WO2016029113A1

WO2016029113A1 - Methods of increasing endogenous production of beta-hydroxy-beta-methylbutyrate

Info

Publication number: WO2016029113A1
Application number: PCT/US2015/046292
Authority: WO
Inventors: María del Pilar BUENO VARGAS; José María LÓPEZ PEDROSA; Manuel Cristóbal MANZANO MARTÍN; Ángela SANTOS-FANDILA; Enrique VÁQUEZ HERNANDEZ
Original assignee: Abbott Laboratories
Current assignee: Abbott Laboratories
Priority date: 2014-08-22
Filing date: 2015-08-21
Publication date: 2016-02-25
Anticipated expiration: 2017-02-22

Abstract

Nutritional compositions and methods of increasing endogenous production of beta-hydroxy-beta-methylbutyrate in a subject are provided. The methods include administering a nutritional composition comprising a prebiotic to a subject. The prebiotic may include one or more of short chain fructooligosaccharides, long chain fructooligosaccharides, galactooligosaccharides, inulins, xylooligosaccharides, and resistant starches or may be a human milk oligosaccharide, optionally selected from 6'-sialyllactose (6'SL), lacto-N-neotetraose (LNnT), 3'- fucosyllactose (3'FL), and 3'-sialyllactose (3'SL), and 2'-fucosyllactose (2'FL).

Description

METHODS OF INCREASING ENDOGENOUS PRODUCTION OF

BETA-HYDROXY-BETA-METHYLBUTYRATE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and any benefit of European Patent Application No. 14382323.5, filed August 22, 2014, the content of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The general inventive concepts relate to nutritional compositions and methods of increasing endogenous production of beta-hydroxy-beta-methylbutyrate (HMB) in a subject. More particularly, the general inventive concepts relate to the administration of a nutritional composition comprising a prebiotic to a subject to increase endogenous production of HMB in the subject.

BACKGROUND

Beta-hydroxy-beta-methylbutyrate (HMB) is a metabolite of the branched-chain amino acid leucine. In general, approximately 5% to 10% of the leucine ingested by a human is metabolized to HMB. Accordingly, humans endogenously synthesize about 0.2 to 0.5 grams of HMB per day, depending on the amount of dietary leucine consumed. Studies have indicated that dietary supplementation with HMB may attenuate muscle protein degradation, increase lean body mass, and increase strength. Prebiotics are typically non-digestible compounds that pass undigested through the upper part of the gastrointestinal tract. These compounds are beneficial to host organisms because they can selectively stimulate the growth and/or activity of bacteria in the colon and thereby improve the health of the gastrointestinal tract.

Human milk oligosaccharides (HMOs) are the third most abundant component in human milk, after lactose and lipids. They are present in large amounts ranging between 5 and 23 g/L and are diverse and complex in nature. HMOs are typically resistant to gastrointestinal digestion and therefore act as the first prebiotics in newborns and infants. HMOs have been identified as having a variety of other important biological effects including antimicrobial effects, immunomodulatory functions, positive effects on learning and memory in infants, and neuroprotective effects. As such, nutritional compositions incorporating HMOs have been developed, as described for example in WO2014/100191 , WO2014/100225, WO2014/100022, WO2014/100126 and WO2014/100225.

SUMMARY OF INVENTION

The general inventive concepts relate to nutritional compositions and methods of increasing endogenous production of HMB in a subject. To illustrate various aspects of the general inventive concepts, several exemplary embodiments of nutritional compositions and methods are provided herein.

In one exemplary embodiment, the invention provides a method of increasing endogenous production of beta-hydroxy-beta-methylbutyrate (HMB) in a subject. The method comprises administering a nutritional composition to the subject, wherein the composition comprises a prebiotic. Consumption of the nutritional composition comprising the prebiotic increases endogenous levels of beta-hydroxy-beta methylbutyrate in the subject. Also provided herein is a composition comprising a prebiotic for use in a method of increasing endogenous production of beta-hydroxy-beta-methylbutyrate in a subject, the method comprising administering the composition to the subject. Further provided herein is use of a nutritional composition comprising a prebiotic for the manufacture of a medicament for increasing endogenous production of beta-hydroxy-beta-methylbutyrate in a subject.

In one exemplary embodiment, the invention provides a method of increasing endogenous production of beta-hydroxy-beta-methylbutyrate in an offspring of a subject. The method comprises administering a nutritional composition comprising a prebiotic to the subject during gestation. Consumption of the nutritional composition comprising the prebiotic by the subject increases endogenous levels of beta-hydroxy-beta methylbutyrate in the offspring in the womb. Also provided herein is a composition comprising a prebiotic for use in a method of increasing endogenous production of beta-hydroxy-beta-methylbutyrate in an offspring of a subject, the method comprising administering the composition to the subject during gestation. Further provided herein is use of a nutritional composition comprising a prebiotic for the manufacture of a medicament for increasing endogenous production of beta-hydroxy- beta-methylbutyrate in an offspring of a subject.

In one exemplary embodiment, the invention provides a method of increasing endogenous production of beta-hydroxy-beta-methylbutyrate in an offspring of a subject. The method comprises administering a nutritional composition comprising a prebiotic to the subject following delivery of the offspring and during breastfeeding of the offspring by the subject. Consumption of the nutritional composition comprising the prebiotic by the subject increases endogenous levels of beta-hydroxy-beta methylbutyrate in the offspring. Also provided herein is a composition comprising a prebiotic for use in a method of increasing endogenous production of beta-hydroxy-beta-methylbutyrate in an offspring of a subject, the method comprising administering the composition to the subject following delivery of the offspring and during breastfeeding of the offspring. Further provided herein is use of a nutritional composition comprising a prebiotic for the manufacture of a medicament for increasing endogenous production of beta-hydroxy-beta-methylbutyrate in an offspring of a subject.

In one exemplary embodiment, the invention provides a method of promoting muscle hypertrophy and/or preventing muscle atrophy in a subject. The method comprises administering a nutritional composition comprising a prebiotic to the subject. Also provided herein is a composition comprising a prebiotic for use in promoting muscle hypertrophy and/or preventing muscle atrophy in a subject, the method comprising administering the composition to the subject. Further provided herein is use of a nutritional composition comprising a prebiotic for the manufacture of a medicament for promoting muscle hypertrophy and/or preventing muscle atrophy in a subject.

In one exemplary embodiment, the invention provides a method of improving brain development and/or cognitive function in a subject. The method comprises administering a nutritional composition comprising a prebiotic to the subject. Also provided herein is a composition comprising a prebiotic for use in improving brain development and/or cognitive function in a subject, the method comprising administering the composition to the subject. Further provided herein is use of a nutritional composition comprising a prebiotic for the manufacture of a medicament for improving brain development and/or cognitive function in a subject. In one exemplary embodiment, the invention provides a method of treating a disease or condition selected from: sarcopenia, muscle wasting diseases, hyperglycaemia, glucose intolerance and neurodegenerative diseases. The method comprises administering a nutritional composition comprising a prebiotic to the subject. Also provided herein is a composition comprising a prebiotic for use in the prevention or treatment of a disease or condition selected from: sarcopenia, muscle wasting diseases, hyperglycaemia, glucose intolerance and neurodegenerative diseases, said method comprising administration of a nutritional composition comprising a prebiotic.

In certain embodiments of the invention described herein, the prebiotic may be selected from the group consisting of short chain fructooligosaccharides, long chain

fructooligosaccharides, inulins, galactooligosaccharides, xylooligosaccharides, resistant starch, and combinations thereof. In certain embodiments, the prebiotic comprises fructooligosaccharides having an average degree of polymerization of 4 and high

performance inulin having an average degree of polymerization of 25, and wherein a weight ratio of the fructooligosaccharides to the high performance inulin is about 1 :1 .

In certain embodiments of the invention described herein, the prebiotic is a human milk oligosaccharide, optionally a human milk oligosaccharide selected from the group consisting of 6'-sialyllactose (6'SL), lacto-N-neotetraose (LNnT), 3'- fucosyllactose (3'FL), 3'- sialyllactose (3'SL), and 2'-fucosyllactose (2'FL).

BRIEF DESCRIPTION OF FIGURES

Fig.1 HMB concentration in serum after gavage of 2'-FL. A. Time course study of HMB in serum after gavage of 2'FL. B. Comparison of HMB levels in serum at time 0 (TO) and after 300 minutes (T300). Data are expressed as mean + SEM. Fig.2 HMB concentration in serum after gavage of 6 -SL (A) and LNnT (B). Data are expressed as mean + SEM.

Fig.3 HMB concentration in urine samples after gavage of 2'-FL (A), 6'-SL (B) and LNnT (C). Data are expressed as mean + SEM. DETAILED DESCRIPTION

While the general inventive concepts are susceptible of embodiment in many different forms, described herein in detail are specific embodiments thereof with the understanding that the present disclosure is to be considered as an exemplification of the principles of the general inventive concepts. Accordingly, the general inventive concepts are not intended to be limited to the specific embodiments illustrated and described herein. 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. 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.

Any combination of method or process steps as used herein may be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made. The term "nutritional composition" as used herein, unless otherwise specified, refers to nutritional products in various forms including, but not limited to, liquids, solids, powders, semi-solids, semi-liquids, nutritional supplements, and any other nutritional food product known in the art. A nutritional composition in powder form may be reconstituted (upon addition of water or another liquid) to form a nutritional composition in liquid form. In certain exemplary embodiments, the nutritional compositions disclosed herein include at least one of a source of protein, a source of carbohydrate, and a source of fat. In certain exemplary embodiments, the nutritional compositions disclosed herein include a source of protein, a source of carbohydrate, and a source of fat. The nutritional compositions disclosed herein are generally suitable for oral consumption by a human.

The term "nutritional liquid" as used herein, unless otherwise specified, refers to nutritional compositions in ready-to-drink liquid form, concentrated liquid form, and nutritional liquids made by reconstituting nutritional powders described herein prior to use. The nutritional liquid may also be formulated as a suspension, an emulsion, a solution, and so forth. The terms "nutritional powder" and "reconstitutable powder" as used herein, unless otherwise specified, refer 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 drymixed/dryblended powders.

The term "semi-solid" as used herein to describe a nutritional composition refers to nutritional compositions that are intermediate in properties, such as rigidity, between solids and liquids. Some exemplary semi-solids include puddings, yogurts, gels, gelatins, doughs, and so forth.

The term "semi-liquid" as used herein to describe a nutritional composition refers to nutritional compositions that are intermediate in properties, such as flow properties, between liquids and solids. Some exemplary semi-liquids include thick shakes, liquid yogurts, liquid gels, and so forth.

The terms "administer," "administering," "administered," and "administration," as used herein, unless otherwise specified, should be understood to include providing a nutritional composition to a subject, the act of consuming a nutritional composition (self-administration), and combinations thereof. In addition, it should be understood that the methods disclosed herein may be practised with or without doctor supervision or other medical direction.

The term "serving" as used herein, unless otherwise specified, refers to an amount of the nutritional composition administered, or intended to be administered, to a subject in a single administration event. In certain exemplary embodiments, the nutritional composition is packaged as a single serving. In certain exemplary embodiments, the nutritional composition is packaged in a container containing multiple servings, wherein the container bears instructions on how to separate a single serving of the nutritional composition from the bulk nutritional composition. The term "prebiotic" as used herein refers to a substantially non-digestible food ingredient that selectively stimulates the growth, the activity, or both of certain bacteria (e.g., bifidobacteria, lactobacilli) in the distal portion of the small intestine that includes the ileum, the cecum, and the colon. The term "human milk oligosaccharide" or "HMO" as used herein, unless otherwise specified, refers generally to a number of complex carbohydrates found in human breast milk that can be in acidic or neutral form, and to precursors thereof. Exemplary non-limiting human milk oligosaccharides include 6'-sialyllactose (6'SL), lacto-N-neotetraose (LNnT), 3'- fucosyllactose (3'FL), and 3'-sialyllactose (3'SL), and 2'-fucosyllactose (2'FL).

The term "subject" as used herein, unless otherwise specified, refers to a mammal, including companion animals, livestock, laboratory animals, working animals, sport animals, and humans. In certain exemplary embodiments, the subject is a human.

Beta-hydroxy-beta-methylbutyrate (HMB) is a metabolite of the branched-chain amino acid leucine. As a result of dietary intake of leucine, mammals endogenously produce a small amount of HMB, which is typically in the range of about 0.2 grams to about 0.5 grams per day. Some of the benefits that have been associated with the consumption of HMB include attenuation of muscle protein degradation, reduction in cardiovascular risk factors (e.g., total cholesterol, LDL cholesterol, systolic blood pressure), increased lean body mass, increased muscle cell integrity, increased strength, and reduced muscle soreness, just to name a few. Since the amount of HMB produced via leucine intake is relatively small, and the cost of HMB supplements is relatively high, alternative sources of HMB are desirable.

To that end, it has now been unexpectedly discovered that consumption of prebiotics can increase the endogenous production of HMB in a mammal.

The term "endogenous production" is used herein to mean the production of HMB in the body following the ingestion of any suitable precursor molecule.

Due to their ability to resist digestion, prebiotics can reach the colon and serve as a substrate for fermentation by colonic microbiota. Short-chain fatty acids (SCFAs) are the primary products generated from prebiotic fermentation and have various effects on gut morphology and function, such as supply of energy to the intestinal mucosa, lowering of the pH, and stimulation of mineral (e.g., sodium, potassium, calcium, magnesium, phosphorous), and water absorption. Such SCFAs produced by prebiotic fermentation in the colon include acetic acid, propionic acid, and butyric acid. Among these SCFAs, butyric acid, which is structurally similar to HMB, has been associated with many beneficial functions, not only in the gut, but also in other organs such as bone.

While not wishing to be bound by any particular theory, it is believed that prebiotic fermentation by colonic microbiota produces HMB in addition to SCFAs, and that the HMB produced in the colon by prebiotic fermentation is absorbed by enterocytes and enters circulation where it is available for use by various organs and tissues (e.g., muscle, brain).

Accordingly, nutritional compositions and methods of increasing endogenous production of HMB in a subject are provided. The increase in endogenous production of HMB may provide a variety of benefits including, but not limited to, attenuation of muscle protein degradation, reduction in cardiovascular risk factors (e.g., total cholesterol, LDL cholesterol, systolic blood pressure), increased lean body mass, increased muscle cell integrity, increased strength, and reduced muscle soreness.

In one exemplary embodiment, a nutritional composition comprising a prebiotic for use in increasing endogenous production of HMB in a subject and a method of increasing endogenous production of HMB in a subject is provided. The method includes administering a nutritional composition comprising a prebiotic to the subject. Consumption of the nutritional composition comprising the prebiotic increases endogenous levels of HMB in the subject. In certain embodiments, the subject is a human. In certain embodiments, the human may be an infant, an adolescent, an adult, or an elderly adult. As used herein, the term "infant" refers to a human having an age of up to 12 months. The term "adolescent" as used herein refers to a human having an age of 12 months to 18 years. The term "adult" as used herein refers to a human having an age of 18 years to 50 years. The term "elderly adult" as used herein refers to a human having an age of greater than 50 years. In certain embodiments, the human may be a toddler or a child. As used herein, the term "toddler" refers to a human having an age of greater than one year up to three years of age. The term "child" as used herein refers to a human having an age of greater than three years up to twelve years of age.

In one exemplary embodiment, a nutritional composition comprising a prebiotic for use in increasing endogenous production of HMB in an offspring of a subject and a method of increasing endogenous production of HMB in an offspring of a subject is provided. The method includes administering a nutritional composition comprising a prebiotic to the subject during gestation. Consumption of the nutritional composition comprising the prebiotic during gestation increases endogenous levels of HMB in the offspring in the womb. While not wishing to be bound by any particular theory, it is believed that consumption of the nutritional composition comprising the prebiotic by the subject (i.e., a subject who is pregnant) exposes the developing offspring to beneficial bacterially derived metabolites such as SCFAs and HMB, which facilitates healthy in utero development of the offspring, particularly the gastrointestinal tract of the offspring. In one exemplary embodiment, a nutritional composition comprising a prebiotic for use in increasing endogenous production of HMB in an offspring of a subject and a method of increasing endogenous production of HMB in an offspring of a subject is provided. The method includes administering a nutritional composition comprising a prebiotic to the subject following delivery of the offspring and during breastfeeding of the offspring. Consumption of the nutritional composition comprising the prebiotic by the subject during breastfeeding of the offspring increases endogenous levels of HMB in the offspring. While not wishing to be bound by any particular theory, it is believed that consumption of the nutritional composition comprising the prebiotic by the subject (e.g., a subject who breastfeeds her offspring) results in the endogenous production of HMB that subsequently enters circulation and the breast milk, and is transferred via the breast milk to the breastfeeding offspring to facilitate healthy development of the offspring.

In the exemplary embodiments described herein, the nutritional compositions comprise a prebiotic and may be formulated as and administered in any known or otherwise suitable oral product form. As used herein "oral administration" and "administered orally" refer to any form of administration in which the nutritional composition is introduced into the subject's digestive system, including the stomach and small intestine. For example, oral administration includes nasogastric intubation, in which a tube is run through the nose to the stomach of the subject to administer food or drugs. Any solid, liquid, semi-solid, semi-liquid, or powder product form, including combinations or variations thereof, are suitable for use in the exemplary embodiments described herein, provided that such forms allow for safe and effective oral delivery to the subject via oral consumption of the ingredients as also defined herein. The exemplary nutritional compositions described herein may be formulated to include only the essential ingredients described herein, or may be modified with optional ingredients to produce a variety of different product forms.

The exemplary nutritional compositions include, in addition to a prebiotic, at least one of a source of protein, a source of carbohydrate, and a source of fat. In certain exemplary embodiments, the nutritional compositions include a source of protein, a source of carbohydrate, and a source of fat. In certain exemplary embodiments, the nutritional compositions also include vitamins, minerals, and combinations thereof. The exemplary nutritional compositions described herein may be formulated with sufficient kinds and amounts of nutrients to provide a sole, a primary, or a supplemental source of nutrition. In certain exemplary embodiments, the nutritional compositions may be specifically formulated for pregnant women, lactating women, infants, adolescents, adults, or elderly adults.

In one exemplary embodiment, the nutritional composition is formulated as a solid nutritional composition. Examples of suitable solid nutritional compositions for use herein include snack and meal replacement products, including those formulated as bars; sticks; cookies, breads, cakes, or other baked goods; frozen liquids; candy; breakfast cereals; reconstitutable powders, granulated solids, or other particulates; snack chips or bites; frozen or retorted entrees; and so forth. In certain exemplary embodiments, when the nutritional composition is formulated as a solid nutritional composition, the serving is within a range of about 25 grams to about 150 grams. In certain exemplary embodiments, when the nutritional composition is a solid nutritional composition, the subject is administered a single serving or multiple (e.g., two, three, four, or more) servings per day.

In one exemplary embodiment, the nutritional composition is formulated as a nutritional liquid. Examples of nutritional liquids include snack and meal replacement products, hot or cold beverages, carbonated or non-carbonated beverages, juices or other acidified beverages, milk or soy-based beverages, shakes, coffees, teas, and so forth. These liquid compositions are most typically formulated as suspensions or emulsions, but can also be formulated in any other suitable form such as clear liquids, substantially clear liquids, solutions, and so forth. In certain exemplary embodiments, when the nutritional composition is a liquid, a serving thereof may be about 30 milliliters to about 500 milliliters (-1 fl. oz. to -17 fl. oz.). In certain other exemplary embodiments, when the nutritional composition is a liquid, the serving is about 237 milliliters (~8 fl. oz.). In other exemplary embodiments, when the nutritional composition is a liquid, the serving is about 177 milliliters to about 414 milliliters (-6 fl. oz. to -14 fl. oz.) or about 207 milliliters to about 296 milliliters (~7 fl. oz. to -10 fl. oz.). In other exemplary embodiments, when the nutritional composition is a liquid, the serving is about 30 milliliters to about 75 milliliters (-1 fl. oz. to -2.5 fl. oz.). In certain exemplary embodiments, when the nutritional composition is a liquid, the subject is administered a single serving or multiple (e.g., two, three, four, or more) servings per day. In certain exemplary embodiments, the nutritional composition is formulated as one of a liquid, a liquid reconstituted from a powder, and a bar. In certain exemplary embodiments, the nutritional composition is formulated as an emulsion. The emulsion generally comprises a source of protein, a source of carbohydrate, and a source of fat. The emulsions are 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. The emulsion may have a pH ranging from about 3.0 to about 8.0, but is generally formulated with a pH in a range of from about 5.0 to about 8.0, including from about 6.0 to about 8.0, and also including from about 6.5 to about 7.5.

In certain exemplary embodiments, the nutritional composition may be formulated as a semisolid composition (e.g., puddings, yogurts, gels, gelatins, doughs) or a semi-liquid composition (e.g., thick shakes, liquid yogurts, liquid gels), as well as product forms such as capsules, tablets, caplets, pills, and so forth. In certain exemplary embodiments, the nutritional composition may be formulated as lozenges, tablets (e.g., chewable, coated), pastes, gels, and so forth.

The nutritional compositions described herein may serve as a sole or supplemental source of nutrition. Accordingly, the nutritional compositions described herein may be formulated to provide a certain amount of energy per serving of the nutritional composition. For example, in certain exemplary embodiments, the nutritional composition provides up to 500 kcal of energy per serving, including from 20 kcal to 500 kcal, from 75 kcal to 500 kcal, from 150 kcal to 500 kcal, from 200 kcal to 500 kcal, from 300 kcal to 500 kcal, or from 400 kcal to 500 kcal per serving. The exemplary nutritional compositions described herein include a prebiotic. Exemplary prebiotics that may be utilized in the nutritional compositions and methods described herein include short chain fructooligosaccharides (FOS), long chain FOS, inulins, galactooligosaccharides (GOS), xylooligosaccharides (XOS), resistant starch, and combinations thereof. Fructooligosaccharides (FOS) refer to oligosaccharides comprised of D-fructose and D-glucose. Short chain FOS generally contain two to six monosaccharide units, whereas long chain FOS generally contain seven or more monosaccharide units. Generally, FOS are resistant to digestion in the upper gastrointestinal tract, and stimulate the growth of Bifidobacterium species in the colon.

Inulins refer to a group of naturally-occurring fructose-containing oligosaccharides. Inulins belong to a class of carbohydrates known as fructans. They are derived from the roots of chicory (Cichorium intybus) and Jerusalem artichokes. Inulins are mainly comprised of fructose units and typically have a terminal glucose. The bond between fructose units in inulins is a beta-(2-1 ) glycosidic linkage. The average degree of polymerization of inulins marketed as nutritional supplements is 10 to 12. Inulins stimulate the growth of Bifidobacterium species in the colon. Galactooligosaccharides (GOS) are a mixture of oligosaccharides consisting of D-glucose and D-galactose. The GOS may be produced from lactose via the action of the enzyme beta-galactosidase. In general, GOS are resistant to digestion in the upper gastrointestinal tract and stimulate the growth of Bifidobacteria in the colon. Xylooligosaccharides (XOS) are comprised of oligosaccharides containing beta-linked xylose residues. The degree of polymerization of XOS is from two to four. XOS may be obtained by the enzymatic hydrolysis of the polysaccharide xylan.

Other suitable prebiotics for use in the exemplary nutritional compositions and methods described herein include, but are not limited to, resistant starch, acacia gum, and dextrans.

In one exemplary embodiment, the prebiotic is a combination of FOS having an average degree of polymerization of four and high performance inulin having an average degree of polymerization of twenty-five, wherein a weight ratio of the FOS to the high performance inulin is about 1 :1 . A suitable commercially available prebiotic for use in the nutritional compositions and methods described herein is Synergyl^® high-performance inulin enriched with FOS (available from Beneo-Orafti S.A., Belgium). Exemplary prebiotics that may be utilized in the nutritional compositions and methods described herein also include prebiotics selected from the group of oligosaccharides referred to as "human milk oligosaccharides". Human milk oligosaccharides are one of the main components of human breast milk, which contains, on average, 10 grams per liter of neutral oligosaccharides and 1 gram per liter of acidic oligosaccharides. The composition of human milk oligosaccharides is very complex and more than 200 different oligosaccharide- like structures are known.

Specific non-limiting examples of HMOs that may be included individually or in combination in the nutritional compositions used in the methods of the present disclosure include: sialic acid (i.e., free sialic acid, lipid-bound sialic acid, protein-bound sialic acid); D-glucose (Glc); D-galactose (Gal); N-acetylglucosamine (GlcNAc); L-fucose (Fuc); fucosyl

oligosaccharides (i.e., lacto-N-fucopentaose I; lacto-N-fucopentaose II; lacto-N- fucopentaose III; lacto-N-difucohexaose I; and lactodifucotetraose); non-fucosylated, non- sialylated

oligosaccharides (i.e., lacto-N-tetraose); sialyl oligosaccharides (i.e., 3'-sialyl-3- fucosyllactose; disialomonofucosyllacto-N-neohexaose; monofucosylmonosialyllacto-N- octaose (sialyl Lea); sialyllacto-N-fucohexaose II; disialyllacto-N-fucopentaose II;

monofucosyldisialyllacto-N- tetraose); and sialyl fucosyl oligosaccharides (i.e., 2'- sialyllactose; 2-sialyllactosamine; 3'- 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 II; sialyllacto-N- tetraose a; disialyllacto-N-hexaose I; and sialyllacto-N-tetraose b). Also useful are variants in which the glucose (Glc) at the reducing end is replaced by 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. Pat. No. 8,197,872, issued June 12, 2012, to Mills, et al, which is herein incorporated by reference in its entirety. Other suitable examples of HMOs that may be included in the compositions of the present disclosure include lacto-N-fucopentaose V, lacto-N-hexaose, para-lacto-N-hexaose, lacto-N- neohexaose, para-lacto-N-neohexaose, monofucosyllacto-N-hexaose II, isomeric

fucosylated lacto-N-hexaose (1 ), isomeric fucosylated lacto-N-hexaose (3), isomeric fucosylated lacto-N-hexaose (2), difucosyl-para-lacto-N-neohexaose, difucosyl-para-lacto-N- hexaose, difucosyllacto-N-hexaose, lacto-N-neoocataose, para-lacto-N-octanose, iso-lacto- N-octaose, lacto-N-octaose, monofucosyllacto-neoocataose, monofucosyllacto-N-ocataose, difucosyllacto-N-octaose I, difucosyllacto-N-octaose II, difucosyllacto-N-neoocataose II, difucosyllacto-N-neoocataose I, lacto-N-decaose, trifucosyllacto-N-neooctaose,

trifucosyllacto-N-octaose, trifucosyl-iso-lacto-N-octaose, lacto-N-difuco-hexaose II, sialyl- lacto-N-tetraose a, sialyl-lacto-N-tetraose b, sialyl-lacto-N-tetraose c, sialyl-fucosyl-lacto-N- tetraose I, sialyl-fucosyl-lacto-N-tetraose II, and disialyl-lacto-N-tetraose, and combinations thereof. In certain exemplary embodiments, the HMOs that may be included individually or in combination in the nutritional compositions used in the methods of the present disclosure are selected from the group consisting of: 6'-sialyllactose (6'SL); lacto-N-neotetraose (LNnT); 3'- fucosyllactose (3'FL); 3'-sialyllactose (3'SL); and 2'-fucosyl lactose (2'FL), preferably selected from the group consisting of: 6'-sialyllactose (6'SL); lacto-N-neotetraose (LNnT); and 2'-fucosyl lactose (2'FL), more preferably 2'-fucosyllactose (2'FL).

The HMO or HMOs may be isolated or enriched from milk(s) secreted by mammals including, but not limited to: human, bovine, ovine, porcine, or caprine species. The HMOs may also be produced via microbial fermentation, enzymatic processes, chemical synthesis, or combinations thereof.

In certain exemplary embodiments, the prebiotic comprises from about 1 % to about 20% by weight of the nutritional composition. In certain exemplary embodiments, the total amount of prebiotic may be provided by one or multiple prebiotics, including any one or more of the prebiotics disclosed herein. In certain exemplary embodiments, the prebiotic comprises from about 1 % to about 15% by weight of the nutritional composition, including from about 5% to about 12%, from about 5% to about 10%, or from about 7% to about 10% by weight of the nutritional composition. In certain exemplary embodiments, the nutritional composition is a liquid and the prebiotic is present in an amount from about 0.001 mg/ml to about 20 mg/ml, including from about 0.001 mg/mL to about 10 mg/mL, including from about 0.001 mg/mL to about 5 mg/mL, including from about 0.001 mg/mL to less than 2 mg/mL, and also including from about 0.01 mg/mL to about 20 mg/mL, including from about 0.01 mg/mL to less than 2 mg/mL. The amount of prebiotic may be provided by one or multiple prebiotics including any one or more of the prebiotics disclosed herein, particularly one or multiple human milk oligosaccharides described herein, preferably wherein the one or multiple human milk oligosaccharides are selected from the group consisting of 6'-sialyllactose (6'SL), lacto-N-neotetraose (LNnT), 3'- fucosyllactose (3'FL), 3'-sialyllactose (3'SL), and 2'-fucosyllactose (2'FL), preferably 6'- sialyllactose (6'SL), lacto-N-neotetraose (LNnT) and 2'-fucosyllactose (2'FL).

In certain exemplary embodiments, the nutritional composition is a powder and the prebiotic is present in an amount from about 0.0005% to about 5% by weight of the powder, including from about 0.01 % to about 1 %, by weight of the powder. The amount of prebiotic may be provided by one or multiple prebiotics including any one or more of the prebiotics disclosed herein, particularly one or multiple human milk oligosaccharides described herein, preferably wherein the one or multiple human milk oligosaccharides are selected from the group consisting of 6'-sialyllactose (6'SL), lacto-N-neotetraose (LNnT), 3'- fucosyllactose (3'FL), 3'- sialyllactose (3'SL), and 2'-fucosyllactose (2'FL), preferably 6'-sialyllactose (6'SL), lacto-N- neotetraose (LNnT) and 2'-fucosyllactose (2'FL).

In certain exemplary embodiments, the nutritional composition is a bar and the prebiotic is present in an amount from about 0.0005% to about 10% by weight of the bar, including from about 0.01 % to about 5%, by weight of the bar. The amount of prebiotic may be provided by one or multiple prebiotics including any one or more of the prebiotics disclosed herein, particularly one or multiple human milk oligosaccharides described herein, preferably wherein the one or multiple human milk oligosaccharides are selected from the group consisting of 6'-sialyllactose (6'SL), lacto-N-neotetraose (LNnT), 3'- fucosyllactose (3'FL), 3'- sialyllactose (3'SL), and 2'-fucosyllactose (2'FL), preferably 6'-sialyllactose (6'SL), lacto-N- neotetraose (LNnT) and 2'-fucosyllactose (2'FL).

In addition to the prebiotic, the exemplary nutritional compositions described herein may include at least one of a protein, a carbohydrate, and a fat. In certain exemplary embodiments, the nutritional composition comprises a protein, a carbohydrate, and a fat. In certain exemplary embodiments, the nutritional composition comprises a protein, a carbohydrate, and is substantially free of fat. As used herein "substantially free of fat" refers to a nutritional composition containing less than 0.5%, including less than 0.1 %, fat by weight of the nutritional composition. "Substantially free of fat" may also refer to a nutritional composition that contains no added fat, although the nutritional composition may contain some amount of fat resulting from an added ingredient (e.g., a protein source that inherently has some fat).

In one exemplary embodiment, the nutritional composition comprises from about 6 grams to about 50 grams of protein per serving of the nutritional composition. In certain exemplary embodiments, the nutritional composition comprises from about 6 grams to about 45 grams of protein per serving, including from about 10 grams to about 40 grams, from about 15 grams to about 35 grams, and also including from about 15 grams to about 25 grams of protein per serving of the nutritional composition. In certain exemplary embodiments, the nutritional composition comprises from about 5% to about 40% by weight protein, including from about 10% to about 30%, and also including from about 15% to about 25% by weight protein. The protein present in the exemplary nutritional compositions described herein may be provided by one source of protein or multiple sources of protein, so long as the source of protein is suitable for use in oral nutritional compositions and is otherwise compatible with any other selected ingredients or features in the nutritional composition.

In certain exemplary embodiments, the protein present in the exemplary nutritional compositions may be provided by protein sources including, but not limited to, intact protein, hydrolyzed protein, and partially hydrolyzed protein, which may be derived from any known or otherwise suitable source such as milk (e.g., casein, whey), animal (e.g., meat, fish), cereal (e.g., rice, corn), vegetable (e.g., soy, potato, pea), and combinations thereof. In certain exemplary embodiments, the protein present in the exemplary nutritional compositions described herein is provided by any one or more of the following protein sources: a whey protein concentrate, a whey protein isolate, a whey protein hydrolysate, an acid casein, a sodium caseinate, a calcium caseinate, a potassium caseinate, a casein hydrolysate, a milk protein concentrate, a milk protein isolate, a milk protein hydrolysate, nonfat dry milk, condensed skim milk, skim milk powder, a soy protein concentrate, a soy protein isolate, a soy protein hydrolysate, a pea protein concentrate, a pea protein isolate, a pea protein hydrolysate, a collagen protein, a collagen protein isolate, potato protein, insect protein, earthworm protein, rice protein, quinoa protein, legume protein, grain protein, and combinations thereof. In one exemplary embodiment, the nutritional composition comprises from about 15 grams to about 1 10 grams of carbohydrates per serving of the nutritional composition, and the prebiotic comprises from about 1 % to about 25% by weight of the total carbohydrates. In certain exemplary embodiments, the nutritional composition comprises from about 25 grams to about 90 grams of carbohydrates per serving, including from about 40 grams to about 65 grams, and also including from about 45 grams to about 55 grams of carbohydrates per serving of the nutritional composition. In certain exemplary embodiments, the prebiotic comprises from about 5% to about 20% by weight of the total carbohydrates, including from about 10% to about 15%, from about 5% to about 10%, and also including about 7.5% by weight of the total carbohydrates. In certain exemplary embodiments, the nutritional composition comprises from about 10% to about 80% by weight carbohydrates, including from about 30% to about 60%, and also including from about 50% to about 70% by weight carbohydrates. The carbohydrates present in the exemplary nutritional compositions described herein may be provided by one source of carbohydrates or multiple sources of carbohydrates, so long as the source of carbohydrates is suitable for use in oral nutritional compositions and is otherwise compatible with any other selected ingredients or features in the nutritional composition.

In certain exemplary embodiments, the carbohydrate, in addition to the prebiotic, present in the exemplary nutritional compositions may be provided by carbohydrate sources such as simple carbohydrates, complex carbohydrates, or variations or combinations thereof. In certain exemplary embodiments, the carbohydrate present in the exemplary nutritional compositions described herein is provided by any one or more of the following carbohydrate sources: maltodextrin, hydrolyzed starch, modified starch, cornstarch, glucose polymers, corn syrup, corn syrup solids, rice-derived carbohydrate, sucrose, glucose, fructose, lactose, high fructose corn syrup, honey, sugar alcohol, isomaltulose, sucromalt, gum arabic, sodium carboxymethyl cellulose, guar gum, citrus pectin, low methoxy pectin, high methoxy pectin, oat beta-glucan, barley beta-glucan, carrageenan, psyllium, oat hull fiber, pea hull fiber, soy hull fiber, soy cotyledon fiber, sugar beet fiber, cellulose, corn bran, and combinations thereof. In one exemplary embodiment, the nutritional composition comprises from about 0.5 grams to about 45 grams of fat per serving of the nutritional composition. In certain exemplary embodiments, where the nutritional composition includes fat, the nutritional composition comprises from about 2 grams to about 40 grams of fat per serving of the nutritional composition, including from about 5 grams to about 35 grams, from about 10 grams to about 30 grams, and from about 15 grams to about 25 grams of fat per serving of the nutritional composition. In certain exemplary embodiments, where the nutritional composition includes fat, the nutritional composition comprises from about 0.5% to about 30% by weight fat, including from about 1 % to about 25%, from about 5% to about 20%, or from about 10% to about 15% by weight fat. When present in the exemplary nutritional compositions described herein, the fat may be provided by one source of fat or multiple sources of fat, so long as the source of fat is suitable for use in oral nutritional compositions and is otherwise compatible with any other selected ingredients or features in the nutritional composition.

In certain exemplary embodiments where the nutritional composition includes fat, the fat may be derived from fat sources such as plants, animals, and combinations thereof. In certain exemplary embodiments where fat is included in the nutritional composition, the fat may be provided by any one or more of the following fat sources: coconut oil, fractionated coconut oil, soy oil, corn oil, olive oil, safflower oil, high oleic safflower oil, high gamma- linolenic acid (GLA) safflower oil, MCT (medium chain triglycerides) oil, sunflower oil, high oleic sunflower oil, palm oil, palm kernel oil, palm olein, canola oil, marine oil, cottonseed oil, eicosapentaenoic acid, docosahexaenoic acid, gamma-linolenic acid, algal oil, and combinations thereof.

In certain exemplary embodiments, the nutritional composition is formulated as a clear liquid having a pH of about 2.0 to about 5.0, and also having no more than about 0.5% fat by weight of the nutritional composition. The limited amount of fat contributes to the desired clarity of the nutritional composition. Typically, liquid nutritional compositions that are clear, or at least substantially translucent, are substantially free of fat. Furthermore, certain exemplary embodiments of liquid nutritional compositions that have a desired acidic pH in the range of about 2.0 to 5.0 (e.g., juices, fruit juices, fruit-flavored beverages) are typically substantially free of fat. Liquid nutritional compositions that are both clear and have a pH ranging from about 2.0 to about 5.0 are also typically substantially free of fat. In certain of the preceding embodiments, the pH of the nutritional composition may be from about 2.5 to about 4.6, including a pH of about 3.0 to about 3.5. In those embodiments of the nutritional compositions that are substantially free of fat but have some amount of fat present, the fat may be present as a result of being inherently present in another ingredient (e.g., a protein source).

In certain exemplary embodiments, the nutritional composition may also comprise optional components or ingredients that modify the physical, chemical, aesthetic, or processing characteristics of the nutritional composition, or serve as additional nutritional components. Many such optional ingredients are known or otherwise suitable for use in medical food or other nutritional products and may also be used in the exemplary nutritional compositions described herein, provided that such optional ingredients are safe for oral administration and are compatible with the essential and other ingredients in the selected product form. Non-limiting examples of such optional ingredients include vitamins, minerals, preservatives, emulsifying agents, buffers, creatine, probiotics, anti-inflammatory agents, colorants, flavorants, thickening agents, stabilizers, lubricants, flowing or anti-caking agents, masking agents, and so forth. In certain exemplary embodiments, the nutritional composition may comprise any of a variety of vitamins or related nutrients, non-limiting examples of which include vitamin A, vitamin E, vitamin D₂, vitamin D₃, vitamin A palmitate, vitamin E acetate, vitamin C palmitate (ascorbyl palmitate), vitamin K, thiamine, riboflavin, pyridoxine, vitamin B₁₂, carotenoids (e.g., beta-carotene, zeaxanthin, lutein, lycopene), niacin, folic acid, pantothenic acid, biotin, vitamin C, choline, inositol, salts and derivatives thereof, and combinations thereof.

In certain exemplary embodiments, the nutritional composition comprises any of a variety of minerals. Non-limiting examples of suitable minerals include, but are not limited to, calcium, selenium, potassium, iodine, phosphorus, magnesium, iron, zinc, manganese, copper, sodium, molybdenum, chromium, chloride, and combinations thereof.

The various exemplary embodiments of the nutritional compositions described herein may be prepared by any process or suitable method (now known or known in the future) for making a selected product form, such as a nutritional liquid or a nutritional powder. Many such techniques are known for any given product form, such as nutritional liquids or nutritional powders, and can readily be applied by one of ordinary skill in the art to the various exemplary embodiments of the nutritional composition described herein. In one suitable manufacturing process, an exemplary liquid nutritional composition is prepared utilizing at least three separate slurries, including a protein-in-fat (PIF) slurry, a carbohydrate-mineral (CHO-MIN) slurry, and a protein-in-water (PIW) slurry. The PIF slurry is formed by heating and mixing the selected oil (e.g., canola oil, corn oil, fish oil) and then adding an emulsifier (e.g., lecithin), fat soluble vitamins, and a portion of the total protein (e.g., milk protein concentrate) with continued heat and agitation. The CHO-MIN slurry is formed by adding with heated agitation to water: prebiotic, minerals (e.g., potassium citrate, dipotassium phosphate, sodium citrate), trace and ultra trace minerals (TM/UTM premix), thickening or suspending agents (e.g., microcrystalline cellulose, gellan, carrageenan). The resulting CHO-MIN slurry is held for 10 minutes with continued heat and agitation before adding additional minerals (e.g., potassium chloride, magnesium carbonate, potassium iodide), or carbohydrates (e.g., sucrose, corn syrup), or combinations thereof. The PIW slurry is formed by mixing with heat and agitation the remaining protein (e.g., sodium caseinate, soy protein concentrate) into water.

In accordance with this process, the three resulting slurries are blended together with heated agitation and the pH adjusted to the desired range (e.g., 6.6 to 7.0) after which the blended composition is subjected to high-temperature short-time (HTST) processing during which the blended composition is heat treated, emulsified, homogenized, and then allowed to cool. Water soluble vitamins and ascorbic acid are added (if applicable), the pH is again adjusted (if necessary), flavorants are added, and any additional water can be added to adjust the solids content of the liquid nutritional composition to the desired range. At this point, the liquid nutritional composition may be packaged and sterilized according to any suitable sterilization technique, such as aseptic, retort, or hot-fill sterilization.

A nutritional powder suitable for use in the exemplary methods described herein, such as a spray dried nutritional powder or dry-blended nutritional powder, may be prepared by any collection of known or otherwise effective techniques suitable for making and formulating a nutritional powder. 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 nutritional powders described herein.

One method of preparing a nutritional powder comprises forming and homogenizing an aqueous slurry or liquid comprising predigested fat, and optionally protein, carbohydrate, and other sources of fat, and then spray drying the slurry or liquid to produce a nutritional powder. The method may further comprise the step of spray drying, dry-blending, or otherwise adding additional nutritional or functional ingredients, including any one or more of the ingredients described herein, to the nutritional powder.

The exemplary methods of increasing endogenous production of HMB described herein include administering a nutritional composition comprising a prebiotic to a subject. Any of the embodiments of the nutritional composition described above may be utilized in the exemplary methods.

In one exemplary embodiment, the nutritional composition comprising a prebiotic is administered to a subject, and consumption of the nutritional composition increases endogenous levels of HMB in the subject. In one exemplary embodiment, the subject is a human. In certain exemplary embodiments, the human may be an infant, an adolescent, an adult, or an elderly adult. In certain exemplary embodiments, the subject has a condition that promotes protein degradation, a loss of lean body mass, a decrease in muscle strength, and combinations thereof. Such conditions include, but are not limited to, cachexia, cancer, end stage renal disease, chronic obstructive pulmonary disease (COPD), diabetes, and so forth. The increase in endogenous levels of HMB as a result of consuming the nutritional composition comprising a prebiotic can treat these conditions by attenuating protein degradation, attenuating losses of lean body mass, attenuating decreases in muscle strength, and combinations thereof.

In one exemplary embodiment, a method of increasing endogenous production of HMB in an offspring of a subject is provided. In accordance with this embodiment, the nutritional composition comprising a prebiotic is administered to a subject during gestation. In other words, the nutritional composition comprising a prebiotic is administered to a subject who is pregnant. Consumption of the nutritional composition comprising a prebiotic by the subject during gestation increases endogenous levels of HMB in the offspring in the womb of the subject. As mentioned above, it is believed that consumption of the nutritional composition comprising the prebiotic by the subject during gestation exposes the developing offspring to beneficial bacterially derived metabolites such as SCFAs and HMB, which facilitates healthy in utero development of the offspring, particularly the gastrointestinal tract of the offspring. In one exemplary embodiment, a method of increasing endogenous production of HMB in an offspring of a subject includes administering the nutritional composition comprising a prebiotic to the subject following delivery of the offspring and during breastfeeding of the offspring. Consumption of the nutritional composition comprising a prebiotic by the subject during breastfeeding increases endogenous levels of HMB in the offspring. It is believed that consumption of the nutritional composition comprising a prebiotic by the subject following delivery and during breastfeeding results in the endogenous production of HMB that subsequently enters circulation and the breast milk, and the endogenously produced HMB is then transferred via the breast milk to the breastfeeding offspring to facilitate healthy development of the offspring. In certain exemplary embodiments, the nutritional composition comprising a prebiotic is administered to a subject during gestation and is also administered to the subject following delivery of the offspring and during breastfeeding of the offspring. In certain exemplary embodiments, the nutritional composition comprising a prebiotic is administered to the offspring from weaning through adolescence. In other words, the nutritional composition is administered directly to the offspring after breastfeeding of the offspring is discontinued. It should be understood that the phrase "through adolescence" means for a period of time during adolescence, which could be, for example, one month, three months, six months, one year, two years, and so forth. In one exemplary embodiment, the nutritional composition comprising a prebiotic is administered orally to the subject. In certain exemplary embodiments, the nutritional composition comprising a prebiotic is administered to the subject at least once daily.

In certain exemplary embodiments, the nutritional composition comprising a prebiotic is chronically administered to the subject. The phrases "chronically administered," "chronic administration," and "chronically administering" as used herein refers to regular administration which is provided indefinitely or to regular administration for a significant period of time. For example, in certain exemplary embodiments, chronic administration can include regular administration for at least two weeks, regular administration for at least one month, regular administration for at least 6 weeks, regular administration for at least two months, regular administration for at least 3 months, regular administration for at least 4 months, regular administration for at least 5 months, regular administration for at least 6 months, or regular administration for at least 9 months. In other exemplary embodiments, chronic administration refers to regular administration for at least 1 year, regular administration for at least 1 .5 years, regular administration for at least 2 years, or regular administration for more than 2 years. "Regular administration," as used herein, refers to administration according to a schedule whereby the subject consumes the exemplary nutritional composition at regular intervals.

As used herein, "regular intervals" refers to administration in a repeating, periodic fashion where the time between administrations is approximately (or intended to be approximately) the same. In various exemplary embodiments, administration at regular intervals includes daily administration or weekly administration. In other exemplary embodiments, administration at regular intervals includes administration 1-2 times per week, administration 1 -3 times per week, administration 2-3 times per week, administration 1-4 times per week, administration 1-5 times per week, administration 2-5 times per week, administration 3-5 times per week, administration 1-6 times per week, administration 1-7 times per week, administration 2-6 times per week, administration 2-7 times per week, administration 1-2 times per day, administration 1 -3 times per day, administration 1-4 times per day, administration 2-3 times per day, administration 2-4 times per day, administration 3-4 times per day, administration 2-5 times per day, administration 3-5 times per day, or administration 4-5 times per day. In certain exemplary embodiments where the nutritional composition comprising a prebiotic is consumed by a subject during gestation, the consumption takes place every day for: at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months during gestation, or in some instances daily during the entire period of gestation. Taking into account that some subjects will not recognize that they are pregnant on day 1 of their pregnancy, it should be understood that the exemplary methods are intended to encompass consumption of the nutritional composition comprising a prebiotic on less than every day of the period of pregnancy or gestation. In certain exemplary embodiments, the nutritional composition comprising a prebiotic is consumed by the subject on at least 1 day per week, at least 2 days per week, at least 3 days per week, at least 4 days per week, at least 5 days per week, or at least 6 days per week during gestation. This periodic consumption may take place for: at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months during gestation or in some instances during the entire period of gestation.

In certain exemplary embodiments, the nutritional composition comprising a prebiotic is consumed daily by the subject following delivery of the offspring and during breastfeeding of the offspring. In certain exemplary embodiments where the nutritional composition comprising a prebiotic is consumed by the subject following delivery of the offspring and during breastfeeding of the offspring, the consumption takes place every day for: at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months, and up to the period of time when the offspring is weaned. It should be understood that the exemplary methods are intended to encompass consumption of the nutritional composition comprising a prebiotic on less than every day following delivery of the offspring and during breastfeeding of the offspring. In certain exemplary embodiments, the nutritional composition comprising a prebiotic is consumed by the subject on at least 1 day per week, at least 2 days per week, at least 3 days per week, at least 4 days per week, at least 5 days per week or at least 6 days per week following delivery and during breastfeeding of the offspring. This periodic consumption may take place for: at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months, and up to 24 months or up to the period of time when the offspring is weaned (i.e., the entire period of time when the subject breastfeeds the offspring).

In certain exemplary embodiments, the nutritional composition comprising a prebiotic is consumed daily by the subject during gestation and also following delivery and during breastfeeding of the offspring. In certain exemplary embodiments where the nutritional composition comprising a prebiotic is consumed by the subject during gestation and following delivery and during breastfeeding of the offspring, the consumption takes place every day for: at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months during gestation or in some instances daily during the entire period of gestation. Taking into account that some subjects will not recognize that they are pregnant on day 1 of their pregnancy, it should be understood that the exemplary methods are intended to encompass consumption of the nutritional composition comprising a prebiotic on less than every day during gestation and on less than every day during breastfeeding of the offspring. In other embodiments, the nutritional composition comprising a prebiotic is consumed by the subject on at least 1 day per week, at least 2 days per week, at least 3 days per week, at least 4 days per week, at least 5 days per week, or at least 6 days per week during gestation and during breastfeeding of the offspring. This periodic consumption may take place for: at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months during gestation or in some instances during the entire period of gestation. Similarly, the periodic consumption described may take place during similar periods or during the entire period of breastfeeding the offspring. It should be understood that the schedule of administration or consumption during the period of gestation may be the same or different than the schedule of administration or consumption during the period of breastfeeding the offspring. In certain exemplary embodiments, the nutritional composition comprising a prebiotic is administered such that the amount of prebiotic consumed by the subject ranges from about 0.001 g/day to about 15 g/day, including from about 0.001 g/day to about 10 g/day, and including from about 0.1 g/day to about 5 g/day. In certain exemplary embodiments, the nutritional composition comprising a prebiotic is provided in one or multiple (e.g., two, three, four) servings per day to achieve the desired amount of prebiotic. In certain exemplary embodiments, the nutritional composition comprising a prebiotic is provided in two servings per day to achieve the desired amount of prebiotic.

In certain exemplary embodiments, administration of a nutritional composition comprising a prebiotic may result in a chronic increase in endogenous HMB production such that HMB levels remain elevated for a sustained period. In order to achieve a chronic increase in endogenous HMB production, the nutritional composition may be chronically administered according to any of the embodiments described above. For embodiments wherein administration of a nutritional composition results in a chronic increase in endogenous HMB production, the one or more prebiotics of the composition may be selected from short chain fructooligosaccharides (FOS), long chain FOS, inulins, galactooligosaccharides (GOS), xylooligosaccharides (XOS), resistant starch, and combinations thereof. In certain exemplary embodiments, administration of a nutritional composition comprising a prebiotic may result in an acute increase in endogenous HMB production such that HMB levels are elevated rapidly, typically after a single administration of composition. The term "acute increase" may be taken to mean an increase in endogenous HMB production occurring within a time period directly after administration of the composition of approximately 1 hour, approximately 2 hours, approximately 3 hours, or a time period up to 5 hours (300 minutes) after administration. For embodiments wherein administration of a nutritional composition results in an acute increase in endogenous HMB production, the one or more prebiotics of the composition may be human milk oligosaccharides selected from: 6'-sialyllactose (6'SL); lacto-N-neotetraose (LNnT); 3'- fucosyllactose (3'FL); 3'-sialyllactose (3'SL); and 2'-fucosyllactose (2'FL), preferably selected from the group consisting of: 6'- sialyllactose (6'SL); lacto-N-neotetraose (LNnT); and 2'-fucosyllactose (2'FL), more preferably 2'-fucosyllactose (2'FL). The level of endogenous HMB in a subject may be determined by a variety of suitable methods (now known or known in the future). For example, the level of HMB in the serum and urine of a subject can be determined by ultra-high performance liquid chromatography- tandem mass spectrometry (UHPLC-MS/MS). Accordingly, to determine the amount of increase in the endogenous production of HMB in a subject, a first measurement of endogenous HMB may be obtained from the subject within 1 week prior to initiation of the administration of the nutritional composition comprising a prebiotic disclosed herein. The first measurement may take place within 1 day to 7 days prior to initiation of the administration. Subsequently, a second measurement of endogenous HMB may be obtained from the subject. The second measurement may be obtained within a few hours after administration of the nutritional composition, for example within 8 hours, within 5 hours. Alternatively or in addition, the second measurement may be obtained at least two weeks after administration of the nutritional composition comprising a prebiotic has commenced, including at least one month, at least three months, at least six months, or at least one year after administration of the nutritional composition comprising a prebiotic has commenced. The second measurement of endogenous HMB is compared to the first measurement of endogenous HMB to determine the amount of increase in the endogenous production of HMB in the subject. In certain exemplary embodiments, the endogenous levels of HMB in the subject may increase at least 5%, including at least 9%, including at least 15%, including at least 25%, including at least 35%, including at least 40%, including at least 50% or more as a result of consuming a nutritional composition comprising a prebiotic as described herein.

As noted above, HMB has previously been recognized as an important metabolite, and compositions containing HMB as a dietary supplement have been described for a variety of applications linked to the control of skeletal muscle metabolism, glucose metabolism, brain development and cognitive function, see for example International patent applications: WO2012/092035, WO2012/097061 , WO2012/097064 and WO2012/1 12419, the contents of which are incorporated herein in their entirety. The observation reported herein that prebiotics are able to increase endogenous production of HMB gives rise to the possibility of using nutritional compositions comprising prebiotics in situations where increased HMB levels are desirable or have therapeutic benefit.

Accordingly, provided herein are methods of promoting muscle hypertrophy and/or preventing muscle atrophy in a subject, said methods comprising administering a nutritional composition comprising a prebiotic to a subject. Also provided herein are compositions comprising a prebiotic for use in promoting muscle hypertrophy and/or preventing muscle atrophy in a subject. As used herein, the term "muscle hypertrophy" means the increase in the size or mass of one or more skeletal muscles as a consequence of muscle cell growth. The term "muscle atrophy" is used herein to mean the opposite of muscle hypertrophy i.e. a decrease in the size or mass of one or more skeletal muscles. Muscle atrophy is typically caused by a change in the normal balance between protein synthesis and protein degradation in favour of destructive protein pathways. In the context of the present methods, the "promotion" of muscle hypertrophy may be measured or determined by an increase in muscle mass over a defined period of time and the "prevention" of muscle atrophy may be measured or determined by the absence of a decrease in muscle mass over a defined period of time.

In exemplary embodiments, the methods of promoting muscle hypertrophy and/or preventing muscle atrophy involve administering a nutritional composition comprising a prebiotic to a healthy individual i.e. an individual who is not affected by a disease condition and in particular an individual who is not affected by a disease condition affecting skeletal muscle. In certain embodiments, the nutritional compositions described herein are administered to healthy adult subjects to promote muscle hypertrophy and/or prevent muscle atrophy. Such methods may be performed for the purposes of increasing muscle strength and/or performance and/or for the purposes of regulating or maintaining lean body mass. The methods described herein as applied to healthy subjects may be regarded as non-therapeutic methods of promoting muscle hypertrophy and/or preventing muscle atrophy.

In exemplary embodiments, the methods of promoting muscle hypertrophy and/or preventing muscle atrophy involve administering a nutritional composition comprising a prebiotic to a subject having muscle atrophy caused by a period of muscle disuse, for example an extended period of muscle disuse consisting of at least four weeks, at least six weeks, at least six months or at least one year. The period of muscle disuse may be caused by immobilization of a part of the body, for example the immobilization of a broken limb in a cast, or may be caused by an extended period of enforced bedrest. It will be recognized by one skilled in the art that the rate of muscle atrophy for individuals may differ on the basis of, for example, age; that is elderly adults may experience more rapid muscle atrophy as compared with adolescents. In exemplary embodiments, the methods of promoting muscle hypertrophy and/or preventing muscle atrophy involve administering a nutritional composition comprising a prebiotic to a subject having cachexia, particularly wherein the cachexia is a co-morbidity of a disease or condition selected from cancer, congestive heart failure, chronic obstructive pulmonary disease (COPD) and/or liver failure.

Further provided herein are methods of improving brain development and/or cognitive function in a subject, said methods comprising administering a nutritional composition comprising a prebiotic to a subject. Also provided are compositions comprising a prebiotic for use in improving brain development and/or cognitive function in a subject. In exemplary embodiments, the nutritional composition is administered to a subject selected from an infant, a toddler, a child, an adolescent or an elderly adult as defined elsewhere herein.

In certain exemplary embodiments, the method is carried out by administration of nutritional composition comprising a prebiotic to an infant, toddler, child or adolescent who is otherwise healthy i.e. is not affected by a disease condition, particularly an infant or adolescent who is not affected by a condition affecting the brain and/or cognition. The nutritional composition is administered to improve brain development and/or cognitive function in said infant or adolescent. As used herein, the term "brain development" means the growth, maturation and remodeling of neurons that occurs in the brain, particularly in infants and adolescents. An improvement in brain development may be measured or determined by an improvement in the mental performance of a subject, as assessed by standard tests routinely carried out to gauge mental performance. As used herein, the term "cognitive function" means the level of cognition displayed by a given individual. By "cognition" is meant the mental processes involved in gaining knowledge and comprehension, including thinking, knowing,

remembering, judging and problem solving. Cognition includes higher-level functions of the brain and encompasses language, imagination, perception, and planning. An improvement in "cognitive function" may be measured or determined by an improvement in the cognition of a subject, as assessed by standard tests routinely used to test the various mental skills falling under the term cognition as defined above.

Improvements in brain development and/or cognitive function may be determined over a defined period of time and/or may be compared to any improvement determined for one or more control subjects, particularly wherein the control subjects do not receive a nutritional composition comprising prebiotic.

In certain exemplary embodiments, methods of improving brain development and/or cognitive function are carried out by administration of a nutritional composition comprising a prebiotic to a preterm infant. As used herein, the term "preterm infant" refers to a person born prior to 36 weeks of gestation. In such embodiments, the method may be carried out to improve brain development and/or cognitive function such that the preterm infant, when assessed using standard tests for cognition, performs at the standard expected of an average term infant at the same age. In certain exemplary embodiments, methods of improving brain development and/or cognitive function are carried out by administration of a nutritional composition comprising a prebiotic to an elderly subject, optionally wherein the elderly adult is determined to be risk of cognitive decline or is already diagnosed as having cognitive impairment. For individuals diagnosed as having cognitive impairment, this may be the result of a neurodegenerative disorder, for example a neurodegenerative disorder selected from Alzheimer's disease, Huntington's disease, Parkinson's disease, dementia, amyotrophic lateral sclerosis, stroke and schizophrenia. Further provided herein are methods of preventing or treating a disease or condition selected from sarcopenia, muscle wasting diseases, hyperglycaemia, glucose intolerance and neurodegenerative diseases, said methods comprising administering a nutritional composition comprising a prebiotic to a subject. Also provided herein are compositions comprising a prebiotic for use in preventing or treating a disease or condition selected from sarcopenia, muscle wasting diseases, hyperglycaemia, glucose intolerance and neurodegenerative diseases.

Sarcopenia is the term typically used to describe the degenerative loss of skeletal muscle mass and strength associated with aging. It is a condition that is typically not caused by or associated with other pathological conditions and therefore can affect otherwise healthy individuals. In certain exemplary embodiments, methods of preventing or treating sarcopenia involve administering a composition comprising a prebiotic to an elderly subject who is otherwise healthy, particularly an elderly subject who does not suffer from any other muscle-wasting disease or condition.

The term "muscle wasting diseases" is used herein to mean any disease wherein the patient suffers from muscle atrophy as a cause or consequence of the pathology. "Cachexia" is a term used to describe the muscle wasting that occurs in individuals suffering from a number of different diseases. Thus, in certain embodiments, provided herein are methods of preventing or treating cachexia, optionally cachexia associated with one or more of the following diseases: cancer, human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS), autoimmune disease including multiple sclerosis, chronic obstructive pulmonary disease (COPD), end stage renal disease (ESRD), tuberculosis, congestive heart failure and familial amyloid polyneuropathy.

The term "hyperglycaemia" means high blood glucose levels, typically a blood glucose level of higher than 1 1.1 mmol/l (200 mg/dl). Hyperglycaemia is the characteristic defining individuals having the condition "glucose intolerance". Both hyperglycaemia and glucose intolerance are metabolic defects associated with a number of diseases. Thus, in certain embodiments, provided herein are methods for preventing or treating the hyperglycaemia and/or glucose intolerance in subjects suffering from a disease selected from COPD, obesity, diabetes and cancer. In certain exemplary embodiments, provided herein are methods of preventing and/or treating a neurodegenerative disease, optionally wherein the neurodegenerative disease is selected from Alzheimer's disease, Huntington's disease, Parkinson's disease, dementia, amyotrophic lateral sclerosis, stroke and schizophrenia. In the context of such methods, the "prevention" of a neurodegenerative disease may involve preventing the onset of clinical symptoms typically associated with the disease, for example, the onset of clinical dementia. Methods for the "treatment" of neurodegenerative disease may include methods intended to alleviate symptoms of disease or methods that are aimed at stabilizing or reversing the underlying pathology. The methods of preventing neurodegenerative disease as described herein may involve administering a composition comprising a prebiotic to a subject identified as "at risk" of disease, wherein a subject "at risk" is one identified as having a higher probability of developing the disease as compared with an average individual in the general population, or a given population. The methods of treating neurodegenerative disease as described herein may involve administering a composition comprising a prebiotic to any individual diagnosed as having the neurodegenerative disease.

All embodiments of the invention pertaining to a nutritional composition comprising a prebiotic are equally applicable to the methods described herein above. In particular, the nutritional compositions for use in any of the methods described herein can include any features or combination of features described herein. Furthermore, in all of the methods described above, the nutritional composition is administered to the subject at a dose effective to produce an increase in HMB production in said subject. In certain exemplary embodiments, the increase in HMB production may be measured as an increase in endogenous HMB levels, optionally endogenous plasma HMB levels, optionally wherein the increase in endogenous HMB levels is an increase of at least 5%, at least 10%, at least 25%, at least 50%.

The present invention is also set out in the following clauses: 1 . A method of increasing endogenous production of beta-hydroxy-beta-methylbutyrate in a subject, the method comprising:

administering a nutritional composition comprising a prebiotic to the subject, whereby consumption of the nutritional composition increases endogenous levels of beta-hydroxy- beta methylbutyrate in the subject.

2. The method of clause 1 , wherein the prebiotic is selected from the group consisting of short chain fructooligosaccharides, long chain fructooligosaccharides, inulins,

galactooligosaccharides, xylooligosaccharides, resistant starch, and combinations thereof.

3. The method of clause 1 , wherein the prebiotic comprises fructooligosaccharides having an average degree of polymerization of 4 and high performance inulin having an average degree of polymerization of 25, and wherein a weight ratio of the

fructooligosaccharides to the high performance inulin is about 1 :1 .

4. The method of any one of clauses 1 -3, wherein the prebiotic comprises from about 1 % to about 20% by weight of the nutritional composition.

5. The method of any one of clauses 1 -4, wherein the nutritional composition further comprises at least one of a protein, a carbohydrate, and a fat.

6. The method of clause 5, wherein the nutritional composition comprises from about 6 grams to about 50 grams of protein per serving. 7. The method of any one of clauses 5-6, wherein the protein is one of a whey protein concentrate, a whey protein isolate, a whey protein hydrolysate, an acid casein, a sodium caseinate, a calcium caseinate, a potassium caseinate, a casein hydrolysate, a milk protein concentrate, a milk protein isolate, a milk protein hydrolysate, nonfat dry milk, condensed skim milk, skim milk powder, a soy protein concentrate, a soy protein isolate, a soy protein hydrolysate, a pea protein concentrate, a pea protein isolate, a pea protein hydrolysate, a collagen protein, collagen protein isolates, potato protein, insect protein, earthworm protein, rice protein, quinoa protein, legume protein, grain protein, and combinations thereof. 8. The method of any one of clauses 5-7, wherein the nutritional composition comprises from about 15 grams to about 1 10 grams of carbohydrates per serving, and wherein the prebiotic comprises from about 1 % to about 25% by weight of the total carbohydrates. 9. The method of any one of clauses 5-8, wherein the carbohydrate is one of maltodextrin, hydrolyzed starch, modified starch, cornstarch, glucose polymers, corn syrup, corn syrup solids, rice-derived carbohydrate, sucrose, glucose, fructose, lactose, high fructose corn syrup, honey, sugar alcohol, isomaltulose, sucromalt, gum arabic, sodium carboxymethyl cellulose, guar gum, citrus pectin, low methoxy pectin, high methoxy pectin, oat beta-glucan, barley beta-glucan, carrageenan, psyllium, oat hull fiber, pea hull fiber, soy hull fiber, soy cotyledon fiber, sugar beet fiber, cellulose, corn bran, and combinations thereof.

10. The method of any one of clauses 5-9, wherein the nutritional composition comprises from about 0.5 grams to about 45 grams of fat per serving.

1 1 . The method of any one of clauses 5-10, wherein the fat is one of coconut oil, fractionated coconut oil, soy oil, corn oil, olive oil, safflower oil, high oleic safflower oil, high gamma-linolenic acid (GLA) safflower oil, MCT (medium chain triglycerides) oil, sunflower oil, high oleic sunflower oil, palm oil, palm kernel oil, palm olein, canola oil, marine oil, cottonseed oil, eicosapentaenoic acid, docosahexaenoic acid, gamma-linolenic acid, algal

011. and combinations thereof.

12. The method of any one of clauses 1 -1 1 , wherein the nutritional composition is one of a liquid, a liquid reconstituted from a powder, and a solid.

13. The method of any one of clauses 1 -12, wherein the nutritional composition is an emulsion. 14. The method of any one of clauses 1 -13, wherein the nutritional composition is administered orally.

15. The method of any one of clauses 1 -14, wherein the nutritional composition is administered to the subject at least once daily. 16. A method of increasing endogenous production of beta-hydroxy-beta-methylbutyrate in an offspring of a subject, the method comprising:

administering a nutritional composition comprising a prebiotic to the subject during gestation, whereby consumption of the nutritional composition by the subject during gestation increases endogenous levels of beta-hydroxy-beta methylbutyrate in the offspring in the womb.

17. A method of increasing endogenous production of beta-hydroxy-beta-methylbutyrate in an offspring of a subject, the method comprising:

administering a nutritional composition comprising a prebiotic to the subject following delivery of the offspring and during breastfeeding of the offspring, whereby consumption of the nutritional composition by the subject during breastfeeding of the offspring increases endogenous levels of beta-hydroxy-beta methylbutyrate in the offspring.

18. The method of clause 16, further comprising administering the nutritional composition to the subject following delivery of the offspring and during breastfeeding of the offspring. 19. The method of any one of clauses 17-18, further comprising administering a nutritional composition comprising a prebiotic to the offspring from weaning through adolescence.

20. The method of any one of clauses 16-18, wherein the nutritional composition is administered to the subject at least once daily.

21 . The method of clause 19, wherein the nutritional composition is administered to the offspring at least once daily. 22. The method of any one of clauses 16-21 , wherein the prebiotic is selected from the group consisting of short chain fructooligosaccharides, long chain fructooligosaccharides, inulins, galactooligosaccharides, xylooligosaccharides, resistant starch, and combinations thereof. 23. The method of any one of clauses 16-21 , wherein the prebiotic comprises

fructooligosaccharides having an average degree of polymerization of 4 and high

24. The method of any one of clauses 16-23, wherein the prebiotic comprises from about 1 % to about 20% by weight of the nutritional composition.

25. The method of any one of clauses 16-24, wherein the nutritional composition further comprises at least one of a protein, a carbohydrate, and a fat.

26. The method of clause 25, wherein the nutritional composition comprises from about 6 grams to about 50 grams of protein per serving. 27. The method of any one of clauses 25-26, wherein the protein is one of a whey protein concentrate, a whey protein isolate, a whey protein hydrolysate, an acid casein, a sodium caseinate, a calcium caseinate, a potassium caseinate, a casein hydrolysate, a milk protein concentrate, a milk protein isolate, a milk protein hydrolysate, nonfat dry milk, condensed skim milk, skim milk powder, a soy protein concentrate, a soy protein isolate, a soy protein hydrolysate, a pea protein concentrate, a pea protein isolate, a pea protein hydrolysate, a collagen protein, collagen protein isolates, potato protein, insect protein, earthworm protein, rice protein, quinoa protein, legume protein, grain protein, and combinations thereof. 28. The method of any one of clauses 25-27, wherein the nutritional composition from about 15 grams to about 1 10 grams of carbohydrates per serving, and wherein the prebiotic comprises from about 1 % to about 25% by weight of the total carbohydrates.

29. The method of any one of clauses 25-28, wherein the carbohydrate is one of maltodextrin, hydrolyzed starch, modified starch, cornstarch, glucose polymers, corn syrup, corn syrup solids, rice-derived carbohydrate, sucrose, glucose, fructose, lactose, high fructose corn syrup, honey, sugar alcohol, isomaltulose, sucromalt, gum arabic, sodium carboxymethyl cellulose, guar gum, citrus pectin, low methoxy pectin, high methoxy pectin, oat beta-glucan, barley beta-glucan, carrageenan, psyllium, oat hull fiber, pea hull fiber, soy hull fiber, soy cotyledon fiber, sugar beet fiber, cellulose, corn bran, and combinations thereof.

30. The method of any one of clauses 25-29, wherein the nutritional composition comprises from about 0.5 grams to about 45 grams of fat per serving.

31 . The method of any one of clauses 25-30, wherein the fat is one of coconut oil, fractionated coconut oil, soy oil, corn oil, olive oil, safflower oil, high oleic safflower oil, high gamma-linolenic acid (GLA) safflower oil, MCT (medium chain triglycerides) oil, sunflower oil, high oleic sunflower oil, palm oil, palm kernel oil, palm olein, canola oil, marine oil, cottonseed oil, eicosapentaenoic acid, docosahexaenoic acid, gamma-linolenic acid, algal oil, and combinations thereof.

32. The method of any one of clauses 16-31 , wherein the nutritional composition is one of a liquid, a liquid reconstituted from a powder, and a solid.

33. The method of any one of clauses 16-32, wherein the nutritional composition is an emulsion. 34. The method of any one of clauses 16-33, wherein the nutritional composition is administered orally.

35. A nutritional composition comprising a prebiotic for use in increasing endogenous production of beta-hydroxy-beta-methylbutyrate in a subject.

36. A nutritional composition comprising a prebiotic for use in increasing endogenous production of beta-hydroxy-beta-methylbutyrate in an offspring of a subject, wherein consumption of the nutritional composition by the subject during gestation increases endogenous levels of beta-hydroxy-beta methylbutyrate in the offspring in the womb.

37. A nutritional composition comprising a prebiotic for use in increasing endogenous production of beta-hydroxy-beta-methylbutyrate in an offspring of a subject, whereby consumption of the nutritional composition by the subject following delivery and during breastfeeding of the offspring increases endogenous levels of beta-hydroxy-beta methylbutyrate in the offspring.

38. The composition of clause 36, wherein the nutritional composition is also consumed by the subject following delivery of the offspring and during breastfeeding of the offspring.

39. The composition of clause 37 or 38, wherein the nutritional composition is also administered to the offspring from weaning through adolescence. 40. The composition of any one of clauses 35-39, wherein the nutritional composition is administered to the subject at least once daily.

41 . The composition of any one of clauses 35-40, wherein the prebiotic is selected from the group consisting of short chain fructooligosaccharides, long chain

fructooligosaccharides, inulins, galactooligosaccharides, xylooligosaccharides, resistant starch, and combinations thereof.

42. The composition of any one of clauses 35-41 , wherein the prebiotic comprises fructooligosaccharides having an average degree of polymerization of 4 and high performance inulin having an average degree of polymerization of 25, and wherein a weight ratio of the fructooligosaccharides to the high performance inulin is about 1 :1 .

43. The composition of any one of clauses 35-42, wherein the prebiotic comprises from about 1 % to about 20% by weight of the nutritional composition.

44. The composition of any one of clauses 35-43, wherein the nutritional composition further comprises at least one of a protein, a carbohydrate, and a fat.

45. The composition of clause 44, wherein the nutritional composition comprises from about 6 grams to about 50 grams of protein per serving.

46. The composition of clause 44 or 45, wherein the protein is one of a whey protein concentrate, a whey protein isolate, a whey protein hydrolysate, an acid casein, a sodium caseinate, a calcium caseinate, a potassium caseinate, a casein hydrolysate, a milk protein concentrate, a milk protein isolate, a milk protein hydrolysate, nonfat dry milk, condensed skim milk, skim milk powder, a soy protein concentrate, a soy protein isolate, a soy protein hydrolysate, a pea protein concentrate, a pea protein isolate, a pea protein hydrolysate, a collagen protein, collagen protein isolates, potato protein, insect protein, earthworm protein, rice protein, quinoa protein, legume protein, grain protein, and combinations thereof.

47. The composition of any one of clauses 44-46, wherein the nutritional composition comprises from about 15 grams to about 1 10 grams of carbohydrates per serving, and wherein the prebiotic comprises from about 1 % to about 25% by weight of the total carbohydrates.

48. The composition of any one of clauses 44-47, wherein the carbohydrate is one of maltodextrin, hydrolyzed starch, modified starch, cornstarch, glucose polymers, corn syrup, corn syrup solids, rice-derived carbohydrate, sucrose, glucose, fructose, lactose, high fructose corn syrup, honey, sugar alcohol, isomaltulose, sucromalt, gum arabic, sodium carboxymethyl cellulose, guar gum, citrus pectin, low methoxy pectin, high methoxy pectin, oat beta-glucan, barley beta-glucan, carrageenan, psyllium, oat hull fiber, pea hull fiber, soy hull fiber, soy cotyledon fiber, sugar beet fiber, cellulose, corn bran, and combinations thereof.

49. The composition of any one of clauses 44 to 48, wherein the nutritional composition comprises from about 0.5 grams to about 45 grams of fat per serving.

50. The composition of any one of clauses 44 to 49, wherein the fat is one of coconut oil, fractionated coconut oil, soy oil, corn oil, olive oil, safflower oil, high oleic safflower oil, high gamma-linolenic acid (GLA) safflower oil, MCT (medium chain triglycerides) oil, sunflower oil, high oleic sunflower oil, palm oil, palm kernel oil, palm olein, canola oil, marine oil, cottonseed oil, eicosapentaenoic acid, docosahexaenoic acid, gamma-linolenic acid, algal oil, and combinations thereof.

51 . The composition of any one of claims 35 to 50, wherein the nutritional composition is one of a liquid, a liquid reconstituted from a powder, and a solid. 52. The composition of any one of claims 35 to 51 , wherein the nutritional composition is an emulsion.

53. The composition of any one of claims 35 to 52, wherein the nutritional composition is administered orally.

EXAMPLES The following examples illustrate certain exemplary embodiments of the methods and nutritional compositions described herein. The examples are given solely for the purpose of illustration and are not to be construed as limitations of the general inventive concepts, as many variations thereof are possible without departing from the spirit and scope of the general inventive concepts.

Example 1

Example 1 describes an animal study that was conducted to determine the effect of prebiotic supplementation of growing Sprague-Dawley rats as a nutritional approach to improve the health, particularly the bone health, of the rats. It was unexpectedly discovered that the rats that received prebiotic supplementation had significantly higher levels of HMB in the plasma and urine as compared to the rats that did not receive prebiotic supplementation.

Twenty five day-old Sprague-Dawley rats (Charles Rivers Laboratories, Orleans Cedex, France) were housed in groups of four per cage under standardized environmental conditions (22 °C, relative humidity of 50%, artificial lighting on for 12 hours/day) and were given free access to de-ionized water during the study period. The rats were randomly divided into two feeding groups (n=10/group): (1 ) Control Group ("Control"), which received a purified rodent diet (AIN-93G); and (2) Prebiotic group ("Prebiotic"), which received a purified rodent diet (AIN-93G) containing 7.5% (w/w) of the total carbohydrates as inulin- type fructans (Synergy-1™, Orafti, Belgium). The prebiotic is a 1 :1 mixture of fructooligosaccharides with an average degree of polymerization (DP) of 4 and high performance inulin with an average DP of 25. After twenty days, the rats were individualized. The Control group rats were fed with a purified rodent diet (AIN-93M) and the Prebiotic group rats were fed with a purified rodent diet (AIN-93G) containing 7.5% (w/w) of the total carbohydrates as inulin-type fructans (Synergy-1™, Orafti, Belgium). Both the Control group rats and the Prebiotic group rats were fed their respective diet until the end of their adolescence period (90 days).

At the end of the adolescent period and prior to the rats being sacrificed, urine samples were collected from the rats in acidified tubes under postprandial condition. Subsequently, the rats were sacrificed after overnight fasting. Blood samples from the rats were obtained by cardiac puncture. Serum was isolated, frozen, and stored at -80 °C until analysis. The level of HMB in the serum and urine samples was determined by ultra high-performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS).

Statistical Analysis - The data presented in Table 1 are expressed as mean ± standard error of the mean (SEM) of up to eight measurements. To evaluate the differences in the means between the Control group rats and the Prebiotic group rats, t-test or Mann-Whitney non- parametric test, when variance was not homogenously distributed, were performed.

TABLE 1

Discussion - Under fasting conditions at the time of sacrifice, the Control group rats exhibited basal serum levels of HMB typically associated with leucine catabolism. On the other hand, the Prebiotic group rats had approximately 9% higher basal serum levels of HMB as compared to the Control group, as shown in Table 1. These data suggest that, in addition to leucine catabolism, HMB can be endogenously produced by a pathway associated with prebiotic fermentation in the bowel by intestinal microbiota. The HMB endogenously produced via prebiotic fermentation by intestinal microbiota could be easily absorbed by enterocytes and reach the blood circulation to contribute to the final concentration of HMB in the serum. The increase in serum HMB exhibited by the Prebiotic group rats was in spite of the blood samples being collected under fasting conditions (e.g., after an approximately 12 hour overnight fast). Kinetic studies in animals have reported that the ingestion of HMB results in a rise of plasma HMB, which peaks between one and two hours. Additionally, these studies have reported that plasma HMB values are not different from baseline values after 8 or 9 hours from the time the HMB was orally administered.

The urine samples collected were also analyzed for HMB concentration under non-fasting conditions over a four day period. It is estimated that approximately 40% of circulating HMB is eliminated through the urine, and that the concentration of HMB in the urine is directly dependent on the daily physiological kinetics of HMB. Accordingly, a higher amount of HMB excreted in the urine would be directly associated with higher concentrations of HMB in the plasma as a consequence of either an increase in the endogenous production of HMB or the exogenous consumption of HMB. As can be determined from Table 1 , the Prebiotic group rats excreted 75% more HMB through the urine as compared to the Control group rats. The increase in HMB excretion by the Prebiotic group was calculated by considering the absolute amount of HMB ^g) excreted in the urine over a four day collection period and is expressed as a percentage change with respect to the Control group. Since neither the Prebiotic group rat diet nor the Control group rat diet included HMB, the data suggests that consumption of prebiotics may increase endogenous production of HMB via microbiota fermentation of the prebiotics.

Example 2 Example 2 describes an animal study that was conducted to determine the effect of prebiotic supplementation of Sprague-Dawley rat dams during their gestation and lactation periods as a nutritional approach to improve the health, particularly the bone health, of the offspring. It was unexpectedly discovered that the offspring of rat dams that received prebiotic supplementation had significantly higher levels of HMB in the plasma and urine as compared to the offspring of rat dams that did not receive prebiotic supplementation.

Twenty 10-week-old pregnant Sprague-Dawley rats (Charles Rivers Laboratories, Orleans Cedex, France) at the eleventh day of gestation were housed under standardized environmental conditions (22 °C, relative humidity of 50%, artificial lighting on for 12 hours/day) and were given free access to de-ionized water during the study period. The rat dams were randomly divided into two feeding groups (n=10/group): (1 ) Control Group ("Control"), which received a purified rodent diet (AIN-93G); and (2) Prebiotic group ("Prebiotic"), which received a purified rodent diet (AIN-93G) containing 7.5% (w/w) of the total carbohydrates as inulin-type fructans (Synergy-1™, Orafti, Belgium). The prebiotic is a 1 :1 mixture of fructooligosaccharides with an average degree of polymerization (DP) of 4 and high performance inulin with an average DP of 25. All rat dams were fed their respective diet ad libitum during the periods of gestation and lactation.

After delivery, to standardize and minimize variation of the offspring during suckling, litters from the same feeding group were mixed and 8 offspring were randomly housed to one dam from the same feeding group. During their suckling period, the offspring received only the dam's milk. The dam's nutritional treatment was finished at the end of the lactation period (offspring 28-days-old).

At weaning, offspring were separated from the dams and were housed in groups of four offspring per cage. Forty-three days after delivery, the offspring were individualized. During the period from weaning until the end of adolescence (90 days), the offspring of the Control group were fed with a purified rodent diet (AIN-93M) and the offspring of the Prebiotic group were fed with a purified rodent diet (AIN-93M) containing 7.5% (w/w) of the total carbohydrates as inulin-type fructans (Synergy-1™, Orafti, Belgium).

At the end of the adolescent period, the offspring were housed in metabolic cages over a four day period and urine samples were collected in acidified tubes under postprandial condition. Subsequently, the offspring were sacrificed after overnight fasting. Blood samples from the offspring were obtained by cardiac puncture. Serum was isolated, frozen, and stored at -80 °C until analysis. The level of HMB in the serum and urine samples was determined by ultra high-performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS). Statistical Analysis - The data presented in Table 2 are expressed as means ± standard error of the mean (SEM) of up to eight measurements. To evaluate the differences in the means between the Control group and the Prebiotic group, t-test or Mann-Whitney non- parametric test, when variance was not homogenously distributed, were performed.

TABLE 2

^* significantly different from control group (p < 0.05) Discussion - Under fasting conditions at the time of sacrifice, the Control group offspring exhibited basal serum levels of HMB typically associated with leucine catabolism. On the other hand, the Prebiotic group offspring had significantly higher basal serum levels of HMB as compared to the Control group offspring, as shown in Table 2. These data suggest that, in addition to leucine catabolism, HMB can be endogenously produced by a pathway associated with prebiotic fermentation in the bowel by intestinal microbiota. The HMB endogenously produced via prebiotic fermentation by intestinal microbiota could be easily absorbed by enterocytes and contribute to the final concentration of HMB in the blood circulation. In addition, urine samples were also analyzed for HMB concentration under non-fasting conditions. It is estimated that approximately 40% of circulating HMB is eliminated through the urine, and that the concentration of HMB in the urine reflects the daily physiological kinetics of HMB. Accordingly, a higher amount of HMB excreted in the urine would be directly associated with higher concentrations of HMB in the plasma as a consequence of either an increase in the endogenous production of HMB or the exogenous consumption of HMB.

As illustrated in Table 2, the Prebiotic group offspring had a significantly higher level of HMB in urine as compared to the Control group offspring. Since neither the Prebiotic group offspring diet nor the Control group offspring diet included HMB, the data suggests that consumption of prebiotic by the rat dams during gestation and lactation, as well as consumption of prebiotic by the offspring, may exert a beneficial effect on the offspring microbiota that results in an accumulative increase in the endogenous production of HMB. Accordingly, the data suggests that prebiotics may be used as an alternative source of HMB.

Examples 3-5

Examples 3-5 illustrate exemplary embodiments of a nutritional composition in the form of a nutritional powder and formulated for consumption by a pregnant woman or a lactating woman. The nutritional powder may be prepared by spray drying methods or dry blending, and may be reconstituted with water prior to use to the desired target ingredient concentrations. All ingredient amounts are listed as kilogram per 1000 kilogram batch of product, unless otherwise specified.

Example 6

Example 6 illustrates an exemplary embodiment of a nutritional composition formulated as a nutritional powder. The nutritional powder may be prepared by spray drying methods or dry blending, and may be reconstituted with water prior to use to the desired target ingredient concentrations. All ingredient amounts are approximate and listed as kilogram per 1000 kilogram batch of product, unless otherwise specified.

Folic Acid 1.6 g

Biotin 247.4 mg

Cyanocobalamin 15.4 mg

Diluent Dextrose Anhydrous q.s.

Ascorbic Acid 800.0 g

Ultra Trace Mineral/Trace Mineral 475.0 g Premix

Zinc Sulfate 1 16.2 g

Ferrous Sulfate 44.4 g

Manganese Sulfate 40.3 g

Cupric Sulfate 6.0 g

Sodium Molybdate 927.9 mg

Chromium Chloride 766.7 mg

Sodium Selenate 613.7 mg

Diluent Maltodextrin q.s.

Vanillin 300.0 g

Potassium Phosphate Dibasic 185.6 g

Ascorbyl Palmitate 164.6 g

Vitamin ADEK Premix 105.0 g dl-Alpha Tocopheryl Acetate 30.4 g

Vitamin A Palmitate 3.3 g

Phylloquinone 178.6 mg

Vitamin D₃ 30.5 mg

Diluent Oil (Corn or Sunflower) q.s.

Tocopherol-2 Food Grade Antioxidant 82.3 g

Mixed Tocopherols 57.6 g

Diluent Soy Oil q.s. dl-Alpha Tocopheryl Acetate 44.7 g

Vitamin A Palmitate 14.9 g

Vitamin A Palmitate 8.2 g

Diluent Oil (Corn or Sunflower) q.s.

Ferrous Sulfate 14.0 g

Beta Carotene 30% 5.5 g Beta Carotene 1.6 g

Diluent Corn Oil q.s.

Vitamin D₃ Oil Soluble 1.0 g

Vitamin D₃ 25.7 mg

Diluent MCT Oil q.s.

Potassium Iodide 913.3 mg

Example 7

Example 7 illustrates an exemplary embodiment of a nutritional composition formulated as a nutritional liquid. The nutritional liquid may be prepared according to any of a variety of manufacturing processes, including those described herein. All ingredient amounts are approximate and listed as kilogram per 1000 kilogram batch of product, unless otherwise specified.

Sodium Hydroxide (20%) 2.3

Avicel CL-61 1 1 .5

Cellulose Gel 1 .3

Cellulose Gum 225.0 g

Soy Lecithin 1 .2

Potassium Phosphate Dibasic 1 .2

Sodium Citrate 1 .0

Magnesium Phosphate Dibasic 772.6 g

Choline Chloride 500.1 g

Artificial Flavors 800.0 g

Artificial Cream Nut Flavor

Ultra Trace Mineral/Trace Mineral 363.7 g Premix

Ferrous Sulfate 53.4 g

Zinc Sulfate 45.6 g

Citric Acid 22.4 g

Manganese Sulfate 17.6 g

Cupric Sulfate 9.2 g

Chromium Chloride 561.1 mg

Sodium Molybdate 440.7 mg

Sodium Selenate 195.2 mg

Diluent Maltodextrin q.s.

Ascorbic Acid 351 .6 g

Potassium Hydroxide (processing aid) 157.5 g

Carrageenan 150.0 g

Potassium Chloride 101 .0 g

Vitamin Premix (water soluble) 68.6 g

Niacinamide 25.7 g

Calcium Pantothenate 16.6 g

Thiamine Chloride Hydrochloride 4.3 g

Pyridoxine Hydrochloride 4.1 g

Riboflavin 3.3 g

Folic Acid 633.9 mg Biotin 501.4 mg

Cyanocobalamin 1 1.4 mg

Diluent Dextrose q.s.

Vitamin Premix (oil soluble) 62.9 g

dl-Alpha Tocopheryl Acetate 52.7 g

Phylloquinone 89.2 mg

Vitamin D₃ 12.8 mg

Diluent Coconut Oil q.s.

Vitamin A Palmitate (55% in Vegetable 8.4 g

Oil)

Vitamin A Palmitate 4.6 g

Potassium Iodide 224.1 mg

Example 8

Example 8 describes an animal study that was conducted to determine the effect of human milk oligosaccharide (HMO) supplementation on growing Sprague-Dawley rats. The protocol for this experimental procedure was carried out according to ethical guidelines for animal experimentation at the Spanish National Research Council (RD 53/2013). It was unexpectedly discovered that the rats that received HMO supplementation had significantly higher levels of HMB in the plasma and urine as compared to the rats that did not receive HMO supplementation.

Healthy adult female rats (Charles Rivers Laboratories, Orleans Cedex, France) were used for the experiments. Three groups of rats (8 rats per group) having an age between 13 and 15 weeks were each administered a single bolus of human milk oligosaccharides via intragastric gavage: Group 1 rats received 2'-fuscosyllactose (2'-FL); Group 2 rats received 6'-sialyllactose (6^'-SL); and Group 3 rats received lacto-N-neotetraose (LNnT). The dose was 1 g/kg body weight for 2^'-FL. The other two HMOs, 6^'-SL and LNnT, were given at an equimolecular amount with respect to the 2FL; thus, 6^'-SL was given at 1.29 g/kg body weight and LnNT was administered at 1 .45 g/kg body weight.

Prior to intragastric gavage, a blood sample was taken from the rats in each group. This was used to determine basal HMB levels at time zero (Time 0 or TO). Repeated blood samples were taken at 30, 60, 90, 120, 180, 240 and 300 minutes after the gavage. Serum was obtained and stored frozen for subsequent analysis. Urine samples were also collected at TO and at 300 minutes post gavage (T300). HMB levels were determined in both serum and urine samples by ultra-high-performance-liquid-chromatography-tandem mass spectrometry (UHPLC-MS/MS).

Statistical Analysis - The data presented in Figures 1 -3 are expressed as mean ± standard error of the mean (SEM). To evaluate the differences in the means between groups, t-test or Mann-Whitney non-parametric test, when variance was not homogenously distributed, were performed.

Discussion - Before gavage with 2'-FL, the rats showed basal circulating levels of HMB in serum that should mainly be associated with leucine catabolism (see Figures 1A and B at TO). After receiving 2'-FL orally, a gradual increase in circulating HMB levels was observed during the kinetic curve, which was found to be significantly different to baseline at 300 minutes post-gavage (see Figure 1A). The increase seen at 300 minutes as compared with TO was around 168.5%. These data suggest that HMB may be generated not only by leucine degradation but also could be produced by a different pathway i.e. enzymatic fermentation of HMOs or specific chemical hydrolysis in the bowel. This HMB produced in the intestine could be easily absorbed by enterocytes, reaching the blood circulation.

Kinetic studies in animals have reported that the ingestion of HMB results in a rise of serum HMB which peaks between one and two hours (around 120 minutes). The data reported herein show a higher amount of HMB through 300 minutes that could be due to cecal fermentation of HMOs.

Similar results as reported herein for rats administered 2'-FL were obtained using the HMOs 6'-SL and LNnT. For both 6'-SL and LNnT, the maximum peak of HMB in serum appeared earlier (around 90-180 minutes) as compared with the results seen using 2'-FL (see Figure 2).

It is estimated that approximately 40% of circulating HMB is eliminated through the urine, and that the concentration of HMB in the urine reflects the daily physiological kinetics of HMB. Accordingly, a higher amount of HMB excreted in the urine would be directly associated with higher concentrations of HMB in the plasma as a consequence of either an increase in the endogenous production of HMB or the exogenous consumption of HMB. HMB concentration was measured in urine samples taken from the rats pre- and post- gavage. As shown in Figure 3, around 300 minutes after gavage, the rats excreted a significantly higher amount of HMB as compared with the amount of HMB excreted at baseline. These data suggest that consumption of HMOs may exert a beneficial effect on the intestinal microbiota that results in an accumulative increase in the endogenous production of HMB. Accordingly, the data suggest that HMOs may be used as an alternative source of HMB.

The following examples describe exemplary shelf stable nutritional compositions for use according to the methods of the invention. These exemplified compositions, unless otherwise specified, includes an aseptically processed embodiment and a retort packaged embodiment.

Examples 9-13

Prophetic examples 9-13 illustrate ready-to-feed nutritional emulsions, the ingredients of which are listed in the table below. All ingredient amounts listed are in kilograms, unless otherwise specified. Table 3: Examples 9-13

Ingredient Ex. 9 Ex. 10 Ex. 11 Ex. 12 Ex. 13

Water Q.S. Q.S. Q.S. Q.S. Q.S.

Condensed Skim Milk 86.64 86.64 86.64 86.64 86.64

Lactose 54.80 54.80 54.80 54.80 54.80

High oleic safflower oil 14.10 14.10 14.10 14.10 14.10

Soybean oil 10.6 10.6 10.6 10.6 10.6

Coconut oil 10.1 10.1 10.1 10.1 10.1 lacto-N-neotetraose (LNnT) 0.049 0.097 0.245 0.490 3.92

Galactooligosaccharides (GOS) 3.92 3.92 3.92 3.92 0

Whey protein concentrate 6.40 6.40 6.40 6.40 6.40

Potassium citrate 478.9 g 478.9 g 478.9 g 478.9 g 478.9 g

Calcium carbonate 448.28 g 448.28 g 448.28 g 448.28 g 448.28 g

Soy lecithin 355.74 g 355.74 g 355.74 g 355.74 g 355.74 g

Stabilizer 355.74 g 355.74 g 355.74 g 355.74 g 355.74 g

ARA oil 368.01 g 368.01 g 368.01 g 368.01 g 368.01 g

Nucleotide/chloride premix 293.26 g 293.26 g 293.26 g 293.26 g 293.26 g

Potassium chloride 226.45 g 226.45 g 226.45 g 226.45 g 226.45 g

Ascorbic acid 445.94 g 445.94 g 445.94 g 445.94 g 445.94 g

Vitamin mineral premix 142.88 g 142.88 g 142.88 g 142.88 g 142.88 g

DHA oil 137.8 g 137.8 g 137.8 g 137.8 g 137.8 g

Carrageenan 180.0 g 180.0 g 180.0 g 180.0 g 180.0 g

Magnesium chloride 55.0 g 55.0 g 55.0 g 55.0 g 55.0 g

Ferrous sulfate 58.0 g 58.0 g 58.0 g 58.0 g 58.0 g

Choline chloride 53.9 g 53.9 g 53.9 g 53.9 g 53.9 g

Vitamin A, D3, E, K1 premix 47.4 g 47.4 g 47.4 g 47.4 g 47.4 g

Citric acid 29.77 g 29.77 g 29.77 g 29.77 g 29.77 g

Mixed carotenoid premix 26.40 g 26.40 g 26.40 g 26.40 g 26.40 g

Sodium chloride AN AN AN AN AN

L-carnitine 3.31 g 3.31 g 3.31 g 3.31 g 3.31 g

Tricalcium phosphate 15.65 g 15.65 g 15.65 g 15.65 g 15.65 g

Potassium phosphate monobasic 13.67 g 13.67 g 13.67 g 13.67 g 13.67 g

Riboflavin 2.42 g 2.42 g 2.42 g 2.42 g 2.42 g

Potassium hydroxide AN AN AN AN AN

AN = as needed

Examples 14-18 Prophetic examples 14-18 illustrate ready-to-feed nutritional emulsions of the present disclosure, the ingredients of which are listed in the table below. All ingredient amounts listed are in kilograms, unless otherwise specified. Table 4: Examples 14-18

AN = as needed Examples 19-23

Prophetic examples 19-23 illustrate concentrated liquid emulsions, the ingredients of which are listed in the table below. All ingredient amounts listed are in kilograms, unless otherwise specified. Table 5: Examples 19-23

AN = as needed

Examples 24-28

Prophetic examples 24-28 illustrate ready-to-feed nutritional emulsions, the ingredients of which are listed in the table below. All ingredient amounts listed are in kilograms, unless otherwise specified. Table 6: Examples 24-28

AN = as needed

Examples 29-33

Prophetic examples 29-33 illustrate concentrated liquid emulsions, the ingredients of which are listed in the table below. All ingredient amounts listed are in kilograms, unless otherwise specified. Table 7: Examples 29-33

AN = as needed

Examples 34-38

Prophetic examples 34-38 illustrate human milk fortifier liquids, the ingredients of which are listed in the table below. All ingredient amounts listed are in kilograms, unless otherwise specified. Table 8: Examples 34-38

Examples 39-43 Prophetic examples 39-43 illustrate spray dried nutritional powders, the ingredients of which are listed in the table below. All ingredient amounts listed are in kilograms, unless otherwise specified. Table 9: Examples 39-43

AN = as needed

Examples 44-48

Prophetic examples 44-48 illustrate nutritional bars, the ingredients of which are listed in the table below. All ingredient amounts listed are in kilograms, unless otherwise specified. Table 10: Examples 44-48

Examples 49-53

Prophetic examples 49-53 illustrate liquid formulations, the ingredients of which are listed in the table below. All ingredient amounts listed are in kilograms, unless otherwise specified.

Table 11: Examples 49-53

Biotin 476.5 mg 476.5 mg 476.5 mg 476.5 mg 476.5 mg

Sodium Molybdate, Dihydrate 247.2 mg 247.2 mg 247.2 mg 247.2 mg 247.2 mg

Sodium Selenate, Anhydrous 21 1 .5 mg 21 1 .5 mg 21 1 .5 mg 21 1.5 mg 21 1 .5 mg

Cyanocobalamin 12.5 mg 12.5 mg 12.5 mg 12.5 mg 12.5 mg

Sucralose 33.0 g 33.0 g 33.0 g 33.0 g 33.0 g

Acesulfame Potassium 76.0 g 76.0 g 76.0 g 76.0 g 76.0 g dl-Alpha-Tocopheryl Acetate 54.5 g 54.5 g 54.5 g 54.5 g 54.5 g

Phylloquinone 92.4 mg 92.4 mg 92.4 mg 92.4 mg 92.4 mg

Vitamin D3 13.2 mg 13.2 mg 13.2 mg 13.2 mg 13.2 mg

Vitamin A Palmitate 4.5 g 4.5 g 4.5 g 4.5 g 4.5 g

Potassium Iodide 220.5 mg 220.5 mg 220.5 mg 220.5 mg 220.5 mg

Vitamin B12 (86.4%

Cyanocobalamin) 31.7 mg 31.7 mg 31.7 mg 31 .7 mg 31.7 mg lacto-N-neotetraose (LNnT) 0.392 1.96 3.92 7.84 15.68

Examples 54-58

Prophetic examples 54-58 illustrate liquid formulations, the ingredients of which are listed in the table below. All ingredient amounts listed are in kilograms, unless otherwise specified.

Table 12: Examples 54-58

Cyanocobalamin 1 1.4 mg 1 1.4 mg 1 1.4 mg 1 1 .4 mg 1 1.4 mg lacto-N-neotetraose (LNnT) 0.392 1.96 3.92 7.84 15.68

To the extent that the term "includes" or "including" is used in the specification 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. Also, to the extent that the terms "in" or "into" are used in the specification or the claims, it is intended to additionally mean "on" or "onto." Furthermore, to the extent the term "connect" is used in the specification or claims, it is intended to mean not only "directly connected to," but also "indirectly connected to" such as connected through another component or components.

While the present application has been illustrated by the description of embodiments thereof, and while the embodiments 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 and processes, 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 inventive concepts.

Claims

1 . A method of increasing endogenous production of beta-hydroxy-beta-methylbutyrate in a subject, the method comprising administering a nutritional composition comprising a prebiotic to the subject, whereby consumption of the nutritional composition increases endogenous levels of beta-hydroxy-beta methylbutyrate in the subject.

2. A method of increasing endogenous production of beta-hydroxy-beta-methylbutyrate in an offspring of a subject, the method comprising administering a nutritional composition comprising a prebiotic to the subject during gestation, whereby consumption of the nutritional composition by the subject during gestation increases endogenous levels of beta-hydroxy-beta methylbutyrate in the offspring in the womb.

3. A method of increasing endogenous production of beta-hydroxy-beta-methylbutyrate in an offspring of a subject, the method comprising administering a nutritional composition comprising a prebiotic to the subject following delivery of the offspring and during breastfeeding of the offspring, whereby consumption of the nutritional composition by the subject during breastfeeding of the offspring increases endogenous levels of beta-hydroxy-beta methylbutyrate in the offspring.

4. The method of any one of claims 1-3 wherein the prebiotic is selected from the group consisting of short chain fructooligosacchandes, long chain fructooligosacchandes, inulins, galactooligosaccharides, xylooligosaccharides, resistant starch, and combinations thereof.

5. The method of any one of claims 1 -3 wherein the prebiotic comprises fructooligosacchandes having an average degree of polymerization of 4 and high performance inulin having an average degree of polymerization of 25, and wherein a weight ratio of the fructooligosacchandes to the high performance inulin is about 1 :1 .

6. The method of any one of claims 1-5, wherein the prebiotic comprises from about 1 % to about 20% by weight of the nutritional composition.

7. The method of any one of claims 1-3, wherein the prebiotic is a human milk oligosaccharide (HMO).

8. The method of claim 7, wherein the human milk oligosaccharide is selected from the group consisting of: 6'-sialyllactose (6'SL); lacto-N-neotetraose (LNnT); 3'- fucosyllactose (3'FL); 3'-sialyllactose (3'SL); and 2'-fucosyllactose (2'FL).

9. The method of claim 7 wherein the human milk oligosaccharide is selected from: 6'- sialyllactose (6'SL); lacto-N-neotetraose (LNnT); and 2'-fucosyllactose (2'FL).

10. The method of any one of claims 1-3 or 7-9 wherein the prebiotic is a human milk oligosaccharide and the nutritional composition is a liquid comprising the human milk oligosaccharide in an amount from about 0.001 mg/ml to about 20 mg/ml.

1 1 . The method of any one of claims 1-3 or 7-9 wherein the prebiotic is a human milk oligosaccharide and the nutritional composition is a powder comprising the human milk oligosaccharide in an amount from about 0.0005% to about 5% by weight of the powder.

12. The method of any one of claims 1-3 or 7-9 wherein the prebiotic is a human milk oligosaccharide and the nutritional composition is a bar comprising the human milk oligosaccharide in an amount from about 0.0005% to about 10% by weight of the bar.

13. The method of any one of claims 1-12, wherein the nutritional composition further comprises at least one of a protein, a carbohydrate, and a fat.

14. The method of claim 13, wherein the nutritional composition comprises from about 6 grams to about 50 grams of protein per serving.

15. The method of claim 13 or claim 14, wherein the protein is one of a whey protein concentrate, a whey protein isolate, a whey protein hydrolysate, an acid casein, a sodium caseinate, a calcium caseinate, a potassium caseinate, a casein hydrolysate, a milk protein concentrate, a milk protein isolate, a milk protein hydrolysate, nonfat dry milk, condensed skim milk, skim milk powder, a soy protein concentrate, a soy protein isolate, a soy protein hydrolysate, a pea protein concentrate, a pea protein isolate, a pea protein hydrolysate, a collagen protein, collagen protein isolates, potato protein, insect protein, earthworm protein, rice protein, quinoa protein, legume protein, grain protein, and combinations thereof.

16. The method of any one of claims 13-15, wherein the nutritional composition comprises from about 15 grams to about 1 10 grams of carbohydrates per serving.

17. The method of claim 16 wherein the prebiotic comprises from about 1 % to about 25% by weight of the total carbohydrates.

18. The method of any one of claims 13-17, wherein the carbohydrate is one of maltodextrin, hydrolyzed starch, modified starch, cornstarch, glucose polymers, corn syrup, corn syrup solids, rice-derived carbohydrate, sucrose, glucose, fructose, lactose, high fructose corn syrup, honey, sugar alcohol, isomaltulose, sucromalt, gum arabic, sodium carboxymethyl cellulose, guar gum, citrus pectin, low methoxy pectin, high methoxy pectin, oat beta-glucan, barley beta-glucan, carrageenan, psyllium, oat hull fiber, pea hull fiber, soy hull fiber, soy cotyledon fiber, sugar beet fiber, cellulose, corn bran, and combinations thereof.

19. The method of any one of claims 13-18, wherein the nutritional composition comprises from about 0.5 grams to about 45 grams of fat per serving.

20. The method of any one of claims 13-19, wherein the fat is one of coconut oil, fractionated coconut oil, soy oil, corn oil, olive oil, safflower oil, high oleic safflower oil, high gamma-linolenic acid (GLA) safflower oil, MCT (medium chain triglycerides) oil, sunflower oil, high oleic sunflower oil, palm oil, palm kernel oil, palm olein, canola oil, marine oil, cottonseed oil, eicosapentaenoic acid, docosahexaenoic acid, gamma-linolenic acid, algal oil, and combinations thereof.

21 . The method of any one of claims 1 -9 and 13-20, wherein the nutritional composition is one of a liquid, a liquid reconstituted from a powder, and a solid.

22. The method of any one of claims 1 -9 and 13-20, wherein the nutritional composition is an emulsion.

23. The method of any one of claims 1 -22, wherein the nutritional composition is administered orally.

24. The method of any one of claims 1 -23, wherein the nutritional composition is administered to the subject at least once daily.

25. A method of promoting muscle hypertrophy and/or preventing muscle atrophy in a subject, the method comprising administering a nutritional composition comprising a prebiotic to the subject.

26. The method of claim 25 wherein the subject is a healthy adult subject.

27. The method of claim 25 wherein the method is for promoting muscle hypertrophy in a subject having muscle atrophy caused by a period of muscle disuse.

28. A method of improving brain development and/or cognitive function in a subject, the method comprising administering a nutritional composition comprising a prebiotic to the subject.

29. The method of claim 28 wherein the subject is selected from: an infant; a toddler; a child; an adolescent; an adult; and an elderly adult.

30. A method of treating a disease or condition, the method comprising administering to a subject a nutritional composition comprising a prebiotic, wherein the disease or condition is selected from: sarcopenia; muscle wasting diseases; hyperglycaemia; glucose intolerance; and neurodegenerative diseases.

31. The method of claim 30 wherein the neurodegenerative disease is selected from: Alzheimer's disease; Huntington's disease; Parkinson's disease; dementia; amyotrophic lateral sclerosis; stroke; and schizophrenia.

32. The method of any one of claims 25-31 wherein the nutritional composition comprises a prebiotic selected from the group consisting of short chain fructooligosaccharides, long chain fructooligosaccharides, inulins, galactooligosaccharides, xylooligosaccharides, resistant starch, and combinations thereof.

33. The method of any one of claims 25-32 wherein the nutritional composition comprises a prebiotic and the prebiotic comprises fructooligosaccharides having an average degree of polymerization of 4 and high performance inulin having an average degree of polymerization of 25, and wherein a weight ratio of the fructooligosaccharides to the high performance inulin is about 1 :1.

34. The method of any one of claims 25-33, wherein the prebiotic comprises from about 1 % to about 20% by weight of the nutritional composition.

35. The method of any one of claims 25-31 , wherein the prebiotic is a human milk oligosaccharide.

36. The method of claim 35, wherein the human milk oligosaccharide is selected from the group consisting of: 6'-sialyllactose (6'SL); lacto-N-neotetraose (LNnT); 3'- fucosyllactose (3'FL); 3'-sialyllactose (3'SL); and 2'-fucosyllactose (2'FL).

37. The method of claim 35, wherein the human milk oligosaccharide is selected from: 6'- sialyllactose (6'SL); lacto-N-neotetraose (LNnT); and 2'-fucosyllactose (2'FL).

38. The method of any one of claims 25-31 or 35-37 wherein the prebiotic is a human milk oligosaccharide and the nutritional composition is a liquid comprising the human milk oligosaccharide in an amount from about 0.001 mg/ml to about 20 mg/ml.

39. The method of any one of claims 25-31 or 35-37 wherein the prebiotic is a human milk oligosaccharide and the nutritional composition is a powder comprising the human milk oligosaccharide in an amount from about 0.0005% to about 5% by weight of the powder.

40. The method of any one of claims 25-31 or 35-37 wherein the prebiotic is a human milk oligosaccharide and the nutritional composition is a bar comprising the human milk oligosaccharide in an amount from about 0.0005% to about 10% by weight of the bar.

41 . The method of any one of claims 25-40, wherein the nutritional composition further comprises at least one of a protein, a carbohydrate, and a fat.