536, B. longum AH1205, and B. longum AH1206), and B.
lactis (e.g., B. lactis HN019 and B. lactis Bi07); the genus
Pediococcus including: P. acidilactici; the genus Propionibacterium
including: P. acidipropionici, P. freudenreichii, P. jensenii, and
P. theonii; and the genus Streptococcus including: S. cremoris, S.
lactis, and S. thermophilus. Particularly preferred probiotics
include probiotics of human infant origin such as B. infantis M-63
and B. infantis ATCC 15697.
[0127] The probiotic is present in the nutritional compositions in
a total amount of at least about 10.sup.3 CFU/g, including from
about 10.sup.3 CFU/g to about 10.sup.12 CFU/g, and including from
about 10.sup.6 CFU/g to about 10.sup.7 CFU/g.
[0128] In some embodiments, the nutritional composition includes a
probiotic in combination with a first oligosaccharide including
fructooligosaccharide and/or a galactooligosaccharide further in
combination with a second oligosaccharide including at least one
HMO such as 2'FL, 3'FL, 3'SL, 6'SL, and/or LNnT. In these
embodiments, the first oligosaccharide and the second
oligosaccharide are present in the compositions in a weight ratio
of first oligosaccharide:second oligosaccharide of about 10:1, or
even from about 11:1 to about 8:1.
Macronutrients
[0129] The nutritional compositions including the HMO or HMOs may
be formulated to include at least one of protein, fat, and
carbohydrate. In many embodiments, the nutritional compositions
will include the HMO or HMOs with protein, carbohydrate and
fat.
[0130] Although total concentrations or amounts of the fat,
protein, and carbohydrates may vary depending upon the product type
(i.e., human milk fortifier, preterm infant formula, infant
formula, toddler formula, pediatric formula, follow-on formula,
adult nutritional, etc.), product form (i.e., nutritional solid,
powder, ready-to-feed liquid, or concentrated liquid), and targeted
dietary needs of the intended user, such concentrations or amounts
most typically fall within one of the following embodied ranges,
inclusive of any other essential fat, protein, and/or carbohydrate
ingredients as described herein.
[0131] For the liquid preterm and term infant formulas,
carbohydrate concentrations (including both HMOs and any other
carbohydrate/oligosaccharide sources) most typically range from
about 5% to about 40%, including from about 7% to about 30%,
including from about 10% to about 25%, by weight of the preterm or
term infant formula; fat concentrations most typically range from
about 1% to about 30%, including from about 2% to about 15%, and
also including from about 3% to about 10%, by weight of the preterm
or term infant formula; and protein concentrations most typically
range from about 0.5% to about 30%, including from about 1% to
about 15%, and also including from about 2% to about 10%, by weight
of the preterm or term infant formula.
[0132] For the liquid human milk fortifiers, carbohydrate
concentrations (including both HMOs and any other
carbohydrate/oligosaccharide sources) most typically range from
about 10% to about 75%, including from about 10% to about 50%,
including from about 20% to about 40%, by weight of the human milk
fortifier; fat concentrations most typically range from about 10%
to about 40%, including from about 15% to about 37%, and also
including from about 18% to about 30%, by weight of the human milk
fortifier; and protein concentrations most typically range from
about 5% to about 40%, including from about 10% to about 30%, and
also including from about 15% to about 25%, by weight of the human
milk fortifier.
[0133] For the adult nutritional liquids, carbohydrate
concentrations (including both HMOs and any other
carbohydrate/oligosaccharide sources) most typically range from
about 5% to about 40%, including from about 7% to about 30%,
including from about 10% to about 25%, by weight of the adult
nutritional; fat concentrations most typically range from about 2%
to about 30%, including from about 3% to about 15%, and also
including from about 5% to about 10%, by weight of the adult
nutritional; and protein concentrations most typically range from
about 0.5% to about 30%, including from about 1% to about 15%, and
also including from about 2% to about 10%, by weight of the adult
nutritional.
[0134] The amount of carbohydrates, fats, and/or proteins in any of
the liquid nutritional compositions described herein may also be
characterized in addition to, or in the alternative, as a
percentage of total calories in the liquid nutritional composition
as set forth in the following table. These macronutrients for
liquid nutritional compositions of the present disclosure are most
typically formulated within any of the caloric ranges (embodiments
A-F) described in the following table (each numerical value is
preceded by the term "about").
TABLE-US-00001 Nutrient % Total Cal. Embodiment A Embodiment B
Embodiment C Carbohydrate 0-98 2-96 10-75 Protein 0-98 2-96 5-70
Fat 0-98 2-96 20-85 Embodiment D Embodiment E Embodiment F
Carbohydrate 30-50 25-50 25-50 Protein 15-35 10-30 5-30 Fat 35-55
1-20 2-20
[0135] In one specific example, liquid infant formulas (both
ready-to-feed and concentrated liquids) include those embodiments
in which the protein component may comprise from about 7.5% to
about 25% of the caloric content of the formula; the carbohydrate
component (including both HMOs and any other
carbohydrate/oligosaccharide sources) may comprise from about 35%
to about 50% of the total caloric content of the infant formula;
and the fat component may comprise from about 30% to about 60% of
the total caloric content of the infant formula. These ranges are
provided as examples only, and are not intended to be limiting.
Additional suitable ranges are noted in the following table (each
numerical value is preceded by the term "about").
TABLE-US-00002 Nutrient % Total Cal. Embodiment G Embodiment H
Embodiment I Carbohydrates: 20-85 30-60 35-55 Fat: 5-70 20-60 25-50
Protein: 2-75 5-50 7-40
[0136] When the nutritional composition is a powdered preterm or
term infant formula, the protein component is present in an amount
of from about 5% to about 35%, including from about 8% to about
12%, and including from about 10% to about 12% by weight of the
preterm or term infant formula; the fat component is present in an
amount of from about 10% to about 35%, including from about 25% to
about 30%, and including from about 26% to about 28% by weight of
the preterm or term infant formula; and the carbohydrate component
(including both HMOs and any other carbohydrate/oligosaccharide
sources) is present in an amount of from about 30% to about 85%,
including from about 45% to about 60%, including from about 50% to
about 55% by weight of the preterm or term infant formula.
[0137] For powdered human milk fortifiers, the protein component is
present in an amount of from about 1% to about 55%, including from
about 10% to about 50%, and including from about 10% to about 30%
by weight of the human milk fortifier; the fat component is present
in an amount of from about 1% to about 30%, including from about 1%
to about 25%, and including from about 1% to about 20% by weight of
the human milk fortifier; and the carbohydrate component (including
both HMOs and any other carbohydrate/oligosaccharide sources) is
present in an amount of from about 15% to about 75%, including from
about 15% to about 60%, including from about 20% to about 50% by
weight of the human milk fortifier.
[0138] For powdered adult nutritionals, the protein component is
present in an amount of from about 10% to about 90%, including from
about 30% to about 80%, and including from about 40% to about 75%
by weight of the adult nutritional; the fat component is present in
an amount of from about 0.5% to about 20%, including from about 1%
to about 10%, and including from about 2% to about 5% by weight of
the adult nutritional; and the carbohydrate component (including
both HMOs and any other carbohydrate/oligosaccharide sources) is
present in an amount of from about 5% to about 40%, including from
about 7% to about 30%, including from about 10% to about 25% by
weight of the adult nutritional.
[0139] The total amount or concentration of fat, carbohydrate, and
protein, in the powdered nutritional compositions of the present
disclosure can vary considerably depending upon the selected
composition and dietary or medical needs of the intended user.
Additional suitable examples of macronutrient concentrations are
set forth below. In this context, the total amount or concentration
refers to all fat, carbohydrate, and protein sources in the
powdered composition. For powdered nutritional compositions, such
total amounts or concentrations are most typically and preferably
formulated within any of the embodied ranges described in the
following table (each numerical value is preceded by the term
"about`).
TABLE-US-00003 Nutrient % Total Cal. Embodiment J Embodiment K
Embodiment L Carbohydrate 1-85 30-60 35-55 Fat 5-70 20-60 25-50
Protein 2-75 5-50 7-40
Fat
[0140] The nutritional compositions of the present disclosure may
optionally comprise any source or sources of fat. Suitable sources
of fat for use herein include any fat or fat source that is
suitable for use in an oral nutritional composition and is
compatible with the essential elements and features of such
composition. For example, in one specific embodiment, the fat is
derived from long chain polyunsaturated fatty acids (LCPUFAs).
[0141] Exemplary LCPUFAs for use in the nutritional compositions
include, for example, .omega.-3 LCPUFAs and .omega.-6 LCPUFAs.
Specific LCPUFAs include docosahexaenoic acid (DHA),
eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA),
arachidonic acid (ARA), linoleic acid, linolenic acid (alpha
linolenic acid) and gamma-linolenic acid derived from oil sources
such as plant oils, marine plankton, fungal oils, and fish oils. In
one particular embodiment, the LCPUFAs are derived from fish oils
such as menhaden, salmon, anchovy, cod, halibut, tuna, or herring
oil. Particularly preferred LCPUFAs for use in the nutritional
compositions with the HMOs include DHA, ARA, EPA, and combinations
thereof.
[0142] In order to reduce potential side effects of high dosages of
LCPUFAs in the nutritional compositions, the content of LCPUFAs
preferably does not exceed 3% by weight of the total fat content,
including below 2% by weight of the total fat content, and
including below 1% by weight of the total fat content in the
nutritional composition.
[0143] The LCPUFA may be provided as free fatty acids, in
triglyceride form, in diglyceride form, in monoglyceride form, in
phospholipid form, in esterified form or as a mixture of one or
more of the above, preferably in triglyceride form. In another
specific embodiment, the fat is derived from short chain fatty
acids.
[0144] Additional non-limiting examples of suitable fats or sources
thereof for use in the nutritional compositions described herein
include coconut oil, fractionated coconut oil, soybean oil, corn
oil, olive oil, safflower oil, high oleic safflower oil, oleic
acids (EMERSOL 6313 OLEIC ACID, Cognis Oleochemicals, Malaysia),
MCT oil (medium chain triglycerides), sunflower oil, high oleic
sunflower oil, palm and palm kernel oils, palm olein, canola oil,
marine oils, fish oils, fungal oils, algae oils, cottonseed oils,
and combinations thereof.
Protein
[0145] The nutritional compositions of the present disclosure may
optionally further comprise protein. Any protein source that is
suitable for use in oral nutritional compositions and is compatible
with the essential elements and features of such compositions is
suitable for use in the nutritional compositions.
[0146] Non-limiting examples of suitable proteins or sources
thereof for use in the nutritional compositions include hydrolyzed,
partially hydrolyzed or non-hydrolyzed proteins or protein sources,
which may be derived from any known or otherwise suitable source
such as milk (e.g., casein, whey), animal (e.g., meat, fish),
cereal (e.g., rice, corn), vegetable (e.g., soy) or combinations
thereof. Non-limiting examples of such proteins include milk
protein isolates, milk protein concentrates as described herein,
casein protein isolates, extensively hydrolyzed casein, whey
protein, sodium or calcium caseinates, whole cow milk, partially or
completely defatted milk, soy protein isolates, soy protein
concentrates, and so forth. In one specific embodiment, the
nutritional compositions include a protein source derived from milk
proteins of human and/or bovine origin.
[0147] In one embodiment, the protein source is a hydrolyzed
protein hydrolysate. In this context, the terms "hydrolyzed
protein" or "protein hydrolysates" are used interchangeably herein
and include extensively hydrolyzed proteins, wherein the degree of
hydrolysis is most often at least about 20%, including from about
20% to about 80%, and also including from about 30% to about 80%,
even more preferably from about 40% to about 60%. The degree of
hydrolysis is the extent to which peptide bonds are broken by a
hydrolysis method. The degree of protein hydrolysis for purposes of
characterizing the extensively hydrolyzed protein component of
these embodiments is easily determined by one of ordinary skill in
the formulation arts by quantifying the amino nitrogen to total
nitrogen ratio (AN/TN) of the protein component of the selected
liquid formulation. The amino nitrogen component is quantified by
USP titration methods for determining amino nitrogen content, while
the total nitrogen component is determined by the Tecator Kjeldahl
method, all of which are well known methods to one of ordinary
skill in the analytical chemistry art.
[0148] Suitable hydrolyzed proteins may include soy protein
hydrolysate, casein protein hydrolysate, whey protein hydrolysate,
rice protein hydrolysate, potato protein hydrolysate, fish protein
hydrolysate, egg albumen hydrolysate, gelatin protein hydrolysate,
combinations of animal and vegetable protein hydrolysates, and
combinations thereof. Particularly preferred protein hydrolysates
include whey protein hydrolysate and hydrolyzed sodium
caseinate.
[0149] When used in the nutritional compositions, the protein
source may include at least about 20% (by weight total protein)
protein hydrolysate, including from about 30% to 100% (by weight
total protein) protein hydrolysate, and including from about 40% to
about 80% (by weight total protein) protein hydrolysate, and
including about 50% (by weight total protein) protein hydrolysate.
In one particular embodiment, the nutritional composition includes
100% (by weight total protein) protein hydrolysate.
Carbohydrate
[0150] The nutritional compositions of the present disclosure may
further optionally comprise any carbohydrates that are suitable for
use in an oral nutritional composition and are compatible with the
essential elements and features of such compositions.
[0151] Non-limiting examples of suitable carbohydrates or sources
thereof for use in the nutritional compositions described herein
may include maltodextrin, hydrolyzed or modified starch or
cornstarch, glucose polymers, corn syrup, corn syrup solids,
rice-derived carbohydrates, pea-derived carbohydrates,
potato-derived carbohydrates, tapioca, sucrose, glucose, fructose,
lactose, high fructose corn syrup, honey, sugar alcohols (e.g.,
maltitol, erythritol, sorbitol), artificial sweeteners (e.g.,
sucralose, acesulfame potassium, stevia) and combinations thereof.
A particularly desirable carbohydrate is a low dextrose equivalent
(DE) maltodextrin.
Other Optional Ingredients
[0152] The nutritional compositions of the present disclosure may
further comprise other optional components that may modify the
physical, chemical, aesthetic or processing characteristics of the
compositions or serve as pharmaceutical or additional nutritional
components when used in the targeted population. Many such optional
ingredients are known or otherwise suitable for use in medical food
or other nutritional products or pharmaceutical dosage forms and
may also be used in the compositions herein, provided that such
optional ingredients are safe for oral administration and are
compatible with the essential and other ingredients in the selected
product form.
[0153] Non-limiting examples of such optional ingredients include
preservatives, emulsifying agents, buffers, postbiotics,
pharmaceutical actives, anti-inflammatory agents, additional
nutrients as described herein, colorants, flavors, thickening
agents and stabilizers, emulsifying agents, lubricants, and so
forth.
[0154] The nutritional compositions may further comprise a
sweetening agent, preferably including at least one sugar alcohol
such as maltitol, erythritol, sorbitol, xylitol, mannitol,
isolmalt, and lactitol, and also preferably including at least one
artificial or high potency sweetener such as acesulfame K,
aspartame, sucralose, saccharin, stevia, and tagatose. These
sweetening agents, especially as a combination of a sugar alcohol
and an artificial sweetener, are especially useful in formulating
liquid beverage embodiments of the present disclosure having a
desirable favor profile. These sweetener combinations are
especially effective in masking undesirable flavors sometimes
associated with the addition of vegetable proteins to a liquid
beverage. Optional sugar alcohol concentrations in the nutritional
composition may range from at least 0.01%, including from 0.1% to
about 10%, and also including from about 1% to about 6%, by weight
of the nutritional composition. Optional artificial sweetener
concentrations may range from about 0.01%, including from about
0.05% to about 5%, also including from about 0.1% to about 1.0%, by
weight of the nutritional composition.
[0155] A flowing agent or anti-caking agent may be included in the
nutritional compositions as described herein to retard clumping or
caking of the powder over time and to make a powder embodiment flow
easily from its container. Any known flowing or anti-caking agents
that are known or otherwise suitable for use in a nutritional
powder or product form are suitable for use herein, non-limiting
examples of which include tricalcium phosphate, silicates, and
combinations thereof. The concentration of the flowing agent or
anti-caking agent in the nutritional composition varies depending
upon the product form, the other selected ingredients, the desired
flow properties, and so forth, but most typically range from about
0.1% to about 4%, including from about 0.5% to about 2%, by weight
of the nutritional composition.
[0156] A stabilizer may also be included in the nutritional
compositions. Any stabilizer that is known or otherwise suitable
for use in a nutritional composition is also suitable for use
herein, some non-limiting examples of which include gums such as
xanthan gum. The stabilizer may represent from about 0.1% to about
5.0%, including from about 0.5% to about 3%, including from about
0.7% to about 1.5%, by weight of the nutritional composition.
[0157] Additionally, the nutritional compositions may comprise one
or more antioxidants to provide nutritional support, as well as to
reduce oxidative stress. Any antioxidants suitable for oral
administration may be included for use in the nutritional
compositions of the present disclosure, including, for example,
ascorbyl palmitate, vitamin A, vitamin E, vitamin C, retinol,
tocopherol, carotenoids, polyphenols (e.g., curcumin), glutathione,
and superoxide dismutase.
[0158] In one specific embodiment, the antioxidants for use in the
nutritional compositions include carotenoids such as lutein,
zeaxanthin, lycopene, beta-carotene, and combinations thereof, and
particularly, combinations of the carotenoids lutein, lycopene, and
beta-carotene. Nutritional compositions containing these
combinations, as selected and defined herein, can be used to
modulate inflammation and/or levels of C-reactive protein in
preterm and term infants.
[0159] The nutritional compositions may further comprise any of a
variety of other water or fat soluble vitamins or related
nutrients, non-limiting examples of which include vitamin D,
vitamin K, thiamine, riboflavin, pyridoxine, vitamin B.sub.12,
niacin, folic acid, pantothenic acid, biotin, choline, inositol,
salts and derivatives thereof, and combinations thereof.
[0160] The nutritional compositions may further comprise any of a
variety of other additional minerals and trace elements,
non-limiting examples of which include calcium, phosphorus,
magnesium, iron, zinc, manganese, copper, sodium, potassium,
molybdenum, chromium, chloride, and combinations thereof.
[0161] The nutritional compositions of the present disclosure may
additionally comprise nucleotides and/or nucleotide precursors
selected from the group consisting of nucleoside, purine base,
pyrimidine base, ribose and deoxyribose to further improve
intestinal barrier integrity and/or maturation. The nucleotide may
be in monophosphate, diphosphate, or triphosphate form. The
nucleotide may be a ribonucleotide or a deoxyribonucleotide. The
nucleotides may be monomeric, dimeric, or polymeric (including RNA
and DNA). The nucleotide may be present in the nutritional
composition as a free acid or in the form of a salt, preferably a
monosodium salt.
[0162] Suitable nucleotides and/or nucleosides for use in the
nutritional compositions include one or more of cytidine
5'-monophosphate, uridine 5'-monophosphate, adenosine
5'-monophosphate, guanosine 5'-1-monophosphate, and/or inosine
5'-monophosphate, more preferably cytidine 5'-monophosphate,
uridine 5'-monophosphate, adenosine 5'-monophosphate, guanosine
5'-monophosphate, and inosine 5'-monophosphate.
[0163] The nutritional compositions of the present disclosure may
additionally comprise bioactive factors, such as growth hormones or
cytokines, of human and/or bovine milk origin, tributyrin, other
SCFA-containing mono-, di-, or triglycerides, or human milk-derived
lipids.
Methods of Manufacture
[0164] The nutritional compositions of the present disclosure may
be prepared by any known or otherwise effective manufacturing
technique for preparing the selected product solid or liquid form.
Many such techniques are known for any given product form such as
nutritional liquids or powders and can easily be applied by one of
ordinary skill in the art to the nutritional compositions described
herein.
[0165] The nutritional compositions of the present disclosure can
therefore be prepared by any of a variety of known or otherwise
effective formulation or manufacturing methods. In one suitable
manufacturing process, for example, at least three separate
slurries are prepared, including a protein-in-fat (PIF) slurry, a
carbohydrate-mineral (CHO-MIN) slurry, and a protein-in-water (PIW)
slurry. The PIF slurry is formed by heating and mixing the oil
(e.g., canola oil, corn oil, etc.) and then adding an emulsifier
(e.g., lecithin), fat soluble vitamins, and a portion of the total
protein (e.g., milk protein concentrate, etc.) with continued heat
and agitation. The CHO-MIN slurry is formed by adding with heated
agitation to water: minerals (e.g., potassium citrate, dipotassium
phosphate, sodium citrate, etc.), trace and ultra trace minerals
(TM/UTM premix), thickening or suspending agents (e.g. avicel,
gellan, carrageenan). The resulting CHO-MIN slurry is held for 10
minutes with continued heat and agitation before adding additional
minerals (e.g., potassium chloride, magnesium carbonate, potassium
iodide, etc.), and/or carbohydrates (e.g., HMOs,
fructooligosaccharide, sucrose, corn syrup, etc.). The PIW slurry
is then formed by mixing with heat and agitation the remaining
protein, if any.
[0166] The resulting slurries are then blended together with heated
agitation and the pH adjusted to 6.6-7.0, after which the
composition is subjected to high-temperature short-time (HTST)
processing during which the composition is heat treated, emulsified
and homogenized, and then allowed to cool. Water soluble vitamins
and ascorbic acid are added, the pH is adjusted to the desired
range if necessary, flavors are added, and water is added to
achieve the desired total solid level. The composition is then
aseptically packaged to form an aseptically packaged nutritional
emulsion. This emulsion can then be further diluted, heat-treated,
and packaged to form a ready-to-feed or concentrated liquid, or it
can be heat-treated and subsequently processed and packaged as a
reconstitutable powder, e.g., spray dried, drymixed,
agglomerated.
[0167] The nutritional solid, such as a spray dried nutritional
powder or drymixed nutritional powder, may be prepared by any
collection of known or otherwise effective techniques, suitable for
making and formulating a nutritional powder.
[0168] For example, when the nutritional powder is a spray dried
nutritional powder, the spray drying step may likewise include any
spray drying technique that is known for or otherwise suitable for
use in the production of nutritional powders. Many different spray
drying methods and techniques are known for use in the nutrition
field, all of which are suitable for use in the manufacture of the
spray dried nutritional powders herein.
[0169] One method of preparing the spray dried nutritional powder
comprises forming and homogenizing an aqueous slurry or liquid
comprising predigested fat, and optionally protein, carbohydrate,
and other sources of fat, and then spray drying the slurry or
liquid to produce a spray dried nutritional powder. The method may
further comprise the step of spray drying, drymixing, or otherwise
adding additional nutritional ingredients, including any one or
more of the ingredients described herein, to the spray dried
nutritional powder.
[0170] Other suitable methods for making nutritional compositions
are described, for example, in U.S. Pat. No. 6,365,218 (Borschel,
et al.), U.S. Pat. No. 6,589,576 (Borschel, et al.), U.S. Pat. No.
6,306,908 (Carlson, et al.), U.S. Patent Application No.
20030118703 A1 (Nguyen, et al.), which descriptions are
incorporated herein by reference to the extent that they are
consistent herewith.
Methods of Use
[0171] The nutritional compositions as described herein can be used
to address one or more of the diseases, disorders, or conditions
discussed herein, or can be used to provide one or more of the
benefits described herein, to preterm infants, infants, toddlers,
children, and adults, including pregnant women. The preterm infant,
infant, toddler, child, adult and pregnant women utilizing the
nutritional compositions described herein may actually have or be
afflicted with the disease or condition described, or may be
susceptible to, or at risk of, getting the disease or condition
(that is, may not actually yet have the disease or condition, but
is at elevated risk as compared to the general population for
getting it due to certain conditions, family history, etc.) Whether
the preterm infant, infant, toddler, child, adult, and pregnant
women actually have the disease or condition, or is at risk or
susceptible to the disease or condition, the preterm infant,
infant, toddler, child, adult, and pregnant women are classified
herein as "in need of" assistance in dealing with and combating the
disease or condition. For example, the preterm infant, infant,
toddler, child, adult and pregnant women may actually have
respiratory inflammation or may be at risk of getting respiratory
inflammation (susceptible to getting respiratory inflammation) due
to family history or other medical conditions, for example. Whether
the preterm infant, infant, toddler, child, adult, and pregnant
women actually has the disease or condition, or is only at risk or
susceptible to getting the disease or condition, it is within the
scope of the present disclosure to assist the preterm infant,
infant, toddler, child, adult and pregnant women with the
nutritional compositions described herein.
[0172] Based on the foregoing, because some of the method
embodiments of the present disclosure are directed to specific
subsets or subclasses of identified individuals (that is, the
subset or subclass of individuals "in need" of assistance in
addressing one or more specific diseases or specific conditions
noted herein), not all preterm infants, infants, toddlers,
children, adults and pregnant women will fall within the subset or
subclass of preterm infants, infants, toddlers, children, adults,
and pregnant women as described herein for certain diseases or
conditions.
[0173] The nutritional compositions as described herein comprise
HMOs, alone or in combination with one or more additional
components, to provide a nutritional source for improving at least
the intestinal/gut function. Specifically, the nutritional
compositions can stimulate enteric nerve cells in the
gastrointestinal tract of an individual to improve intestinal/gut
barrier integrity; improve feeding tolerance (e.g., reduce feeding
intolerance, reduce diarrhea, loose stools, gas, and bloating);
reduce colic in infants; promote tolerance to enteral feeding,
decrease time to full enteral feeding, increase the rate of
advancement of enteral feeding, decrease the amount and duration of
partial or total parenteral nutrition, protect against necrotizing
enterocolitis and other disorders of prematurity; address
gastrointestinal diseases and disorders associated with the enteric
nervous system; address gastrointestinal diseases and disorders of
gut contractility and inflammation; correct effects of gut
dysbiosis; and affect long-term modulation of allergic
tolerance.
[0174] More particularly, in some embodiments, the nutritional
compositions may be administered to an individual having,
susceptible to, or at risk of, gastrointestinal diseases and
disorders associated with the enteric nervous system and/or
associated with gut contractility and inflammation, which may
include, for example, irritable bowel syndrome, colitis (e.g.,
necrotizing enterocolitis, Crohn's disease, ischemic colitis,
cryptosporidium enterocolitis, pseudomembranous colitis,
cytomegalovirus, ulcerative colitis), food intolerance, and food
allergies.
[0175] Along with improved growth and maturation of an individual's
immune system as described above, the use of the nutritional
compositions of the present disclosure may also function to enhance
the individual's ability to resist microbial infection and to
promote the growth of beneficial microbiota in the gastrointestinal
tract of an infant, toddler, child, or adult.
[0176] Additionally, the nutritional compositions of the present
disclosure may also be used to improve cognition in individuals,
particularly in individuals susceptible to, or at risk of,
neurodegenerative diseases, which may include, for example,
Alzheimer's disease, Huntington's disease, Parkinson's disease, and
schizophrenia, or in individuals suffering from conditions caused
by impaired cognitive development, or neurodevelopmental
conditions, such as attention deficit hyperactivity disorder and
autism.
EXAMPLES
[0177] The following examples illustrate specific embodiments
and/or features of the nutritional compositions and methods of the
present disclosure. The examples are given solely for the purpose
of illustration and are not to be construed as limitations of the
present disclosure, as many variations thereof are possible without
departing from the spirit and scope of the disclosure. All
exemplified amounts are weight percentages based upon the total
weight of the composition, unless otherwise specified.
[0178] The exemplified compositions are shelf stable nutritional
compositions prepared in accordance with the manufacturing methods
described herein, such that each exemplified composition, unless
otherwise specified, includes an aseptically processed embodiment
and a retort packaged embodiment.
[0179] The nutritional liquid embodiments are aqueous oil-in-water
emulsions that are packaged in 240 mL plastic containers and remain
physically stable for 12-18 months after composition/packaging at
storage temperatures ranging from 1-25.degree. C.
Examples 1-5
[0180] Examples 1-5 illustrate ready-to-feed nutritional emulsions
of the present disclosure, the ingredients of which are listed in
the table below. All ingredient amounts are listed as kilogram per
1000 kilogram batch of product, unless otherwise specified.
TABLE-US-00004 Ingredient Ex. 1 Ex. 2 Ex. 3 Ex. 4 Ex. 5 Water Q.S.
Q.S. Q.S. Q.S. Q.S. Condensed Skim Milk 86.64 86.64 86.64 86.64
86.64 Lactose 54.80 54.80 54.80 54.80 54.80 High oleic safflower
oil 14.10 14.10 14.10 14.10 14.10 Soybean oil 10.6 10.6 10.6 10.6
10.6 Coconut oil 10.1 10.1 10.1 10.1 10.1 2' fucosyllactose (2'FL)
0.1896 0.1801 0.1706 0.1991 0.2086 Galactooligosaccharides (GOS)
8.630 8.630 8.630 8.630 8.630 Whey protein concentrate 6.40 6.40
6.40 6.40 6.40 Potassium citrate 478.9 g 478.9 g 478.9 g 478.9 g
478.9 g Calcium carbonate 448.28 g 448.28 g 448.28 g 448.28 g
448.28 g Soy lecithin 355.74 g 355.74 g 355.74 g 355.74 g 355.74 g
Stabilizer 355.74 g 355.74 g 355.74 g 355.74 g 355.74 g ARA oil
368.01 g 368.01 g 368.01 g 368.01 g 368.01 g Nucleotide/chloride
premix 293.26 g 293.26 g 293.26 g 293.26 g 293.26 g Potassium
chloride 226.45 g 226.45 g 226.45 g 226.45 g 226.45 g Ascorbic acid
445.94 g 445.94 g 445.94 g 445.94 g 445.94 g Vitamin mineral premix
142.88 g 142.88 g 142.88 g 142.88 g 142.88 g DHA oil 137.8 g 137.8
g 137.8 g 137.8 g 137.8 g Carrageenan 180.0 g 180.0 g 180.0 g 180.0
g 180.0 g Magnesium chloride 55.0 g 55.0 g 55.0 g 55.0 g 55.0 g
Ferrous sulfate 58.0 g 58.0 g 58.0 g 58.0 g 58.0 g Choline chloride
53.9 g 53.9 g 53.9 g 53.9 g 53.9 g Vitamin A, D.sub.3, E, K.sub.1
premix 47.40 g 47.40 g 47.40 g 47.40 g 47.40 g Citric acid 29.77 g
29.77 g 29.77 g 29.77 g 29.77 g Probiotic 1.0 1.0 1.0 1.0 1.0 Mixed
carotenoid premix 26.40 g 26.40 g 26.40 g 26.40 g 26.40 g Sodium
chloride AN AN AN AN AN L-carnitine 3.31 g 3.31 g 3.31 g 3.31 g
3.31 g Tricalcium phosphate 15.65 g 15.65 g 15.65 g 15.65 g 15.65 g
Potassium phosphate monobasic 13.67 g 13.67 g 13.67 g 13.67 g 13.67
g Riboflavin 2.42 g 2.42 g 2.42 g 2.42 g 2.42 g Potassium hydroxide
AN AN AN AN AN AN = as needed
Examples 6-10
[0181] Examples 6-10 illustrate ready-to-feed nutritional emulsions
of the present disclosure, the ingredients of which are listed in
the table below. All ingredient amounts are listed as kilogram per
1000 kilogram batch of product, unless otherwise specified.
TABLE-US-00005 Ingredient Ex. 6 Ex. 7 Ex. 8 Ex. 9 Ex. 10 Water Q.S.
Q.S. Q.S. Q.S. Q.S. Condensed Skim Milk 86.64 86.64 86.64 86.64
86.64 Lactose 54.80 54.80 54.80 54.80 54.80 High oleic safflower
oil 14.10 14.10 14.10 14.10 14.10 Soybean oil 10.6 10.6 10.6 10.6
10.6 Coconut oil 10.1 10.1 10.1 10.1 10.1 2' fucosyllactose (2'FL)
0.0182 0.18 4.5455 1.818 18.0 Galactooligosaccharides (GOS) 0.1818
0.02 4.5455 18.182 2.0 Whey protein concentrate 6.40 6.40 6.40 6.40
6.40 Potassium citrate 478.9 g 478.9 g 478.9 g 478.9 g 478.9 g
Calcium carbonate 448.28 g 448.28 g 448.28 g 448.28 g 448.28 g Soy
lecithin 355.74 g 355.74 g 355.74 g 355.74 g 355.74 g Stabilizer
355.74 g 355.74 g 355.74 g 355.74 g 355.74 g ARA oil 368.01 g
368.01 g 368.01 g 368.01 g 368.01 g Nucleotide/chloride premix
293.26 g 293.26 g 293.26 g 293.26 g 293.26 g Potassium chloride
226.45 g 226.45 g 226.45 g 226.45 g 226.45 g Ascorbic acid 445.94 g
445.94 g 445.94 g 445.94 g 445.94 g Vitamin mineral premix 142.88 g
142.88 g 142.88 g 142.88 g 142.88 g DHA oil 137.8 g 137.8 g 137.8 g
137.8 g 137.8 g Carrageenan 180.0 g 180.0 g 180.0 g 180.0 g 180.0 g
Magnesium chloride 55.0 g 55.0 g 55.0 g 55.0 g 55.0 g Ferrous
sulfate 58.0 g 58.0 g 58.0 g 58.0 g 58.0 g Choline chloride 53.9 g
53.9 g 53.9 g 53.9 g 53.9 g Vitamin A, D.sub.3, E, K.sub.1 premix
47.40 g 47.40 g 47.40 g 47.40 g 47.40 g Citric acid 29.77 g 29.77 g
29.77 g 29.77 g 29.77 g Probiotic 1.0 1.0 1.0 1.0 1.0 Mixed
carotenoid premix 26.40 g 26.40 g 26.40 g 26.40 g 26.40 g Sodium
chloride AN AN AN AN AN L-carnitine 3.31 g 3.31 g 3.31 g 3.31 g
3.31 g Tricalcium phosphate 15.65 g 15.65 g 15.65 g 15.65 g 15.65 g
Potassium phosphate monobasic 13.67 g 13.67 g 13.67 g 13.67 g 13.67
g Riboflavin 2.42 g 2.42 g 2.42 g 2.42 g 2.42 g Potassium hydroxide
AN AN AN AN AN AN = as needed
Examples 11-15
[0182] Examples 11-15 illustrate ready-to-feed nutritional
emulsions of the present disclosure, the ingredients of which are
listed in the table below. All ingredient amounts are listed as
kilogram per 1000 kilogram batch of product, unless otherwise
specified.
TABLE-US-00006 Ingredient Ex. 11 Ex. 12 Ex. 13 Ex. 14 Ex. 15 Water
Q.S. Q.S. Q.S. Q.S. Q.S. Condensed Skim Milk 86.64 86.64 86.64
86.64 86.64 Lactose 54.80 54.80 54.80 54.80 54.80 High oleic
safflower oil 14.10 14.10 14.10 14.10 14.10 Soybean oil 10.6 10.6
10.6 10.6 10.6 Coconut oil 10.1 10.1 10.1 10.1 10.1 2'
fucosyllactose (2'FL) 0.0948 0.09005 0.0853 0.0995 0.1043
Lacto-N-neotetraose (LNnT) 0.0948 0.09005 0.0853 0.0995 0.1043
Galactooligosaccharides (GOS) 8.630 8.630 8.630 8.630 8.630 Whey
protein concentrate 6.40 6.40 6.40 6.40 6.40 Potassium citrate
478.9 g 478.9 g 478.9 g 478.9 g 478.9 g Calcium carbonate 448.28 g
448.28 g 448.28 g 448.28 g 448.28 g Soy lecithin 355.74 g 355.74 g
355.74 g 355.74 g 355.74 g Stabilizer 355.74 g 355.74 g 355.74 g
355.74 g 355.74 g ARA oil 368.01 g 368.01 g 368.01 g 368.01 g
368.01 g Nucleotide/chloride premix 293.26 g 293.26 g 293.26 g
293.26 g 293.26 g Potassium chloride 226.45 g 226.45 g 226.45 g
226.45 g 226.45 g Ascorbic acid 445.94 g 445.94 g 445.94 g 445.94 g
445.94 g Vitamin mineral premix 142.88 g 142.88 g 142.88 g 142.88 g
142.88 g DHA oil 137.8 g 137.8 g 137.8 g 137.8 g 137.8 g
Carrageenan 180.0 g 180.0 g 180.0 g 180.0 g 180.0 g Magnesium
chloride 55.0 g 55.0 g 55.0 g 55.0 g 55.0 g Ferrous sulfate 58.0 g
58.0 g 58.0 g 58.0 g 58.0 g Choline chloride 53.9 g 53.9 g 53.9 g
53.9 g 53.9 g Vitamin A, D.sub.3, E, K.sub.1 premix 47.40 g 47.40 g
47.40 g 47.40 g 47.40 g Citric acid 29.77 g 29.77 g 29.77 g 29.77 g
29.77 g Probiotic 1.0 0.95 0.90 1.05 1.10 Mixed carotenoid premix
26.40 g 26.40 g 26.40 g 26.40 g 26.40 g Sodium chloride AN AN AN AN
AN L-carnitine 3.31 g 3.31 g 3.31 g 3.31 g 3.31 g Tricalcium
phosphate 15.65 g 15.65 g 15.65 g 15.65 g 15.65 g Potassium
phosphate monobasic 13.67 g 13.67 g 13.67 g 13.67 g 13.67 g
Riboflavin 2.42 g 2.42 g 2.42 g 2.42 g 2.42 g Potassium hydroxide
AN AN AN AN AN AN = as needed
Examples 16-20
[0183] Examples 16-20 illustrate concentrated liquid emulsions of
the present disclosure, the ingredients of which are listed in the
table below. All ingredient amounts are listed as kilogram per 1000
kilogram batch of product, unless otherwise specified.
TABLE-US-00007 Ingredient Ex. 16 Ex. 17 Ex. 18 Ex. 19 Ex. 20 Water
Q.S. Q.S. Q.S. Q.S. Q.S. Condensed Skim Milk 166.6 166.6 166.6
166.6 166.6 Lactose 106.1 106.1 106.1 106.1 106.1 High oleic
safflower oil 27.16 27.16 27.16 27.16 27.16 Soybean oil 20.42 20.42
20.42 20.42 20.42 Coconut oil 19.48 19.48 19.48 19.48 19.48 2'
fucosyllactose (2'FL) 0.1896 0.1188 0.0853 0.2414 0.2560
Galactooligosaccharides (GOS) 16.71 16.71 16.71 16.71 16.71 Whey
protein concentrate 12.20 12.20 12.20 12.20 12.20 Potassium citrate
894.5 g 894.5 g 894.5 g 894.5 g 894.5 g Calcium carbonate 1.072
1.072 1.072 1.072 1.072 Monoglycerides 690.0 g 690.0 g 690.0 g
690.0 g 690.0 g Soy lecithin 690.0 g 690.0 g 690.0 g 690.0 g 690.0
g ARA oil 684.2 g 684.2 g 684.2 g 684.2 g 684.2 g
Nucleotide/chloride premix 568.9 g 568.9 g 568.9 g 568.9 g 568.9 g
Potassium chloride 480.8 g 480.8 g 480.8 g 480.8 g 480.8 g Ascorbic
acid 958.6 g 958.6 g 958.6 g 958.6 g 958.6 g Vitamin mineral premix
276.9 g 276.9 g 276.9 g 276.9 g 276.9 g DHA oil 256.1 g 256.1 g
256.1 g 256.1 g 256.1 g Carrageenan 200.0 g 200.0 g 200.0 g 200.0 g
200.0 g Magnesium chloride 174.7 g 174.7 g 174.7 g 174.7 g 174.7 g
Ferrous sulfate 112.7 g 112.7 g 112.7 g 112.7 g 112.7 g Choline
chloride 104.8 g 104.8 g 104.8 g 104.8 g 104.8 g Vitamin A,
D.sub.3, E, K.sub.1 premix 86.90 g 86.90 g 86.90 g 86.90 g 86.90 g
Citric acid 64.55 g 64.55 g 64.55 g 64.55 g 64.55 g Mixed
carotenoid premix 45.63 g 45.63 g 45.63 g 45.63 g 45.63 g Sodium
chloride AN AN AN AN AN L-carnitine 6.371 g 6.371 g 6.371 g 6.371 g
6.371 g Riboflavin 2.921 g 2.921 g 2.921 g 2.921 g 2.921 g Vitamin
A Palmitate 1.504 g 1.504 g 1.504 g 1.504 g 1.504 g Potassium
hydroxide 659.8 g 659.8 g 659.8 g 659.8 g 659.8 g Tricalcium
phosphate AN AN AN AN AN Potassium phosphate monobasic AN AN AN AN
AN AN = as needed
Examples 21-25
[0184] Examples 21-25 illustrate spray dried nutritional powders of
the present disclosure, the ingredients of which are listed in the
table below. All ingredient amounts are listed as kilogram per 1000
kilogram batch of product, unless otherwise specified.
TABLE-US-00008 Ingredient Ex. 21 Ex. 22 Ex. 23 Ex. 24 Ex. 25 Nonfat
dry milk 456.9 456.9 456.9 456.9 456.9 Lactose 259.0 259.0 259.0
259.0 259.0 High oleic sunflower oil 93.9 93.9 93.9 93.9 93.9 Soy
oil 70.4 70.4 70.4 70.4 70.4 Coconut oil 67.1 67.1 67.1 67.1 67.1
2' fucosyllactose (2'FL) 0.7584 0.7204 0.6824 0.7964 0.8344
Galactooligosaccharide (GOS) 53.5 53.5 53.5 53.5 53.5 Probiotic 1.0
0.95 0.90 1.05 1.10 Flavoring agent 6.2 6.2 6.2 6.2 6.2 Calcium
carbonate 4.8 4.8 4.8 4.8 4.8 Potassium citrate 4.7 4.7 4.7 4.7 4.7
Oligofructose 2.9 2.9 2.9 2.9 2.9 Ascorbic acid 2.0 2.0 2.0 2.0 2.0
Nucleotide/Choline Premix 1.8 1.8 1.8 1.8 1.8 ARA oil 1.8 1.8 1.8
1.8 1.8 Vitamin/Trace Mineral Premix 1.5 1.5 1.5 1.5 1.5 Sodium
chloride 1.3 1.3 1.3 1.3 1.3 Lecithin 1.2 1.2 1.2 1.2 1.2 Sodium
citrate 982.2 g 982.2 g 982.2 g 982.2 g 982.2 g DHA oil 882.1 g
882.1 g 882.1 g 882.1 g 882.1 g Magnesium chloride 477.4 g 477.4 g
477.4 g 477.4 g 477.4 g Vitamin A, D3, E, K1 Premix 314.7 g 314.7 g
314.7 g 314.7 g 314.7 g Ascorbyl Palmitate 278.8 g 278.8 g 278.8 g
278.8 g 278.8 g Antioxidant 137.3 g 137.3 g 137.3 g 137.3 g 137.3 g
Tocopheryl acetate 32.0 g 32.0 g 32.0 g 32.0 g 32.0 g Beta-carotene
30% 11.0 g 11.0 g 11.0 g 11.0 g 11.0 g Potassium iodide 2.5 g 2.5 g
2.5 g 2.5 g 2.5 g Riboflavin 2.0 g 2.0 g 2.0 g 2.0 g 2.0 g
Magnesium sulfate .sup. 499.5 mg .sup. 499.5 mg .sup. 499.5 mg
.sup. 499.5 mg .sup. 499.5 mg Potassium phosphate dibasic AN AN AN
AN AN Potassium chloride AN AN AN AN AN Tricalcium phosphate AN AN
AN AN AN Potassium hydroxide AN AN AN AN AN Calcium hydroxide AN AN
AN AN AN Sodium hydroxide AN AN AN AN AN Water Q.S. Q.S. Q.S. Q.S.
Q.S. AN = as needed
Examples 26-30
[0185] Examples 26-30 illustrate spray dried nutritional powders of
the present disclosure, the ingredients of which are listed in the
table below. All ingredient amounts are listed as kilogram per 1000
kilogram batch of product, unless otherwise specified.
TABLE-US-00009 Ingredient Ex. 26 Ex. 27 Ex. 28 Ex. 29 Ex. 30 Water
Q.S. Q.S. Q.S. Q.S. Q.S. Corn syrup 308.9 308.9 308.9 308.9 308.9
Maltodextrin 297.1 297.1 297.1 297.1 297.1 Sucrose 112.4 112.4
112.4 112.4 112.4 High Oleic sunflower oil 84.9 84.9 84.9 84.9 84.9
Sodium caseinate 73.0 73.0 73.0 73.0 73.0 Calcium caseinate 50.2
50.2 50.2 50.2 50.2 2' fucosyllactose (2'FL) 0.7584 0.7204 0.6824
0.7964 0.8344 Inulin, oligofructose 47.0 47.0 47.0 47.0 47.0 Soy
oil 38.3 38.3 38.3 38.3 38.3 Isolated soy protein 35.9 35.9 35.9
35.9 35.9 Milk protein isolate 16.3 16.3 16.3 16.3 16.3 Canola oil
13.7 13.7 13.7 13.7 13.7 Sodium citrate 9.8 9.8 9.8 9.8 9.8
Potassium citrate 9.7 9.7 9.7 9.7 9.7 Tricalcium phosphate 9.0 9.0
9.0 9.0 9.0 Flavoring agent 7.3 7.3 7.3 7.3 7.3 Magnesium chloride
6.2 6.2 6.2 6.2 6.2 Potassium chloride 5.5 5.5 5.5 5.5 5.5 Choline
chloride 1.7 1.7 1.7 1.7 1.7 Vitamin premix 950.0 g 950.0 g 950.0 g
950.0 g 950.0 g Ascorbic acid 755.0 g 755.0 g 755.0 g 755.0 g 755.0
g Vitamin/trace mineral premix 465.0 g 465.0 g 465.0 g 465.0 g
465.0 g Potassium hydroxide 215.9 g 215.9 g 215.9 g 215.9 g 215.9 g
Potassium phosphate dibasic 185.8 g 185.8 g 185.8 g 185.8 g 185.8 g
Ascorbyl palmitate 164.7 g 164.7 g 164.7 g 164.7 g 164.7 g
Antioxidant 82.3 g 82.3 g 82.3 g 82.3 g 82.3 g Vitamin A, D3, E, K1
premix 82.3 g 82.3 g 82.3 g 82.3 g 82.3 g Vitamin A palmitate 16.5
g 16.5 g 16.5 g 16.5 g 16.5 g Ferrous sulfate 12.0 g 12.0 g 12.0 g
12.0 g 12.0 g Beta carotene 30% 5.5 g 5.5 g 5.5 g 5.5 g 5.5 g
Vitamin D3 oil 1.0 g 1.0 g 1.0 g 1.0 g 1.0 g Potassium iodide .sup.
800.0 mg .sup. 800.0 mg .sup. 800.0 mg .sup. 800.0 mg .sup. 800.0
mg Citric acid AN AN AN AN AN Potassium hydroxide 40% AN AN AN AN
AN Maltodextrin AN AN AN AN AN Magnesium sulfate AN AN AN AN AN
Sodium chloride AN AN AN AN AN Calcium carbonate AN AN AN AN AN AN
= as needed
Examples 31-35
[0186] Examples 31-35 illustrate ready-to-feed nutritional
emulsions of the present disclosure, the ingredients of which are
listed in the table below. All ingredient amounts are listed as
kilogram per 1000 kilogram batch of product, unless otherwise
specified.
TABLE-US-00010 Ingredient Ex. 31 Ex. 32 Ex. 33 Ex. 34 Ex. 35 Water
Q.S. Q.S. Q.S. Q.S. Q.S. Condensed Skim Milk 86.64 86.64 86.64
86.64 86.64 Lactose 54.80 54.80 54.80 54.80 54.80 High oleic
safflower oil 14.10 14.10 14.10 14.10 14.10 Soybean oil 10.6 10.6
10.6 10.6 10.6 Coconut oil 10.1 10.1 10.1 10.1 10.1 2'
fucosyllactose (2'FL) 0.0948 0.09005 0.0853 0.0995 0.1043
6'-sialyllactose (6'SL) 0.0948 0.09005 0.0853 0.0995 0.1043
Galactooligosaccharides (GOS) 8.630 8.630 8.630 8.630 8.630 Whey
protein concentrate 6.40 6.40 6.40 6.40 6.40 Potassium citrate
478.9 g 478.9 g 478.9 g 478.9 g 478.9 g Calcium carbonate 448.28 g
448.28 g 448.28 g 448.28 g 448.28 g Soy lecithin 355.74 g 355.74 g
355.74 g 355.74 g 355.74 g Stabilizer 355.74 g 355.74 g 355.74 g
355.74 g 355.74 g ARA oil 368.01 g 368.01 g 368.01 g 368.01 g
368.01 g Nucleotide/chloride premix 293.26 g 293.26 g 293.26 g
293.26 g 293.26 g Potassium chloride 226.45 g 226.45 g 226.45 g
226.45 g 226.45 g Ascorbic acid 445.94 g 445.94 g 445.94 g 445.94 g
445.94 g Vitamin mineral premix 142.88 g 142.88 g 142.88 g 142.88 g
142.88 g DHA oil 137.8 g 137.8 g 137.8 g 137.8 g 137.8 g
Carrageenan 180.0 g 180.0 g 180.0 g 180.0 g 180.0 g Magnesium
chloride 55.0 g 55.0 g 55.0 g 55.0 g 55.0 g Ferrous sulfate 58.0 g
58.0 g 58.0 g 58.0 g 58.0 g Choline chloride 53.9 g 53.9 g 53.9 g
53.9 g 53.9 g Vitamin A, D.sub.3, E, K.sub.1 premix 47.40 g 47.40 g
47.40 g 47.40 g 47.40 g Citric acid 29.77 g 29.77 g 29.77 g 29.77 g
29.77 g Probiotic 1.0 0.95 0.90 1.05 1.10 Mixed carotenoid premix
26.40 g 26.40 g 26.40 g 26.40 g 26.40 g Sodium chloride AN AN AN AN
AN L-carnitine 3.31 g 3.31 g 3.31 g 3.31 g 3.31 g Tricalcium
phosphate 15.65 g 15.65 g 15.65 g 15.65 g 15.65 g Potassium
phosphate monobasic 13.67 g 13.67 g 13.67 g 13.67 g 13.67 g
Riboflavin 2.42 g 2.42 g 2.42 g 2.42 g 2.42 g Potassium hydroxide
AN AN AN AN AN AN = as needed
Examples 36-39
[0187] Examples 36-39 illustrate concentrated liquid human milk
fortifiers of the present disclosure, the ingredients of which are
listed in the table below. All ingredient amounts are listed as
kilogram per 1000 kilogram batch of product, unless otherwise
specified.
TABLE-US-00011 Ingredient (Per 1000 Kg) Ex. 36 Ex. 37 Ex. 38 Ex. 39
Water Q.S. Q.S. Q.S. Q.S. Casein Hydrolysate 108 108 125 150
Maltodextrin 104 104 104 104 MCT Oil 17.3 17.3 17.3 17.3 Tricalcium
Phosphate 16.0 16.0 16.0 16.0 Soy Oil 10.4 10.4 10.4 10.4 6'
sialyllactose (6'SL) 0.0948 0.09005 0.0853 0.0995
Lacto-N-neotetraose (LNnT) 0.0948 0.09005 0.0853 0.0995
Galactooligosaccharides 6.7704 6.7704 6.7704 6.7704 (GOS) Gum
Arabic 12.0 10.0 15.0 2.031 Starch 12.0 10.0 35.0 6.0 Coconut Oil
6.3 6.3 6.3 6.3 Potassium Citrate 6.9 6.9 6.9 6.9 Ascorbic Acid 2.9
2.9 2.9 2.9 Magnesium Chloride 4.0 4.0 4.0 4.0 ARA oil 2.6 2.6 2.6
2.6 Leucine 1.8 1.8 1.8 1.8 DHA oil 2.1 2.1 2.1 2.1 Potassium
Chloride 1.1 1.1 1.1 1.1 Tyrosine 1.4 1.4 1.4 1.4 Monoglycerides
800 g 800 g 800 g 800 g Mixed Carotenoid Premix 551 g 551 g 551 g
551 g M-Inositol 529 g 529 g 529 g 529 g Sodium Chloride 861 g 861
g 861 g 861 g L-Carnitine 221 g 221 g 221 g 221 g Tryptophan 331 g
331 g 331 g 331 g Zinc Sulfate 309 g 309 g 309 g 309 g Niacinamide
320 g 320 g 320 g 320 g Tocopheryl Acetate 364 g 364 g 364 g 364 g
Gellan Gum 200 g 300 g 400 g 600 g Ferrous Sulfate 106 g 106 g 106
g 106 g Choline Chloride 353 g 353 g 353 g 353 g Calcium
Pantothenate 132 g 132 g 132 g 132 g Vitamin A Palmitate 77 g 77 g
77 g 77 g Riboflavin 33 g 33 g 33 g 33 g Vitamin D3 13 g 13 g 13 g
13 g Copper Sulfate 18 g 18 g 18 g 18 g Pyridoxine Hydrochloride 20
g 20 g 20 g 20 g Thiamin Hydrochloride 24 g 24 g 24 g 24 g Folic
Acid 3.3 g 3.3 g 3.3 g 3.3 g Biotin 2.5 g 2.5 g 2.5 g 2.5 g
Manganese Sulfate 1.8 g 1.8 g 1.8 g 1.8 g Phylloquinone 880 mg 880
mg 880 mg 880 mg Sodium Selenate 90 mg 90 mg 90 mg 90 mg
Cyanocobalamin 88 mg 88 mg 88 mg 88 mg Potassium Hydroxide Q.S.
Q.S. Q.S. Q.S.
Examples 40-43
[0188] Examples 40-43 illustrate concentrated liquid human milk
fortifiers of the present disclosure, the ingredients of which are
listed in the table below. All ingredient amounts are listed as
kilogram per 1000 kilogram batch of product, unless otherwise
specified.
TABLE-US-00012 Ingredient (Per 1000 Kg) Ex. 40 Ex. 41 Ex. 42 Ex. 43
Water Q.S. Q.S. Q.S. Q.S. Casein Hydrolysate 108 108 125 150
Maltodextrin 104 104 104 104 MCT Oil 17.3 17.3 17.3 17.3 Tricalcium
Phosphate 16.0 16.0 16.0 16.0 Soy Oil 10.4 10.4 10.4 10.4 2'
fucosyllactose (2'FL) 0.0948 0.09005 0.0853 0.0995
Lacto-N-neotetraose (LNnT) 0.0948 0.09005 0.0853 0.0995
Galactooligosaccharides 6.7704 6.7704 6.7704 6.7704 (GOS) Gum
Arabic 12.0 10.0 15.0 2.031 Starch 12.0 10.0 35.0 6.0 Coconut Oil
6.3 6.3 6.3 6.3 Potassium Citrate 6.9 6.9 6.9 6.9 Ascorbic Acid 2.9
2.9 2.9 2.9 Magnesium Chloride 4.0 4.0 4.0 4.0 ARA oil 2.6 2.6 2.6
2.6 Leucine 1.8 1.8 1.8 1.8 DHA oil 2.1 2.1 2.1 2.1 Potassium
Chloride 1.1 1.1 1.1 1.1 Tyrosine 1.4 1.4 1.4 1.4 Monoglycerides
800 g 800 g 800 g 800 g Mixed Carotenoid Premix 551 g 551 g 551 g
551 g M-Inositol 529 g 529 g 529 g 529 g Sodium Chloride 861 g 861
g 861 g 861 g L-Carnitine 221 g 221 g 221 g 221 g Tryptophan 331 g
331 g 331 g 331 g Zinc Sulfate 309 g 309 g 309 g 309 g Niacinamide
320 g 320 g 320 g 320 g Tocopheryl Acetate 364 g 364 g 364 g 364 g
Gellan Gum 200 g 300 g 400 g 600 g Ferrous Sulfate 106 g 106 g 106
g 106 g Choline Chloride 353 g 353 g 353 g 353 g Calcium
Pantothenate 132 g 132 g 132 g 132 g Vitamin A Palmitate 77 g 77 g
77 g 77 g Riboflavin 33 g 33 g 33 g 33 g Vitamin D3 13 g 13 g 13 g
13 g Copper Sulfate 18 g 18 g 18 g 18 g Pyridoxine Hydrochloride 20
g 20 g 20 g 20 g Thiamin Hydrochloride 24 g 24 g 24 g 24 g Folic
Acid 3.3 g 3.3 g 3.3 g 3.3 g Biotin 2.5 g 2.5 g 2.5 g 2.5 g
Manganese Sulfate 1.8 g 1.8 g 1.8 g 1.8 g Phylloquinone 880 mg 880
mg 880 mg 880 mg Sodium Selenate 90 mg 90 mg 90 mg 90 mg
Cyanocobalamin 88 mg 88 mg 88 mg 88 mg Potassium Hydroxide Q.S.
Q.S. Q.S. Q.S.
Example 44
[0189] In this Example, the effect of 2'-fucosyllactose (2'FL) and
3'-fucosyllactose (3'FL) on stimulating enteric nerve cells in the
gastrointestinal tract of rodents is analyzed.
[0190] A peristalsis model using luminally perfused mouse colon is
used to test the stimulation effect of 2'FL and 3'FL on enteric
nerve cells. Colon muscle is perfused with 2'FL or 3'FL, at
concentrations of 1 mg/mL, 0.5 mg/mL, and 0.1 mg/mL, for 15
minutes. The frequency and amplitude of contractions of the muscle
are analyzed. The results are shown in FIG. 1.
[0191] As shown in FIG. 1, there is a direct stimulation of nerve
cells by 2'FL or 3'FL without involving gut microbiota and/or their
metabolites. Specifically, the frequency and amplitude of
contraction are reduced consistently and in a dose response
fashion. Further, the data show that 3'FL is more effective than
2'FL in reducing the frequency and amplitude of contraction at a
level of 0.5 mg/mL.
Example 45
[0192] In this Example, the fermentation rates of several
oligosaccharide substrates are measured in an in vitro model using
infant feces. Additionally, the levels of various bacteria species
in the presence of the oligosaccharide substrates are measured
using quantitative polymerase chain reactions to determine whether
the substrates act in a prebiotic manner to facilitate the growth
of beneficial bacteria and possibly retard the growth of harmful
bacteria.
[0193] Eight infant participants for feces donation were selected
based on the following criteria: whether the infant: (1) was full
term at birth with a gestational age of 38 to 42 weeks; (2) was at
or above the fifth percentile for weight at birth; (3) has no
maternal medical history of diabetes, tuberculosis, or perinatal
infection with proven adverse effects on the fetus; (4) was a
vaginal birth; (5) was at least 2 months of age at study entry, but
not older than 4 months of age; (6) has no known cardiac,
respiratory, gastrointestinal, or other systemic disease such as
urinary tract infection or otitis media; (7) is free of history of
blood group incompatibility serious enough to result in
hematological problems; and (8) is not receiving any medications
(except for supplemental vitamins) and has never received
antibiotics. The eight infants are allowed to consume their normal
diet of breast milk or infant formula. Four infants are exclusively
breast fed and four infants are exclusively formula fed one of four
commercially available infant formulas.
[0194] On the day of the in vitro experiments, a fecal sample is
collected in the diaper and prepped within 15 min of defecation.
For prepping, the sample is placed in a container with tepid water
and analyzed. Fecal samples are diluted 1:10 (wt/vol) in anaerobic
dilution solution prepared by blending the solution for 15 seconds
in a blender under a stream of CO.sub.2. Blended, diluted feces are
filtered through four layers of cheesecloth and sealed in 125-mL
serum bottles under CO.sub.2. Inoculum is stored at 37.degree. C.
until inoculation of in vitro tubes.
[0195] Oligosaccharide test substrates evaluated for fermentation
and growing of bacterium include (1) galactooligosaccharides 95
(GOS; Inalco Pharmaceuticals, San Luis, Calif.); (2)
.alpha.-(2-6')-N-Acetylneuraminyl-lactose sodium salt (6'SL; Inalco
Pharmaceuticals, San Luis, Calif.); (3)
2'-.alpha.-L-Fucopyranosyl-D-Lactose (2'FL; Inalco group, Italy);
(4) Lacto-N-neotetraose (LNnT; Inalco Pharmaceuticals, San Luis,
Calif.); (5) Orafti.RTM. HP inulin (HP inulin; BENEO-Orafti,
Belgium); and (6) gum arabic (Fisher Scientific, Pittsburgh,
Pa.).
In Vitro Substrate Fermentation Model
[0196] Approximately 80 mg of each test substrate (1)-(6) is
weighed in triplicate into 16-mL Balch tubes that are used in a
conventional model that simulates large bowel fermentation. An
aliquot (7.2 mL) of medium (Table 1; FIG. 2) is aseptically
transferred into the Balch tubes, capped with butyl rubber
stoppers, and sealed with aluminum caps. Tubes containing HP inulin
and gum arabic are stored at 4.degree. C. for approximately 12 h to
enable hydration of the substrates before initiating fermentation.
These tubes are placed in a 37.degree. C. water bath approximately
30 min before inoculation. Tubes containing GOS, 6'SL, 2'FL, and
LNnT are hydrated upon obtaining a fecal sample and placed in a
37.degree. C. water bath until inoculation.
[0197] Sample and blank tubes are aseptically inoculated with 0.8
ml of diluted feces. Tubes are incubated at 37.degree. C. with
periodic mixing every 2 h for up to 12 h. At 0, 3, 6, and 12 h
after inoculation, tubes are removed from the 37.degree. C.
incubator and processed immediately for analyses. The pH of the
tube contents is measured with a standard pH meter (Denver
Instrument Co., Arvada, Colo.). A 3-ml subsample of fluid is
collected and used for short-chain fatty acid and lactate analyses,
all of which are individual indicators of fermentation as described
further below. A 2-mL subsample is taken and frozen at -80.degree.
C. for bacterial analyses.
Short-Chain Fatty Acid (SCFA) and Lactate Analyses
[0198] SCFA Analysis: measurement of total SCFA production over
time indicates how quickly the substrate is fermented. The
measurement of the concentration of individual SCFAs (acetate,
propionate, and butyrate) allows for the calculation of ratios of
the various SCFAs, which allows determination of whether the
various ratios (and specifically the proportions of acetate and
lactate versus other organic acids) is similar to that of breast
milk, which may be desirable.
[0199] Lactate Analysis: provides an indication of two things: (1)
it is an indirect indicator of the rate of fermentation; and (2) it
is suggestive that bifidobacteria and/or lactobacilli are present
in significant numbers because both genera characteristically
produce large amounts of lactate.
[0200] The 3-mL aliquot of fluid removed from the sample tubes for
SCFA and lactate analyses is immediately added to 0.75 mL of 25%
metaphosphoric acid. Concentrations of acetate, propionate, and
butyrate are determined using a Hewlett-Packard 5890A series II gas
chromatograph (Palo Alto, Calif.) and a glass column (180
cm.times.4 mm i.d.) packed with 10% SP-1200/1% H.sub.3PO.sub.4 on
80/100+ mesh Chromosorb WAW (Supelco Inc., Bellefonte, Pa.). Oven
temperature, detector temperature, and injector temperature are
125, 175, and 180.degree. C., respectively. The supernatants are
analyzed for lactate concentration by a spectrophotometric method.
SCFA and lactate concentration values are corrected for blank tube
production of SCFA and 0 h concentrations for each substrate. Total
SCFA are calculated as the total amount of acetate, propionate, and
butyrate.
Quantitative Polymerase Chain Reaction
[0201] The 2-ml subsample of the in vitro material at each time
point is used for determination of bacterial species. Two tubes
from each substrate at each time point are processed. Genomic DNA
is extracted and isolated using a repeated bead beating plus column
(RBB+C) method. Escherichia coli, Bifidobacterium spp.,
Lactobacillus spp., and Clostridum perfringens are quantified via
qPCR using specific primers. DNA from each serial dilution is
amplified along with in vitro DNA samples using a Taqman ABI PRISM
7900HT Sequence Detection System (Applied BioSystems, Foster City,
Calif.) and colony forming units, based on the standard curves, are
determined as described. Due to the small concentrations of DNA
extracted, only 2 ng of DNA is amplified during qPCR. Bacterial
population values are corrected for blank tube production and 0 h
values for each substrate.
[0202] Data is analyzed as a split-split-plot in a completely
randomized block design using the Mixed procedure of SAS (SAS
Inst., Inc., Cary, N.C.). Block is defined as the diet of the baby
(breast milk or formula). Fixed effects tested include diet
(formula fed or breast fed), substrate ((1)-(6)), and time, and the
interactions are investigated if significant. Infant, period, and
the interaction of infant and substrate are included as random
effects in the model. Means are separated using a protected LSD
with a Tukey adjustment to control for experiment-wise error. Least
square means are reported along with the pooled SEM for all
response criteria. A probability of P<0.05 is accepted as
statistically significant.
Substrates
[0203] Substrates are analyzed for dry matter, organic matter, and
free and hydrolyzed monosaccharide concentrations. HMOs are
quantified using pure standards (V-Labs, Inc., Covington, La.), and
all other compounds are quantified using standards of sugars and
monosaccharides. Chemical composition of the sugars is provided in
Table 2 (FIG. 3). Dry matter is similar among substrates, except
GOS, which is a syrup and, therefore, has a lower dry matter
concentration. As expected, only the HMO substrates contain any
milk oligosaccharides.
Results and Discussion
pH and Fermentative End-Products
[0204] The interaction of diet by time by substrate for pH tends to
be significant (P=0.07); however, there are minor alterations in
the change of pH at various hours, which are likely not
biologically significant, with the largest difference between
breast fed and formula fed infants at any time point for any
substrate being less than a one pH unit change. pH change from
baseline decreases (P=0.005) more in formula fed infants versus
breast fed infants (FIG. 4), and this is driven by the lower pH
change at 6 h (P=0.03) and 12 h (P=0.07) after inoculation.
Specifically, the more rapid decline in pH for formula fed infants
versus breast fed infants indicates that formula fed infants
ferment non-digestible carbohydrates more rapidly than breast fed
infants and have higher SCFA production than breast fed infants.
Because low pHs may discourage the growth of many enteric
pathogens, it is generally desirable to have lower pH.
[0205] The pH change from baseline decreases (P<0.0001) over
time for all substrates except gum arabic (FIG. 5). At 3, 6, and 12
h after inoculation, pH change from baseline is smallest
(P<0.0001) with the gum arabic substrate, and greatest in the
LNnT, 2'FL, and GOS substrates. A decrease in pH indicates
fermentation is occurring, and these data are reflective of SCFA
and lactate accumulation. The lack of pH decrease for the gum
arabic indicates that fermentation is not occurring.
[0206] The interaction of diet by time by substrate for acetate
production is significant (P=0.03). Evaluation of this interaction
indicates that at 6 h, formula fed infants have greater (P<0.01)
acetate production when paired with the HMO substrates. This same
effect occurs at 12 h, where formula fed infants have greater
(P<0.02) acetate production when paired with 6'SL and LNnT
substrates, but not for 2'FL. Overall, acetate production tends to
be greater (P=0.10) with formula fed infants, and at 6 and 12 h
after inoculation, formula fed infants produce more (P<0.03)
acetate (FIG. 6), which is absorbed by the host for energy.
[0207] Acetate production differs over time (P<0.0001) among
substrates (FIG. 7). Gum arabic does not produce (P=0.88) any
appreciable amounts of acetate after 12 h of fermentation. At 3 and
6 h after fermentation, gum arabic and HP inulin produce the
smallest (P<0.01) amounts of acetate compared to all other
substrates. Acetate production by 6'SL is intermediate, but
different (P<0.01) from all other substrates. 2'FL and LNnT
produce similar amounts of acetate. Acetate production by 2'FL is
lower (P=0.02) than GOS at 3 h, and both 2'FL and LNnT produce less
acetate than GOS at 6 h. After 12 h of fermentation, GOS has the
greatest (P<0.01) acetate production, followed by 6'SL and LNnT,
2'FL, HP inulin, with gum arabic producing no acetate.
[0208] Propionate production within diet (FIG. 8) is affected by
time (P<0.0001), where production is similar between breast fed
and formula fed infants at 0 and 3 h, but is increased after 6 and
12 h (P=0.02, P<0.0001, respectively) of fermentation. This led
to an overall greater (P=0.03) propionate production for formula
fed infants. Additionally, propionate production is different
(P<0.0001) over time among substrates (FIG. 9). This interaction
is due to the large increase (P<0.0001) of propionate from 6'SL
after 12 h of fermentation. Propionate concentration at 12 h is
lowest (P<0.0001) with gum arabic compared to all other
substrates. Overall, 6'SL has greater (P<0.0001) propionate
production than all other substrates. 2'FL, GOS, and LNnT have
greater (P<0.0001) propionate production than gum Arabic.
[0209] Butyrate production differed (P=0.01) over time between
diets (FIG. 10). Formula-fed infants have greater (P=0.03) butyrate
production after 12 h of fermentation compared to breast fed
infants. But overall, butyrate production is not different (P=0.35)
between diets. Butyrate production is similar over time within each
substrate (P=0.73), with a general increase (P<0.0001) in
butyrate over time, except for GOS (FIG. 11). Butyrate production
is not affected by substrate (P=0.42).
[0210] Lactate production is not affected by diet (P=0.73), and the
formula fed and breast fed infants respond the same over time
(P=0.19) (FIG. 12). The substrates are affected differently
(P<0.0001) over time (FIG. 13). Three hours after fermentation,
GOS produces greater (P<0.0001) lactate compared to HP inulin,
6'SL, and gum Arabic with this trend continuing through 12 h.
Lactate production is greater (P<0.0001) with the GOS, 2'FL, and
LNnT substrates as compared to HP inulin, 6'SL, and gum arabic.
This trend is similar at 12 h after fermentation, as GOS, 2'FL, and
LNnT lactate production is greater (P<0.0001) compared to gum
Arabic. There is no lactate accumulation for 6'SL, HP inulin, and
gum Arabic.
[0211] Total SCFA production is affected by diet, time, and
substrate (P=0.01). This interaction is due to the greater (P=0.01)
fermentation by formula fed infants when evaluating 2'FL and 6'SL
after 6 and 12 h of fermentation, HP inulin after 12 h of
fermentation, and LNnT after 3, 6, and 12 h of fermentation. Total
SCFA production is greater (P=0.04) in formula fed infants compared
to breast fed infants (FIG. 14), but this is affected by time, as
this difference is only noted at 6 and 12 h of fermentation (P=0.01
and P=0.002, respectively). Further, the molar ratios of SCFA
produced by formula fed infants more closely resemble that of
adults than the molar ratios of SCFA produced by breast fed
infants. Total SCFA production differs among substrates (FIG. 15)
at 3, 6, and 12 h of fermentation (P<0.0001). Gum arabic
produces the least amount of SCFA and does not change over time.
After 3 and 6 h of fermentation, total SCFA production is lower
(P<0.05) with HP inulin compared to all other substrates and is
lower (P<0.05) with 6'SL compared to GOS. By 12 h of
fermentation, total SCFA production remains lower (P<0.05) with
HP inulin relative to 2'FL, 6'SL, GOS, and LNnT substrates. Also,
after 12 h of fermentation, total SCFA production is greater
(P<0.05) for the 6'SL and GOS substrates compared to 2'FL.
[0212] Overall, from the data it is apparent that the three HMO
substrates (2'FL, 6'SL, and LNnT) and GOS are highly fermentable,
producing mostly acetate and propionate, with acetate being the
most prevalent SCFA and having ratios and production rates similar
to total SCFA. Although some butyrate is produced, none of the
substrates produced significantly more butyrate than the others.
There is, however, a main effect of time indicating that there is
an increase in butyrate production. This is likely due to the
substrates chosen as controls for this in vitro study as GOS and HP
inulin are prebiotics and result in production of butyrate, thereby
not allowing substrate differences to be noted.
Conclusions from Fermentation Analysis
[0213] As shown in the data and Figures discussed above, 2'FL,
6'SL, LNnT, and GOS were readily fermented by infant fecal
bacteria. The fermentation generated primarily acetate and
propionate, although some butyrate was also produced. Specifically,
2'FL, LNnT, and GOS were fermented more rapidly than 6'SL, and
their fermentation generated significant amounts of lactate (both
2'FL and LNnT had fermentation rates similar to GOS). The
fermentation of 6'SL also resulted in substantial amounts of SCFA
at a time period of 12 hours, but little lactate had accumulated in
the media. Finally, infant fecal bacteria appear to have some
ability to ferment HP Inulin, but are incapable of fermenting gum
arabic.
Bacterial Species Analysis
[0214] Lactobacilli populations are greater in formula fed infants
as compared to breast fed infants after 3 and 6 h of fermentation
(P=0.03 and P=0.04, respectively) and tend to be greater (P=0.09)
after 12 h of fermentation (FIG. 16). Lactobacilli populations are
not affected (P=0.83) by substrate (FIG. 17). Bifidobacteria
populations tend to be greater (P=0.09) in formula fed infants as
compared to breast fed infants after 12 h of fermentation (FIG.
18). All the substrates change in the same manner over time, with
an increase in bifidobacteria for each substrate (FIG. 19).
Overall, however, GOS and 2'FL result in greater (P=0.01)
bifidobacteria populations as compared to HP inulin.
[0215] E. coli populations decrease (P<0.0001) over time
regardless of diet. E. coli populations tend to be greater (P=0.06)
in breast fed infants as compared to formula fed infants after 6 h
of fermentation (FIG. 20), but these are still below baseline
values. E. coli populations are not affected (P=0.49) by substrate
(FIG. 21). C. perfringens populations decrease (P=0.04) over time
regardless of diet. While C. perfringens populations are noted to
change differently within time based on diet, there are no
differences (P>0.21) between diets after 3, 6, or 12 h of
fermentation (FIG. 22), C. perfringens populations are not affected
(P=0.57) by substrate, and all substrates responded similarly over
time (FIG. 23).
[0216] Overall, the strongest influence on bacteria is the original
diet of the infant. Formula-fed infants have greater population
growth of the two potentially beneficial bacterial species
(lactobacilli and bifidobacteria), while they result in less growth
of potentially pathogenic species (E. coli and C. perfringens) as
compared to breast fed infants. There are no differences in
bacteria between diets at initial evaluation of the inoculum (at
time 0) for bifidobacteria, lactobacilli, or C. perfringens. E.
coli is, however, greater (P=0.04) in formula fed as compared to
breast fed infants (7.1 log CFU/mL and 6.8 log CFU/mL,
respectively). There are very few changes noted in bacterial
populations due to substrate. It is noted, however, that GOS, a
known prebiotic, and 2'FL, which exerts a bifidogenic effect
similar to that of GOS, lead to greater bifidobacteria, with an
approximate average 0.5 log increase indicating 2'FL's potential
role as a prebiotic in infant formulas and more effectiveness as a
prebiotic than other HMOs. Further, the data suggests that LNnT,
2'FL, and 6'SL are highly fermentable and may also be
bifidogenic.
Conclusions from Bacterial Species Analysis
[0217] As shown in the data and Figures discussed above, LNnT, 2'FL
and 6'SL all tended to generally increase bifidobacteria levels,
indicating that all three of these HMOs may have some prebiotic
effect, although 2'FL was the only HMO to provide a statistically
significant difference is bifidobacteria levels. This indicates
that 2'FL has significant prebiotic properties.
Example 46
[0218] In this Example, probiotic fermentation parameters are
determined for purified HMOs, HMO precursors, and other prebiotic
oligosaccharides.
Bacterial Cultures
[0219] All bifidobacteria strains are initially inoculated from
frozen stocks, grown in deMan Rogosa Sharpe (MRS) broth (Difco,
Detroit, Mich.) supplemented with 0.5 g/L L-cysteine/HCl and
incubated at 37.degree. C. for 24 h in an anaerobic chamber (90%
N.sub.2, 5% CO.sub.2 and 5% H.sub.2; Coy Laboratory Products, Grass
Lake, Mich.). Subsequently, the cultures are passed twice on a
semi-synthetic MRS medium (sMRS)+0.5 g/L L-cysteine which is
supplemented with 1% (w/v) filter-sterilized glucose as the sole
carbohydrate source. After the 2nd pass, cultures are prepared to
use as inoculums for growth assays described below. For
bifidobacteria strains, the same procedure is followed except all
media are supplemented with 0.5 g/l L-cysteine/HCl. All bacterial
strains for use in this Example are listed in the table below.
TABLE-US-00013 TABLE Microorganisms Culture Collection # Number
Genus Species Strain 1 MJM29 Bifidobacterium Adolescentis ATCC
15703 2 MJM30 Bifidobacterium infantis S12; ATCC 15697 3 MJM32
Bifidobacterium animalis subsp. lactis DSM 10140 4 MJM22
Bifidobacterium animalis subsp. ATCC 25527 Animalis 5 MJM34
Bifidobacterium bifidum ATCC 29521 6 MJM35 Bifidobacterium breve
ATCC 15700 7 MJM37 Bifidobacterium bifidum ATCC 11617 8 MJM88
Bifidobacterium lactis Bf-6 (Cargill) 9 MJM92 Bifidobacterium
longum BB536 (Morinaga) 10 MJM93 Bifidobacterium infantis M-63
(Morinaga) 11 MJM94 Bifidobacterium breve M-16V (Morinaga) 12 MJM95
Bifidobacterium lactis Bb12; (Chr. Hansen)
Bacterial Growth Assays
[0220] After the 2nd pass in sMRS+glucose+cysteine, the cultures
are washed once with 10 mL of sterile sMRS+cysteine (no
carbohydrate), resuspended in 10 ml of sterile sMRS+cysteine (no
carbohydrate) and then used as a 1% inoculum. Carbohydrates for use
in this Example are shown in the table below. The carbohydrates are
sterilized with a 0.22 micron filter and used at a 1% final
concentration. Cell growth is performed in 250 .mu.L of
sMRS+cysteine covered with 50 .mu.L of mineral oil in a Bioscreen
100-well Honeycomb plate. Cell growth is monitored by measuring
optical density at 600 nm (OD600) using a Bioscreen C Automated
Microbiology Growth Curve Analysis System. The plate reader is
operated in discontinuous mode, with absorbance readings performed
in 30-minute intervals, and preceded by 30-second shaking intervals
at maximum speed. Controls consist of inoculated medium lacking
carbohydrate. Due to space limitations on the microtitre plate, the
carbohydrates are divided into three separate groups: plate A (HMO
precursors: glucose, galactose, lactose, NAG, fucose, fructose and
sialic acid), plate B (Prebiotics: glucose, Purimune.TM. GOS,
purified Purimune.TM. GOS, Vivinal.RTM. GOS, purified Vivinal.RTM.
GOS, scFOS and PDX), and plate C(HMOs: glucose, 6'-SL, 3'-SL,
2'-FL, 3'-FL and LNnT). All three plates include a positive control
(glucose) and negative control (no carbohydrate).
TABLE-US-00014 TABLE Carbohydrates Carbohydrate Source Dextrose
(D-Glucose) Fisher Scientific D(+)-Galatose ACROS-ORGANICS
.alpha.-Lactose Fisher Scientific L-(-) Fucose SIGMA D-Fructose
ALDRICH Sialic acid (N-acetylneuraminic acid) CALBIOCHEM NAG
(N-acetyl-D-glucosamine) SIGMA GOS (Purimune .TM.
Galactooligosaccharide) GTC Nutrition Purified GOS (Purimune .TM.
GTC Nutrition Galactooligosaccharide) Vivinal .RTM. GOS
(Galactooligosaccharide) Friesland Foods Purified Vivinal .RTM. GOS
Friesland Foods (Galactooligosaccharide) scFOS (Short-Chain
Fructooligosaccharide) Nutraflora .RTM. P-95 (GTC Nutrition) PDX
(Litesse .RTM. Polydextrose) DANISCO 6'SL (6'-sialyllactose)
V-labs; SL 306 Lot#HGDX 21-163-1 3'SL (3'-sialyllactose) V-labs; SL
302 Lot#HGDX 76-161-1 2'FL (2'-fucosyllactose) V-labs; Lot# DX103
3'FL (3'-fucosyllactose) V-labs; Lot# DX807 LNnT
(Lacto-N-Neotetraose) Abbott Nutrition
Kinetic Analysis of Bacterial Growth
[0221] The OD600 data for each carbohydrate is corrected by
subtracting the OD600 of the basal media (sMRS)+cysteine from the
sample plate for each probiotic. Maximum OD is determined by
inspection of the corrected growth data. OD is determined by
subtracting the initial corrected OD (time point 0) from the
maximum corrected OD. Samples are grown in biologically independent
triplicates and the resulting growth kinetic data are expressed as
the mean of these replicates.
[0222] For the growth curve plots, OD600 vs. time is first plotted
for the bacteria grown on medium lacking carbohydrate (sMRS). For
all other carbohydrates, the OD600 data is corrected by subtracting
the OD600 of sMRS.
Purification of GOS
[0223] Purified GOS is obtained by purification of Purimune.TM. GOS
(GTC Nutrition) and Vivinal.RTM. GOS (Friesland Foods Domo). Stock
solutions of 1.5 g/100 mL are applied to a XK column (XK 50/100
column, 5.0.times.100 cm, GE healthcare) packed with Sephadex G25
medium (Sigma). The column is eluted with pure distilled water at a
rate of 8 ml/min and is collected in 12-mL fractions by a Gilson FC
203B fraction collector.
[0224] Detection of carbohydrate in every 2-3 fractions is
performed using the phenol-sulfuric acid assay. Briefly, 50 .mu.L
of sample (2 .mu.L of fraction and 48 of distilled water in a well)
is added to 150 n1 of concentrated sulfuric acid rapidly in a
96-well microtitre plate Immediately thereafter, 30 n1 of 5% phenol
is added and the plate is kept in a static water bath for 30
minutes at 80.degree. C. After cooling to room temperature for 5
minutes, it is wiped dry and absorbance at 490 nm is measured by a
SpectraMax Plus384 Spectrophotometer. Based on carbohydrate
analysis, fractions containing minimal di- and monosaccharides are
pooled and freeze dried (Freeze dry system/Freezezone 4.5/LABCONCO)
for bacterial fermentation experiments. In addition, freeze dried
GOS is pooled from multiple runs in order to generate enough
purified GOS for growth experiments (5 runs with Purimune.TM. GOS
and 3 runs with Vivinal.RTM. GOS).
Results & Discussion:
GOS Purification
[0225] GOS is produced by the transgalactosylation of lactose and
has been used as a prebiotic supplement in pediatric nutrition. Due
to issues with GOS synthesis, commercial GOS products are a mixture
of many different carbohydrates which may include mono- and
disaccharides. In order to test the fermentation parameters of GOS
and not the mono- and disaccharides which would not normally reach
the colon, a purified GOS fraction, essentially free of mono- and
disaccharides is obtained. Glucose (monosaccharide), lactose
(disaccharide) and raffinose (trisaccharide) are used as standards.
Consistent with information from the suppliers, Purimune.TM. GOS
has less mono- and disaccharides than Vivinal.RTM. GOS. For
example, the Purimune.TM. GOS peaks before the raffinose peak
suggesting that Purimune.TM. GOS consists primarily of
trisaccharides or larger. For Vivinal.RTM. GOS, the peak is
observed at a similar fraction number as lactose. Since lactose
begins to appear in fraction 55, fractions 30 through 55 are used
as the purified GOS from both suppliers.
HMO Precursor Fermentation
[0226] All bifidobacteria tested grow very little in the basal
media (sMRS+cysteine), whereas they all grow well in glucose (FIGS.
24A-24H). In general, the bifidobacteria, which is not able to
ferment galactose, also has reduced growth on lactose. None of the
bifidobacteria are able to ferment L-fucose or sialic acid, two key
constituents of HMOs and mucin. Only B. breve ATCC 15700 is able to
ferment NAG, a key component of HMOs and mucin. Lastly, the
majority of bifidobacteria is able to ferment fructose.
Prebiotic Fermentation
[0227] Removal of mono- and disaccharides from Purimune.TM. GOS
results in a decrease in growth for all bifidobacteria (FIGS.
25A-25H). In fact, B. lactis DSM 10140, B. animalis ATCC 25527, B.
bifidum ATCC 29521, B. lactis Bf-6 and B. longum are not able to
ferment the purified Purimune.TM. GOS. A similar pattern is seen
with purified Vivinal.RTM. GOS, except more growth is seen with
Vivinal.RTM. GOS than Purimune.TM. GOS. In order to mimic the
colonic situation, the free mono- and disaccharides present in
these products need to be removed. Also, it is clear that
Purimune.TM. GOS has a higher relative concentration of
oligosaccharides. Both B. infantis strains are among the best
growers on purified GOS as determined by 40D, confirming that GOS
is a reasonable prebiotic to add to infant formula if the goal is
to increase B. infantis. All bifidobacteria tested, except for B.
animalis ATCC 25527, are able to ferment scFOS, whereas no
bifidobacteria are able to ferment polydextrose (PDX).
HMO Fermentation
[0228] Only B. infantis ATCC 15697 and B. infantis M-63 are able to
ferment 6'-SL, 3'-SL, 2'-FL and 3'-FL (FIGS. 26A-26G). In all
cases, B. infantis M-63 grows better than B. infantis ATCC 15697.
On the more complex LNnT, B. breve ATCC 15700 and the two B.
infantis strains grow well but not B. breve M-16V. In addition, the
ability of the two B. infantis strains to ferment HMOs correlates
with the abundance of B. infantis found in breast fed infants.
Curiously, both B. infantis strains are not able to ferment fucose
or sialic acid.
Conclusions
[0229] There are significant differences amongst the tested
bifidobacteria strains regarding their abilities to ferment HMO
precursors, prebiotics and HMOs. Of the 12 bifidobacteria strains
tested, none are able to ferment sialic acid. Regarding prebiotics,
most of the bifidobacteria are able to ferment GOS and scFOS, but
they are not able to ferment PDX. Amongst the bifidobacteria
strains tested, only B. infantis ATCC 15697 and B. infantis M-63
are able to ferment 6'-SL, 3'-SL, 2'-FL and 3'-FL. B. breve ATCC
15700, B. infantis ATCC 15697 and B. infantis M-63 are able to
ferment LNnT.
Example 47
[0230] In this Example, the ability of lacto-N-neotetraose (LNnT)
to induce epithelial cell differentiation is evaluated using cell
culture models of the human small intestine. The induction of
epithelial differentiation by administration of LNnT is evaluated
using in vitro cultures representing various phases of the
differentiated intestinal epithelium. Epithelial cells are cultured
in the presence of various concentrations of LNnT or a control
oligosaccharide and the impact of the LNnT or control on cell
differentiation was measured.
[0231] In a first experiment, HT-29 cells, which model the immature
epithelial cells of the small intestine, are incubated in a
humidified atmosphere of 5% carbon dioxide at 37 C in the presence
of LNnT at concentrations of 0 mg/L ("0"), 100 mg/L ("100"), 200
mg/L ("200"), and 400 mg/L ("400") for either 48 hours or 72 hours.
The culture medium utilized is Dulbecco's Modified Eagle Medium
(Life Technologies, Foster City Calif.) supplemented with 10% fetal
calf serum and 2 mM glutamine. The control consists of a
combination of 91.5 mg lactose and 62.4 mg galactosamine ("LG") per
Liter of the Dulbecco's Modified Eagle Medium set forth above. The
impact of the LNnT at various levels and the control on HT-29 cell
proliferation is measured using a conventional BrdU assay, which
measures the number of cells that have recently synthesized DNA.
The results of the measurements are shown in FIGS. 27 and 28, which
indicate that LNnT reduced HT-29 cell proliferation across a wide
spectrum of concentration and time values.
[0232] In a second experiment, Caco-2 cells, which model more
mature epithelial cells of the small intestine, are incubated in a
humidified atmosphere of 5% carbon dioxide at 37 C in the presence
of LNnT at concentrations of 0 mg/L ("0"), 100 mg/L ("100"), 200
mg/L ("200"), and 400 mg/L ("400") for either 48 hours or 72 hours.
The culture medium utilized is Dulbecco's Modified Eagle Medium
(Life Technologies, Foster City Calif.) supplemented with 10% fetal
calf serum and 2 mM glutamine. The control consists of a
combination of 91.5 mg lactose and 62.4 mg galactosamine ("LG") per
Liter of the Dulbecco's Modified Eagle Medium set forth above. The
impact of the LNnT at various levels and the control on Caco-2 cell
proliferation is measured using a conventional BrdU assay, which
measures the number of cells that have recently synthesized DNA.
The results of the measurements are shown in FIGS. 29 and 30, which
indicate that LNnT generally reduces Caco-2 cell proliferation
across a wide spectrum of concentration and time values.
Conclusions
[0233] The data reported in FIGS. 27-30 indicate that LNnT inhibits
intestinal cell proliferation at multiple stages of epithelial cell
development (immature and more mature cells) at concentrations both
equal to and below that of human breast milk. This inhibition of
proliferation promotes and stimulates gastrointestinal maturation
by allowing cells to move into a differentiated state.
Example 48
[0234] In this Example, the ability of Lacto-N-neotetraose (LNnT),
2'-Fucosyllactose (2'FL), and 6'-Sialyllactose (6'SL) to induce
epithelial cell differentiation and barrier function (cell
resistance) is evaluated using cell culture models of the human
small intestine. The induction of epithelial differentiation and
increase in barrier function by administration of LNnT, 2'FL, and
6'SL is evaluated using in vitro cultures representing various
phases of the differentiated intestinal epithelium. Epithelial
cells are cultured in the presence of various concentrations of
LNnT, 2'FL, 6'SL or a control oligosaccharide of each of these
human milk oligosaccharides (HMOs) and the impact of the LNnT,
2'FL, 6'SL or controls on cell proliferation, cell differentiation,
and barrier function was measured.
[0235] In a first experiment, HT-29 cells, which model the immature
epithelial cells of the small intestine, are incubated in a
humidified atmosphere of 5% carbon dioxide at 37.degree. C. in the
presence of LNnT or 2'FL at concentrations of 0 mg/L ("0"), 20 mg/L
("20"), 200 mg/L ("200"), and 2000 mg/L ("2000") or in the presence
of 6'SL at concentrations of 0 mg/mL ("0"), 40 mg/mL ("40"), 400
mg/mL ("400"), and 4000 mg/mL ("4000") for 72 hours. The culture
medium utilized is Dulbecco's Modified Eagle Medium (Life
Technologies, Foster City Calif.) supplemented with 10% fetal calf
serum and 2 mM glutamine. The controls ("energy") consist of 91.5
mg lactose and 64.2 mg N-acetyllactosamine/L for LNnT; 133 mg
lactose and 67 mg fucose/L for 2'FL; and 195 mg lactose and 205
mg/L sialic acid for 6'SL. The impact of the LNnT, 2'FL, and 6'SL
at various levels and the controls on HT-29 cell proliferation is
measured using a conventional BrdU assay, which measures the number
of cells that have recently synthesized DNA. The results of the
measurements are shown in FIGS. 31-33, which indicate that each of
LNnT, 2'FL, and 6'SL is capable of reducing cell proliferation at
higher doses. Additionally, the impact of LNnT, 2'FL, and 6'SL at
various levels and the controls on the alkaline phosphatase
activity per milligram of protein for HT-29 cells, which is an
indicator of cell differentiation, is measured. The results of the
measurements are shown in FIGS. 34-36, which indicate that there is
a significant increase in alkaline phosphatase activity (and thus
an increase in cell differentiation) at the high dose of 2'FL, a
trend toward an increase in cells treated with LNnT, and no
apparent effect on cells treated with 6'SL.
[0236] FIGS. 37-39 illustrate the effect of LNnT, 2'FL, and 6'SL on
cell resistance (transepithelial resistance), which is a marker for
epithelial barrier function, wherein a higher resistance is
associated with a higher barrier function. Epithelial cell
resistance or barrier function is a measure of differentiated
epithelial cell function. Specifically, as the cells mature,
tighter junctions between the cells are formed resulting in a
stronger epithelial cell barrier. This barrier prevents the
movement of large molecules, bacteria, or viruses from one side of
the barrier to the other. Transepithelial resistance is measured
using Transwell Snapwell inserts containing the desired cell
culture are transferred to modified Ussing chambers and bathed in
modified Kreb's solution at 37 C with 95% oxygen and 5% carbon
dioxide. Transepithelial resistance is measured as the passive
transport of ions across the monolayers.
[0237] In a second experiment, Caco-2 cells, which model more
mature epithelial cells of the small intestine, are incubated in a
humidified atmosphere of 5% carbon dioxide at 37.degree. C. in the
presence of LNnT or 2'FL at concentrations of 0 mg/L ("0"), 20 mg/L
("20"), 200 mg/L ("200"), and 2000 mg/L ("2000") or in the presence
of 6'SL at concentrations of 0 mg/mL ("0"), 40 mg/mL ("40"), 400
mg/mL ("400"), and 4000 mg/mL ("4000") for 72 hours. The culture
medium utilized is Dulbecco's Modified Eagle Medium (Life
Technologies, Foster City Calif.) supplemented with 10% fetal calf
serum and 2 mM glutamine. The controls ("energy") consist of 91.5
mg lactose and 64.2 mg N-acetyllactosamine/L for LNnT; 133 mg
lactose and 67 mg fucose/L for 2'FL; and 195 mg lactose and 205 mg
sialic acid/L for 6'SL. The impact of the LNnT, 2'FL, and 6'SL at
various levels and the control on Caco-2 cell proliferation is
measured using a conventional BrdU assay, which measures the number
of cells that have recently synthesized DNA. The results of the
measurements are shown in FIGS. 40-42, which indicate none of LNnT,
2'FL, or 6'SL have an effect on Caco-2 cell proliferation.
Additionally, the impact of LNnT, 2'FL, and 6'SL at various levels
and the controls on the alkaline phosphatase activity per milligram
of protein for Caco-2 cells, which is an indication of cell
differentiation, is measured. The results of the measurements are
shown in FIGS. 43-45, which indicate that there is a trend toward
increased alkaline phosphatase activity (and thus an increase in
cell differentiation) in 2'FL treated cultures, a trend toward an
increase in cells treated with LNnT, and no apparent effect on
cells treated with 6'SL.
[0238] FIGS. 46-48 illustrate the effect of LNnT, 2'FL, and 6'SL on
cell resistance (transepithelial resistance), which is a marker for
epithelial barrier function, wherein a higher resistance is
associated with a higher barrier function. Epithelial cell
resistance or barrier function is a measure of differentiated
epithelial cell function. Specifically, as the cells mature,
tighter junctions between the cells are formed resulting in a
stronger epithelial cell barrier. This barrier prevents the
movement of large molecules, bacteria, or viruses from one side of
the barrier to the other. The results indicate that LNnT can have a
positive effect on cell resistance for more mature Caco-2 cells.
Transepithelial resistance is measured using Transwell Snapwell
inserts containing the desired cell culture were transferred to
modified Ussing chambers and bathed in modified Kreb's solution at
37 C with 95% oxygen and 5% carbon dioxide. Transepithelial
resistance was measured as the passive transport of ions across the
monolayers.
CONCLUSIONS
[0239] The data reported in FIGS. 31-42 indicate that LNnT, 2'FL,
and 6'SL each inhibits intestinal cell proliferation in immature
epithelial cells at concentrations both equal to and below that of
human breast milk. This inhibition promotes and stimulates
gastrointestinal maturation by allowing cells to move into a
differentiated state. Further, the data reported in FIGS. 43-48
indicate that LNnT can positively affect barrier function of more
mature cells. The development of a strong epithelial cell barrier
is characteristic of a differentiated and mature cell culture and
models the strengthening of the intestinal epithelial cell barrier
that develops in human infants during the first weeks of postnatal
life. Combined, these data support that neutral oligosaccharides,
including LNnT, can promote maturation of the gastrointestinal
tract through inhibition of proliferation as well as direct
promotion of differentiation and barrier function of intestinal
epithelial cells.
* * * * *