HUMAN MILK OLIGOSACCHARIDES TO PROMOTE GROWTH OF
BENEFICIAL BACTERIA
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No.
61/428,867 filed on December 31, 2010; and U.S. Provisional Application No. 61/474,691
filed on April 12, 201 1, 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 gastrointestinalrelated
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.
[001 1] 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,
fructoohgosaccharides, 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,
fructoohgosaccharides, 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.
[00 18] 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.
[003 1] 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. 1 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. 2 1 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] FIG. 24 depicts growth curves of various Bifidobacterium spp. as
evaluated in Example 46.
[0046] FIG. 25 depicts growth curves of various Bifidobacterium spp. as
evaluated in Example 46.
[0047] FIG. 26 depicts 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 nonlimiting
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°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 1 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.
[008 1] 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°C to about 25°C and are typically in the form of oil-inwater,
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-Ntetraose
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-Nneohexaose
II; Disialyllacto-N-neohexaose; Disialyllacto-N-tetraose; Disialyllacto-Nhexaose
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 Nacetylglucosamine
(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-Nneohexaose,
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, isolacto-
N-octaose, lacto-N-octaose, monofucosyllacto-neoocataose, monofucosyllacto-Nocataose,
difucosyllacto-N-octaose I, difucosyllacto-N-octaose II, difucosyllacto-Nneoocataose
II, difucosyllacto-N-neoocataose I, lacto-N-decaose, trifucosyllacto-Nneooctaose,
trifucosyllacto-N-octaose, trifucosyl-iso-lacto-N-octaose, lacto-N-difucohexaose
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-Ntetraose,
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 1mg/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 0.00 1% to
about 0.15%, including from about 0.01% to about 0.10%, and further including from
about 0.0 1% 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.
[01 10] 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.
[01 11] 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
[01 12] 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.
[01 13] 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.
[01 14] 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.
[01 15] The oligosaccharide blend is present in the nutritional compositions in a
total amount of at least about 1 mg/mL, including from about 1mg/mL to about 20 mg/mL,
including from about 1mg/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 1mg/mL to about 4 mg/mL.
[01 16] 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). Rapidlyfermented
oligosaccharides generally have a fermentation rate of greater than 4,000 mg/g of
dry matter/hour; medium-fermented oligosaccharides generally have a fermentation rate of
from 1,500 mg/g of dry matter/hour to 4,000 mg/g of dry matter/hour; and slowly-fermented
oligosaccharides generally have a fermentation rate of less than 1,500 mg/g of dry
matter/hour.
[01 17] By way of specific example, rapidly-fermented oligosaccharides include
FOS, GOS (about 9,304 mg/g of dry matter/hour), LNnT (about 4,488 mg/g of dry
matter/hour), 2'FL (about 4,872 mg/g of dry matter/hour), and combinations thereof.
Medium-fermented oligosaccharides include 6'SL (about 1,809 mg/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 mg/g of dry matter/hour),
gum fibers (e.g., gum arabic (about 785 mg/g of dry matter/hour)), and combinations
thereof.
[01 18] 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.
[01 19] 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 . 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 103 CFU/g, including from about 103 CFU/g to about 10 1 CFU/g, and
including from about 106 CFU/g to about 107 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%o, 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").
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").
[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%o, 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').
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, w-3 LCPUFAs and w-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 esterfied 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 casemates, 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 anticaking
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 , 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 R A 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.
[0 162] 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 milkderived
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 (CHOMIN)
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.
[0 169] 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. Patent No. 6,589,576
(Borschel, et al), U.S. Pat. No. 6,306,908 (Carlson, et al), U.S. Patent Application No.
200301 18703 Al (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°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.
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) 0.1 896 0.1 801 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, D3, E, ¾ 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 3.3 1 g 3.3 1 g 3.3 1 g 3.3 1 g 3.3 1 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.
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) 0.01 82 0.18 4.5455 1.81 8 18.0
Galactooligosaccharides (GOS) 0.1 8 18 0.02 4.5455 18.1 82 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, D3, E, ¾ 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 3.3 1 g 3.3 1 g 3.3 1 g 3.3 1 g 3.3 1 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
[0 182] 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.
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, D3, E, ¾ 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 3.3 1 g 3.3 1 g 3.3 1 g 3.3 1 g 3.3 1 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.
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) 0.1 896 0.1 188 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 112.7 112.7 g 112.7 112.7 g 112.7 s
Choline chloride 104.8 g 104.8 g 104.8 g 104.8 g 104.8 g
Vitamin A, D3, E, ¾ 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.
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.
AN = as needed
EXAMPLES 31-35
[0 186] 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.
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.
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.
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 C0 2. Blended, diluted feces are filtered through four layers of
cheesecloth and sealed in 125-mL serum bottles under C0 2. Inoculum is stored at 37°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,
California); (2) a-(2-6')-N-Acetylneuraminyl-lactose sodium salt (6'SL; Inalco
Pharmaceuticals, San Luis, California); (3) 2'- a-L-Fucopyranosyl -D -Lactose (2'FL; Inalco
group, Italy); (4) Lacto-N-neotetraose (LNnT; Inalco Pharmaceuticals, San Luis,
California); (5) Orafti® HP inulin (HP inulin; BENEO-Orafti, Belgium); and (6) gum
arabic (Fisher Scientific, Pittsburgh, Pennsylvania).
In vitro substratefermentation 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°C for
approximately 12 h to enable hydration of the substrates before initiating fermentation.
These tubes are placed in a 37°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°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°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°C incubator and processed
immediately for analyses. The pH of the tube contents is measured with a standard pH
meter (Denver Instrument Co., Arvada, CO). 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°C
for bacterial analyses.
Short-chainfatty 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, CA) and a glass column (180 cm x 4 mm
i.d.) packed with 10% SP-1200/1% H3P0 4 on 80/100+ mesh Chromosorb WAW (Supelco
Inc., Bellefonte, PA). Oven temperature, detector temperature, and injector temperature
are 125, 175, and 180°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, California) 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, NC). 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 (VLabs,
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 andfermentative 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.
[021 1] 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, MI) supplemented with 0.5 g/L Lcysteine/
HCl and incubated at 37°C for 24 h in an anaerobic chamber (90% N2, 5% C0 2
and 5% H2; Coy Laboratory Products, Grass Lake, MI). 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/1 L-cysteine/HCl. All bacterial strains for use in this Example are
listed in the table below.
Table: Microorganisms
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
m of sMRS + cysteine covered with 50 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™ GOS, purified Purimune™ GOS, Vivinal® GOS,
purified Vivinal® 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: Carbohydrates
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™ GOS (GTC
Nutrition) and Vivinal® GOS (Friesland Foods Domo). Stock solutions of 1.5 g/100 mL
are applied to a XK column (XK 50/100 column, 5.0 x 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 m of sample (2 m of fraction and 48 m of
distilled water in a well) is added to 150 mΐ of concentrated sulfuric acid rapidly in a 96-
well microtitre plate. Immediately thereafter, 30 mΐ of 5% phenol is added and the plate is
kept in a static water bath for 30 minutes at 80°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™ GOS and 3 runs with Vivinal® 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 raffmose (trisaccharide) are used as
standards. Consistent with information from the suppliers, Purimune™ GOS has less
mono- and disaccharides than Vivinal® GOS. For example, the Purimune™ GOS peaks
before the raffmose peak suggesting that Purimune™ GOS consists primarily of
trisaccharides or larger. For Vivinal® 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 (FIG. 24). 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™ GOS results in a
decrease in growth for all bifidobacteria (FIG. 25). 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™ GOS. A similar pattern is seen with purified Vivinal®
GOS, except more growth is seen with Vivinal® GOS than Purimune™ 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™ GOS has a higher relative
concentration of oligosaccharides. Both B. infantis strains are among the best growers on
purified GOS as determined by AOD, 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 (FIG. 26). 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.
[023 1] 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 37C 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 California)
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 37C 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 California)
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°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 California) supplemented with 10% fetal calf serum and 2
mM glutamine. The controls ("energy") consist of 9 1.5 mg lactose and 64.2 mg Nacetyllactosamine/
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 37C
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°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 California) supplemented with 10% fetal calf serum and 2
mM glutamine. The controls ("energy") consist of 9 1.5 mg lactose and 64.2 mg Nacetyllactosamine/
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
37C 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.

WHAT IS CLAIMED IS:
1. A method of stimulating enteric nerve cells in the gastrointestinal tract of an
individual, the method comprising administering to the individual a nutritional composition
comprising a neutral human milk oligosaccharide.
2. The method of claim 1, wherein the neutral human milk oligosaccharide is selected
from the group consisting of 2'-fucosyllactose, 3'-fucosyllactose, L-fucose, and
combinations thereof.
3. The method of claim 2, wherein the neutral human milk oligosaccharide is 2'-
fucosyllactose.
4. The method of claim 2, wherein the 2'-fucosyllactose is present in a concentration
of from about 0.001 mg/mL to less than 2.0 mg/mL.
5. The method of claim 3, wherein the 2'-fucosyllactose is present in a concentration
of from greater than 2.5 mg/mL to 20 mg/mL.
6. A method of improving cognition in an individual, the method comprising
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.
7. The method of claim 6, wherein the neutral human milk oligosaccharide is selected
from the group consisting of 2'-fucosyllactose, 3'-fucosyllactose, L-fucose, and
combinations thereof.
8. The method of claim 7, wherein the neutral human milk oligosaccharide is 2'-
fucosyllactose.
9. The method of claim 8, wherein the 2'-fucosyllactose is present in a concentration
of from about 0.5 mg/mL to about 1mg/mL.
10. The method of claim 7, wherein the human milk oligosaccharide is 3'-
fucosyllactose.
11. A method of promoting the growth of beneficial bacteria in an individual, the
method comprising administering to the individual a synthetic nutritional composition
comprising 2'-fucosyllactose.
12. The method of claim 11, wherein the beneficial bacteria is grown in the
gastrointestinal tract of the individual.
13. The method of claim 11, wherein the individual in need of beneficial bacteria
suffers from necrotizing enterocolitis.
14. A method of reducing the incidence of colic in an infant, the method comprising
administering to the infant a synthetic infant formula comprising 2'-fucosyllactose.
15. The method of claim 14, wherein the synthetic infant formula further comprises a
probiotic.