NUTRITIONAL COMPOSITIONS COMPRISING HUMAN MILK
OLIGOSACCHARIDES AND NUCLEOTIDES AND USES THEREOF FOR
TREATING AND/OR PREVENTING ENTERIC VIRAL INFECTION
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No.
61/551,758 filed on October 26, 201 1; and U.S. Provisional Application No. 61/428,866
filed on December 31, 2010, which disclosures are incorporated by reference in their
entirety.
FIELD OF THE DISCLOSURE
[0002] The present disclosure relates to human milk oligosaccharides (HMOs) for
modulating inflammation in an infant, toddler, or child. More particularly, the present
disclosure relates to human milk fortifiers, preterm and term infant formulas and pediatric
formulas comprising HMOs and nucleotides that can reduce inflammation and thereby
prevent and/or treat inflammatory conditions and diseases, such as enteric viral infections.
BACKGROUND OF THE DISCLOSURE
[0003] The inflammatory response is an attempt by the body to restore and
maintain homeostasis after invasion by an infectious agent, antigen challenge, or physical,
chemical or traumatic damage. While the inflammatory response is generally considered a
healthy response to injury, the immune system can present an undesirable physiological
response if it is not appropriately regulated. Specifically, unregulated oxidation and
associated inflammation are major causes of tissue damage and clinically significant
disease in preterm and term infants. This is due in large part to the immaturity in function
of the natural immune system of infants, and especially preterm infants.
[0004] Breastfeeding has been associated with enhanced development and
balanced growth and maturation of the infant's respiratory, gastrointestinal and immune
systems, thereby providing protection of the infant to infection and inflammatory diseases.
Breast milk appears to contain endogenous antioxidants, such as superoxide dismutase,
glutathione peroxidase and catalase, or other non-enzymatic antioxidants such as
glutathione, lactoferrin and polyphenols, in addition to exogenous antioxidants, such as
vitamins A, C, E and selenium. Further, breast milk includes HMOs that not only act as
pathogen receptor analogues, but activate immune factors by infant intestinal epithelial
cells and/or associated immune cell populations. The function of these breast milk
components, functioning as antioxidants and as immune modulators, includes not only the
protection of breast milk lipids by peroxidation, but may also assist in the regulation of
inflammatory responses to infection or other injury.
[0005] Not all infants receive human breast milk. Further, no vaccines are
currently available for the prevention of inflammatory diseases. Therefore, development of
safe and efficacious preventative or therapeutic methods would be beneficial, especially for
infants.
[0006] It would therefore be desirable to provide nutritional compositions, and
synthetic infant formulas in particular, that can produce nutritional benefits including
improved immune system growth and development. It would additionally be beneficial if
the nutritional compositions could modulate inflammation and enhance immunity against
microbial infections, including bacterial and viral infections, and other inflammatory
diseases.
SUMMARY OF THE DISCLOSURE
[0007] 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 one or more of long chain
polyunsaturated fatty acids (LCPUFAs), antioxidants, and/or nucleotides, for reducing
inflammation in an infant, toddler, or child, as well as methods of using the compositions.
[0008] One embodiment is a synthetic pediatric formula comprising from about
0.001 mg/mL to about 20 mg/mL of a human milk oligosaccharide and from about 5 mg/L
to about 350 mg/L of a nucleotide.
[0009] Another embodiment is a method for treating and/or preventing enteric
viral infection in an infant, toddler, or child in need thereof. The method comprises
administering to an infant, toddler, or child a composition comprising from about 0.001
mg/mL to about 20 mg/mL of a human milk oligosaccharide and from about 5 mg/L to
about 350 mg/L of a nucleotide.
[0010] It has been discovered that specific HMOs, such as 3'-sialyllactose, 6'-
sialyllactose and others as noted herein, are highly effective in dampening inflammation
generally in infants, toddlers, and children, and specifically in dampening virus-induced
inflammation, including respiratory syncytial virus, human parainfluenza, and influenza A,
in infants, toddlers, and children by reducing the production of some key cytokines from
human immune cells without increasing viral load, which may lead to faster recovery from
infections. Surprisingly, it was determined that the HMOs demonstrate the desirable
dampening effects even at very low concentrations, including concentrations lower than
those found in breast milk. Also, it was unexpectedly found that 6'-sialyllactose is
immunomodulatory even in the absence of a virus, and induces the production of
monocyte-derived cytokines. It has further been discovered that although biological
reactions often occur within a 30 to 60 minute period, and thus a 30 to 60 minute
incubation is generally used for in vitro procedures, a 24 hour pre-treatment of cells
provides a closer reflection of the daily pre-exposure to HMOs that a breast-fed infant
would receive from breast milk.
[00 11] Additionally, it has been found that fucosyllated HMOs, including 3' -
fucosyllactose, alone or in combination with sialic acid, are highly effective in inhibiting
respiratory viruses. Even at very low concentrations, the 3'-fucosyllactose and sialic acid
are effective.
[0012] Moreover, it has been discovered that specific HMOs act in a synergistic
manner against respiratory viruses, including RSV, when combined with a long chain
polyunsaturated fatty acid and/or a carotenoid. These synergistic actions dampen virusinduced
inflammatory cytokines, and specifically interferon-inducible protein 10 (IP- 10).
Additional components including antioxidants, such as vitamin A and vitamin E, or
nucleotides, may also be added to the HMO and long chain polyunsaturated fatty acid
and/or carotenoid combinations.
[0013] In addition, it has been discovered that a combination of either neutral or
acidic HMOs and nucleotides is effective in decreasing enteric viral infectivity.
Specifically, this combination decreases replication of enteric viruses, such as rotavirus.
[0014] It has further been found that a combination of HMOs including
acidic/sialylated (e.g., 6'-sialyllactose) and/or neutral/fucosylated (e.g., 2'-fucosyllactose)
and/or n-acetylglucosylated (e.g., LNnT) prevents the development of necrotizing
entercolitis. Also, these HMOs have been found to decrease the oxidative stress in infants.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a graph depicting H1N1 virus infectivity of MDCK cells in the
presence of various HMOs as tested in Example 47.
[0016] FIG. 2 is a graph depicting blood plasma levels of glutathione from piglets
as measured in Example 48.
[0017] FIG. 3 is a graph depicting IP- 10 levels resulting from administration of
3'SL and 6'SL as measured in Example 49.
[0018] FIG. 4 is a graph depicting IP- 10 levels resulting from administration of
3'SL and 6'SL as measured in Example 49.
[0019] FIG. 5 is a graph depicting IP- 10 levels resulting from administration of
LNnT as measured in Example 49.
[0020] FIG. 6 is a graph depicting IP- 10 levels resulting from administration of
LNnT as measured in Example 49.
[0021] FIG. 7 is a graph depicting IL-10 levels resulting from administration of
3'SL and 6'SL as measured in Example 49.
[0022] FIG. 8 is a graph depicting IL-10 levels resulting from administration of
3'SL and 6'SL as measured in Example 49.
[0023] FIG. 9 is a graph depicting IL-10 levels resulting from administration of
LNnT as measured in Example 49.
[0024] FIG. 10 is a graph depicting IL-10 levels resulting from administration of
LNnT as measured in Example 49.
[0025] FIG. 11 is a graph depicting NSP4 levels resulting from the treatment of
ileal loops with various combinations of HMOs, nucleotides, and rotavirus as measured in
Example 50.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0026] The nutritional compositions and methods described herein utilize HMOs
alone or in combination with long chain polyunsaturated fatty acids, and/or antioxidants,
and in particular carotenoids, and/or nucleotides for controlling and reducing a number of
diseases and conditions related to inflammation. 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] The term "human milk oligosaccharide" or "HMO", as used herein, unless
otherwise specified, refers generally to a number of complex carbohydrates found in
human breast milk that can be in acidic or neutral form, and to precursors thereof.
Exemplary non-limiting human milk oligosaccharides include 3'-sialyllactose, 6'-
sialyUactose, 3'-fucosyllactose, 2'-fucosyllactose, and lacto-N-neo-tetraose. An exemplary
human milk oligosaccharide precursor includes sialic acid.
[003 1] 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.
[0032] 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 powders, nutritional semi-solids, nutritional semiliquids,
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.
[0033] The term "nutritional liquid" as used herein, unless otherwise specified,
refers to nutritional products in ready-to-drink liquid form, concentrated form, and
nutritional liquids made by reconstituting the nutritional powders described herein prior to
use.
[0034] The term "nutritional powder" as used herein, unless otherwise specified,
refers to nutritional products 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] The term "newborn" as used herein, unless otherwise specified, refers to a
person from birth up to four weeks of age.
[0041] The terms "infant formula" or "synthetic infant formula" as used herein,
unless otherwise specified, are used interchangeably and refer to liquid, solid, semi-solid,
and semi-liquid 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.
[0042] 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 nutritional formula" does not include human
breast milk.
[0043] 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 nutritional formula" does not include human breast milk.
[0044] 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.
[0045] 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.
[0046] The terms "absence of a virus" or "absent a virus" as used herein with
respect to inducing production of monocyte-derived cytokines, unless otherwise specified,
refer to an individual (e.g., an infant) without the virus or having the virus in an amount
less than the amount required to illicit an immune response; that is, an amount that is less
than required for the body's natural immune response to increase the production of
cytokines and other immune factors.
[0047] 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.
[0048] The terms "inflammatory disease" or "inflammatory condition" as used
herein, unless otherwise specified, refer to any disease, disorder, or condition characterized
by inflammation. The term "infection-mediated inflammatory disease" as used herein,
unless otherwise specified, refers to an inflammatory disease associated or induced by
microbial infection, including viral and bacterial infection.
[0049] 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.
[0050] The terms "modulating" or "modulation" or "modulate" as used herein,
unless otherwise specified, refer to the targeted movement of a selected characteristic.
[005 1] 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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 product applications.
Product Form
[0056] 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-solid, semi-liquid 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 as also defined herein.
[0057] The nutritional compositions of the present disclosure include one or more
HMOs as described herein. The compositions may include one or more HMOs alone or in
combination with other immune enhancing factors including, but not limited, to long chain
polyunsaturated acids (LCPUFAs), nucleotides, and antioxidants, such as carotenoids and
vitamins, as discussed below.
[0058] The nutritional compositions may be in any product form comprising the
ingredients described herein, and which is safe and effective for oral administration. The
nutritional compositions may be formulated to include only the ingredients described
herein, or may be modified with optional ingredients to form a number of different product
forms.
[0059] 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 HMOs,
desirably in combination with at least one of protein, fat, vitamins, and minerals, to
produce a nutritional composition.
[0060] The nutritional compositions 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 product for use in individuals afflicted with specific
diseases or conditions or with a targeted nutritional benefit as described below.
[0061] 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, and liquid and powdered follow-on formulas
suitable for use with infants and children.
Nutritional Liquids
[0062] 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.
[0063] 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.
[0064] 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%, of water by weight of the nutritional emulsions. 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.
[0065] 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.
[0066] 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.
[0067] 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 1mL 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
[0068] 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.
[0069] 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)
[0070] 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 composition of human milk oligosaccharides is very complex and more than 200
different oligosaccharide-like structures are known.
[0071] The HMOs may be included in the nutritional compositions alone, or in
some embodiments, in combination with other immune enhancing factors (e.g., LCPUFAs,
antioxidants, nucleotides, etc.) as described herein. 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.
[0072] Suitable HMOs for use in the nutritional compositions may include acidic
oligosaccharides, neutral 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 (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
b). 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); 3'-Sialyllactose
(3'SL); 6'-Sialyllactose (6'SL); 2'-Fucosyllactose (2'FL); 3'-Fucosyllactose (3'FL); Lacto-
N-tetraose and Lacto-N-neotetraose (LNnT), and in particular, combinations of 6'SL and
3'SL; combinations of 3'FL and SA; combinations of 2'FL and 3'FL; combinations of
2'FL, 3'SL, and 6'SL; combinations of 3'SL, 3'FL, and LNnT; combinations of 6'SL,
2'FL, and LNnT; combinations of 3'SL, 6'SL, and LNnT; and LNnT alone.
[0073] 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.
[0074] 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 0.001
mg/mL to 10 mg/mL, including from 0.01 mg/mL to 10 mg/mL, including from 0.001
mg/mL to 5 mg/mL, including from 0.01 mg/mL to 5 mg/mL, including from 0.001 mg/mL
to 0.23 mg/mL, and including from 0.01 mg/mL to 0.23 mg/mL of total HMO in the
nutritional composition. 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.
[0075] In one specific embodiment when the nutritional product 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).
[0076] In another specific embodiment, when the nutritional product is a readyto-
feed nutritional liquid, the total concentration of HMOs in the ready-to-feed nutritional
liquid is from about 0.000 1% 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).
[0077] In another specific embodiment when the nutritional product is a
concentrated nutritional liquid, the total concentration of HMOs in the concentrated
nutritional 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).
[0078] In some embodiments, the HMOs are used in combination to provide the
desired immune enhancing effect. For example, in one embodiment, the nutritional
composition includes 6'SL in combination with 3'SL in a total amount of HMO 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 0.001 mg/mL to 0.23 mg/mL, including from 0.01 mg/mL to 0.23
mg/mL, including from 0.001 mg/mL to less than 0.15 mg/mL, and including from 0.01
mg/mL to less than 0.15 mg/mL of the nutritional composition. In another embodiment,
the nutritional composition includes 6'SL in combination with 3'SL in a total amount of
HMO of from about 0.001 mg/mL to about 20 mg/mL, including from about 0.01 mg/mL
to about 20 mg/mL and including greater than 0.65 mg/mL to 20 mg/mL.
[0079] 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 0.001
mg/mL to less than 0.25 mg/mL, including 0.01 mg/mL to less than 0.25 mg/mL, including
from greater than 0.4 mg/mL to 20 mg/mL, and including from 0.1 mg/mL to 0.5 mg/mL.
[0080] 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.
[0081] 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 0.001
mg/mL to less than 0.15 mg/mL, including from 0.01 mg/mL to less than 0.15 mg/mL, and
including from greater than 0.25 mg/mL to 20 mg/mL.
[0082] 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 85% (by total weight HMOs) to about 100% (by
total weight HMOs), and including about 100% (by total weight HMOs) 3'SL.
[0083] 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 0.001
mg/mL to less than 0.2 mg/mL, including 0.01 mg/mL to less than 0.2 mg/mL, including
from greater than 0.32 mg/mL to 20 mg/mL, and including about 2 mg/mL.
[0084] In another specific embodiment, the nutritional composition includes
3'FL, 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 0.001
mg/mL to less than 1 mg/mL, including 0.01 mg/mL to less than 1 mg/mL, and including
from greater than 1.7 mg/mL to 20 mg/mL.
[0085] In one specific embodiment, the nutritional composition includes 3'FL in
combination with SA in a total amount of HMO of from about 0.001 mg/mL to about 20
mg/mL, including from about 0.01 mg/mL to about 20 mg/mL. In one embodiment, the
nutritional composition includes 3'FL in an amount of from 0.001 mg/mL to less than 1
mg/mL, including from 0.01 mg/mL to less than 1mg/mL and SA in an amount of about 1
mg/mL.
[0086] In another embodiment, the nutritional composition includes 2'FL, 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 0.001 mg/mL to
less than 2 mg/mL and including 0.01 mg/mL to less than 2 mg/mL. In another
embodiment, the nutritional composition includes 2'FL, 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 and including greater than 2.5 mg/mL to 20 mg/mL.
[0087] In one specific embodiment, the nutritional composition includes 2'FL in
combination with 3'FL in a total amount of HMO of from about 0.001 mg/mL to about 20
mg/mL, including from about 0.01 mg/mL to about 20 mg/mL.
[0088] In yet another embodiment, the nutritional composition includes a
combination of 6'SL, 2'FL, and LNnT in a total amount of HMO of from about 0.001
mg/mL to about 20 mg/mL, including from about 0.01 mg/mL to about 20 mg/mL.
[0089] In a further embodiment, the nutritional composition includes a
combination of 6'SL, 3'SL, and sialic acid in a total amount of from about 0.001 mg/mL to
about 20 mg/mL, including about 2 mg/mL. In this embodiment, the 6'SL is present in an
amount of about 40% by weight of total HMOs, the 3'SL is present in an amount of about
10% by weight of total HMOs, and the sialic acid is present in an amount of about 50% by
weight of total HMOs.
Long Chain Polyunsaturated Fatty Acids (LCPUFAs)
[0090] In addition to the HMOs described above, the nutritional products of the
present disclosure may include LCPUFAs. LCPUFAs are included in the nutritional
compositions to provide nutritional support, as well as to reduce oxidative stress and
enhance growth and functional development of the intestinal epithelium and associated
immune cell populations. In some embodiments, the nutritional composition includes a
combination of one or more HMOs and one or more LCPUFAs such that the composition
provides a synergistic benefit to the end user, such as a synergistic benefit in modulating
anti-viral immune responses and dampening inflammation. In some embodiments, the
HMO or HMOs used in combination with the LCPUFAs to provide the synergistic effect
are acidic HMOs.
[0091] 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, DPA, and
combinations thereof.
[0092] 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.
[0093] 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.
[0094] The nutritional compositions as described herein will typically comprise
total concentrations of LCPUFA of from about 0.01 mM to about 10 mM and including
from about 0.01 mM to about 1mM. Alternatively, the nutritional compositions comprise
total concentrations of LCPUFA of from about 0.001 g/L to about 1 g/L.
[0095] In one embodiment, the nutritional compositions include total long chain
-6 fatty acids in a concentration of from about 100 to about 425 mg/L or from about 12 to
about 53 mg per 100 kcals and/or further include total long chain -3 fatty acids in a
concentration of from about 40 to about 185 mg/L or from about 5 to about 23 mg per 100
kcals. In one specific embodiment, the ratio of long chain -6 fatty acids to long chain w-
3 fatty acids in the nutritional compositions ranges from about 2:1 to about 3:1, preferably
about 2.5:1.
[0096] In one specific embodiment, the nutritional compositions include DHA in
a concentration of from about 0.025 mg/mL to about 0.130 mg/mL or from about 3 to
about 16 mg per 100 kcals. In another embodiment, the nutritional compositions include
ARA in a concentration of from about 0.080 mg/mL to about 0.250 mg/mL or from about
10 to about 3 1 mg per 100 kcals. In yet another embodiment, the nutritional compositions
include combinations of DHA and ARA such that the ratio of DHA to ARA ranges from
about 1:4 to about 1:2.
Antioxidants
[0097] Additionally, the nutritional compositions may comprise one or more
antioxidants in combination with the HMOs (and optionally LCPUFAs and/or nucleotides
also) to provide nutritional support, as well as to reduce oxidative stress. In some
embodiments, the nutritional composition includes a combination of HMOs and
antioxidants such that the composition provides a synergistic benefit to the end user, such
as a synergistic benefit in modulating anti-viral immune responses and dampening
inflammation. In some embodiments, the HMO or HMOs is used in combination with
carotenoids (and specifically lutein, beta-carotene, zeaxanthin and/or lycopene) to provide
the synergistic effect.
[0098] Any antioxidants suitable for oral administration may be included for use
in the nutritional compositions of the present disclosure, including, for example, vitamin A,
vitamin E, vitamin C, retinol, tocopherol, and carotenoids, including lutein, beta-carotene,
zeaxanthin, and lycopene, and combinations thereof, for example.
[0099] As noted, the antioxidants for use in the nutritional compositions may be
used with the HMOs alone or in combination with HMOs and LCPUFAs and/or
nucleotides. In one specific embodiment, the antioxidants for use in the nutritional
compositions include carotenoids, and particularly, combinations of the carotenoids lutein,
lycopene, zeaxanthin and/or 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.
[0100] It is generally preferable that the nutritional compositions comprise at least
one of lutein, lycopene, zeaxanthin, beta-carotene to provide a total amount of carotenoid
of from about 0.001 mg/mL to about 10 mg/mL. More particularly, the nutritional
compositions comprise lutein in an amount of from 0.001 mg/mL to 10 mg/mL, including
from 0.001 mg/mL to 0.0190 mg/mL, including from 0.001 mg/mL to 0.0140 mg/mL, and
also including from 0.044 mg/mL to 10 mg/mL of lutein. It is also generally preferable that
the nutritional compositions comprise from 0.001 mg/mL to 10 mg/mL, from 0.001 mg/mL
to 0.0130 mg/mL, including from 0.001 mg/mL to 0.0075 mg/mL of lycopene, and also
including from 0.0185 mg/mL to 10 mg/mL of lycopene. It is also generally preferable that
the nutritional compositions comprise from 1 mg/mL to 10 mg/mL, including from 0.001
m /h to 0.025 m /h of beta-carotene, including from 0.001 mg/mL to 0.01 1 mg/mL of
beta-carotene, and also including from 0.034 mg/mL to about 10 mg/mL of beta-carotene.
It should be understood that any combination of these amounts of beta-carotene, lutein,
zeaxanthin, and lycopene can be included in the nutritional compositions of the present
disclosure. Other carotenoids may optionally be included in the nutritional compositions as
described herein. Any one or all of the carotenoids included in the nutritional compositions
described herein may be from a natural source, or artificially synthesized.
[0101] Each of the carotenoids in the selected combinations can be obtained from
any known or otherwise suitable material source for use in nutritional compositions, and
each can be provided individually, or all together, or in any combination and from any
number of sources, including sources such as multivitamin premixes containing other
vitamins or minerals in combination with one or more of the carotenoids as described
herein. Non-limiting examples of some suitable sources of lutein, lycopene, beta-carotene,
or combinations thereof include LycoVit® lycopene (available from BASF, Mount Olive,
NJ), Lyc-O-Mato® tomato extract in oil, powder, or bead form (available from LycoRed
Corp., Orange, NJ), beta-carotene, lutein, or lycopene (available from DSM Nutritional
Products, Parsippany, NJ), FloraGLO® lutein (available from Kemin Health, Des Moines,
IA), Xangold® Natural Lutein Esters (available from Cognis, Cincinnati, OH), and
Lucarotin® beta-carotene (available from BASF, Mount Olive, N.J).
Nucleotides
[0102] In addition to the HMOs, the nutritional compositions of the present
disclosure may additionally comprise nucleotides and/or nucleotide precursors selected
from the group consisting of nucleosides, purine bases, pyrimidine bases, ribose and
deoxyribose. The nucleotides may be in monophosphate, diphosphate, or triphosphate
form. The nucleotides may be a ribonucleotide or a deoxyribonucleotide. The nucleotides
may be monomeric, dimeric, or polymeric (including RNA and DNA). The nucleotides
may be present in the nutritional composition as a free acid or in the form of a salt,
preferably a monosodium salt. In some embodiments, the nutritional composition includes
a combination of HMOs and nucleotides such that the composition provides a synergistic
benefit to the end user, such as a synergistic benefit in modulating anti-viral immune
responses and dampening inflammation and/or improving intestinal barrier integrity.
[0103] Incorporation of nucleotides in the nutritional compositions of the present
disclosure improves intestinal barrier integrity and/or maturation, which is beneficial to
preterm and term infants who have less developed intestinal flora and hence a slower
maturing intestinal barrier.
[0 104] 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. In one embodiment, the nutritional compositions include
cytidine 5'-monophosphate, uridine 5'-monophosphate, adenosine 5'-monophosphate,
guanosine 5'-monophosphate, and inosine 5'-monophosphate. In another embodiment, the
nutritional compositions include cytidine 5'-monophosphate, uridine 5'-monophosphate,
adenosine 5'-monophosphate and guanosine 5'-monophosphate.
[0105] The nucleotides are present in the nutritional products in total amounts of
nucleotides of at least about 5 mg/L, including at least about 10 mg/L, including from
about 5 mg/L to about 350 mg/L, including from about 20 mg/L to about 350 mg/L,
including from about 40 mg/L to about 350 mg/L, including from about 10 mg/L to about
320 mg/L, including from about 72 mg/L to about 320 mg/L, including from about 10
mg/L to about 200 mg/L, including from about 42 mg/L to about 102 mg/L, including
about 72 mg/L, and including about 320 mg/L of the nutritional product.
[0106] In one specific embodiment, the nucleotides are present in the nutritional
products in a total amount of about 72 mg/L of the nutritional product and comprise about
43% cytidine 5'-monophosphate, about 18.5% uridine 5'-monophosphate, about 16.5%
adenosine 5'-monophosphate and about 22% guanosine 5'-monophosphate by total weight
of nucleotides.
[0107] In one specific embodiment, the nucleotides are present in the nutritional
products in a total amount of about 72 mg/L of the nutritional product and comprise about
29 to 39 mg of cytidine 5'-monophosphate; 15 to 2 1 mg of uridine 5'-monophosphate; 10
to 15 mg of adenosine 5'-monophosphate; and 14 to 20 mg of guanosine 5'-
monophosphate.
[0108] In one specific embodiment, the nucleotides are present in the weight ratio
of cytidine 5'-monophosphate: uridine 5'-monophosphate is from about 1.5:1 to about
2.6:1; of cytidine 5'-monophosphate: adenosine 5'-monophosphate is from about 2:1 to
about 3.9:1; and of cytidine 5'-monophosphate: guanosine 5'-monophosphate is from
about 1.75:1 to about 2.8:1.
[0109] In one specific embodiment when the nutritional composition is a
nutritional powder, the nucleotides may be present at a level of at least about 0.007%,
including from about 0.0078% to about 0.1556%, and including about 0.056% (by weight
of the nutritional powder), or at least about 0.007 grams, including from about 0.0078
grams to about 0.1556 grams, and including about 0.056 grams of nucleotide per 100
grams of nutritional powder.
[01 10] In another specific embodiment, when the nutritional composition is a
ready-to-feed nutritional liquid, the nucleotides are present at a level of at least about
0.001%, including from about 0.001% to about 0.0197%, and including about 0.0071% (by
weight of the nutritional liquid), or at least about 0.001 grams, including from about 0.001
grams to about 0.0197 grams, and including about 0.0071 grams of nucleotide per 100
grams of ready-to-feed nutritional liquid.
[01 11] In another specific embodiment when the nutritional composition is a
concentrated nutritional liquid, the nucleotides are present at a level of at least about
0.0019%, including from about 0.0019% to about 0.0382%, and including about 0.0138%
(by weight of the nutritional liquid), or at least about 0.0019 grams, including from about
0.0019 grams to about 0.0382 grams, and including about 0.0138 grams of nucleotide per
100 grams of concentrated nutritional liquid.
Macronutrients
[01 12] 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.
[01 13] 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, 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.
[01 14] For the liquid preterm and term infant formulas, carbohydrate
concentrations 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.
[01 15] For the liquid human milk fortifiers, carbohydrate concentrations most
typically range from about 10% to about 75%, including from about 10% to about 50%,
including from about 20% to about 40%, by weight of the human milk fortifier; fat
concentrations most typically range from about 10% to about 40%, including from about
15% to about 37%, and also including from about 18% to about 30%, by weight of the
human milk fortifier; and protein concentrations most typically range from about 5% to
about 40%, including from about 10% to about 30%, and also including from about 15% to
about 25%, by weight of the human milk fortifier.
[01 16] 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
[01 17] 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 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").
[01 18] When the nutritional product 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 is present in an amount of from about 30% to about 85%,
including from about 45% to about 60%, including from about 50%> to about 55% by
weight of the preterm or term infant formula.
[01 19] 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 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.
[0120] 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 product. 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").
Fat
[0121] The nutritional compositions of the present disclosure may, in addition to
the LCPUFAs described above, comprise an additional source or sources of fat. Suitable
additional sources of fat for use herein include any fat or fat source that is suitable for use
in an oral nutritional product and is compatible with the essential elements and features of
such products. For example, in one specific embodiment, the additional fat is derived from
short chain fatty acids.
[0122] Additional non-limiting examples of suitable fats or sources thereof for
use in the nutritional products 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
[0123] 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 products is
suitable for use in the nutritional compositions.
[0124] Non-limiting examples of suitable proteins or sources thereof for use in
the nutritional products 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.
Carbohydrate
[0125] The nutritional products of the present disclosure may further optionally
comprise any carbohydrates that are suitable for use in an oral nutritional product and are
compatible with the essential elements and features of such products.
[0126] Non-limiting examples of suitable carbohydrates or sources thereof for use
in the nutritional products 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
[0127] 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 products 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.
[0128] Non-limiting examples of such optional ingredients include preservatives,
emulsifying agents, buffers, fiructooligosaccharides, galactooligosaccharides, polydextrose,
and other prebiotics, probiotics, pharmaceutical actives, anti-inflammatory agents,
additional nutrients as described herein, colorants, flavors, thickening agents and
stabilizers, emulsifying agents, lubricants, and so forth.
[0129] 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 product may range from at least 0.01%, including from about 0.1% to about
10% , and also including from about 1% to about 6%, by weight of the nutritional product.
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 product.
[0130] 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.
[0131] 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.
[0132] The nutritional compositions may further comprise any of a variety of
other vitamins or related nutrients, non-limiting examples of which include vitamin A,
vitamin D , vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, vitamin B i2, carotenoids
(e.g., beta-carotene, zeaxanthin, lutein, lycopene), niacin, folic acid, pantothenic acid,
biotin, vitamin C, choline, inositol, salts and derivatives thereof, and combinations thereof.
[0133] The nutritional compositions may further comprise any of a variety of
other additional minerals, non-limiting examples of which include calcium, phosphorus,
magnesium, iron, zinc, manganese, copper, sodium, potassium, molybdenum, chromium,
chloride, and combinations thereof.
Methods of Manufacture
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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
technique, suitable for making and formulating a nutritional powder.
[0138] 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.
[0139] 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.
[0140] Other suitable methods for making nutritional products are described, for
example, in U.S. Pat. No. 6,365,218 (Borschel, et al), U.S. Patent 6,589,576 (Borschel, et
al), U.S. Pat. No. 6,306,908 (Carlson, et al), U.S. Patent Application 200301 18703 Al
(Nguyen, et al), which descriptions are incorporated herein by reference to the extent that
they are consistent herewith.
Methods of Use
[0141] The nutritional compositions as described herein can be used to address
one or more of the diseases or conditions discussed herein, or can be used to provide one or
more of the benefits described herein, to preterm infants, infants, toddlers, and children.
The preterm infant, infant, toddler, or child 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, or child actually has the disease or condition, or is at risk or susceptible to
the disease or condition, the preterm infant, infant, toddler, or child is classified herein as
"in need of assistance in dealing with and combating the disease or condition. For
example, the preterm infant, infant, toddler, or child 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, or child 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, or child with the nutritional
compositions described herein.
[0142] 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, and children will fall within the subset or subclass of preterm infants, infants,
toddlers, and children as described herein for certain diseases or conditions.
[0143] The nutritional compositions as described herein comprise HMOs, alone
or in combination with one or more additional components, to provide a nutritional source
for reducing inflammation, such as respiratory inflammation (e.g., respiratory syncytial
virus-induced inflammation), enteric inflammation, and nasopharyngeal inflammation.
The nutritional compositions of the present disclosure comprising HMOs may also provide
optimal development and balanced growth and maturation of the infant's gastrointestinal
and immune systems, thereby enhancing the infant's ability to resist microbial infection
and modulate inflammatory responses to infection (e.g., increased phagocytosis and
increased production of reactive oxidative species).
[0144] The nutritional compositions also provide growth and maturation of the
intestinal epithelial cells in an infant. In one specific embodiment, the administration of the
nutritional compositions of the present disclosure including HMOs and nucleotides can
further activate immune activity in or by the intestinal epithelial cells in a newborn.
[0145] Further, the use of HMOs in nutritional compositions can reduce the
growth of respiratory viruses (e.g., RSV, human parainfluenza virus type 2, and influenza
A virus), and thus, reduce viral-induced upper respiratory infections. In addition, the use of
HMOs in combination with nucleotides in nutritional compositions can reduce the growth
of enteric viruses (e.g., rotavirus), and thus, reduce viral-induced enteric infections. As
such, by utilizing HMOs, alone or in combination with other immune enhancing factors, in
a nutritional product, such as an infant formula, it is now possible to provide infants with
an alternative, or supplement, to breast milk that more closely mimics the benefits thereof.
[0146] Along with improved growth and maturation of the infant's immune
system as described above, the use of the nutritional compositions of the present disclosure
also functions as an immune modulator, thereby reducing inflammation induced by
infection in infants, toddlers, and children such as respiratory virus-induced infection, and
particularly, RSV-induced inflammation, and other infection-mediated inflammatory
diseases.
[0147] The addition of HMOs can further increase glutathione levels in the body
and blood of an infant, and in specific embodiments, of a preterm infant.
[0148] When used in combination with LCPUFAs and/or antioxidants, and
particularly, with carotenoids, the HMOs can reduce oxidative stress, which is a metabolic
condition in which there is an increased production and accumulation of oxidized
biomolecules such as lipid peroxides and their catabolites, protein carbonyls, and
oxidatively damaged DNA. The outcomes of oxidative stress range from unwanted
changes in metabolism to inflammation and cell and tissue death. Accordingly, by
reducing the incidence of unregulated inflammation and oxidation in the infant, damage to
the tissue lining and cell death is reduced, further reducing the incidence of inflammatory
diseases, such as necrotizing enterocolitis (NEC).
[0149] In addition to the benefits discussed above, it has been discovered that
nutritional products including HMOs can modulate production of monocyte-derived
cytokines in the infant, even in the absence of a virus. This production results in improved
immunity to further prevent microbial infection and reduce the growth of viruses. In one
specific embodiment, monocyte-derived cytokines produced by administration of the
nutritional compositions of the present disclosure include, for example, interleukin-10,
interleukin-8, interleukin-la, interleukin- ΐ b, interleukin-lra, and combinations thereof.
[0150] Another benefit of utilizing HMOs in nutritional compositions is that it has
been discovered that HMOs modulate the production of IP- 10, which is a chemokine that
plays an important role in the inflammatory response to viral infection. Specifically, a
positive correlation exists between RSV clinical infection severity in children and serum
IP- 10 levels. Accordingly, a decrease in IP- 10 signals a decrease in severity of RSV
infection. In one specific embodiment, IP- 10 production is reduced to the level found in
uninfected controls.
[0151] Along with reducing IP- 10, HMOs have been found to reduce plateletneutrophil
complex (PNC) formation, which is present in human blood and consists of up
to 25% of unstimulated neutrophils. As PNCs are present in aggregates, they have a
greater capacity to initiate inflammatory processes and can increase the production of
reactive oxidative species. Accordingly, a decrease in PNC formation can lead to reduced
oxidative stress and inflammation in the infant.
[0152] In addition, the combination of HMOs and nucleotides has been found to
decrease NSP4 mRNA expression, which signals a decrease in rotavirus replication.
EXAMPLES
[0153] The following examples illustrate specific embodiments and/or features of
the nutritional compositions 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.
[0154] 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.
EXAMPLES 1-5
[0155] 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.
AN = as needed
EXAMPLES 6-10
[0156] 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
6' sialyllactose (6'SL) 0.0948 0.0901 0.0853 9.479 9.0047
Galactooligosaccharides (GOS) 8.63 8.63 8.63 8.63 8.63
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, K 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
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
[0157] Examples 11-15 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. 11 Ex. 12 Ex. 13 Ex. 14 Ex. 15
Water Q.S. Q.S. Q.S. Q.S. Q.S.
Condensed Skim Milk 157.67 157.67 157.67 157.67 157.67
Lactose 108.66 108.66 108.66 108.66 108.66
High oleic safflower oil 26.82 26.82 26.82 26.82 26.82
Soybean oil 20. 16 20.16 20. 16 20. 16 20. 16
Coconut oil 19.24 19.24 19.24 19.24 19.24
3' sialyllactose (3 'SL) 0.1 896 0.1 802 0.1706 18.958 18.009
Galactooligosaccharides (GOS) 17.67 17.67 17.67 17.67 17.67
Whey protein concentrate 12.20 12.20 12.20 12.20 12.20
Potassium citrate 1.277 1.277 1.277 1.277 1.277
Calcium carbonate 996.1 g 996.1 g 996.1 g 996.1 g 996.1 g
Soy lecithin 685.0 g 685.0 g 685.0 g 685.0 g 685.0 g
Monoglycerides 685.0 g 685.0 g 685.0 g 685.0 g 685.0 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 429.7 g 429.7 g 429.7 g 429.7 g 429.7 g
Ascorbic acid 293.8 g 293.8 g 293.8 g 293.8 g 293.8 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 173.3 g 173.3 g 173.3 g 173.3 g 173.3 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 57.50 g 57.50 g 57.50 g 57.50 g 57.50 g
Mixed carotenoid premix 41.90 g 4 1.90 g 4 1.90 g 4 1.90 g 4 1.90 g
10% Lycopene in Sunflower Oil 2.10 g 2.10 g 2.10 g 2.10 g 2.10 g
Lycopene 209.50 mg 209.50 mg 209.50 mg 209.50 mg 209.50 mg
High Oleic Sunflower Oil Q.S. Q.S. Q.S. Q.S. Q.S.
5% Lutein in Corn Oil 1.76 g 1.76 g 1.76 g 1.76 g 1.76 g
Lutein 87.99 mg 87.99 mg 87.99 mg 87.99 mg 87.99 mg
Corn Oil Q.S. Q.S. Q.S. Q.S. Q.S.
30% B-carotene in Corn Oil 153.63 mg 153.63 mg 153.63 mg 153.63 mg 153.63 mg
Beta-carotene 46.09 mg 46.09 mg 46.09 mg 46.09 mg 46.09 mg
Corn Oil Q.S. Q.S. Q.S. Q.S. Q.S.
High Oleic Sunflower Oil Q.S. Q.S. Q.S. Q.S. Q.S.
Sodium chloride 23.50 g 23.50 g 23.50 g 23.50 g 23.50 g
L-carnitine 6.40 g 6.40 g 6.40 g 6.40 g 6.40 g
Tricalcium phosphate AN AN AN AN AN
Potassium phosphate monobasic AN AN AN AN AN
Potassium hydroxide AN AN AN AN AN
AN = as needed
EXAMPLES 16-20
[0158] Examples 16-20 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. 16 Ex. 17 Ex. 18 Ex. 19 Ex. 20
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
HMO Mixture 0.0948 0.0901 0.0853 9.479 9.0047
6'sialyllactose (6'SL) 0.03 16 0.0300 0.0284 3.159 3.002
2'fucosyllactose (2'FL) 0.03 16 0.0300 0.0284 3.159 3.002
Lacto-N-neotetraose (LNnT) 0.03 16 0.0300 0.0284 3.159 3.002
Galactooligosaccharides (GOS) 8.63 8.63 8.63 8.63 8.63
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
Mixed carotenoid premix 26.40 g 26.40 g 26.40 g 26.40 g 26.40 g
Lycopene 132.00 mg 132.00 mg 132.00 mg 132.00 mg 132.00 mg
Lutein 55.44 mg 55.44 mg 55.44 mg 55.44 mg 55.44 mg
B-carotene 29.04 mg 29.04 mg 29.04 mg 29.04 mg 29.04 mg
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 21-25
[0159] Examples 21-25 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. 21 Ex. 22 Ex. 23 Ex. 24 Ex. 25
Water Q.S. Q.S. Q.S. Q.S. Q.S.
Condensed Skim Milk 157.67 157.67 157.67 157.67 157.67
Lactose 108.66 108.66 108.66 108.66 108.66
High oleic safflower oil 26.82 26.82 26.82 26.82 26.82
Soybean oil 20. 16 20.16 20. 16 20. 16 20. 16
Coconut oil 19.24 19.24 19.24 19.24 19.24
HMO Mixture 18.957 18.009 17.061 19.905 20.853
6'sialyllactose (6'SL) 6.3 19 6.003 5.687 6.635 6.95 1
2'fucosyllactose (2'FL) 6.3 19 6.003 5.687 6.635 6.95 1
Lacto-N-neotetraose (LNnT) 6.3 19 6.003 5.687 6.635 6.95 1
Galactooligosaccharides (GOS) 17.67 17.67 17.67 17.67 17.67
Whey protein concentrate 12.20 12.20 12.20 12.20 12.20
Potassium citrate 1.277 1.277 1.277 1.277 1.277
Calcium carbonate 996.1 g 996.1 g 996.1 g 996.1 g 996.1 g
Soy lecithin 685.0 g 685.0 g 685.0 g 685.0 g 685.0 g
Monoglycerides 685.0 g 685.0 g 685.0 g 685.0 g 685.0 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 429.7 g 429.7 g 429.7 g 429.7 g 429.7 g
Ascorbic acid 293.8 g 293.8 g 293.8 g 293.8 g 293.8 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 173.3 g 173.3 g 173.3 g 173.3 g 173.3 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 57.50 g 57.50 g 57.50 g 57.50 g 57.50 g
Mixed carotenoid premix 41.90 g 4 1.90 g 4 1.90 g 4 1.90 g 4 1.90 g
10% Lycopene in Sunflower Oil 2.10 g 2.10 g 2.10 g 2.10 g 2.10 g
Lycopene 209.50 mg 209.50 mg 209.50 mg 209.50 mg 209.50 mg
High Oleic Sunflower Oil Q.S. Q.S. Q.S. Q.S. Q.S.
5% Lutein in Corn Oil 1.76 g 1.76 g 1.76 g 1.76 g 1.76 g
Lutein 87.99 mg 87.99 mg 87.99 mg 87.99 mg 87.99 mg
Corn Oil Q.S. Q.S. Q.S. Q.S. Q.S.
30% B-carotene in Corn Oil 153.63 mg 153.63 mg 153.63 mg 153.63 mg 153.63 mg
Beta-carotene 46.09 mg 46.09 mg 46.09 mg 46.09 mg 46.09 mg
Corn Oil Q.S. Q.S. Q.S. Q.S. Q.S.
High Oleic Sunflower Oil Q.S. Q.S. Q.S. Q.S. Q.S.
Sodium chloride 23.50 g 23.50 g 23.50 g 23.50 g 23.50 g
L-carnitine 6.40 g 6.40 g 6.40 g 6.40 g 6.40 g
Tricalcium phosphate AN AN AN AN AN
Potassium phosphate monobasic AN AN AN AN AN
Potassium hydroxide AN AN AN AN AN
AN = as needed
EXAMPLES 26-30
[0160] Examples 26-30 illustrate human milk fortifier liquids 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. 26 Ex. 27 Ex. 28 Ex. 29 Ex. 30
Water Q.S. Q.S. Q.S. Q.S. Q.S.
Non-fat milk (28% solids) 353 353 353 353 353
Corn Syrup Solids 85.3 85.3 85.3 85.3 85.3
Medium Chain Triglycerides 53.2 53.2 53.2 53.2 53.2
Whey Protein Concentrate 47.2 47.2 47.2 47.2 47.2
HMO Mixture 18.957 18.009 17.061 19.905 20.853
6'sialyllactose (6'SL) 6.3 19 6.003 5.687 6.635 6.95 1
2'fucosyllactose (2'FL) 6.3 19 6.003 5.687 6.635 6.95 1
Lacto-N-neotetraose (LNnT) 6.3 19 6.003 5.687 6.635 6.95 1
Calcium Phosphate 25.5 25.5 25.5 25.5 25.5
Ascorbic Acid 5.6 5.6 5.6 5.6 5.6
Potassium Citrate 3.1 3.1 3.1 3.1 3.1
Magnesium Chloride 2.8 2.8 2.8 2.8 2.8
Sodium Citrate 1.4 1.4 1.4 1.4 1.4
Sodium Chloride 1.4 1.4 1.4 1.4 1.4
Soy Lecithin 609 g 609 g 609 g 609 g 609 g
M-Inositol 500 g 500 g 500 g 500 g 500 g
Niacinamide 400 g 400 g 400 g 400 g 400 g
ARA Oil 313 g 313 g 313 g 313 g 313 g
Tocopherol Acetate 310 g 310 g 310 g 310 g 310 g
Zinc Sulfate 300 g 300 g 300 g 300 g 300 g
Calcium Pantothenate 182 g 182 g 182 g 182 g 182 g
Ferrous Sulfate 133 g 133 g 133 g 133 g 133 g
DHA Oil 116 g 116 g 116 g 116 s 116 s
Vitamin A Palmitate 100 g 100 g 100 g 100 g 100 g
Cupric Sulfate 51.0 g 51.0 51.0 51.0 s 51.0 s
Thiamine Hydrochloride 50.0 g 50.0 g 50.0 g 50.0 g 50.0 g
Riboflavin 47.0 g 47.0 g 47.0 g 47.0 g 47.0 g
Pyridoxine Hydrochloride 27.0 g 27.0 g 27.0 g 27.0 g 27.0 g
Vitamin D3 20.0 g 20.0 g 20.0 g 20.0 g 20.0 g
Folic Acid 3.5 g 3.5 g 3.5 g 3.5 g 3.5 g
Biotin 3.4 g 3.4 g 3.4 g 3.4 g 3.4 g
Manganous Sulfate 1.5 g 1.5 g 1.5 g 1.5 g 1.5 g
Phylloquinone 1.2 g 1.2 g 1.2 g 1.2 g 1.2 g
Cyanocobalamin 100 mg 100 mg 100 mg 100 mg 100 mg
Sodium Selenate 43.0 mg 43.0 mg 43.0 mg 43.0 mg 43.0 mg
EXAMPLES 31-35
[0161] Examples 31-35 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.
Ingredient Ex. 31 Ex. 32 Ex. 33 Ex. 34 Ex. 35
Condensed Skim Milk 698.5 698.5 698.5 698.5 698.5
Lactose 386.0 386.0 386.0 386.0 386.0
High oleic safflower oil 114.4 114.4 114.4 114.4 114.4
Soybean oil 85.5 1 85.5 1 85.5 1 85.5 1 85.51
Coconut oil 78.76 78.76 78.76 78.76 78.76
3' sialyllactose (3 'SL) 0.3792 0.3604 0.3412 37.916 36.0188
Galactooligosaccharides (GOS) 69.50 69.50 69.50 69.50 69.50
Whey protein concentrate 51.08 51.08 51.08 51.08 51.08
Potassium citrate 9.168 9.168 9.168 9.168 9.168
Calcium carbonate 4.054 4.054 4.054 4.054 4.054
Soy lecithin 1.120 1.120 1.120 1.120 1.120
ARA oil 2.949 2.949 2.949 2.949 2.949
Nucleotide/chloride premix 2.347 2.347 2.347 2.347 2.347
Potassium chloride 1.295 1.295 1.295 1.295 1.295
Ascorbic acid 1.275 1.275 1.275 1.275 1.275
Vitamin mineral premix 1. 1 16 1. 1 16 1. 1 16 1.116 1. 1 16
DHA oil 1. 1 13 1. 1 13 1.113 1.1 13 1.113
Magnesium chloride 1.038 1.038 1.038 1.038 1.038
Sodium chloride 579.4 g 579.4 g 579.4 g 579.4 g 579.4 g
Ferrous sulfate 453.6 g 453.6 g 453.6 g 453.6 g 453.6 g
Choline chloride 432.1 g 432.1 g 432.1 g 432.1 g 432.1 g
Vitamin A, D3, E, ¾ premix 377.2 g 377.2 g 377.2 g 377.2 g 377.2 g
Ascorbyl Palmitate 361 .3 g 361 .3 g 361 .3 g 361 .3 g 361 .3 g
Mixed carotenoid premix 350.1 g 350.1 g 350.1 g 350.1 g 350. 1 g
Mixed Tocopherols 159.2 g 159.2 g 159.2 g 159.2 g 159.2 g
L-carnitine 26.30 g 26.30 g 26.30 g 26.30 g 26.30 g
Riboflavin 3.1 8 1 g 3.18 1 g 3.1 8 1 g 3.1 8 1 g 3.1 8 1 g
Tricalcium phosphate 0-5.23 0-5.23 0-5.23 0-5.23 0-5.23
Potassium phosphate monobasic 0-5.23 0-5.23 0-5.23 0-5.23 0-5.23
Potassium hydroxide AN AN AN AN AN
AN = as needed
EXAMPLES 36-40
[0162] Examples 36-40 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. 36 Ex. 37 Ex. 38 Ex. 39 Ex. 40
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
HMO Mixture 0.0948 0.0901 0.0853 9.479 9.0047
6'sialyllactose (6'SL) 0.0379 0.0379 0.0379 0.0379 0.0379
3'sialyllactose (3 'SL) 0.0095 0.0095 0.0095 0.0095 0.0095
Sialic Acid 0.0474 0.0474 0.0474 0.0474 0.0474
Galactooligosaccharides (GOS) 8.63 8.63 8.63 8.63 8.63
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, K 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
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 41-45
[0163] Examples 41-45 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
EXAMPLE 46
[0164] In this Example, the effect of purified human milk oligosaccharides
(HMO) on in vitro inhibition of viral infectivity is analyzed.
[0165] Samples are prepared by co-incubation of a uniform virus dose of from
about 500 units/mL to about 1,000 units/mL of one of three respiratory viruses: (1)
respiratory syncytial virus (RSV); (2) human parainfluenza virus (HPIV3); or (3) HlNl
influenza virus with one of the following HMOs: (1) 3'-sialyllactose (3'SL); (2) 6'-
sialyllactose (6'SL); (3) 3'-fucosyllactose (3'FL); (4) 2'-fucosyllactose (2'FL); (5) lacto-Nneotetraose
(LNnT); or (6) sialic acid (SA). The HMOs are added at concentrations of
either 1 mg/mL or 10 mg/mL. The antiviral activities of the various HMOs on the
respiratory viruses are evaluated, and the results are shown in the table below:
NT = Not Tested
[0166] The results show that 3'FL, at a concentration of 1mg/ML (IC50 ~2-5
mg/ML), has anti-viral activity for all three respiratory viruses. This result is unexpected
as previous published reports show only sialylated oligo forms providing antiviral activity
SA significantly inhibits HPIV3 and HlNl viruses at a concentration of 1 mg/mL. HlNl
influenza virus is also inhibited by 3'SL at a concentration of 1 mg/mL.
EXAMPLE 47
[0167] In this Example, the ability of various HMOs to block H1N1 influenza
virus infectivity in vitro is analyzed.
[0168] Virus infectivity is assessed by observing cytopathic effect (CPE) and
quantifying virus focus forming units. To create virus stocks, H1N1 influenza virus is
purchased from ATCC (VR 1469) and expanded in Madin-Darby Canine Kidney (MDCK)
epithelial cells (ATCC CCL-34). Cell-free supernatants are frozen in aliquots to maintain
stock virus. During initial virus culture and expansion to create virus stocks, cell CPE is
observed.
[0169] To quantify virus infectivity, an immunocytochemical focus forming unit
(FFU) assay is developed using commercially purchased mouse monoclonal antibodies
against the virus nucleoprotein coupled with a biotinylated anti-mouse IgG secondary
antibody. To visualize virus-infected cell foci, color development is performed using
Strepavidin HRP (ABC from Vector Laboratories, Inc.). Although the total number of
virus foci appear proportional to the infecting virus concentration, the foci are quite large,
disperse, and there are numerous individually infected cells that do not form foci,
especially at higher virus concentrations. As this makes quantifying of virus infectivity
difficult and time-consuming, the FFU assay is further refined by varying virus
concentration and by applying an overlay medium of Tragacanth gum to help reduce
Brownian movement spread of the virus throughout the cell layer.
[0 170] The use of Tragacanth gum improves the assay by reducing the number of
individually infected cells while still allowing for the formation of readily observable foci.
While the foci vary in size, with some being quite large, they are still easily quantified and
directly proportional to virus concentration or titer by using a grid technique during the
enumeration.
[0171] Once verified, the assay is used with various HMOs for the ability to block
H1N1 virus infectivity. Specifically, the HMOs are added, at concentrations of 0.01
mg/mL, 0.1 mg/mL, 1.0 mg/mL, and 10 mg/mL, to the inoculating virus suspension,
incubated at 37°C for one hour, and then added to MDCK monolayer cells. This mixture is
allowed to bind to the cell layer for thirty minutes at 37°C. The cell layer is then washed,
and the cells are further incubated for approximately 18-24 hours before fixing and
processing for immunocytochemical staining. The results are shown in FIG. 1.
[0172] As shown in FIG. 1, 3'FL, 3'SL, and SA each inhibit virus infectivity by
greater than 90% when used at a concentration of 10 mg/mL. 2'FL and 6'SL inhibit
infectivity by approximately 60% at 10 mg/mL.
EXAMPLE 48
[0173] In this Example, nutritional compositions including various HMOs are
evaluated for their effects on reducing oxidative stress in preterm piglets.
[0174] Preterm piglets are harvested by caesarian section (CS) at 92% of
gestation. Piglets receive total parenteral nutrition (TPN) for 48 hours. After 48 hours,
TPN is ceased and the piglets are randomized into three groups: a formula group (n=7) that
is fed Enfamil® Lacto-Free, commercially available from Mead Johnson, Evansville, IN; a
treatment group (n=9) that is fed Enfamil® Lacto-Free with the addition of a combination
of 400 mg/L 6'SL, 1500 mg/L 2'FL, and 200 mg/L LNnT; and a colostrum group (n=5)
that is fed bovine colostrum. Piglets are fed their respective feeding enterally at a rate of
120 mL formula per kg body weight for the next 48 hours. Piglets are then euthanized after
48 hours of enteral nutrition (EN), or earlier if a piglet develops signs of necrotizing
enterocolitis. Blood is collected via an umbilical artery catheter, and plasma is separated
from the blood and stored at -70°C until analyzed.
[0175] Glutathione (GSH) concentrations are measured in plasma taken from the
piglets just prior to feeding time (time 0), and at 6 hours, 12 hours, 24 hours, 36 hours, and
48 hours after feeding using a commercially available assay (NWLSS Glutathione Assay
#NWK-GSH01, Northwest Life Science Specialties, Vancouver, WA). The results are
shown in FIG. 2.
[0176] As shown in FIG. 2, the concentration of GSH in blood plasma from the
control group declines from time 0 to 6 hours after feeding. GSH remains lower in the
control group 24 hours after EN. In contrast, piglets fed the composition with a
combination of HMOs have a pattern of blood plasma GSH levels that are comparable to
the colostrum piglets.
EXAMPLE 49
[0177] In this Example, the abilities of3'SL, 6'SL, and LNnT to reduce virusinduced
inflammation in vitro are demonstrated.
[0178] Specifically, either 3'SL or 6'SL is added, at concentrations of 0.1 mg/mL,
0.2 mg/mL, or 0.5 mg/mL to fresh peripheral blood mononuclear cells and incubated at
37°C in 5% C0 2 to pretreat the cells for approximately 24 hours. LNnT is added, at
concentrations of 0.1 mg/mL, 0.2 mg/mL, or 1 mg/mL to fresh peripheral blood
mononuclear cells and incubated at 37°C in 5% C0 2 to pretreat the cells for approximately
24 hours. Lactose is included as a carbohydrate control. Matched endotoxin unit
concentration controls are included to allow differentiation of ingredient effects from
inherent low levels of endotoxin. Some variables are then incubated with RSV at a
multiplicity of infection (MOI) of 0.1 for approximately 1 hour at 37°C in 5% in C0 2.
Uninfected control variables are incubated with medium for approximately 1 hour at 37°C
in 5% C0 2. Supernatants are collected at 24 and 48 hours post-infection.
[0179] Cytokines are measured in supernatants for each variable at 24 and 48
hours to assess the effects of HMOs on the early immune response to RSV. Cytokines are
measured using custom Bio-Plex Human cytokine kits from Bio-Rad. Results for
interferon-inducible protein 10 (IP- 10, also known as CXCL 10) are shown in FIGS. 3 and
4 for 3'SL and 6'SL, and in FIGS. 5 and 6 for LNnT. IP-10 is a CXC chemokine that
attracts, binds to and activates the CXCR3 receptor on natural killer cells and memory T
cells. IP-10 is expressed by monocytes and a number of other cells, and is induced by
interferon. A positive correlation exists between RSV clinical disease severity in children
(as measured by: length of hospital stay, fever, and number of days supplemental 0 2 is
required) and serum IP-10. Therefore, a decrease in IP-10 signals a decrease in severity of
RSV disease experienced.
[0180] IP- 10 results for 3'SL and 6'SL are detailed in FIGS. 3 and 4 and show
some variability in donor response, but surprisingly, 6'SL clearly downregulates IP-10 in
virus-infected variables in both donors. Note that 6'SL is able to reduce IP-10 to levels
found in uninfected controls. 3'SL is not effective in Donor B, but downregulates RSVinduced
IP-10 in Donor E. These data show both 3'SL and 6'SL dampen RSV-induced IP-
10, but that 6'SL is more effective at downregulation of IP-10. Results also suggest that
levels below 0.1 mg/mL of 6'SL as well as levels greater than 0.5 mg/mL may be effective
at reducing IP- 10 in some individuals.
[0181] IP- 10 results for LNnT are detailed in FIGS. 5 and 6 and show some
variability in donor response, but surprisingly, LNnT clearly downregulates IP- 10 in virusinfected
variables in both donors. Note that LNnT is able to reduce IP- 10 to levels found in
uninfected controls. Results also suggest that levels between 0.2 and 1mg LNnT/mL as
well as greater than 1 mg/mL may be effective at reducing IP- 10 in some individuals.
Inclusion of matched endotoxin unit concentration controls clearly indicates that the
decrease in IP- 10 is not due to the presence of very low levels of endotoxin in the LNnT.
[0182] In FIGS. 7 and 8, cytokine results also surprisingly show 6'SL increases
interleukin 10 (IL-10) concentration in a dose-dependent manner in the presence or absence
of RSV. IL-10 results for LNnT are shown in FIGS. 9 and 10. Surprisingly, LNnT
increases IL-10 concentration in a dose-dependent manner in the presence or absence of
RSV. IL-10 is produced by activated CD8+ T-cells, by CD4+ T-cells after both antigenspecific
and polyclonal activation, and by monocytes following cell activation by bacterial
lipopolysaccharides. Inclusion of matched endotoxin unit concentration controls clearly
differentiates that the increase in IL-10 is not due to the presence of very low levels of
endotoxin in the 6'SL or the LNnT.
[0183] Surprisingly, it is found that pretreatment for 24 hours by 6'SL, 3'SL, or
LNnT is effective in reducing inflammation caused by RSV. Moreover, 6'SL and LNnT
are shown to be more effective than 3'SL at dampening virus-induced inflammation as
measured by a decrease in IP- 10. Further, it is shown that 6'SL is immunomodulatory in
the absence of the virus, as the inclusion of 6'SL induces and/or modifies the production of
monocyte-derived cytokines such as IL-10, MIR -I b, Interferon-g , IL-8, IL-la, IL- I b, and
IL-lra. Surprisingly, 3'SL is also immunomodulatory in the presence or absence of the
virus, as the inclusion of 3'SL induces and/or modifies the production of monocyte-derived
cytokines such as MIR -I b, Interferon-g , IL-8, and IL-lra. Surprisingly, LNnT is also
immunomodulatory in the presence or absence of the virus, as the inclusion of LNnT
induces and/or modifies the production of monocyte-derived cytokines such as IL-10, MIP-
1b, Interferon-Y, IL-8, IL-la, IL- I b, and IL-lra.
EXAMPLE 50
[0184] In this Example, the ability of the combination of HMOs and nucleotides
to reduce rotavirus replication is demonstrated. Specifically, HMO and nucleotide
treatment within the ileal lumen of both healthy and acutely rotavirus-infected piglets is
analyzed.
[0185] Two groups of piglets (both n=9) are fed from age 48 hours to age 1 days
either a low nucleotide formula comprising about 72 mg/L of a nucleotide mixture or a
high nucleotide formula comprising about 320 mg/L of a nucleotide mixture.
[0186] At day 21, a midline laparotomy is performed and six loops of ilium are
isolated in situ. Each loop is about 10 cm long and is followed by an inter-loop segment of
about 2 cm. Ligation is conducted by an intestinal circumferential ligature through the
mesentery without damaging grossly visible mesenteric vascular arcades, and thus
maintaining full blood supply for both loops and inter-loop segments.
[0187] The following treatments are injected into the loops: media; LNnT; an
acidic HMO mixture comprising about 40 wt% 6'SL, about 10 wt% 3'SL, and about 50
wt% sialic acid; media and rotavirus; LNnT and rotavirus; and the acidic HMO mixture
comprising about 40 wt% 6'SL, about 10 wt% 3'SL, and about 50 wt% sialic acid and
rotavirus. In each treatment containing HMOs, the HMOs are included in a total
concentration of about 2 mg/mL of treatment solution. In each group containing rotavirus,
the rotavirus is included in a concentration of about 107 foci forming units in the treatment
solution. Each loop is treated for 6 hours, and post-treatment reverse transcriptase products
from ilium mucosal cells are analyzed by quantitative real-time PCR in TaqMan gene
expression assays of the target genes NSP4, IL-8, and TNF-a. Reference cDNA ribosomal
protein L19 (RPL19) is used as an endogenous control. The expression levels of genes of
interest are standardized to RPL19 mRNA and expressed as fold differences which
correspond to a ratio to the mean value for the control group.
[0188] As shown in FIG. 11, loops from both the low nucleotide formula fed
group and the high nucleotide formula fed group that are treated with LNnT and rotavirus
or with the acidic HMO mixture and rotavirus have a reduced NSP4 expression, thus
indicating a lower rotavirus replication, as compared to the loops treated with RV and
media (i.e., non-HMO treated groups). In addition, as can be seen from FIG. 11,
surprisingly there was a more significant reduction in the expression of NSP4 in the loops
from the group fed the low nucleotide formula as compared to the loops from the group fed
the high nucleotide formula. Further, ileal loops not treated with rotavirus show no NSP4
expression, and there is no difference in cytokine mRNA expression in ileal mucosa as
analyzed by loop or dietary treatment. As such, it can be concluded that both neutral
HMOs (i.e., LNnT) and acidic HMOs (i.e., 3'SL, 6'SL, and sialic acid) in combination
with nucleotides, even at a low level, reduce rotavirus replication.

WHAT IS CLAIMED IS:
1. A synthetic pediatric formula comprising from about 0.001 mg/mL to about 20
mg/mL of a human milk oligosaccharide and from about 5 mg/L to about 350 mg/L of a
nucleotide.
2. The synthetic pediatric formula of claim 1, wherein the human milk
oligosaccharide is a neutral human milk oligosaccharide.
3. The synthetic pediatric formula of claim 2, wherein the neutral human milk
oligosaccharide is lacto-N-neotetraose.
4. The synthetic pediatric formula of claim 1, wherein the human milk
oligosaccharide is an acidic human milk oligosaccharide.
5. The synthetic pediatric formula of claim 4, wherein the acidic human milk
oligosaccharide is selected from the group consisting of 3'-sialyllactose, 6'sialyllactose,
sialic acid, and combinations thereof.
6. The synthetic pediatric formula of claim 5, wherein the human milk
oligosaccharide comprises about 40 wt% 6'-sialyllactose, about 10 wt% 3'-sialyllactose,
and about 50 wt% sialic acid.
7. The synthetic pediatric formula of claim 1, wherein the nucleotide is selected
from the group consisting of cytidine 5'-monophosphate, uridine 5'-monophosphate,
adenosine 5'-monophosphate, guanosine 5'-monophosphate, and combinations thereof.
8. The synthetic pediatric formula of claim 7, wherein the nucleotide comprises
about 43% cytidine 5'-monophosphate, about 18.5% uridine 5'-monophosphate, about
16 .5% adenosine 5'-monophosphate, and about 22%> guanosine 5'-monophosphate.
9. The synthetic pediatric formula of claim 7, wherein the nucleotide comprises
cytidine 5'-monophosphate in a concentration of about 29-39 mg/L, uridine 5'-
monophosphate in a concentration of about 15-20 mg/L, adenosine 5'-monophosphate in a
concentration of about 10-15 mg/L, and guanosine 5'-monophosphate in a concentration of
about 14-20 mg/L.
10. The synthetic pediatric formula of claim 9, wherein the weight ratio of cytidine
5'-monophosphate to uridine 5'-monophosphate is from about 1.5:1 to about 2.6:1, the
weight ratio of cytidine 5'-monophosphate to adenosine 5'-monophosphate is from about
2:1 to about 3.9:1, and the weight ratio of cytidine 5'-monophosphate to guanosine 5'-
monophosphate is from about 1.75:1 to about 2.8:1.
11. The synthetic pediatric formula of claim 1, wherein the nucleotides are present
in a concentration of from about 40 mg/L to about 320 mg/L.
12. The synthetic pediatric formula of claim 1, wherein the nucleotides are present
in a concentration of from about 72 mg/L to about 320 mg/L.
13. A method for treating and/or preventing enteric viral infection in an infant,
toddler, or child, the method comprising administering to an infant, toddler, or child a
composition comprising from about 0.001 mg/mL to about 20 mg/mL of a human milk
oligosaccharide and from about 5 mg/L to about 350 mg/L of a nucleotide.
14. The method of claim 13, wherein the enteric viral infection is rotavirus induced
infection.
15. The method of claim 13, wherein the human milk oligosaccharide is a neutral
human milk oligosaccharide.
16. The method of claim 15, wherein the neutral human milk oligosaccharide is
lacto-N-neotetraose.
17. The method of claim 13, wherein the human milk oligosaccharide is an acidic
human milk oligosaccharide.
18. The method of claim 17, wherein the acidic human milk oligosaccharide is
selected from the group consisting of 3'-sialyllactose, 6'sialyllactose, sialic acid, and
combinations thereof.
19. The method of claim 13, wherein the nucleotide is selected from the group
consisting of cytidine 5'-monophosphate, uridine 5'-monophosphate, adenosine 5'-
monophosphate, guanosine 5'-monophosphate, and combinations thereof.
20. The method of claim 19, wherein the nucleotide comprises about 43% cytidine
5'-monophosphate, about 18.5% uridine 5'-monophosphate, about 16.5% adenosine 5'-
monophosphate and about 22% guanosine 5'-monophosphate.