WO2026039306A1 - Betaine and folate for promoting infant brain maturation - Google Patents

Abstract

Provided herein are methods and compositions for improving at least one of brain development, brain maturation, and cognition in an infant, toddler, or child. The compositions include betaine, folate, and combinations thereof, in an amount effective to enhance markers of cognition and brain and/or neuronal development.

Description

Atty Ref. 15782WOO1

BETAINE AND FOLATE FOR PROMOTING INFANT BRAIN MATURATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to European Patent Application No. EP24382902.5, filed on August 12, 2024, the entirety of which is incorporated by reference herein.

FIELD

[0002] The present disclosure generally relates to methods and compositions for promoting development and maturation of brain cells in an individual such as an infant, toddler, or child. More particularly, the present disclosure relates to a composition that includes sources of betaine, folate, and combinations thereof, that is useful for promoting the development and maturation of brain cells in an individual such as an infant, toddler, or child.

BACKGROUND

[0003] The brain is the center of the nervous system in all vertebrates. The human brain requires a level of additional development and maturation after birth that is not seen in most animals. As the core of the nervous system and the organ that governs the function of all other organs, less than optimum or diminished brain maturation can impact all aspects of health and well-being. Accordingly, healthy brain development and maturation is a key component to maintaining and further enhancing health and well-being.

[0004] One-Carbon (1C) metabolites are a group of nutrients that are also known as methyl donors, including betaine, S-adcnosylmcthioninc, folate, choline, methionine, and a few others. They are involved in methylation, which is the biochemical reaction of transferring one carbon or methyl-group to DNA, proteins, or other molecules. This mechanism is critical for many important physiological processes, such as epigenetic regulation (DNA methylation), neurotransmitter metabolism, and others. While some methyl donors are synthesized in the body, it is believed nutritional supplementation can enhance some biological functions that rely on methyl donors.

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SUMMARY

[0005] Disclosed herein are compositions that can be used to enhance infant brain function, maturation, and development. The compositions include betaine and folate in an amount effective to impart an improvement of neuronal health, function, maturation, and development. In certain aspects, the general inventive concepts also contemplate nutritional compositions including betaine, folate, and combinations thereof in an amount sufficient to treat or alleviate the symptoms of underdeveloped infant brain function, maturation, and development (i.e., that which is behind milestone development). In other words, the compositions disclosed herein enhance infant brain development and facilitate “catch-up” development and maturation in infants, toddlers, and children in need thereof.

[0006] In accordance with embodiments of the present disclosure, the nutritional composition, such as an infant formula, comprises macronutrients such as protein, fat, and carbohydrate, and betaine in an amount of at least about 3.5 mg/L, at least about 4.0 mg/L, at least about 5.0 mg/L, or at least about 6.0 mg/L, including for example between about 4.0 mg/L and about 14.0 mg/L, between about 4.0 mg/L and about 12.0 mg/L, between about 4.0 mg/L and about 10.0 mg/L, between about 5.0 mg/L and about 14.0 mg/L, between about 5.0 mg/L and about 12.0 mg/L, between about 6.0 mg/L and about 14.0 mg/L, or between about 6.0 and about 12.0 mg/L of the nutritional composition. The nutritional composition also comprises folate, such as in an amount of at least about 100 mcg/L, including for example between about 100 mcg/L and about 300 mcg/L, of the nutritional composition. Administration of the composition improves or enhances brain development and/or cognition in the individual, and more particularly an infant, toddler, or child.

[0007] In accordance with the present disclosure, a method of promoting brain development in an infant, toddler, or child is provided. The method includes administering a nutritional composition comprising an effective amount of betaine and folate to the infant, toddler, or child. Administration of the composition improves or enhances brain function and/or maturation in the individual. The promotion of brain development may be a function of promoting neuronal (brain cell) differentiation and/or maturation.

[0008] In accordance with the present disclosure, a method of promoting cognition in an infant, toddler, or child is provided. The method includes administering a nutritional composition

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4855-0747-4085, v.1 Atty Ref. 15782WOO1 comprising an effective amount of betaine and folate to the infant, toddler, or child. Administration of the composition improves or enhances cognition in the individual. The promotion of cognition may be a function of promoting neuronal (brain cell) differentiation and/or maturation.

[0009] In embodiments of the methods described herein, the nutritional composition may be a liquid or semi-liquid and may contain betaine in an amount of at least about 3.5 mg/L, at least about 4.0 mg/L, at least about 5.0 mg/L, or at least about 6.0 mg/L, including for example between about 4.0 mg/L and about 14.0 mg/L, between about 4.0 mg/L and about 12.0 mg/L, between about 4.0 mg/L and about 10.0 mg/L, between about 5.0 mg/L and about 14.0 mg/L, between about 5.0 mg/L and about 12.0 mg/L, between about 6.0 mg/L and about 14.0 mg/L, or between about 6.0 and about 12.0 mg/L of the nutritional composition.

[0010] In embodiments of the methods described herein, the nutritional composition may be a liquid or semi-liquid and may contain folate in an amount of at least about 100 mcg/L, including for example between about 100 mcg/L and about 300 mcg/L, of the nutritional composition.

[0011] In embodiments of the methods described herein, the nutritional composition may further include at least one of protein, carbohydrate, and fat. In embodiments of the methods described herein, and for instance when the nutritional composition is formulated to be a sole source of nutrition as is the case with an infant formula, the nutritional composition may include protein, carbohydrate, and fat. In some cases, for instance, the nutritional composition may include carbohydrate in an amount of from about 40% to about 60% by weight (on a dry weight basis); fat in an amount of from about 20% to about 40% by weight (on a dry weight basis); and protein in an amount of about 10% to about 25% by weight (on a dry weight basis).

[0012] In embodiments of the methods described herein, the nutritional composition may be administered to an infant. In some embodiments, for instance, the nutritional composition may be an infant formula.

[0013] In embodiments of the methods described herein, the nutritional composition may be a ready-to-drink liquid. In other embodiments of the methods described herein, the nutritional composition may be a liquid that is reconstituted from a reconstitutable powder.

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[0014] In embodiments of the methods described herein, the nutritional composition may further include one or more human milk oligosaccharides (HMOs). In some embodiments, the one or more HMOs may include at least one selected from the group of a fucosylated oligo saccharide, a sialylated oligosaccharide, an N-acetylglucosamine oligosaccharide, and any combination thereof. In some cases, the one or more HMOs may include a combination of at least one fucosylated oligosaccharide, at least one sialylated oligosaccharide, and at least one N-acetylglucosamine oligosaccharide. The fucosylated oligosaccharide may include at least one of 3-fucosyllactose (3- FL) and 2'-fucosyllactose (2'-FL) for example; the sialylated oligosaccharide may include at least one of 3’-sialyllactose (3'-SL) and 6' sialyllactose (6’-SL) for example; and the N- acetylglucosamine oligosaccharide may include at least one of lacto-N-neotetraose (LNnT) and lacto-N-tetraose (LNT) for example. In some instances, the one or more HMOs may include at least one selected from the group of 2'-fucosyllactose, 3-fucosyllactose, 3 '-sialyllactose, 6'- sialyllactose, and lacto-N-tetraose, and any combination thereof. In some cases, the one or more HMOs may include 2'-fucosyllactose, 3-fucosyllactose, 3 ’-sialyllactose, 6'-sialyllactose, and lacto- N-tetraose. In embodiments of the methods described above, the nutritional composition may contain HMOs in an amount between about 0.1 g/L and about 10 g/L.

[0015] In embodiments of the methods described herein, the nutritional composition may further include a long chain polyunsaturated fatty acid selected from arachidonic acid, docosahexaenoic acid, and a combination thereof.

[0016] In accordance with the present disclosure, a nutritional composition for use in promoting brain development, cognition, or both in an infant, toddler, or child is provided. The nutritional composition comprises an effective amount of betaine and folate. The promotion of brain development, cognition, or both may be a function of promoting neuronal (brain cell) differentiation and/or maturation.

[0017] In embodiments of the nutritional compositions for use in promoting brain development, cognition, or both in an infant, toddler, or child as described herein, the nutritional composition may be a liquid or semi-liquid and may contain betaine in an amount of at least about 3.5 mg/L, at least about 4.0 mg/L, at least about 5.0 mg/L, or at least about 6.0 mg/L, including for example between about 4.0 mg/L and about 14.0 mg/L, between about 4.0 mg/L and about 12.0 mg/L, between about 4.0 mg/L and about 10.0 mg/L, between about 5.0 mg/L and about 14.0 mg/L,

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4855-0747-4085, v.1 Atty Ref. 15782WOO1 between about 5.0 mg/L and about 12.0 mg/L, between about 6.0 mg/L and about 14.0 mg/L, or between about 6.0 and about 12.0 mg/L of the nutritional composition. Alternatively, in embodiments of the nutritional compositions for use in promoting brain development, cognition, or both in an infant, toddler, or child as described herein, the nutritional composition may be a reconstitutable powder and may contain betaine in an amount of at least 0.002 wt.%, optionally from about 0.002 wt.% to about 0.008 wt.%, based on the total weight of the composition.

[0018] In embodiments of the nutritional compositions for use in promoting brain development, cognition, or both in an infant, toddler, or child as described herein, the nutritional composition may be a liquid or semi-liquid and may contain folate in an amount of at least about 100 mcg/L, including for example between about 100 mcg/L and about 300 mcg/L, of the nutritional composition. Alternatively, in embodiments of the nutritional compositions for use in promoting brain development, cognition, or both in an infant, toddler, or child as described herein, the nutritional composition may be a reconstitutable powder and may contain folate in an amount of at least about 0.00006 wt.%, optionally from about 0.00006 wt.% to about 0.0003 wt.%, based on the total weight of the composition.

[0019] In embodiments of the nutritional compositions for use in promoting brain development, cognition, or both in an infant, toddler, or child as described herein, the nutritional composition may further include at least one of protein, carbohydrate, and fat. In embodiments of the nutritional compositions for use in promoting brain development, cognition, or both in an infant, toddler, or child as described herein, and for instance when the nutritional composition is formulated to be a sole source of nutrition as is the case with an infant formula, the nutritional composition may include protein, carbohydrate, and fat. In some cases, for instance, the nutritional composition may include carbohydrate in an amount of from about 40% to about 60% by weight (on a dry weight basis); fat in an amount of from about 20% to about 40% by weight (on a dry weight basis); and protein in an amount of about 10% to about 25% by weight (on a dry weight basis).

[0020] In embodiments of the nutritional compositions for use in promoting brain development, cognition, or both in an infant, toddler, or child as described herein, the infant, toddler, or child is an infant. In embodiments of the nutritional compositions for use in promoting brain development, cognition, or both in an infant, toddler, or child as described herein, the nutritional composition may be an infant formula.

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4855-0747-4085, v.1 Atty Ref. 15782WOO1

[0021 ] In embodiments of the nutritional compositions for use in promoting brain development, cognition, or both in an infant, toddler, or child as described herein, the nutritional composition may be a ready-to-drink liquid or a reconstitutable powder.

[0022] In embodiments of the nutritional compositions for use in promoting brain development, cognition, or both in an infant, toddler, or child as described herein, the nutritional composition may further include one or more human milk oligosaccharides (HMOs). In some embodiments, the one or more HMOs may include at least one selected from the group of a fucosylated oligosaccharide, a sialylated oligosaccharide, an N-acetylglucosamine oligosaccharide, and any combination thereof. In some cases, the one or more HMOs may include a combination of at least one fucosylated oligosaccharide, at least one sialylated oligosaccharide, and at least one N- acetylglucosamine oligosaccharide. The fucosylated oligosaccharide may include at least one of 3-fucosyllactose (3-FL) and 2'-fucosyllactose (2'-FL) for example; the sialylated oligosaccharide may include at least one of 3’-sialyllactose (3'-SL) and 6' sialyllactose (6’-SL) for example; and the N-acetylglucosamine oligosaccharide may include at least one of lacto-N-neotetraose (LNnT) and lacto-N-tetraose (LNT) for example. In some instances, the one or more HMOs may include at least one selected from the group of 2'-fucosyllactose, 3-fucosyllactose, 3 '-sialyllactose, 6'- sialyllactose, and lacto-N-tetraose, and any combination thereof. In some cases, the one or more HMOs may include 2'-fucosyllactose, 3-fucosyllactose, 3 ’-sialyllactose, 6'-sialyllactose, and lacto- N-tetraose. In embodiments of the nutritional compositions for use in promoting brain development, cognition, or both in an infant, toddler, or child described above, the nutritional composition may contain HMOs in an amount between about 0.1 g/L and about 10 g/L of the composition when in liquid form (e.g. as a ready-to-drink liquid or reconstituted from a reconstitutable powder).

[0023] In embodiments of the nutritional compositions for use in promoting brain development, cognition, or both in an infant, toddler, or child as described herein, the nutritional composition may further include a long chain polyunsaturated fatty acid selected from arachidonic acid, docosahexaenoic acid, and a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

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[0024] Figure 1 is a schematic showing the experimental design for the instant examples using Neuro 2a cells (N2a).

[0025] Figure 2 is a graphical representation of the results of Example 1, showing N2a proliferation after exposure to various concentrations of betaine.

[0026] Figure 3 is a graphical representation of the results of Example 1, showing N2a cell proliferation after exposure to betaine, folate (5-MTHF), and a combination thereof.

[0027] Figure 4 is a graphical representation of the results of Example 2, showing the percentage of cells with neurite outgrowth after exposure to betaine, folate, and the combination of betaine and folate.

[0028] Figure 5 is a graphical representation of the results of Example 3, showing the relative expression of MAP-2 after exposure to betaine, folate, and the combination of betaine and folate.

[0029] Figure 6 is a graphical representation of the results of Example 3, showing the relative expression of B3-tubulin after exposure to betaine, folate, and the combination of betaine and folate.

DETAILED DESCRIPTION

[0030] Disclosed herein are compositions that are useful for improving infant, toddler, or child cognition and/or brain development. While the present disclosure describes certain aspects of the compositions and associated methods in detail, the present disclosure is to be considered exemplary and is not intended to be limited to the disclosed aspects. Also, certain elements or features of aspects disclosed herein are not limited to a particular embodiment, but instead apply to all aspects of the present disclosure.

[0031] The compositions and methods disclosed herein utilize betaine and folate for improving neuronal function and development, including improving one or more markers of brain function and/or maturation. These and other features of the compositions and methods, as well as some of the many optional variations and additions, are described in detail hereafter.

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[0032] Without wishing to be bound by any particular theory, it is believed that the compositions of the present disclosure uprcgulatc expression of important processes in the infant body leading to increases and enhancements in neuronal development and brain maturation. This can lead to downstream improvements in cognition and memory, for example. The results shown herein demonstrate a role for betaine and folate in improving brain development and cognition for those demonstrating milestone achievement as well as providing catch-up development and downstream cognition benefits for individuals in need thereof. Importantly, existing literature and products do not recognize a role for the combination of betaine and folate in promoting neural differentiation and maturation or for the unexpected results achieved when the two ingredients are combined. As the present disclosure and examples demonstrate, the combination of betaine and folate has been shown to promote neuronal differentiation, neuron maturation and brain cell development.

[0033] The general inventive concepts contemplate administration of synthetic nutritional compositions comprising betaine and folate to enhance brain/neuronal maturation and development (e.g., via an increase in cell differentiation and/or neurite outgrowth, among others) in an individual, and in particular an infant, toddler, or child. These in turn lead to benefits and enhancements in cognition in the individual.

[0034] In certain exemplary aspects, administration of the claimed betaine and folate containing compositions ameliorates or avoids developmental delays on the part of the individual, and in particular infant, toddler, or child. In certain exemplary aspects, administration of the claimed betaine and folate containing compositions enhances cognition in an infant, toddler, or child., In certain exemplary aspects, administration of the claimed betaine and folate containing compositions enhances brain development and/or allows for catch-up development in the individual, and in particular an infant, toddler, or child.

[0035] In certain exemplary aspects, the compositions and methods facilitate an infant, toddler, or child to achieve or surpass cognitive and/or developmental milestones. Such milestones include, but are not limited to, attention, communication and language development, memory milestones, motor development, and social and emotional development among others. This development is demonstrated by e.g., early childhood screening or developmental screening.

Definitions

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[0036] 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 solids, nutritional semi-solids, nutritional semi-liquids, nutritional supplements, and any other nutritional food product as known in the art. The nutritional powders may be reconstituted to form a nutritional liquid, e.g. by the addition of water, all of which comprise one or more of fat, protein and carbohydrate and are suitable for oral consumption by a human and may be used as a sole or supplemental source of nutrition. The terms “nutritional formulation” or “nutritional composition” do not encompass human breast milk and do not refer to supplemented milk, whether of human origin or otherwise.

[0037] The term “nutritional liquid” as used herein, unless otherwise specified, refers to nutritional compositions in ready-to-drink liquid form, concentrated form, and nutritional liquids made by reconstituting the nutritional powders described herein prior to use.

[0038] The term “nutritional powder” as used herein, unless otherwise specified, refers to nutritional compositions in flowable or scoopable form that can be reconstituted with water or another aqueous liquid prior to consumption and includes both spray-dried and dry-mixed/dry- blended powders.

[0039] The term “nutritional semi-solid” as used herein, unless otherwise specified, refers to nutritional compositions that arc intermediate in properties, such as rigidity, between solids and liquids. Some semi-solids examples include puddings, gelatins, and doughs.

[0040] The term “nutritional semi-liquid” as used herein, unless otherwise specified, refers to nutritional compositions that are intermediate in properties, such as flow properties, between liquids and solids. Some semi-liquids examples include thick shakes and liquid gels.

[0041] The term “reconstitute” or various other forms such as “reconstituted” or “reconstituting” all refer to the general act of adding a suitable amount of liquid, typically water, to a composition that is not in a ready-to-drink liquid form, such as a nutritional powder or a concentrated form of a nutritional liquid, thereby making the composition ready-to-drink.

[0042] 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

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4855-0747-4085, v.1 Atty Ref. 15782WOO1 least 3 months, including from about 6 months to about 24 months, and also including from about 12 months to about 18 months.

[0043] The term “infant” as used herein refers to humans 12 months of age or younger. The term “preterm infant” as used herein refers to an infant bom prior to 36 weeks of gestation. The term “toddler” as used herein refers to a human greater than one year of age and up to three years of age. The term “child” as used herein refers to a person greater than three years of age and up to twelve years of age.

[0044] The terms “susceptible” and “at risk” as used herein, unless otherwise specified, mean having little resistance to a certain condition or disease relative to the general population (e.g., individuals in need thereof are those with impaired or slowed neuronal development), including being genetically predisposed, having a family history of, and/or having symptoms of the condition or disease. The term refers to those having a vulnerability higher than the general population. In certain instances, the individual is in need of a therapeutic intervention, e.g., has compromised or otherwise impaired neuronal development.

[0045] The terms “modulating” or “modulation” or “modulate” as used herein, unless otherwise specified, refer to the targeted movement of a selected characteristic.

[0046] The term “ameliorate” as used herein, unless otherwise specified, means to eliminate, delay, or reduce the prevalence or severity of symptoms associated with a condition or disease (e.g., preventing or avoiding developmental delay).

[0047] The term “brain development" or “brain maturation” as used herein, refers to neuronal development and meeting agreed upon milestones of at least one of motor skills, language development, attention, focus, learning, memory, social and emotional interaction, and I.Q., among others.

[0048] The term “an effective amount” is intended to qualify the amount of betaine and folate which will achieve a beneficial enhancement of neuronal (i.e. brain cell) development, brain development, and/or brain maturation and/or prevent hindered neuronal development, brain development, or brain maturation, over what would have been achieved in the absence of the intervention. The effective amount may be administered in one or more doses.

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[0049] The terms “treating” and “treatment” as used herein, unless otherwise specified, includes delaying the onset of a condition, reducing the severity of symptoms of a condition, or eliminating some or all of the symptoms of a condition. In certain aspects, symptoms of impaired neuronal development include, but are not limited to, developmental delays, including lack of reaching cognitive and/or developmental milestones (communication and language development, memory, motor development, and social and emotional development among others).

[0050] As used herein, all concentrations expressed as either “pg/liter,” “mg/liter,” “mg/L,” “g/L,” “mcg/L,” “mg/mL,” etc., refer to ingredient concentrations within the described compositions as calculated on an as-fed basis (e.g., reconstituted for consumption in the case of nutritional powders), unless otherwise specified.

[0051] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting the disclosure as a whole. 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. Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably. Furthermore, as used in the description and the appended claims, the singular forms “a,” “an,” and “the” are inclusive of their plural forms, unless the context clearly indicates otherwise.

[0052] To the extent that the term “includes” or “including” is used in the description or the claims, it is intended to be inclusive in a manner similar to the term “comprising” as that term is interpreted when employed as a transitional word in a claim. Furthermore, to the extent that the term “or” is employed (e.g., A or B) it is intended to mean “A or B or both.” When the applicants intend to indicate “only A or B but not both” then the term “only A or B but not both” will be employed. Thus, use of the term “or” herein is the inclusive, and not the exclusive use. Furthermore, when the phrase “one or more of A and B” is employed, it is intended to mean “only

A, only B, or both A and B.” Similarly, when the phrases “at least one of A, B, and C” or “at least one of A, B, C, and combinations thereof’ arc employed, they are intended to mean “only A, only

B, only C, or any combination of A, B, and C” (e.g., A and B; B and C; A and C; A, B, and C).

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[0053] The compositions and methods of the present disclosure 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 or feature described herein, or which is otherwise useful in nutritional composition applications.

[0054] The compositions of the present disclosure may be substantially free of any optional or selected ingredient or feature described herein, provided that the remaining composition or formula still contains all the required ingredients or features as described herein. In this context, and unless otherwise specified, the term “substantially free” means that less than a functional amount of the optional or selected ingredient is present, which is typically less than 0.1% by weight, and also including zero percent by weight of such optional or selected ingredient.

[0055] 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, by-products, or other components that may be included in commercially available materials, unless otherwise specified.

[0056] 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.

[0057] The term “about” as used herein means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by 10%.

[0058] 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.

[0059] The betaine and folate can be formulated in suitable compositions and then, in accordance with the methods of the invention, administered to an individual orally. Oral administration, as

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4855-0747-4085, v.1 Atty Ref. 15782WOO1 defined herein, includes any form of administration in which the betaine and folate component and associated functional ingredients pass through the esophagus of the individual. For example, oral administration includes nasogastric intubation, in which a tube is run from through the nose to the stomach of the patient to administer food or drugs.

Methyl Donors

[0060] One-Carbon (1C) metabolites are a group of nutrients that are also known as methyl donors, and which include betaine, S-adenosylmethionine, folate, choline, methionine, and a few others. They are involved in methylation, which is the biochemical reaction of transferring methylgroups to DNA, proteins, or other molecules. This mechanism is critical for many important physiological processes such as epigenetic regulation (DNA methylation), neurotransmitter metabolism, and others.

[0061] Breastmilk is a rich source of methyl donors, signifying that these nutrients are important for the growth and development of infants. In addition to supporting overall growth, choline, one of the most extensively studied methyl donors, is particularly recognized for its involvement in the synthesis of phosphatidylcholine and the neurotransmitter acetylcholine. These compounds are vital for brain development and cognitive function.

[0062] The specific mechanisms and benefits underlying the potential cognitive benefits of individual methyl donors, however, remain poorly understood. Additionally, there is limited knowledge regarding the interactions among various methyl donors in supporting neurocognitive development.

[0063] In view of this, Applicant endeavored to elucidate the roles of certain methyl donors in promoting neuronal differentiation and maturation, which is tightly associated with brain development and/or maturation as well as cognitive development. Betaine and folate are integral to a common biochemical pathway. Based on established knowledge of the one-carbon metabolism pathway, supplementing both betaine and folate is expected to increase methionine production, and eventually SAM, and thereby promote the progression of the one-carbon metabolism cycle. This might lead to the belief that simultaneous supplementation of betaine and folate would be redundant due to potential overlap. However, the present disclosure reveals a surprising synergy between betaine and folate to promote certain markers relating to neuronal development and maturation.

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[0064] The general inventive concepts are based, in part, on the discovery that administration of betaine and folate increases neuronal development associated with an improvement in brain development and/or maturation in infants, toddlers, and children. This in turn leads to cognitive benefits in the infants, toddlers, and children.

Betaine

[0065] The general inventive concepts are based, in part, on the discovery that administration of betaine, and more particularly the simultaneous administration of betaine and folate, produces a surprising improvement in neuronal development associated with brain function (e.g. cognition) and/or maturation in infants, toddlers, and children. Betaine, also known as Trimethylglycine (TMG), may be provided in any known form, including for example its natural form of Trimethylglycine anhydrous. While in general it is contemplated that at least a portion of the betaine may be added to the nutritional composition, e.g. infant formula, in some embodiments a portion of the total amount of betaine in the nutritional composition may be present from inherent sources, such as non-fat dry milk (also known as dry skim milk).

[0066] In certain exemplary aspects the nutritional compositions and methods contemplate providing betaine in an amount of at least 3.5 mg/L of nutritional composition, alternatively at least 4.0 mg/L of nutritional composition, alternatively at least 4.5 mg/L of nutritional composition, alternatively at least 5.0 mg/L of nutritional composition, alternatively at least 5.5 mg/L of nutritional composition, alternatively at least 6.0 mg/L of nutritional composition. This includes, for example, providing betaine in any amount between about 4.0 mg/L and about 20.0 mg/L, alternatively any amount between about 4.0 mg/L and about 14.0 mg/L, alternatively any among between about 4.0 mg/L and about 12.0 mg/L, alternatively any amount between about 4.0 mg/L and about 10.0 mg/L, alternatively any amount between about 5.0 mg/L and about 20.0 mg/L, alternatively any amount between about 5.0 mg/L and about 14.0 mg/L, alternatively any amount between about 5.0 mg/L and about 12.0 mg/L, alternatively any amount between about 5.0 mg/L and about 10.0 mg/L, alternatively any amount between about 6.0 mg/L and about 20.0 mg/L, alternatively any amount between about 6.0 mg/L and about 14.0 mg/L, alternatively any amount between about 6.0 mg/L and about 12.0 mg/L, alternatively any amount between about 6.0 mg/L and about 10.0 mg/L.

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[0067] Using a specific reconstitution ratio for an infant formula, one can calculate the amount of betaine in a rcconstitutablc powder composition, c.g. by weight percentage of the powder composition, that would produce a liquid formulation having betaine within the above-recited concentration range. As is understood in the art, higher caloric density formulas can be also prepared by using a higher ratio of powder to water or lower caloric density formulas can be prepared by using a lower ratio of powder to water. The ratio of powder to water to produce a formula of a desired caloric density will also depend on the specific composition, e.g. macronutrient distribution, of the formula.

[0068] Using an example reconstitution ratio of about 145 g/L, one can calculate the amount of betaine in a powdered composition, e.g. by weight percentage, that would provide a liquid concentration of betaine in the above ranges. In certain aspects, the composition of the present disclosure, when in reconstitutable powder form, may comprise betaine of from about 0.002 wt.% to about 0.008 wt.% based on the total weight of the composition. In certain aspects, the composition of the present disclosure, when in powder form, may comprise betaine of from about 0.002 wt.% to about 0.008 wt.% based on the total weight of the nutritional composition, including from about 0.002 wt.% to about 0.007 wt.%, including from about 0.002 wt.% to about 0.006 wt.%, including from about 0.002 wt.% to about 0.005 wt.%, including from about 0.003 wt.% to about 0.008 wt.%, including from about 0.003 wt.% to about 0.007 wt.%, including from about 0.003 wt.% to about 0.006 wt.%, including from about 0.004 wt.% to about 0.008 wt.%, including from about 0.004 wt.% to about 0.007 wt.%, including from about 0.004 wt.% to about 0.006 wt.%, based on the total weight of the nutritional composition.

Folate

[0069] The general inventive concepts are based, in part, on the discovery that simultaneous administration of betaine and folate produces a surprising improvement in neuronal development associated with brain function (e.g. cognition) and/or maturation in infants, toddlers, and children. Folate (also known as vitamin B-9) may be provided in any known form, including for example folic acid, methyl folate, or a combination thereof.

[0070] In certain exemplary aspects the nutritional compositions and methods contemplate providing folate in an amount of at least about 100 mcg/L, including for example between about

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4855-0747-4085, v.1 Atty Ref. 15782WOO1

100 mcg/L and about 500 mcg/L, between about 100 mcg/L and about 400 mcg/L, between about 100 mcg/L and about 350 mcg/L, between about 100 mcg/L and about 300 mcg/L, between about 100 mcg/L and about 250 mcg/L, or between about 100 mcg/L and about 200 mcg/L of the nutritional composition.

[0071] Using a specific reconstitution ratio for an infant formula, one can calculate the amount of folate in a reconstitutable powder composition, e.g. by weight percentage of the powder composition, that would produce a liquid formulation having folate within the above-recited concentration range. As is understood in the ail, higher caloric density formulas can be also prepared by using a higher ratio of powder to water or lower caloric density formulas can be prepared by using a lower ratio of powder to water. The ratio of powder to water to produce a formula of a desired caloric density will also depend on the specific composition, e.g. macronutrient distribution, of the formula.

[0072] Using an example reconstitution ratio of about 145 g/L, one can calculate the amount of folate in a powdered composition, e.g. by weight percentage, that would provide a liquid concentration of folate in the above ranges. In certain aspects, the composition of the present disclosure, when in reconstitutable powder form, may comprise folate of from about 0.00006 wt.% to about 0.0003 wt.% based on the total weight of the composition.

[0073] In certain exemplary aspects, the nutritional compositions and methods of the present disclosure utilize each of betaine and folate in an amount that, when combined with the amount of the other, is effective to promote brain or neuronal development and maturation in infants, toddlers, and children. In certain exemplary aspects the nutritional compositions and methods contemplate providing a combination of betaine and folate in an amount effective to improve cognition, improve brain maturation, improve brain development, improve early brain development, improve brain cell maturation, improve neuronal development, improve neurite outgrowth, improve cognitive development, treat insufficient brain development (by which is meant is facilitating development in the individual that is otherwise behind schedule or lacks milestone achievement), treat insufficient brain maturation, and/or treat insufficient cognitive development.

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4855-0747-4085, v.1 Atty Ref. 15782WOO1

Macronutrients

[0074] In addition to the specific functional ingredients described herein, the nutritional composition may in many embodiments, such as where the nutritional composition is an infant formula, include one or more ingredients that help satisfy the individual’s nutritional requirements. The optional nutrients can provide up to about 1000 kcal of energy per serving or dose, including from about 25 to about 900 kcal, from about 75 to about 700 kcal, from about 150 to about 500 kcal, from about 350 to about 500 kcal, or from about 200 to about 300 kcal.

[0075] 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.

[0076] The nutritional compositions may be formulated to include at least one of protein, fat, and carbohydrate. In many embodiments, the nutritional compositions will include protein, carbohydrate and fat. Although total concentrations or amounts of the fat, protein, and carbohydrates may vary depending upon the product type (i.e., nutritional formula), 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.

Nutritional Compositions

[0077] Nutritional compositions, as discussed herein, include at least one of fat, carbohydrate, and protein in addition to the sources of betaine and folate. Infant formulas, which are configured to provide sole source nutrition, will typically contain a combination of fat, carbohydrate, and protein.

[0078] Where present, carbohydrate concentrations most typically will range from about 10% to about 65%, including from about 20% to about 60%, including from about 30% to about 60%, by dry weight of the nutritional composition. Where present, fat concentrations most typically range from about 10% to about 45%, including from about 15% to about 40%, and also including from

17

4855-0747-4085, v.1 Atty Ref. 15782WOO1 about 20% to about 40%, by dry weight of the nutritional composition. Where present, protein concentrations most typically range from about 5% to about 30%, including from about 10% to about 30%, and also including from about 15% to about 25%, by dry weight of the nutritional composition. In some embodiments, an infant formula may include from about 40% to about 60% carbohydrates, from about 10% to about 25% protein, and from about 20% to about 40% fat by dry weight of the infant formula.

[0079] The amount of any or all of the carbohydrates, fats, and proteins in any of the nutritional compositions (e.g., infant formula) described herein may also be characterized as a percentage of total calories in the nutritional composition. In some embodiments, the nutritional composition (e.g. infant formula) may contain carbohydrates in an amount of from about 35% to about 50% as a percentage of total calories, protein in an amount of from about 5% to about 20% as a percentage of total calories, and fat in an amount of from about 35% to about 55% as a percentage of total calories.

Fat

[0080] The nutritional compositions according to the general inventive concepts may comprise a source or sources of fat. Suitable sources of fat for use herein include any fat or fat source that is suitable for use in an oral nutritional product and is compatible with the essential elements and features of such products. Most typically the fat may be an emulsified fat. In an exemplary embodiment, the fat is derived from short chain fatty acids.

[0081] 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. Lipid sources of arachidonic acid and docosahexaenoic acid include, but are not limited to, marine oil, egg yolk oil, and fungal or algal oil.

[0082] Numerous commercial sources for these fats are readily available and known to one practicing the art. For example, soy and canola oils are available from Archer Daniels Midland

18

4855-0747-4085, v.1 Atty Ref. 15782WOO1 of Decatur, Ill. Com, coconut, palm and palm kernel oils are available from Premier Edible Oils Corporation of Portland, Organ. Fractionated coconut oil is available from Henkel Corporation of LaGrange, Ill. High oleic safflower and high oleic sunflower oils are available from SVO Specialty Products of Eastlake, Ohio. Marine oil is available from Mochida International of Tokyo, Japan. Olive oil is available from Anglia Oils of North Humberside, United Kingdom. Sunflower and cottonseed oils are available from Cargil of Minneapolis, Minn. Safflower oil is available from California Oils Corporation of Richmond, Calif.

[0083] In addition to these food grade oils, structured lipids may be incorporated into the food product if desired. Structured lipids are known in the art. A concise description of structured lipids can be found in INFORM, Vol. 8, No. 10, page 1004; entitled Structured lipids allow fat tailoring (October 1997). Also see U.S. Pat. No. 4,871,768. Structured lipids are predominantly triacylglycerols containing mixtures of medium and long chain fatty acids on the same glycerol nucleus. Structured lipids and their use in enteral formula are also described in U.S. Pat. Nos. 6,194,379 and 6,160,007.

[0084] Optionally, co-3 fatty acids may comprise up to approximately 5% of the oil blend, preferably the co-3 fatty acids largely consist of the longer chain forms, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Dietary oils used in the preparation of the nutritional composition generally contain co-3 fatty acids in the triglyceride form and include, but are not limited to canola, medium chain triglycerides, fish, soybean, soy lecithin, com, safflower, sunflower, high-oleic sunflower, high-oleic safflower, olive, borage, black currant, evening primrose and flaxseed oil. Optionally, the weight ratio of co-6 fatty acids to oo-3 fatty acids in the lipid blend according to the invention is about 0.1 to 3.0. The daily delivery of co-3 fatty acids should be at least 450 mg and may vary depending on body weight, sex, age and medical condition of the individual. As mentioned, higher levels are desired for adult human consumption: for example, from about 0.5 to 50 gm daily, more preferably from about 2.5 to 5 gm daily

Protein

[0085] In certain exemplary embodiments, the nutritional compositions according to the general inventive concepts include protein. Any protein source that is suitable for use in oral

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4855-0747-4085, v.1 Atty Ref. 15782WOO1 nutritional compositions and is compatible with the essential elements and features of the general inventive concepts may be used.

[0086] Non-limiting examples of suitable proteins or sources thereof for use in the nutritional compositions include hydrolyzed, partially hydrolyzed or non-hydrolyzed proteins or protein sources, which may be derived from any known or otherwise suitable source such as milk (e.g., casein, whey), animal (e.g., meat, fish), cereal (e.g., rice, com), vegetable (e.g., soy) or combinations thereof. Non-limiting examples of such proteins include milk protein isolates, milk protein concentrates as described herein, casein protein isolates, extensively hydrolyzed casein, whey protein, sodium or calcium caseinates, whole cow milk, partially or completely defatted milk, soy protein isolates, and soy protein concentrates.

[0087] In an exemplary embodiment, the protein source is a hydrolyzed protein, i.e., a protein hydrolysate. In this context, the terms “hydrolyzed protein” or “protein hydrolysates” are used interchangeably herein and include extensively hydrolyzed proteins, wherein the degree of hydrolysis is most often at least about 20%, including from about 20% to about 80%, and also including from about 30% to about 80%, even more preferably from about 40% to about 60%. The degree of hydrolysis is the extent to which peptide bonds are broken by a hydrolysis method. The degree of protein hydrolysis for purposes of characterizing the extensively hydrolyzed protein component of these embodiments is easily determined by one of ordinary skill in the formulation arts by quantifying the amino nitrogen to total nitrogen ratio (AN/TN) of the protein component of the selected liquid formulation. The amino nitrogen component is quantified by USP titration methods for determining amino nitrogen content, while the total nitrogen component is determined by the Tecator Kjeldahl method, all of which are well known methods to one of ordinary skill in the analytical chemistry art.

[0088] Suitable hydrolyzed proteins include soy protein hydrolysate, casein protein hydrolysate, whey protein hydrolysate, rice protein hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg albumen hydrolysate, gelatin protein hydrolysate, combinations of animal and vegetable protein hydrolysates, and combinations thereof. Particularly preferred protein hydrolysates include whey protein hydrolysate and hydrolyzed sodium caseinate.

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[0089] In some embodiments, the protein source may include at least about 20% (by weight total protein) protein hydrolysate, including from about 30% to 100% (by weight total protein) protein hydrolysate, and including from about 40% to about 80% (by weight total protein) protein hydrolysate, and including about 50% (by weight total protein) protein hydrolysate.

Carbohydrate

[0090] In an exemplary embodiment, the nutritional compositions according to the general inventive concepts include carbohydrates that are suitable for use in an oral nutritional composition and are compatible with the essential elements and features of the general inventive concepts.

[0091] Non-limiting examples of suitable carbohydrates or sources thereof for use in the nutritional compositions described herein include maltodextrin, hydrolyzed or modified starch or cornstarch, glucose polymers, corn syrup, com syrup solids, rice-derived carbohydrates, pea- derived carbohydrates, potato-derived carbohydrates, tapioca, sucrose, glucose, fructose, lactose, high fructose com 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.

Long Chain Polyunsaturated Fatty Acids (LCPUFAs)

[0092] The nutritional compositions according to the general inventive concepts may also include Long Chain Polyunsaturated Fatty Acids (LCPUFAs). LCPUFAs are included in the nutritional compositions to provide nutritional support and other benefits to the end user.

[0093] Exemplary LCPUFAs for use in the nutritional compositions include, for example, co-3 LCPUFAs and co-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, krill oil and fish oils. In one particular aspect, 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 include DHA, ARA, EPA, DPA, and combinations thereof.

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[0094] In order to reduce potential side effects of high dosages of LCPUFAs including DHA, ARA, EPA, DPA, in the nutritional compositions, the content of DHA, ARA, EPA, DPA, preferably does not exceed 5% 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.

[0095] The LCPUFA may be provided as free fatty acids, in triglyceride form, in diglyceride form, in monoglyceride form, in phospholipid form, in esterified form or as a mixture of one or more of the above, preferably in triglyceride form.

[0096] The nutritional compositions as described herein may comprise total concentrations of ARA, DHA, EPA, and DPA of from about 0.001 g/L to about 1 g/L or more, including from about 0.01 g/L to about 1 g/L, and about 0.1 g/L to about 1 g/L.

[0097] In an exemplary aspect, the nutritional compositions may include a long chain polyunsaturated fatty acid component comprising DHA and ARA in a concentration of from about 0.17 mg/mL to about 0.33 mg/mL, including from about 0.17 mg/mL to about 0.26 mg/mL of ARA and DHA. In an exemplary aspect, the nutritional compositions may include DHA in a concentration of from about 0.025 mg/mL to about 0.130 mg/mL. In another aspect, the nutritional compositions may include ARA in a concentration of from about 0.080 mg/mL to about 0.350 mg/mL. In yet another aspect, the nutritional compositions may include combinations of DHA and ARA such that the ratio of DHA to ARA ranges from about 1:4 to about 1:2.

Human Milk Oligosaccharides (HMOs)

[0098] In some embodiments, the compositions of the present disclosure may further include an HMO. In certain aspects, the compositions may include a mixture of HMOs comprising at least one neutral HMO and at least one acidic HMO.

[0099] In certain aspects, the compositions of the present disclosure may include an HMO or a mixture of HMOs (e.g., a neutral HMO and an acidic HMO). The 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.

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[00100] Suitable HMOs for use in the compositions of the present disclosure may include neutral oligosaccharides, n-acctyl glucosylatcd oligosaccharides, acidic HMOs, and HMO precursors. For example, specific non-limiting suitable HMOs that may be included in the compositions according to the present disclosure include: 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), Difucosyllactose (DFL), 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL), lacto-N-neotetraose (LNnT), and lacto-N-tetraose (LNT).

[00101] Other HMOs that may be included in certain aspects of the compositions of the present disclosure include: N-acetylglucosamine (GlcNAc); L-fucose (L-Fuc); D-fucose (D-Fuc); fucosyl oligosaccharides (i.e., Lacto-N-fucopentaose I; Lacto-N-fucopentaose II; Lacto-N-fucopentaose III; Lacto-N-difucohexaose I; and Lactodifucotetraose); sialyl fucosyl 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 oligosaccharides (i.e., 3-Sialyllactosamine; 6'- Sialyllactosamine; Sialyllacto-N-neotetraose c; Monosialyllacto-N-hexaose; Disialyllacto-N- hexaose I; Monosialyllacto-N-neohexaose I; Monosialyllacto-N-neohexaose II; Disialyllacto-N- neohexaose; Disialyllacto-N-tetraose; Disialyllacto-N-hexaose 11; 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 N-acetylglucosamine (e.g., 2'-fucosyl-N- acetylglucosamine (2'-FLNac) is such a variant to 2'-fucosyllactose). These HMOs are described more fully in U.S. Patent Application No. 2009/0098240, which is herein incorporated by reference in its entirety. Other suitable examples of HMOs that may be included in the compositions according to the present disclosure include lacto-N-fucopentaose V, lacto-N- hexaose, para-lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-neohexaose, monofucosyllacto-N-hexaose II, isomeric fucosylated lacto-N-hexaose (1), isomeric fucosylated lacto-N-hexaose (3), isomeric fucosylated lacto-N-hexaose (2), difucosyl-para-lacto-N- neohexaose, difucosyl-para-lacto-N-hexaose, difucosyllacto-N-hexaose, lacto-N-neoocataose, para-lacto-N-octanose, iso-lacto-N-octaose, lacto-N-octaose, monofucosyllacto-neoocataose, monofucosyllacto-N-ocataose, difucosyllacto-N-octaose I, difucosyllacto-N-octaose II, difucosyllacto-N-neoocataose II, difucosyllacto-N-neoocataose I, lacto-N-decaose, trifucosyllacto-N-neooctaose, trifucosyllacto-N-octaose, trifucosyl-iso-lacto-N-octaose, lacto-N- difuco-hexaose II, sialyl-lacto-N-tetraose a, sialyl-lacto-N-tetraose b, sialyl-lacto-N-tetraose c,

23

4855-0747-4085, v.1 Atty Ref. 15782WOO1 sialyl-fucosyl-lacto-N-tetraose I, sialyl-fucosyl-lacto-N-tetraose II, and disialyl-lacto-N-tetraose, and combinations thereof.

[00102] The HMOs may be present in the composition, when in liquid form (e.g., ready-to-feed form), in total amounts of HMO in the composition (mg of HMO per mL of composition) of at least about about 0.1 g/L, alternatively at least about 0.2 g/L, alternatively at least about 0.5 g/L, alternatively at least about 1.0 g/L, alternatively at least about 1.5 g/L, alternatively at least about 2.0 g/L, alternatively at least about 2.5 g/L. In some embodiments, the nutritional composition may contain total HMOs in an amount of from about 0.1 g/L to about 20 g/L, including about 0.2 g/L to about 10 g/L, about 1 g/L to about 10 mg/mL, about 2 g/L to about 10 g/L, about 2.5 g/L to about 10 g/L, about 0.1 to about 7.5 g/L, about 0.2 g/L to about 7.5 g/L, about 1 g/L to about 7.5 g/L, about 2 g/L to about 7.5 g/L, and about 2.5 g/L to about 7.5 g/L, , about 0.1 to about 5 g/L, about 0.2 g/L to about 5 g/L, about 1 g/L to about 5 g/L, about 2 g/L to about 5 g/L, and about 2.5 g/L to about 5 g/L. The exact amount of HMO in the composition may depend on the specific HMO or HMOs present and the amounts of other components in the composition.

[00103J In certain aspects of the present disclosure, the composition may include an acidic HMO, and a neutral HMO comprising a fucosylated HMO and an N-acetylated HMO. In certain aspects of the present disclosure, the composition comprises a combination of 2’-FL, 3-FL, DFL, 3’-SL, 6’-SL, and LNT. In certain aspects of the present disclosure, the composition (when in ready-to-feed liquid form) may comprise 2’-FL in an amount up to 4.15 mg/mL, LNT in an amount up to 2.11 mg/mL, 3-FL in an amount up to 1.17 mg/mL, 3’-SL in an amount up to 0.36 mg/mL, and 6’-SL in an amount up to 0.44 mg/mL.

[00104] In certain aspects, the composition of the present disclosure, when in powder form, may comprise a total amount of HMOs of from about 1 wt.% to about 10 wt.% based on the total weight of the composition. In certain aspects, the composition of the present disclosure, when in powder form, may comprise a total amount of HMOs of from about 1 wt.% to about 8 wt.% based on the total weight of the nutritional composition, including from about 1.5 wt.% to about 7.5 wt.%, including from about 2 wt.% to about 7 wt.%, including from about 2.5 wt.% to about 6.5 wt.%, and also including from about 3 wt.% to about 6 wt.% based on the total weight of the nutritional composition.

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Nucleotides

[00105] In some embodiments, the nutritional compositions according to the general inventive concepts may additionally comprise nucleotides and/or nucleotide precursors selected from the group consisting of nucleosides, purine bases, pyrimidine bases, ribose and deoxyribose. The nucleotide may be in monophosphate, diphosphate, or triphosphate form. The nucleotide may be a ribonucleotide or a deoxyribonucleotide. The nucleotides may be monomeric, dimeric, or polymeric (including RNA and DNA). The nucleotide may be present in the nutritional composition as a free acid or in the form of a salt, preferably a monosodium salt.

[00106] Suitable nucleotides and/or nucleosides for use in the nutritional compositions include one or more of cytidine 5 '-monophosphate, uridine 5 '-monophosphate, adenosine 5'- monophosphate, guanosine 5 '-1 -monophosphate, and/or inosine 5 '-monophosphate, more preferably cytidine 5 '-monophosphate, uridine 5 '-monophosphate, adenosine 5 ’-monophosphate, guanosine 5 '-monophosphate, and inosine 5 '-monophosphate.

[00107] When present, the nutritional composition may include nucleotides in a total amount of about 15 mg/L to about 150 mg/mL, including about 15 mg/L to about 110 mg/L, including about 20 mg/L to about 110 mg/L of the nutritional composition, including about 30 mg/L to about 100 mg/L of the nutritional composition, including about 50 mg/L to about 100 mg/L of the nutritional composition, including about 15 mg/L to about 100 mg/mL, including about 15 mg/L to about 80 mg/mL, including about 30 mg/L to about 90 mg/L, including about 40 mg/L to about 90 mg/mL of the nutritional composition. In order to obtain a concentration within this range when reconstituted with water, the nutritional composition may include nucleotides in an amount of about 0.01 wt. % to about 0.10 wt. % of the nutritional composition when the nutritional composition is a reconstitutable powder.

[00108] In certain exemplary aspects of the present disclosure, the nucleotides may comprise about 40-50% cytidine 5’monophosphate, about 15-20% uridine 5 '-monophosphate, about 12-18% adenosine 5 '-monophosphate and about 20-25% guanosine 5 '-monophosphate by total weight of nucleotides. In certain exemplary aspects of the present disclosure, the nucleotides may comprise about 43% cytidine 5 '-monophosphate, about 18.5% uridine 5 '-monophosphate, about 16.5%

25

4855-0747-4085, v.1 Atty Ref. 15782WOO1 adenosine 5 '-monophosphate and about 22% guanosine 5 '-monophosphate by total weight of nucleotides.

[00109] In an exemplary aspect, the nucleotides may be present in the nutritional compositions in a total amount of about 50 mg/L to about 150 mg/mL or more of the nutritional composition and comprise about 25 to 39 mg/L of cytidine 5 '-monophosphate; 10 to 21 mg/L of uridine 5'- monophosphate; 10 to 15 mg/L of adenosine 5 '-monophosphate; and 12 to 20 mg/L of guanosine 5 '-monophosphate.

[00110] In an exemplary aspect, the nucleotides may be 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.

Antioxidants

[00111] Additionally, the nutritional compositions may comprise one or more antioxidants.

[00112] Any antioxidants suitable for oral administration may be included for use in the nutritional compositions according to the general inventive concepts, including, for example, vitamin A, vitamin E, vitamin C, retinol, tocopherol, and carotenoids, including lutein, betacarotene, zeaxanthin, and lycopene, and combinations thereof, for example.

[00113] The nutritional composition may comprise at least one carotenoid selected from lutein, lycopene, zeaxanthin, and beta-carotene to provide a total amount of carotenoid of from about 0.001 p.g/mL to about 10 pg/mL. More particularly, the nutritional compositions may comprise lutein in an amount of from about 0.001 pg/mL to about 10 pg/mL, including from about 0.001 pg/mL to about 5 pg/mL, including from about 0.001 pg/mL to about 0.0190 pg/mL, including from about 0.001 pg/mL to about 0.0140 pg/mL, and also including from about 0.044 pg/mL to about 5 pg/mL of lutein. The nutritional composition may comprise from about 0.001 pg/mL to about 10 pg/mL, including from about 0.001 pg/mL to about 5 pg/mL, from about 0.001 pg/mL to about 0.0130 pg/mL, including from about 0.001 pg/mL to about 0.0075 pg/mL, and also including from about 0.0185 pg/mL to about 5 pg/mL of lycopene. The nutritional compositions may comprise from about 1 pg/mL to about 10 pg/mL, including from about 1 pg/mL to about 5

26

4855-0747-4085, v.1 Atty Ref. 15782WOO1 pg/mL, including from about 0.001 pg/mL to about 0.025 pg/mL, including from about 0.001 pg/mL to about 0.011 pg/mL. and also including from about 0.034 pg/mL to about 5 pg/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 according to the general inventive concepts. 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.

[00114] 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, N.J.), Lyc-O-Mato® tomato extract in oil, powder, or bead form (available from LycoRed Corp., Orange, N.J.), beta-carotene, lutein, or lycopene (available from DSM Nutritional Products, Parsippany, N.J.), FloraGLO® lutein (available from Kemin Health, Des Moines, Iowa), Xangold® Natural Lutein Esters (available from Cognis, Cincinnati, Ohio), and Lucarotin® beta-carotene (available from BASF, Mount Olive, N.J.).

Other Optional Ingredients

[00115] The nutritional compositions according to the general inventive concepts 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 arc compatible with the essential and other ingredients in the selected product form.

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[00116] Non-limiting examples of such optional ingredients include preservatives, emulsifying agents, buffers, fructooligosaccharidcs, fiber, galactooligosaccharidcs, 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.

[00117] 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, arc especially useful in formulating liquid beverage embodiments according to the general inventive concepts 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.

[00118] The nutritional compositions may further comprise a flowing agent or anti-caking agent to retard clumping or caking of the powder over time and to make a powder embodiment flow easily from its container. Any known flowing or anti-caking agents that are known or otherwise suitable for use in a nutritional powder or product form are suitable for use herein, non-limiting examples of which include tricalcium phosphate, silicates, and combinations thereof. The concentration of the flowing agent or anti-caking agent in the nutritional composition varies depending upon the product form, the other selected ingredients, the desired flow properties, and so forth, but most typically range from about 0.1% to about 4%, including from about 0.5% to about 2%, by weight of the nutritional composition.

[00119] The nutritional compositions may further comprise a stabilizer. 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

28

4855-0747-4085, v.1 Atty Ref. 15782WOO1 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.

[00120] 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 B12, niacin, folic acid, pantothenic acid, biotin, vitamin C, choline, inositol, salts and derivatives thereof, and combinations thereof. The food products preferably include, but are not limited to, the following vitamins and minerals; calcium, phosphorus, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, chromium, molybdenum, conditionally essential nutrients m-inositol, carnitine and taurine, and Vitamins A, C, D, E (including natural vitamin E), K and the B complex, and mixtures thereof.

[00121] 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.

[00122] The nutritional compositions also may contain fiber and fiber-like stabilizers. Suitable sources of fiber and/or stabilizers include, but are not limited to, xanthan gum, guar gum, gum arabic, gum ghatti, gum karaya, gum tracacanth, agar, furccllaran, gcllan gum, locust bean gum, pectin, low and high methoxy pectin, oat and barley glucans, carrageenans, psyllium, gelatin, microcrystalline cellulose, CMC (sodium carboxymethylcellulose), methylcellulose hydroxypropyl methyl cellulose, hydroxypropyl cellulose, DATEM (diacetyl tartaric acid esters of mono- and diglycerides), dextran, carrageenans, FOS (fructooligosaccharides), and mixtures thereof. Numerous commercial sources of soluble dietary fibers are available. For example, gum arabic, hydrolyzed carboxymethylcellulose, guar gum, pectin and the low and high methoxy pectins are available from TIC Gums, Inc. of Belcamp, Md. The oat and barley glucans are available from Mountain Lake Specialty Ingredients, Inc. of Omaha, Nebr. Psyllium is available from the Meer Corporation of North Bergen, N.J. while the carrageenan is available from FMC Corporation of Philadelphia, Pa.

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[00123] In addition to fiber, the nutritional compositions may also contain oligosaccharides that arc not naturally found in human milk such as fructooligosaccharidcs (FOS) or galactooligosaccharides (GOS). These oligosaccharides are rapidly and extensively fermented to short chain fatty acids by anaerobic microorganisms that inhabit the large bowel. These oligosaccharides are preferential energy sources for most Bifidobacterium species, but are not utilized by potentially pathogenic organisms such as Clostridium perfingens, C. difficile, or Eschericia coli.

[00124] The nutritional products may additionally comprise one or more thickeners (z.e., thickening agents). The addition of thickeners reduces the incidences of paresthesia by inducing the feeling of satiety, which prolongs gastric transit time as discussed above.

Product Forms

[00125] The compositions according to the general inventive concepts may be formulated and administered in any known or otherwise suitable oral product form. Any solid, liquid, semi-solid, and 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.

[00126] The compositions may be in any product form comprising the ingredients described herein, and which is safe and effective for oral administration. The 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.

[00127] The compositions according to the general inventive concepts are desirably formulated as dietary product forms, which are defined herein as those embodiments comprising the ingredients according to the general inventive concepts in a product form that then contains at least one of fat, protein, and carbohydrate, and preferably also contains vitamins, minerals, or combinations thereof. The nutritional compositions will comprise at least one or more sources of betaine and one or more sources of folate; desirably in combination with at least one of protein, fat, carbohydrate, vitamins, and minerals, to produce a nutritional composition.

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[00128] Liquid compositions include both concentrated and ready-to-feed liquids. These nutritional liquids arc most typically formulated as suspensions or emulsions, although other liquid forms are within the scope of the general inventive concepts. Nutritional compositions in the form of 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-in-water, water-in-oil, or complex aqueous emulsions, although such emulsions are most typically in the form of oil-in-water emulsions having a continuous aqueous phase and a discontinuous oil phase.

[00129] 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 may 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.

[00130] The nutritional emulsions may have a caloric density tailored to the nutritional needs of the ultimate user, although in most instances the emulsions may 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 an exemplary embodiment, the emulsion may have a caloric density of 25, or 50, or 75, or 100 kcal/liter.

[00131] The nutritional emulsion may have a pH ranging from about 2.5 to about 8, but arc 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.

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[00132] 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 1 mL to about 475 mL, including from about 30 mL to about 360 mL, including from about 60 mL to about 300 mL, and including from about 60 mL to about 240 mL.

[00133] The compositions in the form of 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 dry blended 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.

[00134] 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 an exemplary embodiment, the emulsion may have a caloric density of 25, or 50, or 75, or 100 kcal/liter.

[00135] Compositions, e.g. infant formulas, in the form of a reconstitutable powder may in some embodiments be reconstituted by mixing a measured amount of powder with a measured amount of water to obtain a liquid formula of a desired caloric density. In some embodiments, for instance, a liquid formula having a caloric density of about 20 kcal/fl oz may be obtained by mixing about 8.7 g powder with about 2 fl oz water; a liquid formulation having a caloric density 32

4855-0747-4085, v.1 Atty Ref. 15782WOO1 of about 22 kcal/fl oz may be obtained by mixing about 17.4 g powder with about 3.5 fl oz water; a liquid formulation having a caloric density of about 24 kcal/fl oz may be obtained by mixing about 26.2 g powder with about 5 fl oz water; a liquid formulation having a caloric density of about 26 kcal/fl oz may be obtained by mixing about 8.7 g powder with about 1.5 fl oz water; a liquid formulation having a caloric density of about 27 kcal/fl oz may be obtained by mixing about 26.2 g powder with about 4.25 fl oz water; or the like.

Methods of Manufacture

[00136] The nutritional compositions according to the present disclosure may be prepared by any known or otherwise effective manufacturing technique for preparing such nutritional compositions. Many such techniques are known for any given product form such as nutritional liquids or nutritional powders and can easily be applied by one of ordinary skill in the art to the nutritional compositions described herein.

[00137] Nutritional compositions according to 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) sluny, a carbohydrate-mineral (CHO-MIN) slurry, and a protein-in-water (PIW) slurry. The PIF slurry is formed by heating and mixing the oil (e.g., canola oil, corn oil, phosphatidylcholine and sphingomyelin 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., gellan gum, 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, fructooligosaccharides, sucrose, corn syrup, etc.). The PIW slurry is then formed by mixing with heat and agitation and remaining protein, if any. Betaine and folate, each of which is water soluble, may be added, for example, to the CHO-MIN slurry (e.g. as part of the additional minerals and/or carbohydrates) or to the PIW slurry.

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[00138] The resulting slurries are then blended together with agitation and the pH adjusted to 6.6-7.0, after which the composition is subjected to processing during which the composition is emulsified and homogenized. 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. 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, dry mixed, agglomerated.

[00139] The nutritional solid, such as a spray dried nutritional powder or dry mixed nutritional powder, may be prepared by any collection of known or otherwise effective techniques, suitable for making and formulating a nutritional powder.

[00140] 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.

[00141] One method of preparing a spray dried nutritional powder comprises forming and homogenizing an aqueous slurry or liquid comprising fat, and optionally protein, carbohydrate, and other sources of fat as described above, 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, dry mixing, or otherwise adding any one or more of the ingredients described herein, to form the spray dried nutritional powder.

[00142] Other suitable methods for making nutritional compositions are described, for example, in U.S. Pat. No. 6,365,218 (Borschel, et al.), U.S. Patent No. 6,589,576 (Borschel, et al.), U.S. Pat. No. 6,306,908 (Carlson, et al.), U.S. Patent Application No. 20030118703 Al (Nguyen, et al.), which descriptions are incorporated herein by reference to the extent that they are consistent herewith.

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Methods of Use

[00143] The methods of use according to the present disclosure include the oral administration of the compositions (e.g., nutritional compositions) that include a combination of betaine and folate to improve cognition, brain function/development/maturation in an infant, toddler, or child, including enhancing one or more markers thereof. The methods of use according to the present disclosure can also help to prevent or alleviate the symptoms of developmental delays and/or can facilitate an infant, toddler, or child to achieve cognitive and/or developmental milestones (e.g., communication and language development, memory, motor development, and social and emotional development). In certain exemplary aspects, the compositions and methods may facilitate an infant, toddler, or child to achieve or surpass cognitive and/or developmental milestones. Such milestones include, but are not limited to, attention, communication and language development, memory milestones, motor development, and social and emotional development among others. This development can be demonstrated by e.g., early childhood screening or developmental screening.

[00144J The compositions as described herein can be administered to individuals including infants, toddlers, or children, or may, in certain aspects of the present disclosure, be administered to a specific subclass of individuals that arc “in need thereof;” that is, to specific individuals that would particularly benefit from administration of the composition. For example, a specific individual may be “in need of’ the compositions as described herein if they are susceptible to, or suffering from suboptimal or otherwise compromised brain or neuronal development. This susceptibility may arise, for example, from one or more of a genetic predisposition, an underlying disease or condition, a medication regimen, or a diet that results in deficiencies in neuronal development.

[00145] The individual desirably consumes at least one serving of the composition daily, and in some embodiments, may consume two, three, or more servings per day. Each serving is desirably administered as a single, undivided dose, although the serving may also be divided into two or more partial or divided servings to be taken at two or more times during the day. The methods according to the present disclosure include continuous day after day administration, as well as periodic or limited administration, although continuous day after day administration is generally desirable. The methods according to the present disclosure are preferably applied on a daily basis, 35

4855-0747-4085, v.1 Atty Ref. 15782WOO1 wherein the daily administration is maintained continuously for at least 3 days, including at least 5 days, including at least 1 month, including at least 6 weeks, including at least 8 weeks, including at least 2 months, including at least 6 months, desirably for at least about 18-24 months, desirably as a long-term, continuous, daily, dietary source or supplement.

[00146] In one aspect, the methods of use of the present disclosure include a method of improving or enhancing neuronal development and/or brain maturation. Neuronal development and/or brain maturation may be shown in one or more ways including in vitro testing. The composition comprising a combination of betaine and folate is effective for enhancing brain development and/or brain maturation in an infant, toddler, or child. In certain aspects, the neuronal development or brain development/maturation may be shown by neurite outgrowth, proliferation, length of neurite outgrowth. In certain exemplary aspects, the development and/or maturation may be shown by way of increasing the expression of one or more proteins associated with development. Neuronal development markers include MAP2 or class III beta tubulin. Neurite outgrowth is an important event in neuronal pathfinding and the establishment of synaptic connections during development.

[00147] In another aspect, the methods of use of the present disclosure include a method of enhancing cognition, brain maturation, and brain development in an infant, toddler, or child by administering to the individual a composition comprising a combination of betaine and folate. Such benefits may include, but are not limited to, attention, communication and language development, memory milestones, motor development, and social and emotional development.

Examples

[00148] The following examples illustrate exemplary aspects and/or features of the methods and compositions of the present disclosure. The examples are given solely for the purpose of illustration and are not to be construed as limiting the present disclosure, as many variations thereof are possible without departing from the spirit and scope of the present disclosure.

[00149] Cell line: mouse neuroblastoma cells (N2a, ATCC CCL-131). The neural crest derived N2a cell line has been widely used to study neuronal differentiation, neurite growth, synaptogenesis and signalling pathways. N2a cells have the advantage of responding quickly to serum deprivation and other stimuli in their environment by expressing signalling molecules that 36

4855-0747-4085, v.1 Atty Ref. 15782WOO1 lead to neuronal differentiation and neurite growth. Neurite outgrowth is an important event in neuronal pathfinding and the establishment of synaptic connections during development. Thus, the observed neuronal differentiation and neurite growth demonstrate that the compositions of the present invention can benefit infants, toddlers, and children by enhancing brain development and/or increasing synaptic connections, both in individuals that show age-appropriate cognitive development and/or milestone achievement and those in need of catch up to achieve developmental milestones.

[00150] Cell culture: in vitro assays were performed with Mouse neuroblastoma cells (N2a, ATCC CCL-131). This cell line was grown in alpha-Minimum Essential Medium (aMEM) supplemented with 10% (v/v) (growing medium) FBS, 2 mmol/1 glutamine plus, 100 units/mL penicillin and 0.1 mg/mL streptomycin in an atmosphere of 5% CO2 and 95% air. To differentiate the N2a cells the medium was supplement only with 1% of FBS (differentiation medium).

Example 1

[00151] N2a proliferation was determined through MTT assay. The test is based on enzymatic reduction of the lightly colored tetrazolium salt to its formazan of intense purple blue color, which can be quantified spectrophotometrically. Under properly optimized conditions the obtained absorbance value is directly proportional to the number of living cells. N2a cells were seeded in 48-wcll plates at a density of 15xl0³ cclls/wcll containing growth medium. After 24h, cells were treated with 0.05, 0.1, 0.5, 5, 10, 50 and 100 pM for Folate and/or Betaine for 48h. Then, MTT reagent was diluted (1:100) in aMEM without FBS and 200pL were added per well. Cells were incubated at 37°C for 45 min. The supernatant was replaced with 200pL acidic isopropanol to dissolve the formazan crystals. The absorbance was measured at 595 nm with a microplate reader Multiskan Spectrum (Thermo Labsystems, USA). The average absorbance of treated and control cells were calculated and expressed as percentage of cell proliferation.

[00152] Figure 2 shows the results of Neuro 2a proliferation testing with increasing amounts of betaine, from 0.05 pM up to 100 pM. As shown in Figure 2, compared with the untreated control, the presence of betaine at 50 pM and 100 pM induced a statistically significant increase in N2a proliferation at 48 hours. At a concentration less than 50 pM, on the other hand, betaine had no

37

4855-0747-4085, v.1 Atty Ref. 15782WOO1 statistically significant effect on N2a proliferation at 48 hours. For subsequent experiments, therefore, a dose of 50 pM was chosen for betaine.

[00153] Given the molecular weight of betaine of 117.148 g/mol, the 50 pM concentration in the culture medium is equivalent to 5.8574 mg/L of betaine in the culture medium. The normal blood volume in children is generally known to be about 90 mL/kg (see Bhardwaj, N., Perioperative fluid therapy and intraoperative blood loss in children. Indian J. Anaesthesia, vol. 9, pages 729-736, September 2019). Accordingly, betaine should be administered in an amount of at least about 0.5 mg per kg body weight of the infant, toddler, or child per day in order to obtain the desired 50 pM concentration.

[00154] In order to provide betaine to the brain in an effective amount, e.g. about 50 pM or more as shown from Figure 2, compositions - and in particular infant formulas - of the present disclosure may contain at least about 3.5 g/L of betaine, alternatively at least about 4.0 g/L of betaine, optionally at least about 4.5 g/L, at least about 5.0 g/L, at least about 5.5 g/L, or at least about 6.0 g/L of betaine. These amounts are based on the following calculations.

[00155] First, one can calculate the amount of betaine needed to yield a blood concentration of at least 50 pM. Because the normal blood volume in children is generally known to be about 90 mL/kg (see Bhardwaj, N., Perioperative fluid therapy and intraoperative blood loss in children. Indian J. Anaesthesia, vol. 9, pages 729-736, September 2019), betaine should be administered in an amount of 0.527 mg per kg body weight of the infant, toddler, or child per day or more in order to obtain the desired 50 pM concentration. Additionally, a child of a particular age is known to have a 50^th percentile body weight, as recited in the CDC National Center for Health Statistics data table of infant weight-for-age charts available at https://www.cdc.gOv/growthcharts/html_charts/wtageinf.htm#females and https://www.cdc.gOv/growthcharts/html_chails/wtageinf.htm#male.

[00156] For a newborn, for example, the 50^th percentile body weight is 3.530203 kg. In order to yield a blood concentration of betaine of at least 50 pM (5.8574 mg/L) therefore, the nutritional composition must provide the newborn with at least 1.86 mg of betaine per day. As another example, a six-month old has a 50^th percentile body weight of 8.162951 kg. In order to yield a

38

4855-0747-4085, v.1 Atty Ref. 15782WOO1 blood concentration of betaine of at least 50 pM (5.8574 mg/L) therefore, the nutritional composition must provide the six-month old with at least 4.30 mg of betaine per day.

[00157] Notably, the absorption of betaine by the body is rapid and nearly complete (see, e.g., https://examine.com/supplements/betaine/research/), meaning that it is reasonable to expect that essentially all of the betaine present in a nutritional composition is absorbed into the bloodstream. One can thus also determine the concentration of betaine in a sole-source nutritional composition, e.g. infant formula, necessary to provide an individual, e.g. a newborn or a six-month old, with the calculated amount of betaine, by using the infant feeding chart presented below as Table 1 (data obtained from https://www.parents.com/baby/feeding/baby-feeding-chart-how-much-and-when- to-feed-infants-the-first-yeai7#citation-l).

Table 1

[00158] Based on this feeding chart, a formula-fed newborn is expected to ingest between 8 ounces (0.24 L) and 24 ounces (0.71 L) of formula per day. Taking an average of the two, therefore, yields about 0.475 L of formula per day. In order to provide at least 1.86 mg of betaine, therefore, the infant formula must contain betaine at a concentration of at least about 3.9 mg/L of formula.

[00159] Based on this feeding chart, a formula-fed six-month old is expected to ingest between 28 ounces (0.83 L) and 48 ounces (1.42 L) of formula per day. Taking an average of the two, therefore, yields about 1.125 L of formula per day. In order to provide at least 4.30 mg betaine per day, therefore, the infant formula must contain betaine at a concentration of at least 3.8 mg/L of formula.

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[00160] A dose of 50 pM of methyl folate was also chosen for subsequent experiments. The methyl folate has low stability, meaning that a large percentage of it would be expected to degrade over the course of the in vitro testing. When ingested by an individual, in contrast, folate present in a nutritional composition would be absorbed into the bloodstream and circulated to the brain rapidly. Accordingly, the concentration of methyl folate used in the experiments (50 pM) is much higher than that which would be present in a nutritional composition contemplated by the present disclosure (which would be expected to be between about 100 mcg/L (0.1 mg/L) and about 300 mcg/L (0.3 mg/L)). Rather, the selected 50 pM concentration of methyl folate is intended solely to ensure that an adequate amount of folate was present throughout the testing, e.g. that there is not a lack of folate in the testing. A nutritional composition comprising between about 100 mcg and about 300 mcg folate would be expected to ensure an adequate intake of folate for an infant, as human breastmilk is known to contain about 100 mcg/L.

[00161] Figure 3 shows the results of Neuro 2a proliferation testing with 50 pM of betaine, 50 pM of methyl folate, and a combination of both 50 pM of betaine and 50 pM of methyl folate. Each of the betaine and the methyl folate produced an increase in cell proliferation compared to the control group, with the combination producing a synergistically greater effect than each ingredient individually.

Example 2

[00162] Betaine, folate, and a combination thereof was also tested for their effects on morphological neurite outgrowth in N2a cells. To perform this testing, neurite outgrowth assay N2a cells were seeded in P60mm culture plate at an initial density of 325xl0³ cells per well containing growth medium (3 ml/well) supplemented with FBS at 10% and incubated overnight. To induce cell differentiation, the growth medium was carefully replaced with differentiation medium (1% FBS). Cells with differentiation medium only served as a negative control of treatment. Cells were incubated with the ingredients for 48 h at 37°C, 95% air and 5% CO2 to observe neurogenesis activity. Images have been taken and the number of undifferentiated cells and neurites was counted using the program ImageJ (National Institutes of Health, USA). Neurites were defined as a process with lengths equivalent to one or more diameters of a cell body. The percentage of cells bearing neurites was calculated as the percentage of number of neurites divided by total number of cells. Neurite outgrowth was observed under a phase-contrast light microscope

40

4855-0747-4085, v.1 Atty Ref. 15782WOO1

(Olympus 1X70) at a magnification of lOOx equipped with DP74 camera (Olympus, USA). For quantification of ncuritc bearing cells three random fields (200 - 300 cclls/wcll) were examined in each well. Results were expressed as mean ± standard error of mean (SEM).

[00163] As shown in Figure 4, compared with the untreated control, both folate (50 pM) and betaine (50 pM) induced a statistically significant increase in the percentage of N2a cells with neurite outgrowth at 48 hours, with folate giving rise to an increase in neurite outgrowth of 71% relative to the control and betaine giving rise to an increase in neurite outgrowth of 69% relative to the control. The combination of both ingredients also produced an statistically significant increase, of about 86%, in the number of cells with neurite outgrowth. This increase was greater than the individual effect of each ingredient when tested separately.

Example 3

[00164] Betaine and folate were also tested for their effects, both alone and in combination, on molecular' neurite outgrowth markers. The expression of MAP-2 and B-3 tubulin proteins were determined from N2a cells immunoblotted with anti-MAP-2 and anti-B 3 -tubulin antibodies. To perform this testing, N2a cells were plated in a 60-mm culture plate at a density of 325xl0³ cells/plate and incubated for 24h in growing medium. The cells were then incubated with the ingredients in differentiation medium for 48 hours. After treatment, they were lysed with RIPA buffer supplemented with phosphatase and protease inhibitors. Cells were collected and sonicated. Then, extracts were centrifugated for 10 min at 14.000 rpm. Supernatants were collected and protein concentration was measured in extracts using the bicinchoninic acid method. Proteins (20 pg) were separated by sodium dodecyl sulfate polyacrylamide (SDS-PAGE), transferred onto nitrocellulose membranes, and immunoblotted with specific antibodies: anti-MAP2 (Microtube- associated protein 2) or anti-class III beta tubulin (B3-tubulin) and anti-GAPDH (glyceraldehyde- 3-phosphate dehydrogenase). To determine total levels of these proteins, independent western blots were carried out to avoid stripping of the proved membranes. Immunoblots were developed by an enhanced chemiluminescence detection method using a Chemidoc XRS+ (Biorad, USA) Quantification was performed by densitometry with the Image Lab Software (Biorad, USA).

[00165] Betaine, folate, and a combination thereof was tested to determine whether each had any effect on MAP-2 (microtube-associated protein 2) expression, which is known to be a marker of

41

4855-0747-4085, v.1 Atty Ref. 15782WOO1 brain cell development. More specifically, MAP-2 is a neuron-specific protein whose main function is to interact with tubulin to promote its assembly into microtubules and stabilize the microtubule network.

[00166] As shown in Figure 5, compared with the untreated control, both folate (50 pM) and betaine (50 pM) induced a statistically significant increase in MAP-2 protein expression at 48 hours. Specifically, treatment with betaine brought about an increase in MAP-2 protein expression of about 17% relative to the control and treatment with folate brought about an increase in MAP- 2 protein expression of about 27% relative to the control. Furthermore, unexpectedly, the combination of both ingredients produced a statistically significant increase in the expression of MAP-2 of about 50%. Notably, this increase represents a surprising synergistic effect between betaine and folate on a known brain cell development marker.

[00167] Betaine, folate, and a combination thereof was also tested to determine whether each had any effect on B3-Tubulin protein expression, which is known to be a marker of brain cell differentiation and maturation. More specifically, B3-Tubulin is one of the five families of tubulin molecules whose expression is limited to neurons and is known to play a vital role in neurite extension and vesicle trafficking.

[00168] As shown in Figure 6, treatment with betaine brought about a small, but not statistically significant increase in B3-Tubulin expression of about 4% while treatment with folate brought about a small, but not statistically significant increase in B3-Tubulin expression of about 13%. No statistically significant change was observed in the expression of B3-Tubulin for either of betaine or folate, when tested individually, relative to the untreated control at 48 hours. However, unexpectedly, the combination of betaine and folate produced a statistically significant increase in the expression of B3-Tubulin of about 36%. This represents a surprising synergy between betaine and folate on a known marker of brain cell development and maturation.

[00169] This testing demonstrates that betaine at a (blood) concentration of at least 50 pM was able to increase cellular proliferation of N2a cells. Further it was observed that the combination of betaine with folate promotes a high grade of differentiation of neuronal cells in term of neurite outgrowth morphology and molecular markers during a key stage of the neuronal differentiation process. Unexpectedly, when both ingredients are used in combination, a surprising synergy was

42

4855-0747-4085, v.1 Atty Ref. 15782WOO1 shown on a number of brain cell development and maturation markers. These results demonstrate that betaine, and in particular betaine in combination with folate, promotes neural proliferation, maturation and/or differentiation in an individual, e.g. an infant, toddler, or young child. By promoting neural proliferation, maturation and/or differentiation in an infant, toddler, or young child, embodiments of the present disclosure utilize betaine, and in particular betaine in combination with folate, to promote brain development, cognition, or both in an infant, toddler, or young child.

[00170] Unless otherwise indicated herein, all sub-aspects and optional aspects are respective sub-aspects and optional aspects to all aspects described herein. While the present application has been illustrated by the description of aspects thereof, and while the aspects have been described in considerable detail, it is not the intention of the applicants to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications will readily appear to those skilled in the art. Therefore, the application, in its broader aspects, is not limited to the specific details, the representative compositions or formulations, and illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the spirit or scope of the applicant’s general disclosure herein.

43

4855-0747-4085, v.1

Claims

Atty Ref. 15782WOO1 WHAT IS CLAIMED IS:

1. A non-therapeutic method for promoting brain development, cognition, or both in an infant, toddler, or child, the method comprising administering a nutritional composition comprising an effective amount of betaine and folate to the infant, toddler, or child.

2. The method according to claim 1, wherein betaine is administered in an amount of at least about 0.5 mg per kg body weight of the infant, toddler, or child per day.

3. The method according to any one of claims 1-2, wherein the nutritional composition, in liquid form, comprises betaine in an amount of at least about 3.5 mg/L, alternatively at least about 4 mg/L, optionally between about 4 mg/L and about 14 mg/L.

4. The method according to any one of claims 1 and 3, wherein the nutritional composition, in liquid form, comprises folate in an amount of at least about 100 mcg/L, optionally between about 100 mcg/L and about 300 mcg/L.

5. The method according to any one of claims 1-4, wherein the nutritional composition comprises at least one of protein, carbohydrate, and fat, optionally wherein the nutritional composition comprises protein, carbohydrate, and fat, optionally wherein the nutritional composition is a sole-source nutritional composition.

6. The method according to any one of claims 1-5, wherein the infant, toddler, or child is an infant.

7. The method according to claim 6, wherein the nutritional composition is an infant formula.

8. The method according to any one of claims 1-7, wherein the nutritional composition is a ready-to-drink liquid.

9. The method according to any one of claims 1-7, wherein the nutritional composition is a liquid that is reconstituted from a reconstitutable powder.

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4855-0747-4085, v.1 Atty Ref. 15782WOO1

10. The method according to any one of claims 1-9, wherein the nutritional composition further comprises one or more human milk oligosaccharides (HMOs).

11. The method according to claim 10, wherein the one or more human milk oligosaccharides comprises at least one selected from the group of a fucosy lated oligosaccharide, a sialylated oligosaccharide, an N-acetylglucosamine oligosaccharide, and any combination thereof.

12. The method according to claim 10, wherein the one or more human milk oligosaccharides comprises a combination of at least one fucosylated oligosaccharide, at least one sialylated oligosaccharide, and at least one N-acetylglucosamine oligosaccharide.

13. The method according to any one of claims 11-12, wherein the fucosylated oligosaccharide comprises at least one of 3-fucosyllactose (3-FL) and 2'-fucosyllactose (2'-FL); the sialylated oligosaccharide comprises at least one of 3’-sialyllactose (3'-SL) and 6' sialyllactose (6’-SL); and the N-acetylglucosamine oligosaccharide comprises at least one of lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT).

14. The method according to claim 10, wherein the one or more human milk oligosaccharides comprises at least one selected from the group of 2'-fucosyllactose, 3- fucosyllactosc, 3 '-sialyllactose, 6 ’-sialyllactose, and lacto-N-tctraosc, and any combination thereof.

15. The method according to claim 10, wherein the one or more human milk oligosaccharides comprises 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and lacto-N-tetraose.

16. The method according to any one of claims 10-15, wherein the nutritional composition, in liquid form, contains total HMOs in an amount between 0.1 g/L and 10 g/L.

17. The method according to any one of claims 1-16, wherein the nutritional composition comprises a long chain polyunsaturated fatty acid selected from arachidonic acid, docosahexaenoic acid, and a combination thereof.

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4855-0747-4085, v.1 Atty Ref. 15782WOO1

18. The method according to any one of claims 1-17, wherein the nutritional composition comprises carbohydrate in an amount of from about 40% to about 60% by weight (on a dry weight basis); fat in an amount of from about 20% to about 40% by weight (on a dry weight basis); and protein in an amount of about 10% to about 25% by weight (on a dry weight basis).

19. The method according to any one of claims 1-18, wherein the method promotes brain development.

20. The method according to any one of claims 1-18, wherein the method promotes cognition.

21. The method according to any one of claims 1-20, wherein promoting brain development, cognition, or both comprises promoting neuronal differentiation and/or maturation.

22. A composition for use in promoting brain development, cognition, or both in an infant, toddler, or child, optionally catch-up brain development, cognition, or both, the composition comprising an effective amount of betaine and folate.

23. The composition according to claim 22, wherein the amount of betaine is effective to provide at least about 0.5 mg betaine per kg body weight of the infant, toddler, or child per day.

24. The composition according to any one of claims 22-23, wherein the composition is a liquid comprising betaine in an amount of at least about 3.5 mg/L, alternatively at least about 4 mg/L, optionally between about 4 mg/L and about 14 mg/L, or wherein the composition is a reconstitutable powder comprising betaine in an amount of at least about 0.002 wt.%, optionally from about 0.002 wt.% to about 0.008 wt.%, based on the total weight of the composition.

25. The composition according to any one of claims 22-24, wherein the composition is a liquid comprising folate in an amount of at least about 100 mcg/L, optionally between about 100 mcg/L and about 300 mcg/L, or wherein the composition is a reconstitutable powder comprising folate in an amount of at least about 0.00006 wt.%, optionally between about 0.00006 wt.% and about 0.0003 wt.%, based on the total weight of the composition.

26. The composition according to any one of claims 22-25, wherein the composition comprises at least one of protein, carbohydrate, and fat, optionally wherein the composition

46

4855-0747-4085, v.1 Atty Ref. 15782WOO1 comprises protein, carbohydrate, and fat, optionally wherein the composition is a sole-source nutritional composition.

27. The composition according to any one of claims 22-26, wherein the infant, toddler, or child is an infant.

28. The composition according to claim 27, wherein the composition is an infant formula.

29. The composition according to any one of claims 22-28, wherein the composition is a ready-to-drink liquid.

30. The composition according to any one of claims 22-28, wherein the composition is a reconstitutable powder.

31. The composition according to any one of claims 22-30, further comprising one or more human milk oligosaccharides (HMOs).

32. The composition according to claim 31, wherein the one or more human milk oligosaccharides comprises at least one selected from the group of a fucosy lated oligosaccharide, a sialylated oligosaccharide, an N-acetylglucosamine oligosaccharide, and any combination thereof.

33. The composition according to claim 31, wherein the one or more human milk oligosaccharides comprises a combination of at least one fucosylated oligosaccharide, at least one sialylated oligosaccharide, and at least one N-acetylglucosamine oligosaccharide.

34. The composition according to any one of claims 32-33, wherein the fucosylated oligosaccharide comprises at least one of 3-fucosyllactose (3-FL) and 2'-fucosyllactose (2'-FL); the sialylated oligosaccharide comprises at least one of 3’-sialyllactose (3'-SL) and 6' sialyllactose (6’-SL); and the N-acetylglucosamine oligosaccharide comprises at least one of lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT).

35. The composition according to claim 31, wherein the one or more human milk oligosaccharides comprises at least one selected from the group of 2'-fucosyllactose, 3-

47

4855-0747-4085, v.1 Atty Ref. 15782WOO1 fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and lacto-N-tetraose, and any combination thereof.

36. The composition according to claim 31, wherein the one or more human milk oligosaccharides comprises 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and lacto-N-tetraose.

37. The composition according to any one of claims 31-36, wherein the composition contains total HMOs in an amount between 0.1 g/L and 10 g/L.

38. The composition according to any one of claims 22-37, further comprising a long chain polyunsaturated fatty acid selected from arachidonic acid, docosahexaenoic acid, and a combination thereof.

39. The composition according to any one of claims 22-38, wherein the composition comprises carbohydrate in an amount of from about 40% to about 60% by weight (on a dry weight basis); fat in an amount of from about 20% to about 40% by weight (on a dry weight basis); and protein in an amount of about 10% to about 25% by weight (on a dry weight basis).

40. The composition according to any one of claims 22-39, for use in promoting brain development.

41. The composition according to any one of claims 22-39, for use in promoting cognition.

42. The composition according to any one of claims 22-41, wherein promoting brain development, cognition, or both comprises promoting neuronal differentiation and/or maturation.

43. A combination of betaine and folate for use in promoting brain development, cognition, or both in an infant, toddler, or child, optionally catch-up brain development, cognition, or both, wherein the betaine is in an amount effective to provide at least about 0.5 mg betaine per kg body weight of the infant, toddler, or child per day.

44. The combination of claim 43, wherein the combination of betaine and folate is present in a nutritional composition.

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4855-0747-4085, v.1 Atty Ref. 15782WOO1

45. The nutritional composition according to claim 44, wherein the nutritional composition is a liquid comprising betaine in an amount of at least about 3.5 mg/L, alternatively at least about 4 mg/L, optionally between about 4 mg/L and about 14 mg/L, or wherein the nutritional composition is a reconstitutable powder comprising betaine in an amount of at least about 0.002 wt.%, optionally from about 0.002 wt.% to about 0.008 wt.%, based on the total weight of the composition.

46. The nutritional composition according to any one of claims 44-45, wherein the nutritional composition is a liquid comprising folate in an amount of at least about 100 mcg/L, optionally between about 100 mcg/L and about 300 mcg/L, or wherein the nutritional composition is a reconstitutable powder comprising folate in an amount of at least about 0.00006 wt.%, optionally between about 0.00006 wt.% and about 0.0003 wt.%, based on the total weight of the composition.

47. The nutritional composition according to any one of claims 44-46, wherein the nutritional composition comprises at least one of protein, carbohydrate, and fat, optionally wherein the nutritional composition comprises protein, carbohydrate, and fat, optionally wherein the nutritional composition is a sole-source nutritional composition.

48. The nutritional composition according to any one of claims 44-47, wherein the infant, toddler, or child is an infant.

49. The nutritional composition according to claim 48, wherein the nutritional composition is an infant formula.

50. The nutritional composition according to any one of claims 44-49, wherein the nutritional composition is a ready-to-drink liquid.

51. The nutritional composition according to any one of claims 44-49, wherein the nutritional composition is a reconstitutable powder.

52. The nutritional composition according to any one of claims 44-51, further comprising one or more human milk oligosaccharides (HMOs).

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4855-0747-4085, v.1 Atty Ref. 15782WOO1

53. The nutritional composition according to claim 52, wherein the one or more human milk oligosaccharides comprises at least one selected from the group of a fucosylatcd oligosaccharide, a sialylated oligosaccharide, an N-acetylglucosamine oligosaccharide, and any combination thereof.

54. The nutritional composition according to claim 52, wherein the one or more human milk oligosaccharides comprises a combination of at least one fucosylated oligosaccharide, at least one sialylated oligosaccharide, and at least one N-acetylglucosamine oligosaccharide.

55. The nutritional composition according to any one of claims 53-54, wherein the fucosylated oligosaccharide comprises at least one of 3-fucosyllactose (3-FL) and 2'- fucosyllactose (2'-FL); the sialylated oligosaccharide comprises at least one of 3’-sialyllactose (3’-SL) and 6' sialyllactose (6’-SL); and the N-acetylglucosamine oligosaccharide comprises at least one of lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT).

56. The nutritional composition according to claim 52, wherein the one or more human milk oligosaccharides comprises at least one selected from the group of 2'-fucosyllactose, 3- fucosyllactose, 3 '-sialyllactose, 6 '-sialyllactose, and lacto-N-tetraose, and any combination thereof.

57. The nutritional composition according to claim 52, wherein the one or more human milk oligosaccharides comprises 2'-fucosyllactose, 3-fucosyllactose, 3 '-sialyllactose, 6'- sialyllactose, and lacto-N-tetraose.

58. The nutritional composition according to any one of claims 52-57, wherein the nutritional composition contains total HMOs in an amount between 0.1 g/L and 10 g/L.

59. The nutritional composition according to any one of claims 44-58, further comprising a long chain polyunsaturated fatty acid selected from arachidonic acid, docosahexaenoic acid, and a combination thereof.

60. The nutritional composition according to any one of claims 44-59, wherein the nutritional composition comprises carbohydrate in an amount of from about 40% to about 60% by weight (on a dry weight basis); fat in an amount of from about 20% to about 40% by weight

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4855-0747-4085, v.1 Atty Ref. 15782WOO1

(on a dry weight basis); and protein in an amount of about 10% to about 25% by weight (on a dry weight basis).

61. The combination of betaine and folate according to any one of claims 43-60, for use in promoting brain development.

62. The combination of betaine and folate according to any one of claims 43-60, for use in promoting cognition.

63. The combination of betaine and folate according to any one of claims 43-62, wherein promoting brain development, cognition, or both comprises promoting neuronal differentiation and/or maturation.

4855-0747-4085, v.1