WO2016073132A1 - Nutritional compositions containing a prebiotic and lactoferrin and uses thereof - Google Patents

Nutritional compositions containing a prebiotic and lactoferrin and uses thereof Download PDF

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Publication number
WO2016073132A1
WO2016073132A1 PCT/US2015/054628 US2015054628W WO2016073132A1 WO 2016073132 A1 WO2016073132 A1 WO 2016073132A1 US 2015054628 W US2015054628 W US 2015054628W WO 2016073132 A1 WO2016073132 A1 WO 2016073132A1
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WIPO (PCT)
Prior art keywords
kcal
nutritional composition
composition
lactoferrin
milk
Prior art date
Application number
PCT/US2015/054628
Other languages
French (fr)
Inventor
Maciej CHICHLOWSKI
Brian Berg
Zeina Jouni
Anja Wittke
Rosaline Waworuntu
Original Assignee
Mjn U.S. Holdings Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US14/535,717 external-priority patent/US20150119322A1/en
Priority claimed from US14/636,810 external-priority patent/US10709770B2/en
Application filed by Mjn U.S. Holdings Llc filed Critical Mjn U.S. Holdings Llc
Priority to CN201580060372.6A priority Critical patent/CN107072282A/en
Priority to BR112017008747A priority patent/BR112017008747A2/en
Priority to EP15784863.1A priority patent/EP3214951A1/en
Priority to SG11201702036QA priority patent/SG11201702036QA/en
Priority to CA2962136A priority patent/CA2962136A1/en
Priority to AU2015343613A priority patent/AU2015343613A1/en
Priority to MX2017005783A priority patent/MX2017005783A/en
Publication of WO2016073132A1 publication Critical patent/WO2016073132A1/en
Priority to PH12017500763A priority patent/PH12017500763A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
    • A23V2200/3202—Prebiotics, ingredients fermented in the gastrointestinal tract by beneficial microflora
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/322—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the nervous system or on mental function
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/28—Oligosaccharides
    • A23V2250/284—Oligosaccharides, non digestible
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/54—Proteins
    • A23V2250/542—Animal Protein
    • A23V2250/5424—Dairy protein
    • A23V2250/54248—Lactoferrin

Definitions

  • the present disclosure relates to methods of modulating serotonergic receptor expression in a pediatric subject via administration of the nutritional composition disclosed herein. Some embodiments of the disclosure are directed to enhancing or promoting an increase in the concentration of beneficial bacteria in a pediatric subject, such as the Lactobacillus and Bifidobacterium species, by administering the nutritional composition disclosed herein. Further provided are methods for reducing constipation in a pediatric subject by providing the nutritional composition disclosed herein.
  • the nutritional composition comprises a prebiotic blend which includes polydextrose and galacto-oligosaccharides, and lactoferrin, wherein the foregoing components may exhibit additive and/or synergistic beneficial effects.
  • Lactoferrin an iron-binding glycoprotein
  • lactoferrin is one of the major multifunctional agents present in human milk. It has the capacity to bind two molecules of iron in a reversible fashion and can facilitate the uptake of iron within the intestines. Further, lactoferrin has been shown to be both bacteriostatic and bactericidal, and it aids in preventing intestinal infections in humans, especially in pediatric subjects. Additionally, human lactoferrin appears to have a positive effect on the symptoms of diarrheal diseases.
  • the infant gut microflora is rapidly established in the first few weeks following birth, and it has a great impact on an infant's immune system.
  • the nature of this intestinal colonization is initially determined by early exposure to environmental sources of microbes and by the general state of health of the infant. Whether the infant is breast-fed or formula-fed also has a strong influence on the intestinal bacterial population.
  • the present disclosure is directed, in an embodiment, to a method for modulating serotonergic receptor expression, modulating gut microbiota, for example increasing the concentration of beneficial bacteria, and/or modulating psychological stress, in a pediatric subject, the method comprising administering to the pediatric subject a nutritional composition comprising prebiotics, especially polydextrose (PDX) and galacto-oligosaccharides (GOS), and lactoferrin from a non-human source.
  • PDX polydextrose
  • GOS galacto-oligosaccharides
  • lactoferrin lactoferrin from a non-human source.
  • the disclosure is directed to a method of reducing the incidence of constipation in a target subject via administration of the nutritional composition disclosed herein.
  • the nutritional composition further comprises an enriched lipid fraction derived from milk.
  • the nutritional composition may include an enriched lipid fraction derived from milk that includes milk fat globules. The addition of the milk fat globules provides an enriched fat and lipid source to the infant that may be more fully digested by a pediatric subject.
  • the enriched lipid fraction and/or the milk fat globules may include saturated fatty acids, trans-fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, cholesterol, odd-branched chain fatty acids "OBCFAs”, branched chain fatty acids “BCFAs”, conjugated linoleic acid “CLA”, phospholipids, or milk fat globule membrane protein, and mixtures thereof.
  • OBCFAs odd-branched chain fatty acids
  • BCFAs branched chain fatty acids
  • CLA conjugated linoleic acid
  • phospholipids or milk fat globule membrane protein
  • Fig. 1 illustrates the relative 5HTi a mRNA levels in the brain tissues, specifically the rostral, mid, and caudal regions, of mice fed a control diet versus a diet supplemented with GOS and PDX.
  • Fig. 2 illustrates the amount of Lactobacillus species in the feces of mice fed a control diet, mice fed a diet supplemented with lactoferrin, mice fed a diet supplemented with GOS and PDX, and mice fed a diet supplemented with GOS, PDX, and lactoferrin.
  • Fig. 3 illustrates the amount of Lactobacillus rhamnosus in the feces of mice fed a control diet, mice fed a diet supplemented with lactoferrin, mice fed a diet supplemented with GOS and PDX, and mice fed a diet supplemented with GOS, PDX, and lactoferrin.
  • Fig. 4 illustrates the amount of plasma Hsp70 present after stress and in a non-stressful situation in mice fed mice fed a control diet, mice fed a diet supplemented with lactoferrin, mice fed a diet supplemented with GOS and PDX, and mice fed a diet supplemented with GOS, PDX, and lactoferrin.
  • Fig. 5 illustrates the percentage of freezing considered shock-elicited freezing (anxiety test) in mice fed a control diet, mice fed a diet supplemented with lactoferrin, mice fed a diet supplemented with GOD and PDX, and mice fed a diet supplemented with GOS, PDX, and lactoferrin.
  • Fig. 6 illustrates the percentage of escape latency in mice fed a control diet, mice fed a diet supplemented with lactoferrin, mice fed a diet supplemented with GOD and PDX, and mice fed a diet supplemented with GOS, PDX, and lactoferrin.
  • the present disclosure relates generally to nutritional compositions that are suitable for administration to a pediatric subject. Additionally, the disclosure relates to methods of modulating serotonergic receptor expression, promoting the growth and concentration of beneficial bacteria in the gastrointestinal tract, reducing constipation, and/or promoting stress resistance in a pediatric subject via
  • Nutritional composition means a substance or formulation that satisfies at least a portion of a subject's nutrient requirements.
  • nutritional composition(s) may refer to liquids, powders, gels, pastes, solids, concentrates, suspensions, or ready-to-use forms of enteral formulas, oral formulas, formulas for infants, formulas for pediatric subjects, formulas for children, growing-up milks and/or formulas for adults.
  • Enteral administration includes oral feeding, intragastric feeding, transpyloric administration, or any other administration into the digestive tract.
  • Administration is broader than “enteral administration” and includes parenteral administration or any other route of administration by which a substance is taken into a subject's body.
  • a pediatric subject means a human no greater than 13 years of age. In some embodiments, a pediatric subject refers to a human subject that is between birth and 8 years old. In other embodiments, a pediatric subject refers to a human subject between 1 and 6 years of age. In still further embodiments, a pediatric subject refers to a human subject between 6 and 12 years of age. The term
  • “pediatric subject” may refer to infants (preterm or full term) and/or children, as described below.
  • infant means a human subject ranging in age from birth to not more than one year and includes infants from 0 to 12 months corrected age.
  • corrected age means an infant's chronological age minus the amount of time that the infant was born premature. Therefore, the corrected age is the age of the infant if it had been carried to full term.
  • infant includes low birth weight infants, very low birth weight infants, extremely low birth weight infants and preterm infants.
  • Preterm means an infant born before the end of the 37 th week of gestation.
  • Late preterm means an infant form between the 34 th week and the 36 th week of gestation.
  • Full term means an infant born after the end of the 37 th week of gestation.
  • “Low birth weight infant” means an infant born weighing less than 2500 grams (approximately 5 lbs, 8 ounces). "Very low birth weight infant” means an infant born weighing less than 1500 grams (approximately 3 lbs, 4 ounces).
  • Extremely low birth weight infant means an infant born weighing less than 1000 grams (approximately 2 lbs, 3 ounces).
  • Child means a subject ranging in age from 12 months to 13 years. In some embodiments, a child is a subject between the ages of 1 and 12 years old. In other embodiments, the terms “children” or “child” refer to subjects that are between one and about six years old, or between about seven and about 12 years old. In other embodiments, the terms “children” or “child” refer to any range of ages between 12 months and about 13 years.
  • Children's nutritional product refers to a composition that satisfies at least a portion of the nutrient requirements of a child.
  • a growing-up milk is an example of a children's nutritional product.
  • degree of hydrolysis refers to the extent to which peptide bonds are broken by a hydrolysis method.
  • the degree of protein hydrolysis for purposes of characterizing the hydrolyzed protein component of the nutritional composition is easily determined by one of ordinary skill in the formulation arts by quantifying the amino nitrogen to total nitrogen ratio (AN/TN) of the protein component of the selected formulation.
  • the amino nitrogen component is quantified by USP titration methods for determining amino nitrogen content, while the total nitrogen component is determined by the Tecator Kjeldahl method, all of which are well known methods to one of ordinary skill in the analytical chemistry art.
  • partially hydrolyzed means having a degree of hydrolysis which is greater than 0% but less than about 50%.
  • protein-free means containing no measurable amount of protein, as measured by standard protein detection methods such as sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or size exclusion chromatography.
  • the nutritional composition is substantially free of protein, wherein “substantially free” is defined hereinbelow.
  • infant formula means a composition that satisfies at least a portion of the nutrient requirements of an infant.
  • the content of an infant formula is dictated by the federal regulations set forth at 21 C.F.R. Sections 100, 106, and 107. These regulations define macronutrient, vitamin, mineral, and other ingredient levels in an effort to simulate the nutritional and other properties of human breast milk.
  • growing-up milk refers to a broad category of nutritional compositions intended to be used as a part of a diverse diet in order to support the normal growth and development of a child between the ages of about 1 and about 6 years of age.
  • milk-based means comprising at least one component that has been drawn or extracted from the mammary gland of a mammal.
  • a milk-based nutritional composition comprises components of milk that are derived from domesticated ungulates, ruminants or other mammals or any combination thereof.
  • milk-based means comprising bovine casein, whey, lactose, or any combination thereof.
  • milk-based nutritional composition may refer to any composition comprising any milk-derived or milk- based product known in the art.
  • “Milk” means a component that has been drawn or extracted from the mammary gland of a mammal.
  • the nutritional composition comprises components of milk that are derived from domesticated ungulates, ruminants or other mammals or any combination thereof.
  • Fractionation procedure includes any process in which a certain quantity of a mixture is divided up into a number of smaller quantities known as fractions. The fractions may be different in composition from both the mixture and other fractions. Examples of fractionation procedures include but are not limited to, melt fractionation, solvent fractionation, supercritical fluid fractionation and/or combinations thereof.
  • Fat globule refers to a small mass of fat surrounded by phospholipids and other membrane and/or serum proteins, where the fat itself can be a
  • Nutritionally complete means a composition that may be used as the sole source of nutrition, which would supply essentially all of the required daily amounts of vitamins, minerals, and/or trace elements in combination with proteins, carbohydrates, and lipids. Indeed, “nutritionally complete” describes a nutritional composition that provides adequate amounts of carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals and energy required to support normal growth and development of a subject.
  • a nutritional composition that is "nutritionally complete" for a preterm infant will, by definition, provide qualitatively and quantitatively adequate amounts of carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for growth of the preterm infant.
  • a nutritional composition that is "nutritionally complete" for a full term infant will, by definition, provide qualitatively and quantitatively adequate amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for growth of the full term infant.
  • a nutritional composition that is "nutritionally complete” for a child will, by definition, provide qualitatively and quantitatively adequate amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids,
  • essential refers to any nutrient that cannot be synthesized by the body in amounts sufficient for normal growth and to maintain health and that, therefore, must be supplied by the diet.
  • conditionally essential as applied to nutrients means that the nutrient must be supplied by the diet under conditions when adequate amounts of the precursor compound is unavailable to the body for endogenous synthesis to occur.
  • Probiotic means a microorganism with low or no pathogenicity that exerts a beneficial effect on the health of the host.
  • inactivated probiotic means a probiotic wherein the metabolic activity or reproductive ability of the referenced probiotic has been reduced or destroyed.
  • the "inactivated probiotic” does, however, still retain, at the cellular level, its cell structure or other structure associated with the cell, for example exopolysaccharide and at least a portion its biological glycol-protein and DNA/RNA structure.
  • inactivated is synonymous with “non-viable”.
  • Prebiotic means a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the digestive tract that can improve the health of the host.
  • BCFA Branched Chain Fatty Acid
  • a fatty acid containing a carbon constituent branched off the carbon chain typically the branch is an alkyl branch, especially a methyl group, but ethyl and propyl branches are also known.
  • the addition of the methyl branch lowers the melting point compared with the equivalent straight chain fatty acid.
  • Odd- and Branched-Chain Fatty Acid is a subset of BCFA that has an odd number of carbon atoms and have one or more alkyl branches on the carbon chain.
  • the main odd- and branched-chain fatty acids found in bovine milk include, but are not limited to, the isomers of tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, and heptadecanoic acid.
  • the term "BCFA” includes both branched-chain fatty acids and odd-and-branched chain fatty acids.
  • Trans-fatty acid means an unsaturated fat with a trans-isomer.
  • Trans- fats may be monounsaturated or polyunsaturated.
  • Trans refers to the arrangement of the two hydrogen atoms bonded to the carbon atoms involved in a double bond. In the trans arrangement, the hydrogens are on opposite sides of the bond.
  • a trans-fatty acid is a lipid molecule that contains one or more double bonds in trans geometric configuration.
  • Phospholipids means an organic molecule that contains a diglyceride, a phosphate group and a simple organic molecule.
  • Examples of phospholipids include but are not limited to, phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phsphatidylinositol, phosphatidylinositol phosphate, phosphatidylinositol biphosphate and phosphatidylinositol triphosphate, ceramide phosphorylcholine, ceramide phosphorylethanolamine and ceramide phosphorylglycerol.
  • This definition further includes sphigolipids, glycolipids, and gangliosides.
  • phytonutrient means a chemical compound that occurs naturally in plants. Phytonutrients may be included in any plant-derived substance or extract.
  • the term “phytonutrient(s)” encompasses several broad categories of compounds produced by plants, such as, for example, polyphenols compounds, anthocyanins, proanthocyanidins, and flavan-3-ols (i.e. catechins, epicatechins), and may be derived from, for example, fruit, seed or tea extracts.
  • phytonutrient includes all carotenoids, phytosterols, thiols, and other plant-derived compounds.
  • plant extracts may include
  • phytonutrients such as polyphenols
  • apple or grape seed extract(s) may include beneficial phytonutrient components, such as polyphenols, in addition to other plant- derived substances.
  • ⁇ -glucan means all ⁇ -glucan, including specific types of ⁇ -glucan, such as ⁇ -1 ,3-glucan or ⁇ -1 ,3;1 ,6-glucan. Moreover, ⁇ -1 ,3;1 ,6-glucan is a type of ⁇ -1 ,3- glucan. Therefore, the term " ⁇ -1 ,3-glucan” includes -1 ,3;1 ,6-glucan.
  • Pectin means any naturally-occurring oligosaccharide or
  • pectin polysaccharide that comprises galacturonic acid that may be found in the cell wall of a plant.
  • Different varieties and grades of pectin having varied physical and chemical properties are known in the art. Indeed, the structure of pectin can vary significantly between plants, between tissues, and even within a single cell wall.
  • pectin is made up of negatively charged acidic sugars (galacturonic acid), and some of the acidic groups are in the form of a methyl ester group.
  • the degree of esterification of pectin is a measure of the percentage of the carboxyl groups attached to the galactopyranosyluronic acid units that are esterified with methanol.
  • Pectin having a degree of esterification of less than 50% are classified as low-ester, low methoxyl, or low methylated (“LM”) pectins, while those having a degree of esterification of 50% or greater (i.e., more than 50% of the carboxyl groups are methylated) are classified as high-ester, high methoxyl or high methylated (“HM”) pectins.
  • Very low (“VL”) pectins, a subset of low methylated pectins have a degree of esterification that is less than approximately 15%.
  • lactoferrin from a non-human source means lactoferrin which is produced by or obtained from a source other than human breast milk.
  • lactoferrin for use in the present disclosure includes human lactoferrin produced by a genetically modified organism as well as non-human lactoferrin.
  • organism refers to any contiguous living system, such as animal, plant, fungus or micro-organism.
  • non-human lactoferrin means lactoferrin that has an amino acid sequence that is different than the amino acid sequence of human lactoferrin.
  • Pathogen means an organism that causes a disease state or pathological syndrome.
  • pathogens may include bacteria, viruses, parasites, fungi, microbes or combination(s) thereof.
  • Modulate means exerting a modifying, controlling and/or regulating influence.
  • modulating means exhibiting an increasing or stimulatory effect on the level/amount of a particular component.
  • modulating means exhibiting a decreasing or inhibitory effect on the level/amount of a particular component.
  • the nutritional composition of the present disclosure may be any nutritional composition of the present disclosure.
  • substantially free of any optional or selected ingredients described herein provided that the remaining nutritional composition still contains all of the required ingredients or features described herein.
  • the term “substantially free” means that the selected composition may contain less than a functional amount of the optional ingredient, typically less than 0.1% by weight, and also, including zero percent by weight of such optional or selected ingredient.
  • compositions of the present disclosure can comprise, consist of, or consist essentially of the essential elements and limitations of the embodiments described herein, as well as any additional or optional ingredients, components or limitations described herein or otherwise useful in nutritional compositions.
  • the present disclosure is directed to nutritional compositions comprising a prebiotic component including GOS and PDX, and lactoferrin from a non-human source, to uses thereof, and to methods comprising administration of those nutritional compositions.
  • the nutritional compositions of the present disclosure support overall health and development in a pediatric human subject, such as an infant (preterm and/or term) or a child.
  • the intestinal microflora plays a crucial role in the postnatal
  • the nutritional composition of the present disclosure supports gastrointestinal health and
  • the present disclosure is directed to a method for modulating psychological stress responses.
  • the disclosed combination has additive and/or synergistic beneficial effects that support gastrointestinal development.
  • the disclosure is related to a method for supporting cognitive development and brain function, and gastrointestinal health and/or development in a pediatric subject. The method comprises administering prebiotics, especially PDX and GOS, and lactoferrin from a non-human source to a pediatric subject.
  • the nutritional composition of the present disclosure can reduce or suppress psychological stress and/or modulate plasma stress hormone levels, thereby further promoting healthy brain and cognitive growth and development in a pediatric subject.
  • 5-HT 1a receptor expression and function an important neural mechanism for protection against stress- induced anxiety and depression-like behavior is an increase in 5-HT 1a receptor expression and function.
  • 5-HT- is a subtype of the 5-HT family of receptors that binds the endogenous neurotransmitter serotonin.
  • 5-HTi a is a G protein-coupled receptor that mediates inhibitory neurotransmission. As such, modulation of the function of the 5-HTi a may provide additional cognitive and behavioral benefits in a target individual.
  • the present disclosure includes a method for modulating 5-HTi a receptor expression in a pediatric subject comprising administering to the subject an effective amount of the disclosed nutritional composition. Still, in some embodiments, the present disclosure includes a method for promoting stress resistance in a pediatric subject comprising administering to the subject an effective amount of the disclosed nutritional composition. In other embodiments, the disclosure is directed to a method for modulating serotonergic circuits and/or regulating mood in a pediatric subject by administering an effective amount of the composition to the subject.
  • the present disclosure is directed to a method of promoting the development of or increasing the concentration of beneficial bacteria in the Gl tract, which may provide cognitive benefits. For example, an increase in the amount of Lactobacillus species and/or Bifidobacterium species, may promote stress resistance in a target subject. Moreover, the increase in the beneficial bacterium is observed without introducing these additional bacteria to the gastrointestinal tract via nutritional supplementation.
  • the unique combination of nutrients in the disclosed nutritional composition is believed to be capable of providing novel and unexpected brain- related benefits for infants and children. Moreover, the benefit of this nutritional composition is believed to be obtained during infancy, and also by including it as part of a diverse diet as the child and its brain continues to grow and develop.
  • Lactobacillus species including Lactobacillus rhamnosus, and Bifidobacterium species.
  • An increase in the concentration of these beneficial bacteria in the gastrointestinal tract may provide further health benefits, such as reduction in anxiety-like behavior and stress-induced inflammatory response. Further, an increase in beneficial bacteria may reduce constipation in a target subject.
  • the nutritional composition may also contain one or more prebiotics (also referred to as a prebiotic component) in certain embodiments.
  • prebiotics exert health benefits, which may include, but are not limited to, selective stimulation of the growth and/or activity of one or a limited number of beneficial gut bacteria, stimulation of the growth and/or activity of ingested probiotic microorganisms, selective reduction in gut pathogens, and favorable influence on gut short chain fatty acid profile.
  • Such prebiotics may be naturally-occurring, synthetic, or developed through the genetic manipulation of organisms and/or plants, whether such new source is now known or developed later.
  • Prebiotics useful in the present disclosure may include oligosaccharides, polysaccharides, and other prebiotics that contain fructose, xylose, soya, galactose, glucose and mannose.
  • prebiotics useful in the present disclosure may include polydextrose, polydextrose powder, lactulose, lactosucrose, raffinose, gluco- oligosaccharide, inulin, fructo-oligosaccharide, isomalto-ofigosaccharide, soybean oligosaccharides, lactosucrose, xylo-oligosaccharide, chito-oligosaccharide, manno- oligosaccharide, aribino-oligosaccharide, siallyl-oligosaccharide, fuco-oligosaccharide, galacto-oligosaccharides and gentio-oligosaccharides.
  • the total amount of prebiotics present in the nutritional composition may be from about 1.0 g/L to about 10.0 g/L of the composition. More preferably, the total amount of prebiotics present in the nutritional composition may be from about 2.0 g/L and about 8.0 g/L of the composition. In some embodiments, the total amount of prebiotics present in the nutritional composition may be from about 0.01 g/100 kcal to about 1.5 g/100 kcal. In certain embodiments, the total amount of prebiotics present in the nutritional composition may be from about 0.15 g/100 kcal to about 1.5 g/100 kcal. Moreover, the nutritional composition may comprise a prebiotic component comprising PDX. In some embodiments, the prebiotic component comprises at least 20% w/w PDX, GOS or a mixture thereof.
  • the amount of PDX in the nutritional composition may, in an
  • the prebiotic component also comprises GOS.
  • the amount of GOS in the nutritional composition may, in an embodiment, be from about 0.015 g/100 kcal to about 1.0 g/100 kcal. In another embodiment, the amount of GOS in the nutritional composition may be from about 0.2 g/100 kcal to about 0.5 g/100 kcal.
  • PDX is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-phenyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • GOS and PDX are supplemented into the nutritional composition in a total amount of at least about 0.015 g/100 kcal or about 0.015 g/100 kcal to about 1.5 g/100 kcal.
  • the nutritional composition may comprise GOS and PDX in a total amount of from about 0.1 to about 1.0 g/100 kcal.
  • lactoferrin is also included in the nutritional composition of the present disclosure.
  • Lactoferrins are single chain polypeptides of about 80 kD containing 1 - 4 glycans, depending on the species.
  • the 3-D structures of lactoferrin of different species are very similar, but not identical.
  • Each lactoferrin comprises two homologous lobes, called the N- and C-lobes, referring to the N-terminal and C- terminal part of the molecule, respectively.
  • Each lobe further consists of two sub- lobes or domains, which form a cleft where the ferric ion (Fe 3+ ) is tightly bound in synergistic cooperation with a (bi)carbonate anion. These domains are called N1 ; N2, C1 and C2, respectively.
  • the N-terminus of lactoferrin has strong cationic peptide regions that are responsible for a number of important binding characteristics.
  • Lactoferrin has a very high isoelectric point ( ⁇ pl 9) and its cationic nature plays a major role in its ability to defend against bacterial, viral, and fungal pathogens.
  • ⁇ pl 9 isoelectric point
  • the N-terminal residues 1-47 of human lactoferrin (1- 48 of bovine lactoferrin) are critical to the iron-independent biological activities of lactoferrin.
  • residues 2 to 5 (RRRR) and 28 to 31 (RKVR) are arginine-rich cationic domains in the N-terminus especially critical to the antimicrobial activities of lactoferrin.
  • a similar region in the N-terminus is found in bovine lactoferrin (residues 17 to 42; FKCRRWQWRMKKLGAPSITCVRRAFA).
  • Lactoferrins from different host species may vary in their amino acid sequences though commonly possess a relatively high isoelectric point with positively charged amino acids at the end terminal region of the internal lobe.
  • Suitable non- human lactoferrins for use in the present disclosure include, but are not limited to, those having at least 48% homology with the amino acid sequence of human lactoferrin.
  • bovine lactoferrin (“bLF”) has an amino acid composition which has about 70% sequence homology to that of human lactoferrin.
  • the non-human lactoferrin has at least 55% homology with human lactoferrin and in some embodiments, at least 65% homology.
  • Non-human lactoferrins acceptable for use in the present disclosure include, without limitation, bLF, porcine lactoferrin, equine lactoferrin, buffalo lactoferrin, goat lactoferrin, murine lactoferrin and camel lactoferrin.
  • lactoferrin is present in the nutritional composition in an amount of at least about 15 mg/100 kCal. In certain embodiments, the nutritional composition may include between about 15 and about 300 mg lactoferrin per 100 kCal. In another embodiment, where the nutritional composition is an infant formula, the nutritional composition may comprise lactoferrin in an amount of from about 60 mg to about 150 mg lactoferrin per 100 kCal; in yet another embodiment, the nutritional composition may comprise about 60 mg to about 100 mg lactoferrin per 100 kCal.
  • the nutritional composition can include lactoferrin in the quantities of from about 0.5 mg to about 1.5 mg per milliliter of formula.
  • lactoferrin may be present in quantities of from about 0.6 mg to about 1.3 mg per milliliter of formula.
  • the nutritional composition may comprise between about 0.1 and about 2 grams lactoferrin per liter.
  • composition includes between about 0.6 and about 1.5 grams lactoferrin per liter of formula.
  • the bLF that is used in certain embodiments may be any bLF isolated from whole milk and/or having a low somatic cell count, wherein "low somatic cell count” refers to a somatic cell count less than 200,000 cells/mL.
  • suitable bLF is available from Tatua Co-operative Dairy Co. Ltd., in Morrinsville, New Zealand, from FrieslandCampina Domo in Amersfoort, Netherlands or from Fonterra Co-Operative Group Limited in Auckland, New Zealand.
  • Lactoferrin for use in the present disclosure may be, for example, isolated from the milk of a non-human animal or produced by a genetically modified organism. For example, in U.S. Patent No.
  • Okonogi et al. discloses a process for producing bovine lactoferrin in high purity.
  • the process as disclosed includes three steps.
  • Raw milk material is first contacted with a weakly acidic cationic exchanger to absorb lactoferrin followed by the second step where washing takes place to remove nonabsorbed substances.
  • a desorbing step follows where lactoferrin is removed to produce purified bovine lactoferrin.
  • Other methods may include steps as described in U.S. Patent Nos. 7,368,141 , 5,849,885, 5,919,913 and 5,861 ,491 , the disclosures of which are all incorporated by reference in their entirety.
  • lactoferrin utilized in the present disclosure may be provided by an expanded bed absorption ("EBA") process for isolating proteins from milk sources.
  • EBA also sometimes called stabilized fluid bed adsorption, is a process for isolating a milk protein, such as lactoferrin, from a milk source comprises establishing an expanded bed adsorption column comprising a particulate matrix, applying a milk source to the matrix, and eluting the lactoferrin from the matrix with an elution buffer comprising about 0.3 to about 2.0 M sodium chloride.
  • Any mammalian milk source may be used in the present processes, although in particular embodiments, the milk source is a bovine milk source.
  • the milk source comprises, in some embodiments, whole milk, reduced fat milk, skim milk, whey, casein, or mixtures thereof.
  • the target protein is lactoferrin, though other milk proteins, such as lactoperoxidases or lactalbumins, also may be isolated.
  • the process comprises the steps of establishing an expanded bed adsorption column comprising a particulate matrix, applying a milk source to the matrix, and eluting the lactoferrin from the matrix with about 0.3 to about 2.0M sodium chloride.
  • the lactoferrin is eluted with about 0.5 to about 1.0 M sodium chloride, while in further embodiments, the lactoferrin is eluted with about 0.7 to about 0.9 M sodium chloride.
  • the expanded bed adsorption column can be any known in the art, such as those described in U.S. Patent Nos. 7,812,138, 6,620,326, and 6,977,046, the disclosures of which are hereby incorporated by reference herein.
  • a milk source is applied to the column in an expanded mode, and the elution is performed in either expanded or packed mode.
  • the elution is performed in an expanded mode.
  • the expansion ratio in the expanded mode may be about 1 to about 3, or about 1.3 to about 1.7.
  • EBA technology is further described in international published application nos. WO
  • the isoelectric point of lactoferrin is approximately 8.9.
  • Prior EBA methods of isolating lactoferrin use 200 mM sodium hydroxide as an elution buffer.
  • the pH of the system rises to over 12, and the structure and bioactivity of lactoferrin may be comprised, by irreversible structural changes.
  • a sodium chloride solution can be used as an elution buffer in the isolation of lactoferrin from the EBA matrix.
  • the sodium chloride has a concentration of about 0.3 M to about 2.0 M.
  • the lactoferrin elution buffer has a sodium chloride concentration of about 0.3 M to about 1.5 M, or about 0.5 m to about 1.0 M.
  • the nutritional composition of the disclosure also contains a source of LCPUFAs; especially a source of LCPUFAs that comprises docosahexaenoic acid.
  • a source of LCPUFAs include, but are not limited to, a-linoleic acid, ⁇ -linoleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA) and arachidonic acid (ARA).
  • the nutritional composition is supplemented with both DHA and ARA.
  • the weight ratio of ARA:DHA may be between about 1 :3 and about 9:1. In a particular embodiment, the ratio of ARA:DHA is from about 1 :2 to about 4:1.
  • the amount of long chain polyunsaturated fatty acid in the nutritional composition is advantageously at least about 5 mg/100 kcal, and may vary from about 5 mg/100 kcal to about 100 mg/100 kcal, more preferably from about 10 mg/100 kcal to about 50 mg/100 kcal.
  • the nutritional composition may be supplemented with oils containing DHA and/or ARA using standard techniques known in the art.
  • DHA and ARA may be added to the composition by replacing an equivalent amount of an oil, such as high oleic sunflower oil, normally present in the composition.
  • the oils containing DHA and ARA may be added to the composition by replacing an equivalent amount of the rest of the overall fat blend normally present in the composition without DHA and ARA.
  • the source of DHA and/or ARA may be any source known in the art such as marine oil, fish oil, single cell oil, egg yolk lipid, and brain lipid.
  • the DHA and ARA are sourced from single cell Martek oils, DHASCO ® and ARASCO ® , or variations thereof.
  • the DHA and ARA can be in natural form, provided that the remainder of the LCPUFA source does not result in any substantial deleterious effect on the infant.
  • the DHA and ARA can be used in refined form.
  • sources of DHA and ARA are single cell oils as taught in U.S. Pat. Nos. 5,374,567; 5,550,156; and 5,397,591, the disclosures of which are incorporated herein in their entirety by reference.
  • the present disclosure is not limited to only such oils.
  • the nutritional composition may include an enriched lipid fraction derived from milk.
  • the enriched lipid fraction derived from milk may be produced by any number of fractionation techniques. These techniques include but are not limited to melting point fractionation, organic solvent fractionation, super critical fluid fractionation, and any variants and combinations thereof.
  • the nutritional composition may include an enriched lipid fraction derived from milk that contains milk fat globules.
  • the addition of the enriched lipid fraction or the enriched lipid fraction including milk fat globules may provide a source of saturated fatty acids, trans-fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, OBCFAs, BCFAs, CLA, cholesterol, phospholipids, and/or milk fat globule membrane proteins to the nutritional composition.
  • the milk fat globules may have an average diameter (volume-surface area average diameter) of at least about 2 ⁇ . In some embodiments, the average diameter is in the range of from about 2 ⁇ to about 13 ⁇ . In other embodiments, the milk fat globules may range from about 2.5 ⁇ to about 10 ⁇ . Still in other embodiments, the milk fat globules may range in average diameter from about 3 ⁇ ⁇ ⁇ to about 6 ⁇ .
  • the specific surface area of the globules is, in certain embodiments, less than 3.5 m 2 /g, and in other embodiments is between about 0.9 mVg to about 3 m 2 /g. Without being bound by any particular theory, it is believed that milk fat globules of the aforementioned sizes are more accessible to lipases therefore leading to better digestion lipid digestion.
  • the enriched lipid fraction and/or milk fat globules contain saturated fatty acids.
  • the saturated fatty acids may be present in a concentration from about 0.1 g/100 kcal to about 8.0 g/100 kcal. In certain embodiments the saturated fatty acids may be present from about 0.5 g/100 kcal to about 2.0 g/100 kcal. In still other embodiments the saturated fatty acids may be present from about 3.5 g/100 kcal to about 6.9 g/100 kcal.
  • saturated fatty acids suitable for inclusion include, but are not limited to, butyric, valeric, caproic, caprylic, decanoic, lauric, myristic, palmitic, steraic, arachidic, behenic, alignoceric, tetradecanoic, hexadecanoic, palmitic, and octadecanoic acid, and/or combinations and mixtures thereof.
  • the enriched lipid fraction and/or milk fat globules may comprise, in some embodiments, lauric acid.
  • Lauric acid also known as dodecanoic acid, is a saturated fatty acid with a 12-carbon atom chain and is believed to be one of the main antiviral and antibacterial substances currently found in human breast milk.
  • the milk fat globules may be enriched with triglycerides containing lauric acid at either the Sn-1, Sn-2 and/or Sn-3 positions.
  • the mouth lingual lipase and pancreatic lipase will hydrolyze the triglycerides to a mixture of glycerides including mono-lauric and free lauric acid.
  • the concentration of lauric acid in the globules varies from 80mg/100ml to 800mg/100ml.
  • the concentration of monolauryl n the globules can be in the range of 20mg/100ml to 300mg/100ml feed. In some embodiments, the range is
  • the enriched lipid fraction and/or milk fat globules may contain trans- fatty acids in certain embodiments.
  • the trans-fatty acids included in the milk fat globules may be monounsaturated or polyunsaturated trans-fatty acids.
  • the trans-fatty acids may be present in an amount from about 0.2 g/100 kcal to about 7.0 g/100 kcal.
  • the trans-fatty acids may be present in an amount from about 3.4 g/100 kcal to about 5.2 g/100 kcal.
  • the trans-fatty acids may be present from about 1.2 g/100 kcal to about 4.3 g/100 kcal.
  • trans-fatty acids for inclusion include, but are not limited to, vaccenic, or elaidic acid, and mixtures thererof.
  • mammals when consumed, mammals convert vaccenic acid into rumenic acid, which is a conjugated linoleic acid that exhibits anticarcinogenic properties.
  • a diet enriched with vaccenic acid may help lower total cholesterol, LDL cholesterol and triglyceride levels.
  • the enriched lipid fraction and/or milk fat globules may contain OBCFAs.
  • the OBCFAs may be present in an amount from about 0.3 g/100 kcal to about 6.1 g/100 kcal.
  • OBCFAs may be present in an amount from about 2.2 g/100 kcal to about 4.3 g/100 kcal. In yet another embodiment OBCFAs may be present in an amount from about 3.5 g/ 00 kcal to about 5.7 g/100 kcal. In still other
  • the milk fat globules comprise at least one OBCFA.
  • OBCFAs typically absorb OBCFAs while in utero and from the breast milk of a nursing mother. Therefore, OBCFAs that are identified in human milk are preferred for inclusion in the milk fat globules of the nutritional composition. Addition of OBCFAs to infant or children's formulas allows such formulas to mirror the composition and functionality of human milk and to promote general health and well-being.
  • the enriched lipid fraction and/or milk fat globules may comprise BCFAs.
  • the BCFAs are present at a concentration from about 0.2 g/100 kcal and about 5.82 g/100 kcal.
  • the BCFAs are present in an amount of from about 2.3 g/100 kcal to about 4.2 g/100 kcal.
  • the BCFAs are present from about 4.2 g/100 kcal to about 5.82 g/100 kcal.
  • the milk fat globules comprise at least one BCFA.
  • BCFAs that are identified in human milk are preferred for inclusion in the nutritional composition. Addition of BCFAs to infant or children's formulas allows such formulas to mirror the composition and functionality of human milk and to promote general health and well-being.
  • the enriched lipid fraction and/or milk fat globules may comprise CLA.
  • CLA may be present in a concentration from about 0.4 g/100 kcal to about 2.5 g/100 kcal.
  • CLA may be present from about 0.8 g/100 kcal to about 1.2 g/100 kcal.
  • CLA may be present from about 1.2 g/100 kcal to about 2.3 g/100 kcal.
  • the milk fat globules comprise at least one CLA.
  • CLAs that are identified in human milk are preferred for inclusion in the nutritional composition.
  • CLAs are absorbed by the infant from the human milk of a nursing mother. Addition of CLAs to infant or children's formulas allows such formulas to mirror the composition and functionality of human milk and to promote general health and wellbeing.
  • CLAs found in the milk fat globules for the nutritional composition include, but are not limited to, cis-9, trans-11 CLA, trans-10, cis-12 CLA, cis-9, trans- 2 octadecadienoic acid, and mixtures thereof.
  • the enriched lipid fraction and/or milk fat globules of the present disclosure comprise monounsaturated fatty acids in some embodiments.
  • the enriched lipid fraction and/or milk fat globules may be formulated to include monounsaturated fatty acids from about 0.8 g/100 kcal to about 2.5 g/100 kcal.
  • the milk fat globules may include monounsaturated fatty acids from about 1.2 g/100 kcal to about 1.8 g/100 kcal.
  • Examples of monounsaturated fatty acids suitable include, but are not limited to, palmitoleic acid, cis-vaccenic acid, oleic acid, and mixtures thereof.
  • the enriched lipid fraction and/or milk fat globules of the present disclosure comprise polyunsaturated fatty acids from about 2.3 g/100 kcal to about 4.4 g/100 kcal. In other embodiments, the polyunsaturated fatty acids are present from about 2.7 g/100 kcal to about 3.5 g/100 kcal. In yet another embodiment, the polyunsaturated fatty acids are present from about 2.4 g/100 kcal to about 3.3 g/100 kcal.
  • the enriched lipid fraction and/or milk fat globules of the present disclosure comprise polyunsaturated fatty acids, such as, for example linoleic acid, linolenic acid, octadecatrienoic acid, arachidonic acid (ARA), eicosatetraenoic acid, eicopsapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA).
  • Polyunsaturated fatty acids are the precursors for prostaglandins and eicosanoids, which are known to provide numerous health benefits, including, anti-inflammatory response, cholesterol absorption, and increased bronchial function.
  • the enriched lipid fraction and/or milk fat globules of the present disclosure can also comprise cholesterol in some embodiments from about 100 mg/100 kcal to about 400 mg/100 kal. In another embodiment, cholesterol is present from about 200 mg/100 kcal to about 300 mg/100 kcal. As is similar to human milk and bovine milk, the cholesterol included in the milk fat globules may be present in the outer bilayer membrane of the milk fat globule to provide stability to the globular membrane.
  • the enriched lipid fraction and/or milk fat globules of the present disclosure comprise phospholipids from about 50 mg/100 kcal to about 200 mg/100 kcal. In other embodiments, the phospholipids are present from about 75 mg/100 kcal to about 150 mg/100 kcal. In yet other embodiments, the phospholipids are present at a concentration of from about 100 mg/100 kcal to about 250 mg/100 kcal.
  • phospholipids may be incorporated into the milk fat globules to stabilize the milk fat globule by providing a phospholipid membrane or bilayer phospholipid membrane. Therefore, in some embodiments the milk fat globules may be formulated with higher amounts of phospholipids than those found in human milk.
  • the phospholipid composition of human milk lipids is phosphatidylcholine("PC") 24.9%,
  • PE phosphatidylethanolamine
  • PS phosphatidylserine
  • the milk fat globules comprise one or more of PC, PE, PS, PI, SPGM, and mixtures thereof.
  • the phospholipid composition included in the milk fat globules may be formulated to provide certain health benefits by incorporating desired phospholipids.
  • the enriched lipid fraction and/or milk fat globules of the present disclosure comprise milk fat globule membrane protein.
  • the milk fat globule membrane protein is present from about 50 mg/100 kcal to about 500 mg/100 kcal.
  • Galactolipids may be included, in some embodiments, in the enriched lipid fraction and/or milk fat globules of the present disclosure.
  • galactolipids refer to any glycolipid whose sugar group is galactose. More specifically, galactolipids differ from glycosphingolipids in that they do not have nigtrogen in their composition.
  • Galactolipids play an important role in supporting brain development and overall neuronal health. Additionally, the galactolipids, galactocerebroside and sulfatides constitute about 23% and 4% of total myelin lipid content respectively, and thus may be incorporated into the milk fat globules in some embodiments.
  • the nutritional composition(s) of the disclosure may comprise at least one protein source, by which is meant excluding lactoferrin.
  • the protein source can be any used in the art, e.g., nonfat milk, whey protein, casein, soy protein, hydrolyzed protein, amino acids, and the like.
  • Bovine milk protein sources useful in practicing the present disclosure include, but are not limited to, milk protein powders, milk protein concentrates, milk protein isolates, nonfat milk solids, nonfat milk, nonfat dry milk, whey protein, whey protein isolates, whey protein concentrates, sweet whey, acid whey, casein, acid casein, caseinate [e.g. sodium caseinate, sodium calcium caseinate, calcium caseinate) and any combinations thereof.
  • the proteins of the nutritional composition are provided as intact proteins. In other embodiments, the proteins are provided as a combination of both intact proteins and hydrolyzed proteins. In certain embodiments, the proteins are provided as a combination of both intact proteins and hydrolyzed proteins.
  • the proteins may be partially hydrolyzed or extensively hydrolyzed.
  • the protein source comprises amino acids.
  • the protein source may be supplemented with glutamine-containing peptides.
  • the protein component comprises extensively hydrolyzed protein.
  • the protein component of the nutritional composition consists essentially of extensively hydrolyzed protein in order to minimize the occurrence of food allergy.
  • the protein source may be supplemented with glutamine-containing peptides.
  • the protein component of the nutritional composition comprises either partially or extensively hydrolyzed protein, such as protein from cow's milk.
  • the hydrolyzed proteins may be treated with enzymes to break down some or most of the proteins that cause adverse symptoms with the goal of reducing allergic reactions, intolerance, and sensitization.
  • proteins may be hydrolyzed by any method known in the art.
  • protein hydrolysates or “hydrolyzed protein” are used interchangeably herein and refer to hydrolyzed proteins, wherein the degree of hydrolysis is may be from about 20% to about 80%, or from about 30% to about 80%, or even from about 40% to about 60%.
  • hydrolyzed proteins When a peptide bond in a protein is broken by enzymatic hydrolysis, one amino group is released for each peptide bond broken, causing an increase in amino nitrogen. It should be noted that even non-hydrolyzed protein would contain some exposed amino groups. Hydrolyzed proteins will also have a different molecular weight distribution than the non-hydrolyzed proteins from which they were formed. The functional and nutritional properties of hydrolyzed proteins can be affected by the different size peptides. A molecular weight profile is usually given by listing the percent by weight of particular ranges of molecular weight (in Daltons) fractions (e.g., 2,000 to 5,000 Daltons, greater than 5,000 Daltons).
  • the nutritional composition is protein-free and contains free amino acids as a protein equivalent source.
  • the amino acids may comprise, but are not limited to, histidine, isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, threonine, tryptophan, valine, alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, carnitine, taurine and mixtures thereof.
  • the amino acids may be branched chain amino acids.
  • small amino acid peptides may be included as the protein component of the nutritional composition.
  • Such small amino acid peptides may be naturally occurring or synthesized.
  • the amount of free amino acids in the nutritional composition may vary from about 1 to about 5 g/100 kcal. In an embodiment, 100% of the free amino acids have a molecular weight of less than 500 Daltons.
  • the nutritional formulation may be hypoallergenic.
  • the protein source comprises from about 40% to about 85% whey protein and from about 15% to about 60% casein.
  • the nutritional composition comprises between about 1 g and about 7 g of a protein and/or protein equivalent source per 100 kcal. In other embodiments, the nutritional composition comprises between about 3.5 g and about 4.5 g of protein or protein equivalent per 100 kcal.
  • the nutritional composition of the present disclosure may comprise at least one starch or starch component.
  • a starch is a carbohydrate composed of two distinct polymer fractions: amylose and amylopectin.
  • Amylose is the linear fraction consisting of a-1 ,4 linked glucose units.
  • Amylopectin has the same structure as amylose, but some of the glucose units are combined in an ct-1,6 linkage, giving rise to a branched structure.
  • Starches generally contain 17-24% amylose and from 76-83% amylopectin.
  • Yet special genetic varieties of plants have been developed that produce starch with unusual amylose to amylopectin ratios. Some plants produce starch that is free of amylose.
  • the nutritional compositions of the present disclosure comprise at least one starch that is gelatinized or pregelatinized.
  • gelatinization occurs when polymer molecules interact over a portion of their length to form a network that entraps solvent and/or solute molecules.
  • gels form when pectin molecules lose some water of hydration owing to competitive hydration of cosolute molecules. Factors that influence the occurrence of gelation include pH, concentration of cosolutes, concentration and type of cations,
  • pregelatinization of starch is a process of precooking starch to produce material that hydrates and swells in cold water.
  • the precooked starch is then dried, for example by drum drying or spray drying.
  • the starch of the present disclosure can be chemically modified to further extend the range of its finished properties.
  • the nutritional compositions of the present disclosure may comprise at least one pregelatinized starch.
  • Native starch granules are insoluble in water, but, when heated in water, native starch granules begin to swell when sufficient heat energy is present to overcome the bonding forces of the starch molecules. With continued heating, the granule swells to many times its original volume. The friction between these swollen granules is the major factor that contributes to starch paste viscosity.
  • the nutritional composition of the present disclosure may comprise native or modified starches, such as, for example, waxy corn starch, waxy rice starch, corn starch, rice starch, potato starch, tapioca starch, wheat starch or any mixture thereof.
  • native starches such as, for example, waxy corn starch, waxy rice starch, corn starch, rice starch, potato starch, tapioca starch, wheat starch or any mixture thereof.
  • common corn starch comprises about 25% amylose
  • waxy corn starch is almost totally made up of amylopectin.
  • potato starch generally comprises about 20% amylose
  • rice starch comprises an
  • amylose:amylopectin ratio of about 20:80 and waxy rice starch comprises only about 2% amylose.
  • tapioca starch generally comprises about 15% to about 18% amylose, and wheat starch has an amylose content of around 25%.
  • the nutritional composition comprises
  • the nutritional composition comprises gelatinized and/or pre-gelatinized waxy corn starch.
  • the nutritional composition comprises gelatinized and/or pre-gelatinized tapioca starch.
  • Other gelatinized or pre-gelatinized starches such as rice starch or potato starch may also be used.
  • the nutritional compositions of the present disclosure comprise at least one source of pectin.
  • the source of pectin may comprise any variety or grade of pectin known in the art.
  • the pectin has a degree of esterification of less than 50% and is classified as low methylated ("LM") pectin.
  • the pectin has a degree of esterification of greater than or equal to 50% and is classified as high-ester or high methylated (“HM”) pectin.
  • the pectin is very low (“VL”) pectin, which has a degree of esterification that is less than approximately 15%.
  • the nutritional composition of the present disclosure may comprise LM pectin, HM pectin, VL pectin, or any mixture thereof.
  • the nutritional composition may include pectin that is soluble in water.
  • solubility and viscosity of a pectin solution are related to the molecular weight, degree of esterification, concentration of the pectin preparation and the pH and presence of counterions.
  • pectin has a unique ability to form gels.
  • a pectin's degree of gelation, the gelling temperature, and the gel strength are proportional to one another, and each is generally proportional to the molecular weight of the pectin and inversely proportional to the degree of esterification.
  • the polysaccharide molecules can associate over a portion of their length to form a gel.
  • pectins with increasing degrees of methylation will gel at somewhat higher pH because they have fewer carboxylate anions at any given pH.
  • J.N. Bemiller, An Introduction to Pectins: Structure and Properties, Chemistry and Function of Pectins; Chapter 1 ; 1986. J.N. Bemiller, An Introduction to Pectins: Structure and Properties, Chemistry and Function of Pectins; Chapter 1 ; 1986.
  • the nutritional composition may comprise a gelatinized and/or pregelatinized starch together with pectin and/or gelatinized pectin. While not wishing to be bound by this or any other theory, it is believed that the use of pectin, such as LM pectin, which is a hydrocolloid of large molecular weight, together with starch granules, provides a synergistic effect that increases the molecular internal friction within a fluid matrix.
  • pectin such as LM pectin, which is a hydrocolloid of large molecular weight
  • the carboxylic groups of the pectin may also interact with calcium ions present in the nutritional composition, thus leading to an increase in viscosity, as the carboxylic groups of the pectin form a weak gel structure with the calcium ion(s), and also with peptides present in the nutritional composition.
  • the nutritional composition comprises a ratio of starch to pectin that is between about 12:1 and 20:1 , respectively. In other embodiments, the ratio of starch to pectin is about 17:1.
  • the nutritional composition may comprise between about 0.05 and about 2.0% w/w pectin. In a particular embodiment, the nutritional composition may comprise about 0.5% w/w pectin.
  • Pectins for use herein typically have a peak molecular weight of 8,000 Daltons or greater.
  • the pectins of the present disclosure have a preferred peak molecular weight of between 8,000 and about 500,000, more preferred is between about 10,000 and about 200,000 and most preferred is between about 15,000 and about 100,000 Daltons.
  • the pectin of the present disclosure may be hydrolyzed pectin.
  • the nutritional composition comprises hydrolyzed pectin having a molecular weight less than that of intact or unmodified pectin.
  • the hydrolyzed pectin of the present disclosure can be prepared by any means known in the art to reduce molecular weight.
  • the nutritional composition comprises partially hydrolyzed pectin.
  • the partially hydrolyzed pectin has a molecular weight that is less than that of intact or unmodified pectin but more than 3,300 Daltons.
  • the nutritional composition may contain at least one acidic
  • An acidic polysaccharide such as negatively charged pectin, may induce an anti-adhesive effect on pathogens in a subject's gastrointestinal tract.
  • nonhuman milk acidic oligosaccharides derived from pectin are able to interact with the epithelial surface and are known to inhibit the adhesion of pathogens on the epithelial surface.
  • the nutritional composition comprises at least one pectin-derived acidic oligosaccharide.
  • Pectin-derived acidic oligosaccharide(s) result from enzymatic pectinolysis, and the size of a pAOS depends on the enzyme use and on the duration of the reaction.
  • the pAOS may beneficially affect a subject's stool viscosity, stool frequency, stool pH and/or feeding tolerance.
  • the nutritional composition of the present disclosure may comprise between about 2 g pAOS per liter of formula and about 6 g pAOS per liter of formula.
  • the nutritional composition comprises about 0.2 g pAOS/dL, corresponding to the concentration of acidic oligosaccharides in human milk.
  • the nutritional composition comprises up to about 20% w/w of a mixture of starch and pectin. In some embodiments, the nutritional composition comprises up to about 19% starch and up to about 1% pectin. In other embodiments, the nutritional composition comprises about up to about 15% starch and up to about 5% pectin. In still other embodiments, the nutritional composition comprises up to about 18% starch and up to about 2% pectin. In some embodiments the nutritional composition comprises between about 0.05% w/w and about 20% w/w of a mixture of starch and pectin. Other
  • embodiments include between about 0.05% and about 19% w/w starch and between about 0.05% and about 1% w/w pectin. Further, the nutritional composition may comprise between about 0.05% and about 15% w/w starch and between about 0.05% and about 5% w/w pectin.
  • the nutritional composition comprises at least one additional carbohydrate source, that is, a carbohydrate component provided in addition to the aforementioned starch component.
  • Additional carbohydrate sources can be any used in the art, e.g., lactose, glucose, fructose, corn syrup solids, ma!todextrins, sucrose, starch, rice syrup solids, and the like.
  • the amount of the additional carbohydrate component in the nutritional composition typically can vary from between about 5 g and about 25 g/100 kcal. In some embodiments, the amount of carbohydrate is between about 6 g and about 22 g/ 100 kcal. In other embodiments, the amount of carbohydrate is between about 12 g and about 14 g/100 kcal.
  • corn syrup solids are preferred.
  • hydrolyzed, partially hydrolyzed, and/or extensively hydrolyzed carbohydrates may be desirable for inclusion in the nutritional composition due to their easy digestibility. Specifically, hydrolyzed carbohydrates are less likely to contain allergenic epitopes.
  • Non-limiting examples of carbohydrate materials suitable for use herein include hydrolyzed or intact, naturally or chemically modified, starches sourced from corn, tapioca, rice or potato, in waxy or non-waxy forms.
  • suitable carbohydrates include various hydrolyzed starches characterized as hydrolyzed cornstarch, maltodextrin, maltose, corn syrup, dextrose, corn syrup solids, glucose, and various other glucose polymers and combinations thereof.
  • Non- limiting examples of other suitable carbohydrates include those often referred to as sucrose, lactose, fructose, high fructose corn syrup, indigestible oligosaccharides such as fructooligosaccharides and combinations thereof.
  • the additional carbohydrate component of the nutritional composition is comprised of 100% lactose.
  • the additional carbohydrate component comprises between about 0% and 60% lactose.
  • the additional carbohydrate component comprises between about 15% and 55% lactose.
  • the additional carbohydrate component comprises between about 20% and 30% lactose.
  • the remaining source of carbohydrates may be any carbohydrate known in the art.
  • the carbohydrate component comprises about 25% lactose and about 75% corn syrup solids.
  • the nutritional composition comprises sialic acid.
  • Sialic acids are a family of over 50 members of 9-carbon sugars, all of which are derivatives of neuroaminic acid. The predominant sialic acid family found in humans is from the N-acetylneuraminic acid sub-family. Sialic acids are found in milk, such as bovine and caprine. In mammals, neuronal cell membranes have the highest concentration of sialic acid compared to other body cell membranes. Sialic acid residues are also components of gangliosides.
  • sialic acid may be present in an amount from about 0.5 mg/100 kcals to about 45 mg/100 kcal. In some
  • sialic acid may be present in an amount from about 5 mg/100 kcals to about 30 mg/ 00 kcals. In still other embodiments, sialic acid may be present in an amount from about 10 mg/ 00 kcals to about 25 mg/100 kcals.
  • the nutritional composition may contain one or more probiotics. Any probiotic known in the art may be acceptable in this embodiment.
  • the probiotic may be selected from any Lactobacillus species, Lactobacillus rhamnosus GG (LGG) (ATCC number 53103), Bifidobacterium species, Bifidobacterium longum BB536 (BL999, ATCC: BAA-999), Bifidobacterium longum AH1206 (NCIMB: 41382), Bifidobacterium breve AH 1205 (NCIMB: 41387), Bifidobacterium infantis 35624 (NCIMB: 41003), and Bifidobacterium animalis subsp. lactis BB-12 (DSM No. 10140) or any combination thereof.
  • LGG Lactobacillus rhamnosus GG
  • Bifidobacterium species Bifidobacterium longum BB536 (BL999, ATCC: BAA-999)
  • Bifidobacterium longum AH1206 NCIMB: 41382
  • the amount of the probiotic may vary from about 1 x 10 to about 1.5 x 10 12 cfu of probiotic(s) per 100 kcal. In some embodiments the amount of probiotic may be from about 1 x 10 6 to about 1 x 10 9 cfu of probiotic(s) per 100 kcal. In certain other embodiments the amount of probitic may vary from about 1 x 10 7 cfu/100 kcal to about 1 x 10 8 cfu of probiotic(s) per 100 kcal.
  • the probiotic(s) may be viable or non-viable.
  • viable refers to live microorganisms.
  • non-viable or non-viable probiotic means non-living probiotic microorganisms, their cellular components and/or metabolites thereof. Such non-viable probiotics may have been heat-killed or otherwise inactivated, but they retain the ability to favorably influence the health of the host.
  • the probiotics useful in the present disclosure may be naturally-occurring, synthetic or developed through the genetic manipulation of organisms, whether such source is now known or later developed.
  • the nutritional composition may include a source comprising probiotic cell equivalents, which refers to the level of non-viable, non- replicating probiotics equivalent to an equal number of viable cells.
  • probiotic cell equivalents refers to the level of non-viable, non- replicating probiotics equivalent to an equal number of viable cells.
  • non- replicating is to be understood as the amount of non-replicating microorganisms obtained from the same amount of replicating bacteria (cfu/g), including inactivated probiotics, fragments of DNA, cell wall or cytoplasmic compounds.
  • the quantity of non-living, non-replicating organisms is expressed in terms of cfu as if all the microorganisms were alive, regardless whether they are dead, non-replicating, inactivated, fragmented etc.
  • the amount of the probiotic cell equivalents may vary from about 1 x 10 4 to about 1.5 x 10 10 cell equivalents of probiotic(s) per 100 kcal. In some embodiments the amount of probiotic cell equivalents may be from about 1 x 10 6 to about 1 x 10 9 cell equivalents of probiotic(s) per 100 kcal nutritional composition. In certain other embodiments the amount of probiotic cell equivalents may vary from about 1 x 10 7 to about 1 x 10 8 cell equivalents of probiotic(s) per 100 kcal of nutritional composition.
  • the probiotic source incorporated into the nutritional composition may comprise both viable colony-forming units, and nonviable cell-equivalents.
  • the nutritional composition includes a culture supernatant from a late-exponential growth phase of a probiotic batch-cultivation process.
  • the activity of the culture supernatant can be attributed to the mixture of components (including proteinaceous materials, and possibly including (exo)polysaccharide materials) as found released into the culture medium at a late stage of the exponential (or "log") phase of batch cultivation of the probiotic.
  • culture supernatant includes the mixture of components found in the culture medium. The stages recognized in batch cultivation of bacteria are known to the skilled person.
  • a culture supernatant is obtainable by a process comprising the steps of (a) subjecting a probiotic such as LGG to cultivation in a suitable culture medium using a batch process; (b) harvesting the culture supernatant at a late exponential growth phase of the cultivation step, which phase is defined with reference to the second half of the time between the lag phase and the stationary phase of the batch-cultivation process; (c) optionally removing low molecular weight constituents from the supernatant so as to retain molecular weight constituents above 5-6 kiloDaltons (kDa); (d) removing liquid contents from the culture supernatant so as to obtain the composition.
  • a probiotic such as LGG
  • the culture supernatant may comprise secreted materials that are harvested from a late exponential phase.
  • the late exponential phase occurs in time after the mid exponential phase (which is halftime of the duration of the exponential phase, hence the reference to the late exponential phase as being the second half of the time between the lag phase and the stationary phase).
  • the term "late exponential phase” is used herein with reference to the latter quarter portion of the time between the lag phase and the stationary phase of the LGG batch- cultivation process.
  • the culture supernatant is harvested at a point in time of 75% to 85% of the duration of the exponential phase, and may be harvested at about S of the time elapsed in the exponential phase.
  • the disclosed nutritional composition may comprise a source of ⁇ -glucan.
  • Glucans are polysaccharides, specifically polymers of glucose, which are naturally occurring and may be found in cell walls of bacteria, yeast, fungi, and plants.
  • Beta glucans ( ⁇ -glucans) are themselves a diverse subset of glucose polymers, which are made up of chains of glucose monomers linked together via beta-type glycosidic bonds to form complex carbohydrates.
  • p-1 ,3-glucans are carbohydrate polymers purified from, for example, yeast, mushroom, bacteria, algae, or cereals.
  • Stone BA Clarke AE. Chemistry and Biology of (1 -3)-Beta-Glucans. London:Portland Press Ltd; 1993.
  • the chemical structure of P-1 ,3-glucan depends on the source of the P-1 ,3-glucan.
  • various physiochemical parameters such as solubility, primary structure, molecular weight, and branching, play a role in biological activities of P-1 ,3-glucans.
  • Yadomae T. Structure and biological activities of fungal beta-1 ,3-glucans. Yakugaku Zasshi. 2000;120:413-431.
  • P-1 ,3-glucans are naturally occurring polysaccharides, with or without ⁇ - 1 ,6-glucose side chains that are found in the cell walls of a variety of plants, yeasts, fungi and bacteria.
  • p-1 ,3;1 ,6-glucans are those containing glucose units with (1 ,3) links having side chains attached at the (1 ,6) position(s).
  • ⁇ -1 ,3;1 ,6 glucans are a heterogeneous group of glucose polymers that share structural commonalities, including a backbone of straight chain glucose units linked by a ⁇ -1 ,3 bond with ⁇ - 1 ,6-linked glucose branches extending from this backbone.
  • yeast ⁇ -glucans have additional regions of ⁇ (1 ,3) branching extending from the ⁇ (1 ,6) branches, which add further complexity to their respective structures.
  • ⁇ -glucans derived from baker's yeast, Saccharomyces cerevisiae are made up of chains of D-glucose molecules connected at the 1 and 3 positions, having side chains of glucose attached at the 1 and 6 positions.
  • Yeast-derived ⁇ -glucan is an insoluble, fiber-like, complex sugar having the general structure of a linear chain of glucose units with a ⁇ -1 ,3 backbone interspersed with ⁇ -1 ,6 side chains that are generally 6-8 glucose units in length. More specifically, ⁇ -glucan derived from baker's yeast is poly-(1 ,6) ⁇ -D-glucopyranosyl-(1 ,3) ⁇ -D-glucopyranose.
  • ⁇ -glucans are well tolerated and do not produce or cause excess gas, abdominal distension, bloating or diarrhea in pediatric subjects.
  • Addition of ⁇ -glucan to a nutritional composition for a pediatric subject, such as an infant formula, a growing-up milk or another children's nutritional product, will improve the subject's immune response by increasing resistance against invading pathogens and therefore maintaining or improving overall health.
  • the nutritional composition of the present disclosure comprises ⁇ - glucan.
  • the ⁇ -glucan is -1 ,3;1,6-glucan.
  • the -1,3;1 ,6-glucan is derived from baker's yeast.
  • the nutritional composition may comprise whole glucan particle ⁇ -glucan, particulate ⁇ -glucan, PGG- glucan (poly-1 ,6 ⁇ -D-glucopyranosyl-1 ,3 ⁇ -D-glucopyranose) or any mixture thereof.
  • composition is at between about 0.010 and about 0.080 g per 100g of composition.
  • the nutritional composition comprises between about 10 and about 30 mg ⁇ -glucan per serving.
  • the nutritional composition comprises between about 5 and about 30 mg ⁇ -glucan per 8 fl. oz.
  • the nutritional composition comprises an amount of ⁇ -glucan sufficient to provide between about 15 mg and about 90 mg ⁇ - glucan per day.
  • the nutritional composition may be delivered in multiple doses to reach a target amount of ⁇ -glucan delivered to the subject throughout the day.
  • the amount of ⁇ -glucan in the nutritional composition is between about 3 mg and about 17 mg per 100 kcal. In another embodiment the amount of ⁇ -glucan is between about 6 mg and about 17 mg per 100 kcal.
  • vitamins and/or minerals may also be added in to the nutritional composition in amounts sufficient to supply the daily nutritional
  • vitamin and mineral requirements will vary, for example, based on the age of the child. For instance, an infant may have different vitamin and mineral requirements than a child between the ages of one and thirteen years.
  • the embodiments are not intended to limit the nutritional composition to a particular age group but, rather, to provide a range of acceptable vitamin and mineral components.
  • the nutritional composition may optionally include, but is not limited to, one or more of the following vitamins or derivations thereof: vitamin Bi (thiamin, thiamin pyrophosphate, TPP, thiamin triphosphate, TTP, thiamin hydrochloride, thiamin mononitrate), vitamin B2 (riboflavin, flavin mononucleotide, FMN, flavin adenine dinucleotide, FAD, lactoflavin, ovoflavin), vitamin B3 (niacin, nicotinic acid, nicotinamide, niacinamide, nicotinamide adenine dinucleotide, NAD, nicotinic acid mononucleotide, NicMN, pyridine-3-carboxylic acid), vitamin B3-precursor
  • vitamin B 6 pyridoxine, pyridoxal, pyridoxamine, pyridoxine
  • pantothenic acid pantothenate, panthenol
  • folate folic acid, folacin, pteroylg!utamic acid
  • vitamin B12 cobalamin, methylcobalamin
  • vitamin C deoxyadenosylcobalamin, cyanocobalamin, hydroxycobalamin, adenosylcobalamin), biotin, vitamin C (ascorbic acid), vitamin A (retinol, retinyl acetate, retinyl palmitate, retinyl esters with other long-chain fatty acids, retinal, retinoic acid, retinol esters), vitamin D (calciferol, cholecalciferol, vitamin D3, 1,25,-dihydroxyvitamin D), vitamin E (a-tocopherol, a-tocopherol acetate, a-tocopherol succinate, a-tocopherol nicotinate, ⁇ -tocopherol), vitamin K (vitamin Ki, phylloquinone, naphthoquinone, vitamin K2, menaquinone-7, vitamin K3, menaquinone-4, menadione, menaquinone-8,
  • menaquinone-8H menaquinone-9, menaquinone-9H, menaquinone-10,
  • menaquinone-11 menaquinone-12, menaquinone-13
  • choline inositol
  • ⁇ -carotene any combinations thereof.
  • the nutritional composition may optionally include, but is not limited to, one or more of the following minerals or derivations thereof: boron, calcium, calcium acetate, calcium gluconate, calcium chloride, calcium lactate, calcium phosphate, calcium sulfate, chloride, chromium, chromium chloride, chromium picolonate, copper, copper sulfate, copper gluconate, cupric sulfate, fluoride, iron, carbonyl iron, ferric iron, ferrous fumarate, ferric orthophosphate, iron trituration, polysaccharide iron, iodide, iodine, magnesium, magnesium carbonate, magnesium hydroxide, magnesium oxide, magnesium stearate, magnesium sulfate, manganese, molybdenum, phosphorus, potassium, potassium phosphate, potassium iodide, potassium chloride, potassium acetate, selenium, sulfur, sodium, docusate sodium, sodium chloride, sodium selenate
  • the minerals can be added to nutritional compositions in the form of salts such as calcium phosphate, calcium glycerol phosphate, sodium citrate, potassium chloride, potassium phosphate, magnesium phosphate, ferrous sulfate, zinc sulfate, cupric sulfate, manganese sulfate, and sodium selenite. Additional vitamins and minerals can be added as known within the art.
  • the nutritional composition may contain between about 10 and about 50% of the maximum dietary recommendation for any given country, or between about 10 and about 50% of the average dietary
  • the children's nutritional composition may supply about 10 - 30% of the maximum dietary recommendation for any given country, or about 10 - 30% of the average dietary recommendation for a group of countries, per serving of B-vitamins.
  • the levels of vitamin D, calcium, magnesium, phosphorus, and potassium in the children's nutritional product may correspond with the average levels found in milk.
  • other nutrients in the children's nutritional composition may be present at about 20% of the maximum dietary recommendation for any given country, or about 20% of the average dietary recommendation for a group of countries, per serving.
  • the nutritional compositions of the present disclosure may optionally include one or more of the following flavoring agents, including, but not limited to, flavored extracts, volatile oils, cocoa or chocolate flavorings, peanut butter flavoring, cookie crumbs, vanilla or any commercially available flavoring.
  • flavoring agents including, but not limited to, flavored extracts, volatile oils, cocoa or chocolate flavorings, peanut butter flavoring, cookie crumbs, vanilla or any commercially available flavoring.
  • useful flavorings include, but are not limited to, pure anise extract, imitation banana extract, imitation cherry extract, chocolate extract, pure lemon extract, pure orange extract, pure peppermint extract, honey, imitation pineapple extract, imitation rum extract, imitation strawberry extract, or vanilla extract; or volatile oils, such as balm oil, bay oil, bergamot oil, cedarwood oil, cherry oil, cinnamon oil, clove oil, or peppermint oil; peanut butter, chocolate flavoring, vanilla cookie crumb, butterscotch, toffee, and mixtures thereof.
  • the amounts of flavoring agent can vary greatly depending upon the flavoring agent used. The type and amount of flavoring agent can be selected as is known in the art.
  • the nutritional compositions of the present disclosure may optionally include one or more emulsifiers that may be added for stability of the final product.
  • suitable emulsifiers include, but are not limited to, lecithin ⁇ e.g., from egg or soy), alpha lactalbumin and/or mono- and di-glycerides, and mixtures thereof.
  • Other emulsifiers are readily apparent to the skilled artisan and selection of suitable emulsifier(s) will depend, in part, upon the formulation and final product.
  • the nutritional compositions of the present disclosure may optionally include one or more preservatives that may also be added to extend product shelf life. Suitable preservatives include, but are not limited to, potassium sorbate, sodium sorbate, potassium benzoate, sodium benzoate, calcium disodium EDTA, and mixtures thereof.
  • the nutritional compositions of the present disclosure may optionally include one or more stabilizers.
  • Suitable stabilizers for use in practicing the nutritional composition of the present disclosure include, but are not limited to, gum arabic, gum ghatti, gum karaya, gum tragacanth, agar, furcellaran, guar gum, gellan gum, locust bean gum, pectin, low methoxyl pectin, gelatin, microcrystalline cellulose, CMC (sodium carboxymethylcelfulose), methylcellulose hydroxypropyl methyl cellulose, hydroxypropyl cellulose, DATEM (diacetyl tartaric acid esters of mono- and diglycerides), dextran, carrageenans, and mixtures thereof.
  • the disclosed nutritional composition(s) may be provided in any form known in the art, such as a powder, a gel, a suspension, a paste, a solid, a liquid, a liquid concentrate, a reconstituteable powdered milk substitute or a ready-to-use product.
  • the nutritional composition may, in certain embodiments, comprise a nutritional supplement, children's nutritional product, infant formula, human milk fortifier, growing-up milk or any other nutritional composition designed for an infant or a pediatric subject.
  • Nutritional compositions of the present disclosure include, for example, orally-ingestible, health-promoting substances including, for example, foods, beverages, tablets, capsules and powders.
  • the nutritional composition of the present disclosure may be standardized to a specific caloric content, it may be provided as a ready-to-use product, or it may be provided in a concentrated form.
  • the nutritional composition is in powder form with a particle size in the range of 5 ⁇ to 1500 ⁇ , more preferably in the range of 10 pm to 300pm.
  • the osmolality of the nutritional composition may be between about 100 and about 1100 mOsm/kg water, more typically about 200 to about 700 mOsm/kg water.
  • Suitable fat or lipid sources for the nutritional composition of the present disclosure may be any known or used in the art, including but not limited to, animal sources, e.g., milk fat, butter, butter fat, egg yolk lipid; marine sources, such as fish oils, marine oils, single cell oils; vegetable and plant oils, such as corn oil, canola oil, sunflower oil, soybean oil, palm olein oil, coconut oil, high oleic sunflower oil, evening primrose oil, rapeseed oil, olive oil, flaxseed (linseed) oil, cottonseed oil, high oleic safflower oil, palm stearin, palm kernel oil, wheat germ oil; medium chain triglyceride oils and emulsions and esters of fatty acids; and any combinations thereof.
  • animal sources e.g., milk fat, butter, butter fat, egg yolk lipid
  • marine sources such as fish oils, marine oils, single cell oils
  • vegetable and plant oils such as corn oil, canola oil, sunflower oil, soybean oil, palm
  • the nutritional compositions of the disclosure may provide minimal, partial or total nutritional support.
  • the compositions may be nutritional supplements or meal replacements.
  • the compositions may, but need not, be nutritionally complete.
  • the nutritional composition of the disclosure is nutritionally complete and contains suitable types and amounts of lipid,
  • the amount of lipid or fat typically can vary from about 1 to about 7 g/100 kcal.
  • the amount of protein typically can vary from about 1 to about 7 g/100 kcal.
  • the amount of carbohydrate typically can vary from about 6 to about 22 g/100 kcal.
  • the nutritional composition of the present disclosure may further include at least one additional phytonutrient, that is, another phytonutrient component in addition to the pectin and/or starch components described
  • Phytonutrients, or their derivatives, conjugated forms or precursors, that are identified in human milk are preferred for inclusion in the nutritional composition.
  • dietary sources of carotenoids and polyphenols are absorbed by a nursing mother and retained in milk, making them available to nursing infants. Addition of these phytonutrients to infant or children's formulas allows such formulas to mirror the composition and functionality of human milk and to promote general health and well being.
  • the nutritional composition of the present disclosure may comprise, in an 8 fl. oz. (236.6 ml_) serving, between about 80 and about 300 mg anthocyanins, between about 100 and about 600 mg
  • the nutritional composition comprises apple extract, grape seed extract, or a combination or mixture thereof.
  • the at least one phytonutrient of the nutritional composition may be derived from any single or blend of fruit, grape seed and/or apple or tea extract(s).
  • additional phytonutrients may be added to a nutritional composition in native, purified, encapsulated and/or chemically or enzymatically-modified form so as to deliver the desired sensory and stability properties.
  • encapsulation it is desirable that the encapsulated phytonutrients resist dissolution with water but are released upon reaching the small intestine. This could be achieved by the application of enteric coatings, such as cross-linked alginate and others.
  • phytonutrients suitable for the nutritional composition include, but are not limited to, anthocyanins, proanthocyanidins, flavan- 3-ols (i.e.. catechins, epicatechins, etc.), flavanones, flavonoids, isoflavonoids, stilbenoids (i.e. resveratrol, etc.)proanthocyanidins, anthocyanins, resveratrol, quercetin, curcumin, and/or any mixture thereof, as well as any possible combination of phytonutrients in a purified or natural form. Certain components, especially plant- based components of the nutritional compositions may provide a source of phytonutrients.
  • phytonutrients may be inherently present in known ingredients, such as natural oils, that are commonly used to make nutritional compositions for pediatric subjects. These inherent phytonutrient(s) may be but are not necessarily considered part of the phytonutrient component described in the present disclosure. In some embodiments, the phytonutrient concentrations and ratios as described herein are calculated based upon added and inherent
  • phytonutrient sources In other embodiments, the phytonutrient concentrations and ratios as described herein are calculated based only upon added phytonutrient sources.
  • the nutritional composition comprises anthocyanins, such as, for example, glucosides of aurantinidin, cyanidin, delphinidin, europinidin, luteolinidin, pelargonidin, malvidin, peonidin, petunidin, and rosinidin.
  • anthocyanins such as, for example, glucosides of aurantinidin, cyanidin, delphinidin, europinidin, luteolinidin, pelargonidin, malvidin, peonidin, petunidin, and rosinidin.
  • Non-limiting examples of plants rich in anthocyanins suitable for use in the inventive composition include: berries (acai, grape, bilberry, blueberry, lingonberry, black currant, chokeberry, blackberry, raspberry, cherry, red currant, cranberry, crowberry, cloudberry, whortleberry, rowanberry), purple com, purple potato, purple carrot, red sweet potato, red cabbage, eggplant.
  • the nutritional composition of the present disclosure comprises proanthocyanidins, which include but are not limited to flavan-3- ols and polymers of flavan-3-ols (e.g., catechins, epicatechins) with degrees of polymerization in the range of 2 to 11.
  • proanthocyanidins include but are not limited to flavan-3- ols and polymers of flavan-3-ols (e.g., catechins, epicatechins) with degrees of polymerization in the range of 2 to 11.
  • Such compounds may be derived from a single plant source or a combination of plant sources.
  • Non-limiting examples of plant sources rich in proanthocyanidins suitable for use in the inventive nutritional composition include: grape, grape skin, grape seed, green tea, black tea, apple, pine bark, cinnamon, cocoa, bilberry, cranberry, black currant chokeberry.
  • Non-limiting examples of flavan-3-ols which are suitable for use in the inventive nutritional composition include catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, epicatechin-3-gal!ate, epigaiiocatechin and gallate.
  • Plants rich in the suitable flavan-3-ols include, but are not limited to, teas, red grapes, cocoa, green tea, apricot and apple.
  • Certain polyphenol compounds may improve learning and memory in a human subject by increasing brain blood flow, which is associated with an increase and sustained brain energy/nutrient delivery as well as formation of new neurons.
  • Polyphenols may also provide neuroprotective actions and may increase both brain synaptogenesis and antioxidant capability, thereby supporting optimal brain development in younger children.
  • Preferred sources of flavan-3-ols for the nutritional composition include at least one apple extract, at least one grape seed extract or a mixture thereof.
  • flavan-3-ols are broken down into monomers occurring in the range 4% to 20% and polymers in the range 80% to 96%.
  • flavan- 3-ols are broken down into monomers (about 46%) and polymers (about 54%) of the total favan-3-ols and total polyphenolic content.
  • Preferred degree of polymerization of polymeric flavan-3-ols is in the range of between about 2 and 11.
  • apple and grape seed extracts may contain catechin, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, polymeric proanthocyanidins,
  • stilbenoids i.e. resveratrol
  • flavonols i.e. quercetin, myricetin
  • Plant sources rich in flavan-3-ols include, but are not limited to apple, grape seed, grape, grape skin, tea (green or black), pine bark, cinnamon, cocoa, bilberry, cranberry, black currant, chokeberry.
  • an amount of flavan-3-ols including monomeric flavan-3-ols, polymeric flavan-3-ols or a combination thereof, ranging from between about 0.01 mg and about 450 mg per day may be administered.
  • the amount of flavan-3-ols administered to an infant or child may range from about 0.01 mg to about 170 mg per day, from about 50 to about 450 mg per day, or from about 100 mg to about 300 mg per day.
  • flavan-3-ols are present in the nutritional composition in an amount ranging from about 0.4 to about 3.8 mg/g nutritional composition (about 9 to about 90 mg/100 kcal). In another embodiment, flavan-3-ols are present in an amount ranging from about 0.8 to about 2.5 mg/g nutritional composition (about 20 to about 60 mg/100 kcal).
  • the nutritional composition of the present disclosure comprises flavanones.
  • suitable flavanones include butin, eriodictyol, hesperetin, hesperidin, homeriodictyol, isosakuranetin, naringenin, naringin, pinocembrin, poncirin, sakuranetin, sakuranin, steurbin.
  • Plant sources rich in flavanones include, but are not limited to orange, tangerine, grapefruit, lemon, lime.
  • the nutritional composition may be formulated to deliver between about 0.01 and about 150 mg flavanones per day.
  • the nutritional composition may also comprise flavonols.
  • Flavonols from plant or algae extracts may be used. Flavonols, such as ishrhametin, kaempferol, myricetin, quercetin, may be included in the nutritional composition in amounts sufficient to deliver between about 0.01 and 150 mg per day to a subject.
  • the phytonutrient component of the nutritional composition may also comprise phytonutrients that have been identified in human milk, including but not limited to naringenin, hesperetin, anthocyanins, quercetin, kaempferol, epicatechin, epigallocatechin, epicatechin-gallate, epigallocatechin-gallate or any combination thereof.
  • the nutritional composition comprises between about 50 and about 2000 nmol/L epicatechin, between about 40 and about 2000 nmol/L epicatechin gallate, between about 100 and about 4000 nmol/L
  • epigallocatechin gallate between about 50 and about 2000 nmol/L naringenin, between about 5 and about 500 nmol/L kaempferol, between about 40 and about 4000 nmol/L hesperetin, between about 25 and about 2000 nmol/L anthocyanins, between about 25 and about 500 nmol/L quercetin, or a mixture thereof.
  • the nutritional composition may comprise the metabolite(s) of a phytonutrient or of its parent compound, or it may comprise other classes of dietary phytonutrients, such as glucosinolate or sulforaphane.
  • the nutritional composition comprises carotenoids, such as lutein, zeaxanthin, astaxanthin, lycopene, beta-carotene, alpha- carotene, gamma-carotene, and/or beta-cryptoxanthin.
  • carotenoids such as lutein, zeaxanthin, astaxanthin, lycopene, beta-carotene, alpha- carotene, gamma-carotene, and/or beta-cryptoxanthin.
  • Plant sources rich in carotenoids include, but are not limited to kiwi, grapes, citrus, tomatoes,
  • watermelons such as kale, spinach, turnip greens, coflard greens, romaine lettuce, broccoli, zucchini, garden peas and Brussels sprouts, spinach, carrots.
  • dietary sources of carotenoids and/or polyphenols are absorbed by human subjects, accumulated and retained in breast milk, making them available to nursing infants.
  • addition of phytonutrients to infant formulas or children's products would bring the formulas closer in composition and functionality to human milk.
  • Flavonoids as a whole, may also be included in the nutritional composition, as flavonoids cannot be synthesized by humans. Moreover, flavonoids from plant or algae extracts may be useful in the monomer, dimer and/or polymer forms.
  • the nutritional composition comprises levels of the monomeric forms of flavonoids similar to those in human milk during the first three months of lactation. Although flavonoid aglycones (monomers) have been identified in human milk samples, the conjugated forms of flavonoids and/or their metabolites may also be useful in the nutritional composition. The flavonoids could be added in the following forms: free, glucuronides, methyl glucuronides, sulphates, and methyl sulphates.
  • the nutritional composition may also comprise isoflavonoids and/or isoflavones.
  • isoflavonoids include, but are not limited to, genistein (genistin), daidzein (daidzin), glycitein, biochanin A, formononetin, coumestrol, irilone, orobol, pseudobaptigenin, anagyroidisoflavone A and B, calycosin, glycitein, irigenin, 5-0- methylgenistein, pratensein, prunetin, psi-tectorigenin, retusin, tectorigenin, iridin, ononin, puerarin, tectoridin, derrubone, luteone, wighteone, alpinumisoflavone, barbigerone, di-O-methylalpinumisoflavone, and 4'-methyl-alpinumisoflavone.
  • Plant sources rich in isoflavonoids include, but are not limited to, soybeans, psoralea, kudzu, lupine, fava, chick pea, alfalfa, legumes and peanuts.
  • the nutritional composition may be formulated to deliver between about 0.01 and about 150 mg isoflavones and/or isoflavonoids per day.
  • the nutritional composition(s) of the present disclosure comprises an effective amount of choline.
  • Choline is a nutrient that is essential for normal function of cells. It is a precursor for membrane phospholipids, and it accelerates the synthesis and release of acetylcholine, a neurotransmitter involved in memory storage.
  • DHA docosahexaenoic acid
  • the nutritional composition(s) of the present disclosure includes an effective amount of choline, which is about 20 mg choline per 8 fl. oz. (236.6 mL) serving to about 100 mg per 8 fl. oz. (236.6 mL) serving.
  • the nutritional composition is nutritionally complete, containing suitable types and amounts of lipids,
  • the nutritional composition may optionally include any number of proteins, peptides, amino acids, fatty acids, probiotics and/or their metabolic byproducts, prebiotics, carbohydrates and any other nutrient or other compound that may provide many nutritional and physiological benefits to a subject.
  • the nutritional composition of the present disclosure may comprise flavors, flavor enhancers, sweeteners, pigments, vitamins, minerals, therapeutic ingredients, functional food ingredients, food ingredients, processing ingredients or combinations thereof.
  • the present disclosure further provides a method for providing nutritional support to a subject.
  • the method includes administering to the subject an effective amount of the nutritional composition of the present disclosure.
  • the nutritional composition may be expelled directly into a subject's intestinal tract. In some embodiments, the nutritional composition is expelled directly into the gut. In some embodiments, the composition may be formulated to be consumed or administered enterally under the supervision of a physician and may be intended for the specific dietary management of a disease or condition, such as celiac disease and/or food allergy, for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation.
  • a disease or condition such as celiac disease and/or food allergy
  • the nutritional composition of the present disclosure is not limited to compositions comprising nutrients specifically listed herein. Any nutrients may be delivered as part of the composition for the purpose of meeting nutritional needs and/or in order to optimize the nutritional status in a subject.
  • the nutritional composition may be delivered to an infant from birth until a time that matches full-term gestation.
  • the nutritional composition may be delivered to an infant until at least about three months corrected age. In another embodiment, the nutritional composition may be delivered to a subject as long as is necessary to correct nutritional deficiencies. In yet another embodiment, the nutritional composition may be delivered to an infant from birth until at least about six months corrected age. In yet another embodiment, the nutritional composition may be delivered to an infant from birth until at least about one year corrected age.
  • the nutritional composition of the present disclosure may be any nutritional composition of the present disclosure.
  • the nutritional composition of the present disclosure is a growing-up milk.
  • Growing-up milks are fortified milk-based beverages intended for children over 1 year of age (typically from 1-3 years of age, from 4-6 years of age or from 1-6 years of age). They are not medical foods and are not intended as a meal replacement or a supplement to address a particular nutritional deficiency. Instead, growing-up milks are designed with the intent to serve as a complement to a diverse diet to provide additional insurance that a child achieves continual, daily intake of all essential vitamins and minerals, macronutrients plus additional functional dietary components, such as non-essential nutrients that have purported health-promoting properties.
  • compositions according to the present disclosure can vary from market-to-market, depending on local regulations and dietary intake information of the population of interest.
  • nutritional compositions according to the disclosure consist of a milk protein source, such as whole or skim milk, plus added sugar and sweeteners to achieve desired sensory properties, and added vitamins and minerals.
  • the fat composition is typically derived from the milk raw materials.
  • Total protein can be targeted to match that of human milk, cow milk or a lower value.
  • Total carbohydrate is usually targeted to provide as little added sugar, such as sucrose or fructose, as possible to achieve an acceptable taste.
  • Vitamin A, calcium and Vitamin D are added at levels to match the nutrient contribution of regional cow milk. Otherwise, in some
  • vitamins and minerals can be added at levels that provide nutrients
  • nutrient values can vary between markets depending on the identified nutritional needs of the intended population, raw material
  • the nutritional composition is hypoallergenic. In other embodiments, the nutritional composition is kosher. In still further embodiments, the nutritional composition is a non-genetically modified product. In an embodiment, the nutritional formulation is sucrose-free. The nutritional composition may also be lactose-free. In other embodiments, the nutritional composition does not contain any medium-chain triglyceride oil. In some
  • no carrageenan is present in the composition.
  • the nutritional composition is free of all gums.
  • the disclosure is directed to a staged nutritional feeding regimen for a pediatric subject, such as an infant or child, which includes a plurality of different nutritional compositions according to the present disclosure.
  • Each nutritional composition comprises a hydrolyzed protein, at least one pre- gelatinized starch, and at least one pectin.
  • the nutritional compositions of the feeding regimen may also include a source of long chain polyunsaturated fatty acid, at least one prebiotic, an iron source, a source of ⁇ - glucan, vitamins or minerals, lutein, zeaxanthin, or any other ingredient described hereinabove.
  • the nutritional compositions described herein may be administered once per day or via several administrations throughout the course of a day.
  • This example illustrates an embodiment of a nutritional composition according to the present disclosure.
  • Vitamin K (meg) 20 Thiamin (meg) 300
  • This example illustrates another embodiment of a nutritional composition according to the present disclosure.
  • Vitamin B6 (meg) 100
  • Adenosine monophosphate (mg) 1 Adenosine monophosphate (mg) 1
  • Example 3 illustrates the effect of GOS and PDX on promoting stress resistance in Juvenile Fisher rats, and suggests that the composition of gut bacteria can modulate serotonergic circuits.
  • An important neural mechanism for protection against stress-induced anxiety and depression-like behavior is an increase in 5-HTi A receptor expression and function in the dorsal raphe nucleus (especially rostral DRN).
  • the prebiotic diet including GOS and PDX produces stress resistant behavioral effects in mice.
  • Example 3 tested whether dietary GOS and PDX initiated during the juvenile period, would produce changes in brain serotonin receptors.
  • GOS/PDX 7.0 g/kg each
  • Rats were then either exposed to either inescapable tail shock stress (IS; 100 1.5m A tail shocks; a stressor that reliably produces anxiety-like behavior) or remained undisturbed in their home cages.
  • GOS/PDX treatment increased mRNA expression of 5-HT1 a receptors in the dorsal raphe nucleus (DRN), a brain region involved in regulating anxiety (See FIG. 1). These results show that GOS/PDX promote stress resistance in Juvenile Fisher rats, and suggest. that gut bacteria can modulate serotonergic circuits regulating mood.
  • DNN dorsal raphe nucleus
  • Example 4 addressed a problem of constipation which affects up to 30% of children.
  • PDX prebiotics polydextrose
  • GOS galactooligosaccharides
  • a child was considered constipated if at least 2 of the 3 following symptoms were present for at least 2 uninterrupted weeks: presence of hard stools, difficulty or pain to defecate, and an interval of more than 72 hours without defecation.
  • the children who met these criteria were categorized by age at study entry (12-24 months or 25-48 months) and compared using the Fisher's exact test.
  • Example 5 focused on the effect of GOS/PDX and/or lactoferrin (LAC) on the beneficial microbiota, anxiety-like behavior and stress-induced inflammatory response in rats.
  • GOS/PDX and/or lactoferrin LAC
  • diets containing GOS/PDX or combination of GOS/PDX plus LAC significantly increased Lactobacillus spp. and elevated Bifidobacterium spp. in the fecal samples from rats.
  • the same diets also protected against the anxiety -like behavior produced by stress exposure.
  • Lactobacillus-specific M-RTLV agar (modified- rhamnose-2,3,5-triphenyltetrazolium chloride-LBS-vancomycin agar) was used as a selective medium to distinguish Lactobacillus spp. species.
  • Bifidobacterium spp. was detected using Bifidobacterium Selective Agar (BIFIDO, Anaerobe Systems) containing Reinforced Clostridial Agar as the basal medium and Polymixin,
  • FIGS. 1 and 2 show the significant increase of colony forming units (CFU) of Lactobacillus spp. and Lactobacillus rhamnosus ' m fecal samples collected from rats fed GOS/PDX or combination of GOS/PDX and LAC. (See. Figs. 2 and 3).
  • CFU colony forming units
  • Rats were then either exposed to inescapable tail shock stress (IS; 100 1.5mA tail shocks; a stressor that reliably produces anxiety-like behavior) or remained undisturbed in their home cages (HCC). Subsequently, rats were tested for anxiety/depressive-like behavior using shock-elicited freezing and shuttle-box escape tests 24 h later. Plasma samples were immediately collected from a subset of mice after IS termination. Inflammatory molecule Hsp72 is normally increased by stressor exposure. As seen in FIG. 4, rats fed GOS/PDX showed a tendency to reduce stress- induced Hsp72 responses.

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Abstract

Provided is a composition and method for modulating serotonergic receptor expression, reducing anxiety-like behavior and stress-induced inflammatory response in a pediatric subject, the method including administering to the pediatric subject a nutritional composition including lactoferrin from a non-human source and a prebiotic composition including polydextrose and/or galactooligosaccharides. Further provided is a method for reducing constipation in a pediatric subject by providing a nutritional composition including lactoferrin from a non-human source and a prebiotic composition including polydextrose and/or galactooligosaccharides.

Description

DESCRIPTION
NUTRITIONAL COMPOSITIONS CONTAINING A PREBIOTIC AND LACTOFERRIN AND USES THEREOF
TECHNICAL FIELD
[0001] The present disclosure relates to methods of modulating serotonergic receptor expression in a pediatric subject via administration of the nutritional composition disclosed herein. Some embodiments of the disclosure are directed to enhancing or promoting an increase in the concentration of beneficial bacteria in a pediatric subject, such as the Lactobacillus and Bifidobacterium species, by administering the nutritional composition disclosed herein. Further provided are methods for reducing constipation in a pediatric subject by providing the nutritional composition disclosed herein.
[0002] In some embodiments, the nutritional composition comprises a prebiotic blend which includes polydextrose and galacto-oligosaccharides, and lactoferrin, wherein the foregoing components may exhibit additive and/or synergistic beneficial effects.
BACKGROUND ART
[0003] Lactoferrin, an iron-binding glycoprotein, is one of the major multifunctional agents present in human milk. It has the capacity to bind two molecules of iron in a reversible fashion and can facilitate the uptake of iron within the intestines. Further, lactoferrin has been shown to be both bacteriostatic and bactericidal, and it aids in preventing intestinal infections in humans, especially in pediatric subjects. Additionally, human lactoferrin appears to have a positive effect on the symptoms of diarrheal diseases.
[0004] Moreover, the infant gut microflora is rapidly established in the first few weeks following birth, and it has a great impact on an infant's immune system. The nature of this intestinal colonization is initially determined by early exposure to environmental sources of microbes and by the general state of health of the infant. Whether the infant is breast-fed or formula-fed also has a strong influence on the intestinal bacterial population.
[0005] Both the interaction among dietary components and among the microflora of the intestinal ecosystem are very complex. Consequently, then, the matrix of an infant formula or other pediatric nutritional composition may influence the effectiveness of prebiotics when such ingredients are provided as supplements in the diet of a formula-fed infant. Further, the type and concentration of lipids and proteins used in a formula matrix may also modulate the intestinal microbiota.
[0006] Recently, it has been found that combining prebiotics, especially polydextrose (PDX) and galacto-oligosaccharides (GOS), and lactoferrin, can lead to certain unique benefits. Accordingly, it would be beneficial to provide a nutritional composition for pediatric subjects that contains such a combination.
DISCLOSURE OF THE INVENTION
[0007] Briefly, the present disclosure is directed, in an embodiment, to a method for modulating serotonergic receptor expression, modulating gut microbiota, for example increasing the concentration of beneficial bacteria, and/or modulating psychological stress, in a pediatric subject, the method comprising administering to the pediatric subject a nutritional composition comprising prebiotics, especially polydextrose (PDX) and galacto-oligosaccharides (GOS), and lactoferrin from a non-human source. In some embodiments, the disclosure is directed to a method of reducing the incidence of constipation in a target subject via administration of the nutritional composition disclosed herein.
[0008] In certain embodiments, the nutritional composition further comprises an enriched lipid fraction derived from milk. In some embodiments the nutritional composition may include an enriched lipid fraction derived from milk that includes milk fat globules. The addition of the milk fat globules provides an enriched fat and lipid source to the infant that may be more fully digested by a pediatric subject.
[0009] In certain embodiments, the enriched lipid fraction and/or the milk fat globules may include saturated fatty acids, trans-fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, cholesterol, odd-branched chain fatty acids "OBCFAs", branched chain fatty acids "BCFAs", conjugated linoleic acid "CLA", phospholipids, or milk fat globule membrane protein, and mixtures thereof.
[0010] It is to be understood that both the foregoing general description and the following detailed description present embodiments of the disclosure and are intended to provide an overview or framework for understanding the nature and character of the disclosure as it is claimed. The description serves to explain the principles and operations of the claimed subject matter. Other and further features and advantages of the present disclosure will be readily apparent to those skilled in the art upon a reading of the following disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Fig. 1 illustrates the relative 5HTia mRNA levels in the brain tissues, specifically the rostral, mid, and caudal regions, of mice fed a control diet versus a diet supplemented with GOS and PDX.
[0012] Fig. 2 illustrates the amount of Lactobacillus species in the feces of mice fed a control diet, mice fed a diet supplemented with lactoferrin, mice fed a diet supplemented with GOS and PDX, and mice fed a diet supplemented with GOS, PDX, and lactoferrin.
[0013] Fig. 3 illustrates the amount of Lactobacillus rhamnosus in the feces of mice fed a control diet, mice fed a diet supplemented with lactoferrin, mice fed a diet supplemented with GOS and PDX, and mice fed a diet supplemented with GOS, PDX, and lactoferrin.
[0014] Fig. 4 illustrates the amount of plasma Hsp70 present after stress and in a non-stressful situation in mice fed mice fed a control diet, mice fed a diet supplemented with lactoferrin, mice fed a diet supplemented with GOS and PDX, and mice fed a diet supplemented with GOS, PDX, and lactoferrin.
[0015] Fig. 5 illustrates the percentage of freezing considered shock-elicited freezing (anxiety test) in mice fed a control diet, mice fed a diet supplemented with lactoferrin, mice fed a diet supplemented with GOD and PDX, and mice fed a diet supplemented with GOS, PDX, and lactoferrin.
[0016] Fig. 6 illustrates the percentage of escape latency in mice fed a control diet, mice fed a diet supplemented with lactoferrin, mice fed a diet supplemented with GOD and PDX, and mice fed a diet supplemented with GOS, PDX, and lactoferrin.
BEST MODE FOR CARRYING OUT THE INVENTION
[0017] Reference now will be made in detail to the embodiments of the present disclosure, one or more examples of which are set forth hereinbelow. Each example is provided by way of explanation of the nutritional composition of the present disclosure and is not a limitation. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the teachings of the present disclosure without departing from the scope of the disclosure. For instance, features illustrated or described as part of one embodiment, can be used with another embodiment to yield a still further embodiment.
[0018] Thus, it is intended that the present disclosure covers such
modifications and variations as come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present disclosure are disclosed in or are obvious from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only and is not intended as limiting the broader aspects of the present disclosure.
[0019] The present disclosure relates generally to nutritional compositions that are suitable for administration to a pediatric subject. Additionally, the disclosure relates to methods of modulating serotonergic receptor expression, promoting the growth and concentration of beneficial bacteria in the gastrointestinal tract, reducing constipation, and/or promoting stress resistance in a pediatric subject via
administration of the nutritional composition(s) disclosed herein.
[0020] "Nutritional composition" means a substance or formulation that satisfies at least a portion of a subject's nutrient requirements. The terms
"nutritional(s)", "nutritional formula(s)", "enteral nutritional(s)", and "nutritional supplement(s)" are used as non-limiting examples of nutritional composition(s) throughout the present disclosure. Moreover, "nutritional composition(s)" may refer to liquids, powders, gels, pastes, solids, concentrates, suspensions, or ready-to-use forms of enteral formulas, oral formulas, formulas for infants, formulas for pediatric subjects, formulas for children, growing-up milks and/or formulas for adults.
[0021] The term "enteral" means deliverable through or within the
gastrointestinal, or digestive, tract. "Enteral administration" includes oral feeding, intragastric feeding, transpyloric administration, or any other administration into the digestive tract. "Administration" is broader than "enteral administration" and includes parenteral administration or any other route of administration by which a substance is taken into a subject's body.
[0022] "Pediatric subject" means a human no greater than 13 years of age. In some embodiments, a pediatric subject refers to a human subject that is between birth and 8 years old. In other embodiments, a pediatric subject refers to a human subject between 1 and 6 years of age. In still further embodiments, a pediatric subject refers to a human subject between 6 and 12 years of age. The term
"pediatric subject" may refer to infants (preterm or full term) and/or children, as described below.
[0023] "Infant" means a human subject ranging in age from birth to not more than one year and includes infants from 0 to 12 months corrected age. The phrase "corrected age" means an infant's chronological age minus the amount of time that the infant was born premature. Therefore, the corrected age is the age of the infant if it had been carried to full term. The term infant includes low birth weight infants, very low birth weight infants, extremely low birth weight infants and preterm infants. "Preterm" means an infant born before the end of the 37th week of gestation. "Late preterm" means an infant form between the 34th week and the 36th week of gestation. "Full term" means an infant born after the end of the 37th week of gestation. "Low birth weight infant" means an infant born weighing less than 2500 grams (approximately 5 lbs, 8 ounces). "Very low birth weight infant" means an infant born weighing less than 1500 grams (approximately 3 lbs, 4 ounces).
"Extremely low birth weight infant" means an infant born weighing less than 1000 grams (approximately 2 lbs, 3 ounces).
[0024] "Child" means a subject ranging in age from 12 months to 13 years. In some embodiments, a child is a subject between the ages of 1 and 12 years old. In other embodiments, the terms "children" or "child" refer to subjects that are between one and about six years old, or between about seven and about 12 years old. In other embodiments, the terms "children" or "child" refer to any range of ages between 12 months and about 13 years.
[0025] "Children's nutritional product" refers to a composition that satisfies at least a portion of the nutrient requirements of a child. A growing-up milk is an example of a children's nutritional product.
[0026] The term "degree of hydrolysis" refers to the extent to which peptide bonds are broken by a hydrolysis method. The degree of protein hydrolysis for purposes of characterizing the hydrolyzed protein component of the nutritional composition is easily determined by one of ordinary skill in the formulation arts by quantifying the amino nitrogen to total nitrogen ratio (AN/TN) of the protein component of the selected formulation. The amino nitrogen component is quantified by USP titration methods for determining amino nitrogen content, while the total nitrogen component is determined by the Tecator Kjeldahl method, all of which are well known methods to one of ordinary skill in the analytical chemistry art.
[0027] The term "partially hydrolyzed" means having a degree of hydrolysis which is greater than 0% but less than about 50%.
[0028] The term "extensively hydrolyzed" means having a degree of hydrolysis which is greater than or equal to about 50%.
[0029] The term "protein-free" means containing no measurable amount of protein, as measured by standard protein detection methods such as sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or size exclusion chromatography. In some embodiments, the nutritional composition is substantially free of protein, wherein "substantially free" is defined hereinbelow.
[0030] "Infant formula" means a composition that satisfies at least a portion of the nutrient requirements of an infant. In the United States, the content of an infant formula is dictated by the federal regulations set forth at 21 C.F.R. Sections 100, 106, and 107. These regulations define macronutrient, vitamin, mineral, and other ingredient levels in an effort to simulate the nutritional and other properties of human breast milk.
[0031] The term "growing-up milk" refers to a broad category of nutritional compositions intended to be used as a part of a diverse diet in order to support the normal growth and development of a child between the ages of about 1 and about 6 years of age.
[0032] "Milk-based" means comprising at least one component that has been drawn or extracted from the mammary gland of a mammal. In some embodiments, a milk-based nutritional composition comprises components of milk that are derived from domesticated ungulates, ruminants or other mammals or any combination thereof. Moreover, in some embodiments, milk-based means comprising bovine casein, whey, lactose, or any combination thereof. Further, "milk-based nutritional composition" may refer to any composition comprising any milk-derived or milk- based product known in the art.
[0033] "Milk" means a component that has been drawn or extracted from the mammary gland of a mammal. In some embodiments, the nutritional composition comprises components of milk that are derived from domesticated ungulates, ruminants or other mammals or any combination thereof. [0034] "Fractionation procedure" includes any process in which a certain quantity of a mixture is divided up into a number of smaller quantities known as fractions. The fractions may be different in composition from both the mixture and other fractions. Examples of fractionation procedures include but are not limited to, melt fractionation, solvent fractionation, supercritical fluid fractionation and/or combinations thereof.
[0035] "Fat globule" refers to a small mass of fat surrounded by phospholipids and other membrane and/or serum proteins, where the fat itself can be a
combination of any vegetable or animal fat.
[0036] "Nutritionally complete" means a composition that may be used as the sole source of nutrition, which would supply essentially all of the required daily amounts of vitamins, minerals, and/or trace elements in combination with proteins, carbohydrates, and lipids. Indeed, "nutritionally complete" describes a nutritional composition that provides adequate amounts of carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals and energy required to support normal growth and development of a subject.
[0037] Therefore, a nutritional composition that is "nutritionally complete" for a preterm infant will, by definition, provide qualitatively and quantitatively adequate amounts of carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for growth of the preterm infant.
[0038] A nutritional composition that is "nutritionally complete" for a full term infant will, by definition, provide qualitatively and quantitatively adequate amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for growth of the full term infant.
[0039] A nutritional composition that is "nutritionally complete" for a child will, by definition, provide qualitatively and quantitatively adequate amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids,
conditionally essential amino acids, vitamins, minerals, and energy required for growth of a child. [0040] As applied to nutrients, the term "essential" refers to any nutrient that cannot be synthesized by the body in amounts sufficient for normal growth and to maintain health and that, therefore, must be supplied by the diet. The term
"conditionally essential" as applied to nutrients means that the nutrient must be supplied by the diet under conditions when adequate amounts of the precursor compound is unavailable to the body for endogenous synthesis to occur.
[0041] "Probiotic" means a microorganism with low or no pathogenicity that exerts a beneficial effect on the health of the host.
[0042] The term "inactivated probiotic" means a probiotic wherein the metabolic activity or reproductive ability of the referenced probiotic has been reduced or destroyed. The "inactivated probiotic" does, however, still retain, at the cellular level, its cell structure or other structure associated with the cell, for example exopolysaccharide and at least a portion its biological glycol-protein and DNA/RNA structure. As used herein, the term "inactivated" is synonymous with "non-viable".
[0043] "Prebiotic" means a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the digestive tract that can improve the health of the host.
[0044] "Branched Chain Fatty Acid" ("BCFA") means a fatty acid containing a carbon constituent branched off the carbon chain. Typically the branch is an alkyl branch, especially a methyl group, but ethyl and propyl branches are also known. The addition of the methyl branch lowers the melting point compared with the equivalent straight chain fatty acid. This includes branched chain fatty acids with an even number of carbon atoms in the carbon chain. Examples of these can be isomers of tetradecanoic acid, hexadecanoic acid.
[0045] "Odd- and Branched-Chain Fatty Acid" ("OBCFA") is a subset of BCFA that has an odd number of carbon atoms and have one or more alkyl branches on the carbon chain. The main odd- and branched-chain fatty acids found in bovine milk include, but are not limited to, the isomers of tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, and heptadecanoic acid. For the purposes of this disclosure, the term "BCFA" includes both branched-chain fatty acids and odd-and-branched chain fatty acids. [0046] "Trans-fatty acid" means an unsaturated fat with a trans-isomer. Trans- fats may be monounsaturated or polyunsaturated. Trans refers to the arrangement of the two hydrogen atoms bonded to the carbon atoms involved in a double bond. In the trans arrangement, the hydrogens are on opposite sides of the bond. Thus a trans-fatty acid is a lipid molecule that contains one or more double bonds in trans geometric configuration.
[0047] "Phospholipids" means an organic molecule that contains a diglyceride, a phosphate group and a simple organic molecule. Examples of phospholipids include but are not limited to, phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phsphatidylinositol, phosphatidylinositol phosphate, phosphatidylinositol biphosphate and phosphatidylinositol triphosphate, ceramide phosphorylcholine, ceramide phosphorylethanolamine and ceramide phosphorylglycerol. This definition further includes sphigolipids, glycolipids, and gangliosides.
[0048] "Phytonutrient" means a chemical compound that occurs naturally in plants. Phytonutrients may be included in any plant-derived substance or extract. The term "phytonutrient(s)" encompasses several broad categories of compounds produced by plants, such as, for example, polyphenols compounds, anthocyanins, proanthocyanidins, and flavan-3-ols (i.e. catechins, epicatechins), and may be derived from, for example, fruit, seed or tea extracts. Further, the term phytonutrient includes all carotenoids, phytosterols, thiols, and other plant-derived compounds. Moreover, as a skilled artisan will understand, plant extracts may include
phytonutrients, such as polyphenols, in addition to protein, fiber or other plant- derived components. Thus, for example, apple or grape seed extract(s) may include beneficial phytonutrient components, such as polyphenols, in addition to other plant- derived substances.
[0049] "β-glucan" means all β-glucan, including specific types of β-glucan, such as β-1 ,3-glucan or β-1 ,3;1 ,6-glucan. Moreover, β-1 ,3;1 ,6-glucan is a type of β-1 ,3- glucan. Therefore, the term "β-1 ,3-glucan" includes -1 ,3;1 ,6-glucan.
[0050] "Pectin" means any naturally-occurring oligosaccharide or
polysaccharide that comprises galacturonic acid that may be found in the cell wall of a plant. Different varieties and grades of pectin having varied physical and chemical properties are known in the art. Indeed, the structure of pectin can vary significantly between plants, between tissues, and even within a single cell wall. Generally, pectin is made up of negatively charged acidic sugars (galacturonic acid), and some of the acidic groups are in the form of a methyl ester group. The degree of esterification of pectin is a measure of the percentage of the carboxyl groups attached to the galactopyranosyluronic acid units that are esterified with methanol.
[0051] Pectin having a degree of esterification of less than 50% (i.e., less than 50% of the carboxyl groups are methylated to form methyl ester groups) are classified as low-ester, low methoxyl, or low methylated ("LM") pectins, while those having a degree of esterification of 50% or greater (i.e., more than 50% of the carboxyl groups are methylated) are classified as high-ester, high methoxyl or high methylated ("HM") pectins. Very low ("VL") pectins, a subset of low methylated pectins, have a degree of esterification that is less than approximately 15%.
[0052] As used herein, "lactoferrin from a non-human source" means lactoferrin which is produced by or obtained from a source other than human breast milk. For example, lactoferrin for use in the present disclosure includes human lactoferrin produced by a genetically modified organism as well as non-human lactoferrin. The term "organism", as used herein, refers to any contiguous living system, such as animal, plant, fungus or micro-organism.
[0053] As used herein, "non-human lactoferrin" means lactoferrin that has an amino acid sequence that is different than the amino acid sequence of human lactoferrin.
[0054] "Pathogen" means an organism that causes a disease state or pathological syndrome. Examples of pathogens may include bacteria, viruses, parasites, fungi, microbes or combination(s) thereof.
[0055] "Modulate" or "modulating" means exerting a modifying, controlling and/or regulating influence. In some embodiments, the term "modulating" means exhibiting an increasing or stimulatory effect on the level/amount of a particular component. In other embodiments, "modulating" means exhibiting a decreasing or inhibitory effect on the level/amount of a particular component.
[0056] All percentages, parts and ratios as used herein are by weight of the total formulation, unless otherwise specified. [0057] All amounts specified as administered "per day" may be delivered in one unit dose, in a single serving or in two or more doses or servings administered over the course of a 24 hour period.
[0058] The nutritional composition of the present disclosure may be
substantially free of any optional or selected ingredients described herein, provided that the remaining nutritional composition still contains all of the required ingredients or features described herein. In this context, and unless otherwise specified, the term "substantially free" means that the selected composition may contain less than a functional amount of the optional ingredient, typically less than 0.1% by weight, and also, including zero percent by weight of such optional or selected ingredient.
[0059] All references to singular characteristics or limitations of the present disclosure shall include the corresponding plural characteristic or limitation, and vice versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made.
[0060] All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.
[0061] The methods and compositions of the present disclosure, including components thereof, can comprise, consist of, or consist essentially of the essential elements and limitations of the embodiments described herein, as well as any additional or optional ingredients, components or limitations described herein or otherwise useful in nutritional compositions.
[0062] As used herein, the term "about" should be construed to refer to both of the numbers specified as the endpoint(s) of any range. Any reference to a range should be considered as providing support for any subset within that range.
[0063] The present disclosure is directed to nutritional compositions comprising a prebiotic component including GOS and PDX, and lactoferrin from a non-human source, to uses thereof, and to methods comprising administration of those nutritional compositions. The nutritional compositions of the present disclosure support overall health and development in a pediatric human subject, such as an infant (preterm and/or term) or a child.
[0064] The intestinal microflora plays a crucial role in the postnatal
development of most gastrointestinal functions. Accordingly, the nutritional composition of the present disclosure supports gastrointestinal health and
development. Moreover, a healthy intestinal flora supports an adequate gut-brain communication, affecting brain function and, consequently, psychological stress responses, resulting in modified behavior. Administration of the compositions of the present disclosure together can modulate the intestinal flora of a pediatric subject by increasing beneficial bacteria and/or reducing adhesion of pathogens in the gastrointestinal system. Thus, in some embodiments, the present disclosure is directed to a method for modulating psychological stress responses. And in certain embodiments, the disclosed combination has additive and/or synergistic beneficial effects that support gastrointestinal development. In certain embodiments, the disclosure is related to a method for supporting cognitive development and brain function, and gastrointestinal health and/or development in a pediatric subject. The method comprises administering prebiotics, especially PDX and GOS, and lactoferrin from a non-human source to a pediatric subject.
[0065] Moreover, during early life, infants and children experience many stressful situations due to, for example, a changing environment or being hungry or tired. Consequently, stress hormones are released, which may negatively affect brain development and/or cause other long-term detrimental effects in a pediatric subject. Yet the nutritional composition of the present disclosure can reduce or suppress psychological stress and/or modulate plasma stress hormone levels, thereby further promoting healthy brain and cognitive growth and development in a pediatric subject.
[0066] Indeed, an important neural mechanism for protection against stress- induced anxiety and depression-like behavior is an increase in 5-HT1a receptor expression and function. Physiologically, 5-HT- is a subtype of the 5-HT family of receptors that binds the endogenous neurotransmitter serotonin. 5-HTia is a G protein-coupled receptor that mediates inhibitory neurotransmission. As such, modulation of the function of the 5-HTia may provide additional cognitive and behavioral benefits in a target individual.
[0067] Thus in some embodiments, the present disclosure includes a method for modulating 5-HTia receptor expression in a pediatric subject comprising administering to the subject an effective amount of the disclosed nutritional composition. Still, in some embodiments, the present disclosure includes a method for promoting stress resistance in a pediatric subject comprising administering to the subject an effective amount of the disclosed nutritional composition. In other embodiments, the disclosure is directed to a method for modulating serotonergic circuits and/or regulating mood in a pediatric subject by administering an effective amount of the composition to the subject.
[0068] In infants, immaturity of the gut often causes gastrointestinal symptoms that affect their nutritional status with consequences for their overall health. Thus, in some embodiments, the present disclosure is directed to a method of promoting the development of or increasing the concentration of beneficial bacteria in the Gl tract, which may provide cognitive benefits. For example, an increase in the amount of Lactobacillus species and/or Bifidobacterium species, may promote stress resistance in a target subject. Moreover, the increase in the beneficial bacterium is observed without introducing these additional bacteria to the gastrointestinal tract via nutritional supplementation.
[0069] The unique combination of nutrients in the disclosed nutritional composition is believed to be capable of providing novel and unexpected brain- related benefits for infants and children. Moreover, the benefit of this nutritional composition is believed to be obtained during infancy, and also by including it as part of a diverse diet as the child and its brain continues to grow and develop.
[0070] The combination of nutrients in the nutritional composition combine in synergistic ways to provide the foregoing benefits. For instance, providing lactoferrin with a prebiotic comprising PDX and GOS can synergistically increase specific beneficial species of bacteria in the gastrointestinal tract including
Lactobacillus species, including Lactobacillus rhamnosus, and Bifidobacterium species. An increase in the concentration of these beneficial bacteria in the gastrointestinal tract may provide further health benefits, such as reduction in anxiety-like behavior and stress-induced inflammatory response. Further, an increase in beneficial bacteria may reduce constipation in a target subject.
[0071] The nutritional composition may also contain one or more prebiotics (also referred to as a prebiotic component) in certain embodiments. Prebiotics exert health benefits, which may include, but are not limited to, selective stimulation of the growth and/or activity of one or a limited number of beneficial gut bacteria, stimulation of the growth and/or activity of ingested probiotic microorganisms, selective reduction in gut pathogens, and favorable influence on gut short chain fatty acid profile. Such prebiotics may be naturally-occurring, synthetic, or developed through the genetic manipulation of organisms and/or plants, whether such new source is now known or developed later. Prebiotics useful in the present disclosure may include oligosaccharides, polysaccharides, and other prebiotics that contain fructose, xylose, soya, galactose, glucose and mannose.
[0072] More specifically, prebiotics useful in the present disclosure may include polydextrose, polydextrose powder, lactulose, lactosucrose, raffinose, gluco- oligosaccharide, inulin, fructo-oligosaccharide, isomalto-ofigosaccharide, soybean oligosaccharides, lactosucrose, xylo-oligosaccharide, chito-oligosaccharide, manno- oligosaccharide, aribino-oligosaccharide, siallyl-oligosaccharide, fuco-oligosaccharide, galacto-oligosaccharides and gentio-oligosaccharides.
[0073] In an embodiment, the total amount of prebiotics present in the nutritional composition may be from about 1.0 g/L to about 10.0 g/L of the composition. More preferably, the total amount of prebiotics present in the nutritional composition may be from about 2.0 g/L and about 8.0 g/L of the composition. In some embodiments, the total amount of prebiotics present in the nutritional composition may be from about 0.01 g/100 kcal to about 1.5 g/100 kcal. In certain embodiments, the total amount of prebiotics present in the nutritional composition may be from about 0.15 g/100 kcal to about 1.5 g/100 kcal. Moreover, the nutritional composition may comprise a prebiotic component comprising PDX. In some embodiments, the prebiotic component comprises at least 20% w/w PDX, GOS or a mixture thereof.
[0074] The amount of PDX in the nutritional composition may, in an
embodiment, be within the range of from about 0.015 g/100 kcal to about 1.5 g/100 kcal. In another embodiment, the amount of polydextrose is within the range of from about 0.2 g/100 kcal to about 0.6 g/100 kcal. In some embodiments, PDX may be included in the nutritional composition in an amount sufficient to provide between about 1.0 g/L and 10.0 g/L. In another embodiment, the nutritional composition contains an amount of PDX that is between about 2.0 g/L and 8.0 g/L. And in still other embodiments, the amount of PDX in the nutritional composition may be from about 0.05 g/100 kcal to about 1.5 g/100 kcal. [0075] The prebiotic component also comprises GOS. The amount of GOS in the nutritional composition may, in an embodiment, be from about 0.015 g/100 kcal to about 1.0 g/100 kcal. In another embodiment, the amount of GOS in the nutritional composition may be from about 0.2 g/100 kcal to about 0.5 g/100 kcal.
[0076] In a particular embodiment of the present invention, PDX is
administered in combination with GOS.
[0077] In a particular embodiment, GOS and PDX are supplemented into the nutritional composition in a total amount of at least about 0.015 g/100 kcal or about 0.015 g/100 kcal to about 1.5 g/100 kcal. In some embodiments, the nutritional composition may comprise GOS and PDX in a total amount of from about 0.1 to about 1.0 g/100 kcal.
[0078] As noted, lactoferrin is also included in the nutritional composition of the present disclosure. Lactoferrins are single chain polypeptides of about 80 kD containing 1 - 4 glycans, depending on the species. The 3-D structures of lactoferrin of different species are very similar, but not identical. Each lactoferrin comprises two homologous lobes, called the N- and C-lobes, referring to the N-terminal and C- terminal part of the molecule, respectively. Each lobe further consists of two sub- lobes or domains, which form a cleft where the ferric ion (Fe3+) is tightly bound in synergistic cooperation with a (bi)carbonate anion. These domains are called N1 ; N2, C1 and C2, respectively. The N-terminus of lactoferrin has strong cationic peptide regions that are responsible for a number of important binding characteristics.
Lactoferrin has a very high isoelectric point (~pl 9) and its cationic nature plays a major role in its ability to defend against bacterial, viral, and fungal pathogens. There are several clusters of cationic amino acids residues within the N-terminal region of lactoferrin mediating the biological activities of lactoferrin against a wide range of microorganisms. For instance, the N-terminal residues 1-47 of human lactoferrin (1- 48 of bovine lactoferrin) are critical to the iron-independent biological activities of lactoferrin. In human lactoferrin, residues 2 to 5 (RRRR) and 28 to 31 (RKVR) are arginine-rich cationic domains in the N-terminus especially critical to the antimicrobial activities of lactoferrin. A similar region in the N-terminus is found in bovine lactoferrin (residues 17 to 42; FKCRRWQWRMKKLGAPSITCVRRAFA).
[0079] Lactoferrins from different host species may vary in their amino acid sequences though commonly possess a relatively high isoelectric point with positively charged amino acids at the end terminal region of the internal lobe. Suitable non- human lactoferrins for use in the present disclosure include, but are not limited to, those having at least 48% homology with the amino acid sequence of human lactoferrin. For instance, bovine lactoferrin ("bLF") has an amino acid composition which has about 70% sequence homology to that of human lactoferrin. In some embodiments, the non-human lactoferrin has at least 55% homology with human lactoferrin and in some embodiments, at least 65% homology. Non-human lactoferrins acceptable for use in the present disclosure include, without limitation, bLF, porcine lactoferrin, equine lactoferrin, buffalo lactoferrin, goat lactoferrin, murine lactoferrin and camel lactoferrin.
[0080] In one embodiment, lactoferrin is present in the nutritional composition in an amount of at least about 15 mg/100 kCal. In certain embodiments, the nutritional composition may include between about 15 and about 300 mg lactoferrin per 100 kCal. In another embodiment, where the nutritional composition is an infant formula, the nutritional composition may comprise lactoferrin in an amount of from about 60 mg to about 150 mg lactoferrin per 100 kCal; in yet another embodiment, the nutritional composition may comprise about 60 mg to about 100 mg lactoferrin per 100 kCal.
[0081] In some embodiments, the nutritional composition can include lactoferrin in the quantities of from about 0.5 mg to about 1.5 mg per milliliter of formula. In nutritional compositions replacing human milk, lactoferrin may be present in quantities of from about 0.6 mg to about 1.3 mg per milliliter of formula. In certain embodiments, the nutritional composition may comprise between about 0.1 and about 2 grams lactoferrin per liter. In some embodiments, the nutritional
composition includes between about 0.6 and about 1.5 grams lactoferrin per liter of formula.
[0082] The bLF that is used in certain embodiments may be any bLF isolated from whole milk and/or having a low somatic cell count, wherein "low somatic cell count" refers to a somatic cell count less than 200,000 cells/mL. By way of example, suitable bLF is available from Tatua Co-operative Dairy Co. Ltd., in Morrinsville, New Zealand, from FrieslandCampina Domo in Amersfoort, Netherlands or from Fonterra Co-Operative Group Limited in Auckland, New Zealand. [0083] Lactoferrin for use in the present disclosure may be, for example, isolated from the milk of a non-human animal or produced by a genetically modified organism. For example, in U.S. Patent No. 4,791,193, incorporated by reference herein in its entirety, Okonogi et al. discloses a process for producing bovine lactoferrin in high purity. Generally, the process as disclosed includes three steps. Raw milk material is first contacted with a weakly acidic cationic exchanger to absorb lactoferrin followed by the second step where washing takes place to remove nonabsorbed substances. A desorbing step follows where lactoferrin is removed to produce purified bovine lactoferrin. Other methods may include steps as described in U.S. Patent Nos. 7,368,141 , 5,849,885, 5,919,913 and 5,861 ,491 , the disclosures of which are all incorporated by reference in their entirety.
[0084] In certain embodiments, lactoferrin utilized in the present disclosure may be provided by an expanded bed absorption ("EBA") process for isolating proteins from milk sources. EBA, also sometimes called stabilized fluid bed adsorption, is a process for isolating a milk protein, such as lactoferrin, from a milk source comprises establishing an expanded bed adsorption column comprising a particulate matrix, applying a milk source to the matrix, and eluting the lactoferrin from the matrix with an elution buffer comprising about 0.3 to about 2.0 M sodium chloride. Any mammalian milk source may be used in the present processes, although in particular embodiments, the milk source is a bovine milk source. The milk source comprises, in some embodiments, whole milk, reduced fat milk, skim milk, whey, casein, or mixtures thereof.
[0085] In particular embodiments, the target protein is lactoferrin, though other milk proteins, such as lactoperoxidases or lactalbumins, also may be isolated. In some embodiments, the process comprises the steps of establishing an expanded bed adsorption column comprising a particulate matrix, applying a milk source to the matrix, and eluting the lactoferrin from the matrix with about 0.3 to about 2.0M sodium chloride. In other embodiments, the lactoferrin is eluted with about 0.5 to about 1.0 M sodium chloride, while in further embodiments, the lactoferrin is eluted with about 0.7 to about 0.9 M sodium chloride.
[0086] The expanded bed adsorption column can be any known in the art, such as those described in U.S. Patent Nos. 7,812,138, 6,620,326, and 6,977,046, the disclosures of which are hereby incorporated by reference herein. In some embodiments, a milk source is applied to the column in an expanded mode, and the elution is performed in either expanded or packed mode. In particular embodiments, the elution is performed in an expanded mode. For example, the expansion ratio in the expanded mode may be about 1 to about 3, or about 1.3 to about 1.7. EBA technology is further described in international published application nos. WO
92/00799, WO 02/18237, WO 97/ 7132, which are hereby incorporated by reference in their entireties.
[0087] The isoelectric point of lactoferrin is approximately 8.9. Prior EBA methods of isolating lactoferrin use 200 mM sodium hydroxide as an elution buffer. Thus, the pH of the system rises to over 12, and the structure and bioactivity of lactoferrin may be comprised, by irreversible structural changes. It has now been discovered that a sodium chloride solution can be used as an elution buffer in the isolation of lactoferrin from the EBA matrix. In certain embodiments, the sodium chloride has a concentration of about 0.3 M to about 2.0 M. In other embodiments, the lactoferrin elution buffer has a sodium chloride concentration of about 0.3 M to about 1.5 M, or about 0.5 m to about 1.0 M.
[0088] The nutritional composition of the disclosure also contains a source of LCPUFAs; especially a source of LCPUFAs that comprises docosahexaenoic acid. Other suitable LCPUFAs include, but are not limited to, a-linoleic acid, γ-linoleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA) and arachidonic acid (ARA).
[0089] In an embodiment, especially if the nutritional composition is an infant formula, the nutritional composition is supplemented with both DHA and ARA. In this embodiment, the weight ratio of ARA:DHA may be between about 1 :3 and about 9:1. In a particular embodiment, the ratio of ARA:DHA is from about 1 :2 to about 4:1.
[0090] The amount of long chain polyunsaturated fatty acid in the nutritional composition is advantageously at least about 5 mg/100 kcal, and may vary from about 5 mg/100 kcal to about 100 mg/100 kcal, more preferably from about 10 mg/100 kcal to about 50 mg/100 kcal.
[0091] The nutritional composition may be supplemented with oils containing DHA and/or ARA using standard techniques known in the art. For example, DHA and ARA may be added to the composition by replacing an equivalent amount of an oil, such as high oleic sunflower oil, normally present in the composition. As another example, the oils containing DHA and ARA may be added to the composition by replacing an equivalent amount of the rest of the overall fat blend normally present in the composition without DHA and ARA.
[0092] If utilized, the source of DHA and/or ARA may be any source known in the art such as marine oil, fish oil, single cell oil, egg yolk lipid, and brain lipid. In some embodiments, the DHA and ARA are sourced from single cell Martek oils, DHASCO® and ARASCO®, or variations thereof. The DHA and ARA can be in natural form, provided that the remainder of the LCPUFA source does not result in any substantial deleterious effect on the infant. Alternatively, the DHA and ARA can be used in refined form.
[0093] In an embodiment, sources of DHA and ARA are single cell oils as taught in U.S. Pat. Nos. 5,374,567; 5,550,156; and 5,397,591, the disclosures of which are incorporated herein in their entirety by reference. However, the present disclosure is not limited to only such oils.
[0094] In some embodiments the nutritional composition may include an enriched lipid fraction derived from milk. The enriched lipid fraction derived from milk may be produced by any number of fractionation techniques. These techniques include but are not limited to melting point fractionation, organic solvent fractionation, super critical fluid fractionation, and any variants and combinations thereof. In some embodiments the nutritional composition may include an enriched lipid fraction derived from milk that contains milk fat globules.
[0095] In certain embodiments, the addition of the enriched lipid fraction or the enriched lipid fraction including milk fat globules may provide a source of saturated fatty acids, trans-fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, OBCFAs, BCFAs, CLA, cholesterol, phospholipids, and/or milk fat globule membrane proteins to the nutritional composition.
[0096] The milk fat globules may have an average diameter (volume-surface area average diameter) of at least about 2 μηη. In some embodiments, the average diameter is in the range of from about 2 μιτι to about 13 μηη. In other embodiments, the milk fat globules may range from about 2.5 μιτι to about 10 μηη. Still in other embodiments, the milk fat globules may range in average diameter from about 3 μηι to about 6 μιη. The specific surface area of the globules is, in certain embodiments, less than 3.5 m2/g, and in other embodiments is between about 0.9 mVg to about 3 m2/g. Without being bound by any particular theory, it is believed that milk fat globules of the aforementioned sizes are more accessible to lipases therefore leading to better digestion lipid digestion.
[0097] In some embodiments the enriched lipid fraction and/or milk fat globules contain saturated fatty acids. The saturated fatty acids may be present in a concentration from about 0.1 g/100 kcal to about 8.0 g/100 kcal. In certain embodiments the saturated fatty acids may be present from about 0.5 g/100 kcal to about 2.0 g/100 kcal. In still other embodiments the saturated fatty acids may be present from about 3.5 g/100 kcal to about 6.9 g/100 kcal.
[0098] Examples of saturated fatty acids suitable for inclusion include, but are not limited to, butyric, valeric, caproic, caprylic, decanoic, lauric, myristic, palmitic, steraic, arachidic, behenic, alignoceric, tetradecanoic, hexadecanoic, palmitic, and octadecanoic acid, and/or combinations and mixtures thereof.
[0099] Additionally, the enriched lipid fraction and/or milk fat globules may comprise, in some embodiments, lauric acid. Lauric acid, also known as dodecanoic acid, is a saturated fatty acid with a 12-carbon atom chain and is believed to be one of the main antiviral and antibacterial substances currently found in human breast milk. The milk fat globules may be enriched with triglycerides containing lauric acid at either the Sn-1, Sn-2 and/or Sn-3 positions. Without being bound by any particular theory, it is believed that when the enriched lipid fraction is ingested, the mouth lingual lipase and pancreatic lipase will hydrolyze the triglycerides to a mixture of glycerides including mono-lauric and free lauric acid.
[0100] The concentration of lauric acid in the globules varies from 80mg/100ml to 800mg/100ml. The concentration of monolauryl n the globules can be in the range of 20mg/100ml to 300mg/100ml feed. In some embodiments, the range is
60mg/100ml to 130mg/100ml.
[0101] The enriched lipid fraction and/or milk fat globules may contain trans- fatty acids in certain embodiments. The trans-fatty acids included in the milk fat globules may be monounsaturated or polyunsaturated trans-fatty acids. In some embodiments the trans-fatty acids may be present in an amount from about 0.2 g/100 kcal to about 7.0 g/100 kcal. In other embodiments the trans-fatty acids may be present in an amount from about 3.4 g/100 kcal to about 5.2 g/100 kcal. In yet other embodiments the trans-fatty acids may be present from about 1.2 g/100 kcal to about 4.3 g/100 kcal.
[0102] Examples of trans-fatty acids for inclusion include, but are not limited to, vaccenic, or elaidic acid, and mixtures thererof. Moreover, when consumed, mammals convert vaccenic acid into rumenic acid, which is a conjugated linoleic acid that exhibits anticarcinogenic properties. Additionally, a diet enriched with vaccenic acid may help lower total cholesterol, LDL cholesterol and triglyceride levels.
[0103] In some embodiments the enriched lipid fraction and/or milk fat globules may contain OBCFAs. In certain embodiments, the OBCFAs may be present in an amount from about 0.3 g/100 kcal to about 6.1 g/100 kcal. In other
embodiments OBCFAs may be present in an amount from about 2.2 g/100 kcal to about 4.3 g/100 kcal. In yet another embodiment OBCFAs may be present in an amount from about 3.5 g/ 00 kcal to about 5.7 g/100 kcal. In still other
embodiments, the milk fat globules comprise at least one OBCFA.
[0104] Typically, an infant may absorb OBCFAs while in utero and from the breast milk of a nursing mother. Therefore, OBCFAs that are identified in human milk are preferred for inclusion in the milk fat globules of the nutritional composition. Addition of OBCFAs to infant or children's formulas allows such formulas to mirror the composition and functionality of human milk and to promote general health and well-being.
[0105] In some embodiments, the enriched lipid fraction and/or milk fat globules may comprise BCFAs. In some embodiments the BCFAs are present at a concentration from about 0.2 g/100 kcal and about 5.82 g/100 kcal. In another embodiment, the BCFAs are present in an amount of from about 2.3 g/100 kcal to about 4.2 g/100 kcal. In yet another embodiment the BCFAs are present from about 4.2 g/100 kcal to about 5.82 g/100 kcal. In still other embodiments, the milk fat globules comprise at least one BCFA.
[0106] BCFAs that are identified in human milk are preferred for inclusion in the nutritional composition. Addition of BCFAs to infant or children's formulas allows such formulas to mirror the composition and functionality of human milk and to promote general health and well-being.
[0107] In certain embodiments the enriched lipid fraction and/or milk fat globules may comprise CLA. In some embodiments CLA may be present in a concentration from about 0.4 g/100 kcal to about 2.5 g/100 kcal. In other embodiments CLA may be present from about 0.8 g/100 kcal to about 1.2 g/100 kcal. In yet other embodiments CLA may be present from about 1.2 g/100 kcal to about 2.3 g/100 kcal. In still other embodiments, the milk fat globules comprise at least one CLA.
[0108] CLAs that are identified in human milk are preferred for inclusion in the nutritional composition. Typically, CLAs are absorbed by the infant from the human milk of a nursing mother. Addition of CLAs to infant or children's formulas allows such formulas to mirror the composition and functionality of human milk and to promote general health and wellbeing.
[0109] Examples of CLAs found in the milk fat globules for the nutritional composition include, but are not limited to, cis-9, trans-11 CLA, trans-10, cis-12 CLA, cis-9, trans- 2 octadecadienoic acid, and mixtures thereof.
[0110] The enriched lipid fraction and/or milk fat globules of the present disclosure comprise monounsaturated fatty acids in some embodiments. The enriched lipid fraction and/or milk fat globules may be formulated to include monounsaturated fatty acids from about 0.8 g/100 kcal to about 2.5 g/100 kcal. In other embodiments the milk fat globules may include monounsaturated fatty acids from about 1.2 g/100 kcal to about 1.8 g/100 kcal.
[0111] Examples of monounsaturated fatty acids suitable include, but are not limited to, palmitoleic acid, cis-vaccenic acid, oleic acid, and mixtures thereof.
[0112] In certain embodiments, the enriched lipid fraction and/or milk fat globules of the present disclosure comprise polyunsaturated fatty acids from about 2.3 g/100 kcal to about 4.4 g/100 kcal. In other embodiments, the polyunsaturated fatty acids are present from about 2.7 g/100 kcal to about 3.5 g/100 kcal. In yet another embodiment, the polyunsaturated fatty acids are present from about 2.4 g/100 kcal to about 3.3 g/100 kcal.
[0113] In some embodiments, the enriched lipid fraction and/or milk fat globules of the present disclosure comprise polyunsaturated fatty acids, such as, for example linoleic acid, linolenic acid, octadecatrienoic acid, arachidonic acid (ARA), eicosatetraenoic acid, eicopsapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA). Polyunsaturated fatty acids are the precursors for prostaglandins and eicosanoids, which are known to provide numerous health benefits, including, anti-inflammatory response, cholesterol absorption, and increased bronchial function.
[0114] The enriched lipid fraction and/or milk fat globules of the present disclosure can also comprise cholesterol in some embodiments from about 100 mg/100 kcal to about 400 mg/100 kal. In another embodiment, cholesterol is present from about 200 mg/100 kcal to about 300 mg/100 kcal. As is similar to human milk and bovine milk, the cholesterol included in the milk fat globules may be present in the outer bilayer membrane of the milk fat globule to provide stability to the globular membrane.
[0115] In some embodiments, the enriched lipid fraction and/or milk fat globules of the present disclosure comprise phospholipids from about 50 mg/100 kcal to about 200 mg/100 kcal. In other embodiments, the phospholipids are present from about 75 mg/100 kcal to about 150 mg/100 kcal. In yet other embodiments, the phospholipids are present at a concentration of from about 100 mg/100 kcal to about 250 mg/100 kcal.
[0116] In certain embodiments, phospholipids may be incorporated into the milk fat globules to stabilize the milk fat globule by providing a phospholipid membrane or bilayer phospholipid membrane. Therefore, in some embodiments the milk fat globules may be formulated with higher amounts of phospholipids than those found in human milk.
[0117] The phospholipid composition of human milk lipids, as the weight percent of total phospholipids, is phosphatidylcholine("PC") 24.9%,
phosphatidylethanolamine ("PE") 27.7%, phosphatidylserine ("PS") 9.3%,
phosphatidylinositol ("PI") 5.4%, and sphingomyelin ("SPGM") 32.4%, (Harzer, G. et al., Am. J. Clin. Nutr., Vol. 37, pp. 612-621 (1983)). Thus in one embodiment, the milk fat globules comprise one or more of PC, PE, PS, PI, SPGM, and mixtures thereof. Further, the phospholipid composition included in the milk fat globules may be formulated to provide certain health benefits by incorporating desired phospholipids.
[0118] In certain embodiments, the enriched lipid fraction and/or milk fat globules of the present disclosure comprise milk fat globule membrane protein. In some embodiments, the milk fat globule membrane protein is present from about 50 mg/100 kcal to about 500 mg/100 kcal. [0119] Galactolipids may be included, in some embodiments, in the enriched lipid fraction and/or milk fat globules of the present disclosure. For purposes of this disclosure "galactolipids" refer to any glycolipid whose sugar group is galactose. More specifically, galactolipids differ from glycosphingolipids in that they do not have nigtrogen in their composition. Galactolipids play an important role in supporting brain development and overall neuronal health. Additionally, the galactolipids, galactocerebroside and sulfatides constitute about 23% and 4% of total myelin lipid content respectively, and thus may be incorporated into the milk fat globules in some embodiments.
[0120] In some embodiments, the nutritional composition(s) of the disclosure may comprise at least one protein source, by which is meant excluding lactoferrin. The protein source can be any used in the art, e.g., nonfat milk, whey protein, casein, soy protein, hydrolyzed protein, amino acids, and the like. Bovine milk protein sources useful in practicing the present disclosure include, but are not limited to, milk protein powders, milk protein concentrates, milk protein isolates, nonfat milk solids, nonfat milk, nonfat dry milk, whey protein, whey protein isolates, whey protein concentrates, sweet whey, acid whey, casein, acid casein, caseinate [e.g. sodium caseinate, sodium calcium caseinate, calcium caseinate) and any combinations thereof.
[0121] In some embodiments, the proteins of the nutritional composition are provided as intact proteins. In other embodiments, the proteins are provided as a combination of both intact proteins and hydrolyzed proteins. In certain
embodiments, the proteins may be partially hydrolyzed or extensively hydrolyzed. In still other embodiments, the protein source comprises amino acids. In yet another embodiment, the protein source may be supplemented with glutamine-containing peptides. In another embodiment, the protein component comprises extensively hydrolyzed protein. In still another embodiment, the protein component of the nutritional composition consists essentially of extensively hydrolyzed protein in order to minimize the occurrence of food allergy. In yet another embodiment, the protein source may be supplemented with glutamine-containing peptides.
[0122] Accordingly, in some embodiments, the protein component of the nutritional composition comprises either partially or extensively hydrolyzed protein, such as protein from cow's milk. The hydrolyzed proteins may be treated with enzymes to break down some or most of the proteins that cause adverse symptoms with the goal of reducing allergic reactions, intolerance, and sensitization.
Moreover, the proteins may be hydrolyzed by any method known in the art.
[0123] The terms "protein hydrolysates" or "hydrolyzed protein" are used interchangeably herein and refer to hydrolyzed proteins, wherein the degree of hydrolysis is may be from about 20% to about 80%, or from about 30% to about 80%, or even from about 40% to about 60%.
[0124] When a peptide bond in a protein is broken by enzymatic hydrolysis, one amino group is released for each peptide bond broken, causing an increase in amino nitrogen. It should be noted that even non-hydrolyzed protein would contain some exposed amino groups. Hydrolyzed proteins will also have a different molecular weight distribution than the non-hydrolyzed proteins from which they were formed. The functional and nutritional properties of hydrolyzed proteins can be affected by the different size peptides. A molecular weight profile is usually given by listing the percent by weight of particular ranges of molecular weight (in Daltons) fractions (e.g., 2,000 to 5,000 Daltons, greater than 5,000 Daltons).
[0125] In a particular embodiment, the nutritional composition is protein-free and contains free amino acids as a protein equivalent source. In this embodiment, the amino acids may comprise, but are not limited to, histidine, isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, threonine, tryptophan, valine, alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, carnitine, taurine and mixtures thereof. In some embodiments, the amino acids may be branched chain amino acids. In other embodiments, small amino acid peptides may be included as the protein component of the nutritional composition. Such small amino acid peptides may be naturally occurring or synthesized. The amount of free amino acids in the nutritional composition may vary from about 1 to about 5 g/100 kcal. In an embodiment, 100% of the free amino acids have a molecular weight of less than 500 Daltons. In this embodiment, the nutritional formulation may be hypoallergenic.
[0 26] In an embodiment, the protein source comprises from about 40% to about 85% whey protein and from about 15% to about 60% casein. [0127] In some embodiments, the nutritional composition comprises between about 1 g and about 7 g of a protein and/or protein equivalent source per 100 kcal. In other embodiments, the nutritional composition comprises between about 3.5 g and about 4.5 g of protein or protein equivalent per 100 kcal.
[01 8] Moreover, the nutritional composition of the present disclosure may comprise at least one starch or starch component. A starch is a carbohydrate composed of two distinct polymer fractions: amylose and amylopectin. Amylose is the linear fraction consisting of a-1 ,4 linked glucose units. Amylopectin has the same structure as amylose, but some of the glucose units are combined in an ct-1,6 linkage, giving rise to a branched structure. Starches generally contain 17-24% amylose and from 76-83% amylopectin. Yet special genetic varieties of plants have been developed that produce starch with unusual amylose to amylopectin ratios. Some plants produce starch that is free of amylose. These mutants produce starch granules in the endosperm and pollen that stain red with iodine and that contain nearly 100% amylopectin. Predominant among such amylopectin producing plants are waxy corn, waxy sorghum and waxy rice starch.
[0 29] The performance of starches under conditions of heat, shear and acid may be modified or improved by chemical modifications. Modifications are usually attained by introduction of substituent chemical groups. For example, viscosity at high temperatures or high shear can be increased or stabilized by cross-linking with di- or polyfunctional reagents, such as phosphorus oxychloride.
[0130] In some instances, the nutritional compositions of the present disclosure comprise at least one starch that is gelatinized or pregelatinized. As is known in the art, gelatinization occurs when polymer molecules interact over a portion of their length to form a network that entraps solvent and/or solute molecules. Moreover, gels form when pectin molecules lose some water of hydration owing to competitive hydration of cosolute molecules. Factors that influence the occurrence of gelation include pH, concentration of cosolutes, concentration and type of cations,
temperature and pectin concentration. Notably, LM pectin will gel only in the presence of divalent cations, such as calcium ions. And among LM pectins, those with the lowest degree of esterification have the highest gelling temperatures and the greatest need for divalent cations for crossbridging. [0131] Meanwhile, pregelatinization of starch is a process of precooking starch to produce material that hydrates and swells in cold water. The precooked starch is then dried, for example by drum drying or spray drying. Moreover the starch of the present disclosure can be chemically modified to further extend the range of its finished properties. The nutritional compositions of the present disclosure may comprise at least one pregelatinized starch.
[0 32] Native starch granules are insoluble in water, but, when heated in water, native starch granules begin to swell when sufficient heat energy is present to overcome the bonding forces of the starch molecules. With continued heating, the granule swells to many times its original volume. The friction between these swollen granules is the major factor that contributes to starch paste viscosity.
[0133] The nutritional composition of the present disclosure may comprise native or modified starches, such as, for example, waxy corn starch, waxy rice starch, corn starch, rice starch, potato starch, tapioca starch, wheat starch or any mixture thereof. Generally, common corn starch comprises about 25% amylose, while waxy corn starch is almost totally made up of amylopectin. Meanwhile, potato starch generally comprises about 20% amylose, rice starch comprises an
amylose:amylopectin ratio of about 20:80, and waxy rice starch comprises only about 2% amylose. Further, tapioca starch generally comprises about 15% to about 18% amylose, and wheat starch has an amylose content of around 25%.
[0134] In some embodiments, the nutritional composition comprises
gelatinized and/or pre-gelatinized waxy corn starch. In other embodiments, the nutritional composition comprises gelatinized and/or pre-gelatinized tapioca starch. Other gelatinized or pre-gelatinized starches, such as rice starch or potato starch may also be used.
[0135] Additionally, the nutritional compositions of the present disclosure comprise at least one source of pectin. The source of pectin may comprise any variety or grade of pectin known in the art. In some embodiments, the pectin has a degree of esterification of less than 50% and is classified as low methylated ("LM") pectin. In some embodiments, the pectin has a degree of esterification of greater than or equal to 50% and is classified as high-ester or high methylated ("HM") pectin. In still other embodiments, the pectin is very low ("VL") pectin, which has a degree of esterification that is less than approximately 15%. Further, the nutritional composition of the present disclosure may comprise LM pectin, HM pectin, VL pectin, or any mixture thereof. The nutritional composition may include pectin that is soluble in water. And, as known in the art, the solubility and viscosity of a pectin solution are related to the molecular weight, degree of esterification, concentration of the pectin preparation and the pH and presence of counterions.
[0136] Moreover, pectin has a unique ability to form gels. Generally, under similar conditions, a pectin's degree of gelation, the gelling temperature, and the gel strength are proportional to one another, and each is generally proportional to the molecular weight of the pectin and inversely proportional to the degree of esterification. For example, as the pH of a pectin solution is lowered, ionization of the carboxylate groups is repressed, and, as a result of losing their charge, saccharide molecules do not repel each other over their entire length. Accordingly, the polysaccharide molecules can associate over a portion of their length to form a gel. Yet pectins with increasing degrees of methylation will gel at somewhat higher pH because they have fewer carboxylate anions at any given pH. (J.N. Bemiller, An Introduction to Pectins: Structure and Properties, Chemistry and Function of Pectins; Chapter 1 ; 1986.)
[0137] The nutritional composition may comprise a gelatinized and/or pregelatinized starch together with pectin and/or gelatinized pectin. While not wishing to be bound by this or any other theory, it is believed that the use of pectin, such as LM pectin, which is a hydrocolloid of large molecular weight, together with starch granules, provides a synergistic effect that increases the molecular internal friction within a fluid matrix. The carboxylic groups of the pectin may also interact with calcium ions present in the nutritional composition, thus leading to an increase in viscosity, as the carboxylic groups of the pectin form a weak gel structure with the calcium ion(s), and also with peptides present in the nutritional composition. In some embodiments, the nutritional composition comprises a ratio of starch to pectin that is between about 12:1 and 20:1 , respectively. In other embodiments, the ratio of starch to pectin is about 17:1. In some embodiments, the nutritional composition may comprise between about 0.05 and about 2.0% w/w pectin. In a particular embodiment, the nutritional composition may comprise about 0.5% w/w pectin.
[0138] Pectins for use herein typically have a peak molecular weight of 8,000 Daltons or greater. The pectins of the present disclosure have a preferred peak molecular weight of between 8,000 and about 500,000, more preferred is between about 10,000 and about 200,000 and most preferred is between about 15,000 and about 100,000 Daltons. In some embodiments, the pectin of the present disclosure may be hydrolyzed pectin. In certain embodiments, the nutritional composition comprises hydrolyzed pectin having a molecular weight less than that of intact or unmodified pectin. The hydrolyzed pectin of the present disclosure can be prepared by any means known in the art to reduce molecular weight. Examples of said means are chemical hydrolysis, enzymatic hydrolysis and mechanical shear. A preferred means of reducing the molecular weight is by alkaline or neutral hydrolysis at elevated temperature. In some embodiments, the nutritional composition comprises partially hydrolyzed pectin. In certain embodiments, the partially hydrolyzed pectin has a molecular weight that is less than that of intact or unmodified pectin but more than 3,300 Daltons.
[0139] The nutritional composition may contain at least one acidic
polysaccharide. An acidic polysaccharide, such as negatively charged pectin, may induce an anti-adhesive effect on pathogens in a subject's gastrointestinal tract. Indeed, nonhuman milk acidic oligosaccharides derived from pectin are able to interact with the epithelial surface and are known to inhibit the adhesion of pathogens on the epithelial surface.
[0140] In some embodiments, the nutritional composition comprises at least one pectin-derived acidic oligosaccharide. Pectin-derived acidic oligosaccharide(s) (pAOS) result from enzymatic pectinolysis, and the size of a pAOS depends on the enzyme use and on the duration of the reaction. In such embodiments, the pAOS may beneficially affect a subject's stool viscosity, stool frequency, stool pH and/or feeding tolerance. The nutritional composition of the present disclosure may comprise between about 2 g pAOS per liter of formula and about 6 g pAOS per liter of formula. In an embodiment, the nutritional composition comprises about 0.2 g pAOS/dL, corresponding to the concentration of acidic oligosaccharides in human milk. (Fanaro et al., "Acidic Oligosaccharides from Pectin Hydrolysate as New Component for Infant Formulae: Effect on Intestinal Flora, Stool Characteristics, and pH", Journal of Pediatric Gastroenterology and Nutrition, 41 : 186-190, August 2005)
[0141] In some embodiments, the nutritional composition comprises up to about 20% w/w of a mixture of starch and pectin. In some embodiments, the nutritional composition comprises up to about 19% starch and up to about 1% pectin. In other embodiments, the nutritional composition comprises about up to about 15% starch and up to about 5% pectin. In still other embodiments, the nutritional composition comprises up to about 18% starch and up to about 2% pectin. In some embodiments the nutritional composition comprises between about 0.05% w/w and about 20% w/w of a mixture of starch and pectin. Other
embodiments include between about 0.05% and about 19% w/w starch and between about 0.05% and about 1% w/w pectin. Further, the nutritional composition may comprise between about 0.05% and about 15% w/w starch and between about 0.05% and about 5% w/w pectin.
[0142] In some embodiments, the nutritional composition comprises at least one additional carbohydrate source, that is, a carbohydrate component provided in addition to the aforementioned starch component. Additional carbohydrate sources can be any used in the art, e.g., lactose, glucose, fructose, corn syrup solids, ma!todextrins, sucrose, starch, rice syrup solids, and the like. The amount of the additional carbohydrate component in the nutritional composition typically can vary from between about 5 g and about 25 g/100 kcal. In some embodiments, the amount of carbohydrate is between about 6 g and about 22 g/ 100 kcal. In other embodiments, the amount of carbohydrate is between about 12 g and about 14 g/100 kcal. In some embodiments, corn syrup solids are preferred. Moreover, hydrolyzed, partially hydrolyzed, and/or extensively hydrolyzed carbohydrates may be desirable for inclusion in the nutritional composition due to their easy digestibility. Specifically, hydrolyzed carbohydrates are less likely to contain allergenic epitopes.
[0143] Non-limiting examples of carbohydrate materials suitable for use herein include hydrolyzed or intact, naturally or chemically modified, starches sourced from corn, tapioca, rice or potato, in waxy or non-waxy forms. Non-limiting examples of suitable carbohydrates include various hydrolyzed starches characterized as hydrolyzed cornstarch, maltodextrin, maltose, corn syrup, dextrose, corn syrup solids, glucose, and various other glucose polymers and combinations thereof. Non- limiting examples of other suitable carbohydrates include those often referred to as sucrose, lactose, fructose, high fructose corn syrup, indigestible oligosaccharides such as fructooligosaccharides and combinations thereof. [0144] In one particular embodiment, the additional carbohydrate component of the nutritional composition is comprised of 100% lactose. In another embodiment, the additional carbohydrate component comprises between about 0% and 60% lactose. In another embodiment, the additional carbohydrate component comprises between about 15% and 55% lactose. In yet another embodiment, the additional carbohydrate component comprises between about 20% and 30% lactose. In these embodiments, the remaining source of carbohydrates may be any carbohydrate known in the art. In an embodiment, the carbohydrate component comprises about 25% lactose and about 75% corn syrup solids.
[0145] In some embodiments the nutritional composition comprises sialic acid. Sialic acids are a family of over 50 members of 9-carbon sugars, all of which are derivatives of neuroaminic acid. The predominant sialic acid family found in humans is from the N-acetylneuraminic acid sub-family. Sialic acids are found in milk, such as bovine and caprine. In mammals, neuronal cell membranes have the highest concentration of sialic acid compared to other body cell membranes. Sialic acid residues are also components of gangliosides.
[0146] If included in the nutritional composition, sialic acid may be present in an amount from about 0.5 mg/100 kcals to about 45 mg/100 kcal. In some
embodiments sialic acid may be present in an amount from about 5 mg/100 kcals to about 30 mg/ 00 kcals. In still other embodiments, sialic acid may be present in an amount from about 10 mg/ 00 kcals to about 25 mg/100 kcals.
[0147] In one embodiment, the nutritional composition may contain one or more probiotics. Any probiotic known in the art may be acceptable in this
embodiment. In a particular embodiment, the probiotic may be selected from any Lactobacillus species, Lactobacillus rhamnosus GG (LGG) (ATCC number 53103), Bifidobacterium species, Bifidobacterium longum BB536 (BL999, ATCC: BAA-999), Bifidobacterium longum AH1206 (NCIMB: 41382), Bifidobacterium breve AH 1205 (NCIMB: 41387), Bifidobacterium infantis 35624 (NCIMB: 41003), and Bifidobacterium animalis subsp. lactis BB-12 (DSM No. 10140) or any combination thereof.
[0148] If included in the composition, the amount of the probiotic may vary from about 1 x 10 to about 1.5 x 1012 cfu of probiotic(s) per 100 kcal. In some embodiments the amount of probiotic may be from about 1 x 106 to about 1 x 109 cfu of probiotic(s) per 100 kcal. In certain other embodiments the amount of probitic may vary from about 1 x 107 cfu/100 kcal to about 1 x 108 cfu of probiotic(s) per 100 kcal.
[0149] In an embodiment, the probiotic(s) may be viable or non-viable. As used herein, the term "viable", refers to live microorganisms. The term "non-viable" or "non-viable probiotic" means non-living probiotic microorganisms, their cellular components and/or metabolites thereof. Such non-viable probiotics may have been heat-killed or otherwise inactivated, but they retain the ability to favorably influence the health of the host. The probiotics useful in the present disclosure may be naturally-occurring, synthetic or developed through the genetic manipulation of organisms, whether such source is now known or later developed.
[0150] In some embodiments, the nutritional composition may include a source comprising probiotic cell equivalents, which refers to the level of non-viable, non- replicating probiotics equivalent to an equal number of viable cells. The term "non- replicating" is to be understood as the amount of non-replicating microorganisms obtained from the same amount of replicating bacteria (cfu/g), including inactivated probiotics, fragments of DNA, cell wall or cytoplasmic compounds. In other words, the quantity of non-living, non-replicating organisms is expressed in terms of cfu as if all the microorganisms were alive, regardless whether they are dead, non-replicating, inactivated, fragmented etc. In non-viable probiotics are included in the nutritional composition, the amount of the probiotic cell equivalents may vary from about 1 x 104 to about 1.5 x 1010 cell equivalents of probiotic(s) per 100 kcal. In some embodiments the amount of probiotic cell equivalents may be from about 1 x 106 to about 1 x 109 cell equivalents of probiotic(s) per 100 kcal nutritional composition. In certain other embodiments the amount of probiotic cell equivalents may vary from about 1 x 107 to about 1 x 108 cell equivalents of probiotic(s) per 100 kcal of nutritional composition.
[0151] In some embodiments, the probiotic source incorporated into the nutritional composition may comprise both viable colony-forming units, and nonviable cell-equivalents.
[0152] In some embodiments, the nutritional composition includes a culture supernatant from a late-exponential growth phase of a probiotic batch-cultivation process. Without wishing to be bound by theory, it is believed that the activity of the culture supernatant can be attributed to the mixture of components (including proteinaceous materials, and possibly including (exo)polysaccharide materials) as found released into the culture medium at a late stage of the exponential (or "log") phase of batch cultivation of the probiotic. The term "culture supernatant" as used herein, includes the mixture of components found in the culture medium. The stages recognized in batch cultivation of bacteria are known to the skilled person. These are the "lag," the "log" ("logarithmic" or "exponential"), the "stationary" and the "death" (or "logarithmic decline") phases. In all phases during which live bacteria are present, the bacteria metabolize nutrients from the media, and secrete (exert, release) materials into the culture medium. The composition of the secreted material at a given point in time of the growth stages is not generally predictable.
[0153] In an embodiment, a culture supernatant is obtainable by a process comprising the steps of (a) subjecting a probiotic such as LGG to cultivation in a suitable culture medium using a batch process; (b) harvesting the culture supernatant at a late exponential growth phase of the cultivation step, which phase is defined with reference to the second half of the time between the lag phase and the stationary phase of the batch-cultivation process; (c) optionally removing low molecular weight constituents from the supernatant so as to retain molecular weight constituents above 5-6 kiloDaltons (kDa); (d) removing liquid contents from the culture supernatant so as to obtain the composition.
[0154] The culture supernatant may comprise secreted materials that are harvested from a late exponential phase. The late exponential phase occurs in time after the mid exponential phase (which is halftime of the duration of the exponential phase, hence the reference to the late exponential phase as being the second half of the time between the lag phase and the stationary phase). In particular, the term "late exponential phase" is used herein with reference to the latter quarter portion of the time between the lag phase and the stationary phase of the LGG batch- cultivation process. In some embodiments, the culture supernatant is harvested at a point in time of 75% to 85% of the duration of the exponential phase, and may be harvested at about S of the time elapsed in the exponential phase.
[0155] As noted, the disclosed nutritional composition may comprise a source of β-glucan. Glucans are polysaccharides, specifically polymers of glucose, which are naturally occurring and may be found in cell walls of bacteria, yeast, fungi, and plants. Beta glucans (β-glucans) are themselves a diverse subset of glucose polymers, which are made up of chains of glucose monomers linked together via beta-type glycosidic bonds to form complex carbohydrates.
[0156] p-1 ,3-glucans are carbohydrate polymers purified from, for example, yeast, mushroom, bacteria, algae, or cereals. (Stone BA, Clarke AE. Chemistry and Biology of (1 -3)-Beta-Glucans. London:Portland Press Ltd; 1993. ) The chemical structure of P-1 ,3-glucan depends on the source of the P-1 ,3-glucan. Moreover, various physiochemical parameters, such as solubility, primary structure, molecular weight, and branching, play a role in biological activities of P-1 ,3-glucans. (Yadomae T., Structure and biological activities of fungal beta-1 ,3-glucans. Yakugaku Zasshi. 2000;120:413-431.)
[0157] P-1 ,3-glucans are naturally occurring polysaccharides, with or without β- 1 ,6-glucose side chains that are found in the cell walls of a variety of plants, yeasts, fungi and bacteria. p-1 ,3;1 ,6-glucans are those containing glucose units with (1 ,3) links having side chains attached at the (1 ,6) position(s). β-1 ,3;1 ,6 glucans are a heterogeneous group of glucose polymers that share structural commonalities, including a backbone of straight chain glucose units linked by a β-1 ,3 bond with β- 1 ,6-linked glucose branches extending from this backbone. While this is the basic structure for the presently described class of β-glucans, some variations may exist. For example, certain yeast β-glucans have additional regions of β(1 ,3) branching extending from the β(1 ,6) branches, which add further complexity to their respective structures.
[0158] β-glucans derived from baker's yeast, Saccharomyces cerevisiae, are made up of chains of D-glucose molecules connected at the 1 and 3 positions, having side chains of glucose attached at the 1 and 6 positions. Yeast-derived β-glucan is an insoluble, fiber-like, complex sugar having the general structure of a linear chain of glucose units with a β-1 ,3 backbone interspersed with β-1 ,6 side chains that are generally 6-8 glucose units in length. More specifically, β-glucan derived from baker's yeast is poly-(1 ,6)^-D-glucopyranosyl-(1 ,3)^-D-glucopyranose.
[0159] Furthermore, β-glucans are well tolerated and do not produce or cause excess gas, abdominal distension, bloating or diarrhea in pediatric subjects. Addition of β-glucan to a nutritional composition for a pediatric subject, such as an infant formula, a growing-up milk or another children's nutritional product, will improve the subject's immune response by increasing resistance against invading pathogens and therefore maintaining or improving overall health.
[0160] The nutritional composition of the present disclosure comprises β- glucan. In some embodiments, the β-glucan is -1 ,3;1,6-glucan. In some
embodiments, the -1,3;1 ,6-glucan is derived from baker's yeast. The nutritional composition may comprise whole glucan particle β-glucan, particulate β-glucan, PGG- glucan (poly-1 ,6^-D-glucopyranosyl-1 ,3^-D-glucopyranose) or any mixture thereof.
[0161] In some embodiments, the amount of β-glucan present in the
composition is at between about 0.010 and about 0.080 g per 100g of composition. In other embodiments, the nutritional composition comprises between about 10 and about 30 mg β-glucan per serving. In another embodiment, the nutritional composition comprises between about 5 and about 30 mg β-glucan per 8 fl. oz.
(236.6 mL) serving. In other embodiments, the nutritional composition comprises an amount of β-glucan sufficient to provide between about 15 mg and about 90 mg β- glucan per day. The nutritional composition may be delivered in multiple doses to reach a target amount of β-glucan delivered to the subject throughout the day.
[0162] In some embodiments, the amount of β-glucan in the nutritional composition is between about 3 mg and about 17 mg per 100 kcal. In another embodiment the amount of β-glucan is between about 6 mg and about 17 mg per 100 kcal.
[0163] One or more vitamins and/or minerals may also be added in to the nutritional composition in amounts sufficient to supply the daily nutritional
requirements of a subject. It is to be understood by one of ordinary skill in the art that vitamin and mineral requirements will vary, for example, based on the age of the child. For instance, an infant may have different vitamin and mineral requirements than a child between the ages of one and thirteen years. Thus, the embodiments are not intended to limit the nutritional composition to a particular age group but, rather, to provide a range of acceptable vitamin and mineral components.
[0164] The nutritional composition may optionally include, but is not limited to, one or more of the following vitamins or derivations thereof: vitamin Bi (thiamin, thiamin pyrophosphate, TPP, thiamin triphosphate, TTP, thiamin hydrochloride, thiamin mononitrate), vitamin B2 (riboflavin, flavin mononucleotide, FMN, flavin adenine dinucleotide, FAD, lactoflavin, ovoflavin), vitamin B3 (niacin, nicotinic acid, nicotinamide, niacinamide, nicotinamide adenine dinucleotide, NAD, nicotinic acid mononucleotide, NicMN, pyridine-3-carboxylic acid), vitamin B3-precursor
tryptophan, vitamin B6 (pyridoxine, pyridoxal, pyridoxamine, pyridoxine
hydrochloride), pantothenic acid (pantothenate, panthenol), folate (folic acid, folacin, pteroylg!utamic acid), vitamin B12 (cobalamin, methylcobalamin,
deoxyadenosylcobalamin, cyanocobalamin, hydroxycobalamin, adenosylcobalamin), biotin, vitamin C (ascorbic acid), vitamin A (retinol, retinyl acetate, retinyl palmitate, retinyl esters with other long-chain fatty acids, retinal, retinoic acid, retinol esters), vitamin D (calciferol, cholecalciferol, vitamin D3, 1,25,-dihydroxyvitamin D), vitamin E (a-tocopherol, a-tocopherol acetate, a-tocopherol succinate, a-tocopherol nicotinate, α-tocopherol), vitamin K (vitamin Ki, phylloquinone, naphthoquinone, vitamin K2, menaquinone-7, vitamin K3, menaquinone-4, menadione, menaquinone-8,
menaquinone-8H, menaquinone-9, menaquinone-9H, menaquinone-10,
menaquinone-11 , menaquinone-12, menaquinone-13), choline, inositol, β-carotene and any combinations thereof.
[0165] Further, the nutritional composition may optionally include, but is not limited to, one or more of the following minerals or derivations thereof: boron, calcium, calcium acetate, calcium gluconate, calcium chloride, calcium lactate, calcium phosphate, calcium sulfate, chloride, chromium, chromium chloride, chromium picolonate, copper, copper sulfate, copper gluconate, cupric sulfate, fluoride, iron, carbonyl iron, ferric iron, ferrous fumarate, ferric orthophosphate, iron trituration, polysaccharide iron, iodide, iodine, magnesium, magnesium carbonate, magnesium hydroxide, magnesium oxide, magnesium stearate, magnesium sulfate, manganese, molybdenum, phosphorus, potassium, potassium phosphate, potassium iodide, potassium chloride, potassium acetate, selenium, sulfur, sodium, docusate sodium, sodium chloride, sodium selenate, sodium molybdate, zinc, zinc oxide, zinc sulfate and mixtures thereof. Non-limiting exemplary derivatives of mineral compounds include salts, alkaline salts, esters and chelates of any mineral compound.
[0166] The minerals can be added to nutritional compositions in the form of salts such as calcium phosphate, calcium glycerol phosphate, sodium citrate, potassium chloride, potassium phosphate, magnesium phosphate, ferrous sulfate, zinc sulfate, cupric sulfate, manganese sulfate, and sodium selenite. Additional vitamins and minerals can be added as known within the art. [0167] In an embodiment, the nutritional composition may contain between about 10 and about 50% of the maximum dietary recommendation for any given country, or between about 10 and about 50% of the average dietary
recommendation for a group of countries, per serving of vitamins A, C, and E, zinc, iron, iodine, selenium, and choline. In another embodiment, the children's nutritional composition may supply about 10 - 30% of the maximum dietary recommendation for any given country, or about 10 - 30% of the average dietary recommendation for a group of countries, per serving of B-vitamins. In yet another embodiment, the levels of vitamin D, calcium, magnesium, phosphorus, and potassium in the children's nutritional product may correspond with the average levels found in milk. In other embodiments, other nutrients in the children's nutritional composition may be present at about 20% of the maximum dietary recommendation for any given country, or about 20% of the average dietary recommendation for a group of countries, per serving.
[0168] The nutritional compositions of the present disclosure may optionally include one or more of the following flavoring agents, including, but not limited to, flavored extracts, volatile oils, cocoa or chocolate flavorings, peanut butter flavoring, cookie crumbs, vanilla or any commercially available flavoring. Examples of useful flavorings include, but are not limited to, pure anise extract, imitation banana extract, imitation cherry extract, chocolate extract, pure lemon extract, pure orange extract, pure peppermint extract, honey, imitation pineapple extract, imitation rum extract, imitation strawberry extract, or vanilla extract; or volatile oils, such as balm oil, bay oil, bergamot oil, cedarwood oil, cherry oil, cinnamon oil, clove oil, or peppermint oil; peanut butter, chocolate flavoring, vanilla cookie crumb, butterscotch, toffee, and mixtures thereof. The amounts of flavoring agent can vary greatly depending upon the flavoring agent used. The type and amount of flavoring agent can be selected as is known in the art.
[0169] The nutritional compositions of the present disclosure may optionally include one or more emulsifiers that may be added for stability of the final product. Examples of suitable emulsifiers include, but are not limited to, lecithin {e.g., from egg or soy), alpha lactalbumin and/or mono- and di-glycerides, and mixtures thereof. Other emulsifiers are readily apparent to the skilled artisan and selection of suitable emulsifier(s) will depend, in part, upon the formulation and final product. [0170] The nutritional compositions of the present disclosure may optionally include one or more preservatives that may also be added to extend product shelf life. Suitable preservatives include, but are not limited to, potassium sorbate, sodium sorbate, potassium benzoate, sodium benzoate, calcium disodium EDTA, and mixtures thereof.
[0171] The nutritional compositions of the present disclosure may optionally include one or more stabilizers. Suitable stabilizers for use in practicing the nutritional composition of the present disclosure include, but are not limited to, gum arabic, gum ghatti, gum karaya, gum tragacanth, agar, furcellaran, guar gum, gellan gum, locust bean gum, pectin, low methoxyl pectin, gelatin, microcrystalline cellulose, CMC (sodium carboxymethylcelfulose), methylcellulose hydroxypropyl methyl cellulose, hydroxypropyl cellulose, DATEM (diacetyl tartaric acid esters of mono- and diglycerides), dextran, carrageenans, and mixtures thereof.
[0172] The disclosed nutritional composition(s) may be provided in any form known in the art, such as a powder, a gel, a suspension, a paste, a solid, a liquid, a liquid concentrate, a reconstituteable powdered milk substitute or a ready-to-use product. The nutritional composition may, in certain embodiments, comprise a nutritional supplement, children's nutritional product, infant formula, human milk fortifier, growing-up milk or any other nutritional composition designed for an infant or a pediatric subject. Nutritional compositions of the present disclosure include, for example, orally-ingestible, health-promoting substances including, for example, foods, beverages, tablets, capsules and powders. Moreover, the nutritional composition of the present disclosure may be standardized to a specific caloric content, it may be provided as a ready-to-use product, or it may be provided in a concentrated form. In some embodiments, the nutritional composition is in powder form with a particle size in the range of 5 μητι to 1500 μιη, more preferably in the range of 10 pm to 300pm.
[0173] If the nutritional composition is in the form of a ready-to-use product, the osmolality of the nutritional composition may be between about 100 and about 1100 mOsm/kg water, more typically about 200 to about 700 mOsm/kg water.
[0174] Suitable fat or lipid sources for the nutritional composition of the present disclosure may be any known or used in the art, including but not limited to, animal sources, e.g., milk fat, butter, butter fat, egg yolk lipid; marine sources, such as fish oils, marine oils, single cell oils; vegetable and plant oils, such as corn oil, canola oil, sunflower oil, soybean oil, palm olein oil, coconut oil, high oleic sunflower oil, evening primrose oil, rapeseed oil, olive oil, flaxseed (linseed) oil, cottonseed oil, high oleic safflower oil, palm stearin, palm kernel oil, wheat germ oil; medium chain triglyceride oils and emulsions and esters of fatty acids; and any combinations thereof.
[0175] The nutritional compositions of the disclosure may provide minimal, partial or total nutritional support. The compositions may be nutritional supplements or meal replacements. The compositions may, but need not, be nutritionally complete. In an embodiment, the nutritional composition of the disclosure is nutritionally complete and contains suitable types and amounts of lipid,
carbohydrate, protein, vitamins and minerals. The amount of lipid or fat typically can vary from about 1 to about 7 g/100 kcal. The amount of protein typically can vary from about 1 to about 7 g/100 kcal. The amount of carbohydrate typically can vary from about 6 to about 22 g/100 kcal.
[0176] The nutritional composition of the present disclosure may further include at least one additional phytonutrient, that is, another phytonutrient component in addition to the pectin and/or starch components described
hereinabove. Phytonutrients, or their derivatives, conjugated forms or precursors, that are identified in human milk are preferred for inclusion in the nutritional composition. Typically, dietary sources of carotenoids and polyphenols are absorbed by a nursing mother and retained in milk, making them available to nursing infants. Addition of these phytonutrients to infant or children's formulas allows such formulas to mirror the composition and functionality of human milk and to promote general health and well being.
[0177] For example, in some embodiments, the nutritional composition of the present disclosure may comprise, in an 8 fl. oz. (236.6 ml_) serving, between about 80 and about 300 mg anthocyanins, between about 100 and about 600 mg
proanthocyanidins, between about 50 and about 500 mg flavan-3-ols, or any combination or mixture thereof. In other embodiments, the nutritional composition comprises apple extract, grape seed extract, or a combination or mixture thereof. Further, the at least one phytonutrient of the nutritional composition may be derived from any single or blend of fruit, grape seed and/or apple or tea extract(s). [0178] For the purposes of this disclosure, additional phytonutrients may be added to a nutritional composition in native, purified, encapsulated and/or chemically or enzymatically-modified form so as to deliver the desired sensory and stability properties. In the case of encapsulation, it is desirable that the encapsulated phytonutrients resist dissolution with water but are released upon reaching the small intestine. This could be achieved by the application of enteric coatings, such as cross-linked alginate and others.
[0179] Examples of additional phytonutrients suitable for the nutritional composition include, but are not limited to, anthocyanins, proanthocyanidins, flavan- 3-ols (i.e.. catechins, epicatechins, etc.), flavanones, flavonoids, isoflavonoids, stilbenoids (i.e. resveratrol, etc.)proanthocyanidins, anthocyanins, resveratrol, quercetin, curcumin, and/or any mixture thereof, as well as any possible combination of phytonutrients in a purified or natural form. Certain components, especially plant- based components of the nutritional compositions may provide a source of phytonutrients.
[0180] Some amounts of phytonutrients may be inherently present in known ingredients, such as natural oils, that are commonly used to make nutritional compositions for pediatric subjects. These inherent phytonutrient(s) may be but are not necessarily considered part of the phytonutrient component described in the present disclosure. In some embodiments, the phytonutrient concentrations and ratios as described herein are calculated based upon added and inherent
phytonutrient sources. In other embodiments, the phytonutrient concentrations and ratios as described herein are calculated based only upon added phytonutrient sources.
[0181] In some embodiments, the nutritional composition comprises anthocyanins, such as, for example, glucosides of aurantinidin, cyanidin, delphinidin, europinidin, luteolinidin, pelargonidin, malvidin, peonidin, petunidin, and rosinidin. These and other anthocyanins suitable for use in the nutritional composition are found in a variety of plant sources. Anthocyanins may be derived from a single plant source or a combination of plant sources. Non-limiting examples of plants rich in anthocyanins suitable for use in the inventive composition include: berries (acai, grape, bilberry, blueberry, lingonberry, black currant, chokeberry, blackberry, raspberry, cherry, red currant, cranberry, crowberry, cloudberry, whortleberry, rowanberry), purple com, purple potato, purple carrot, red sweet potato, red cabbage, eggplant.
[0182] In some embodiments, the nutritional composition of the present disclosure comprises proanthocyanidins, which include but are not limited to flavan-3- ols and polymers of flavan-3-ols (e.g., catechins, epicatechins) with degrees of polymerization in the range of 2 to 11. Such compounds may be derived from a single plant source or a combination of plant sources. Non-limiting examples of plant sources rich in proanthocyanidins suitable for use in the inventive nutritional composition include: grape, grape skin, grape seed, green tea, black tea, apple, pine bark, cinnamon, cocoa, bilberry, cranberry, black currant chokeberry.
[0183] Non-limiting examples of flavan-3-ols which are suitable for use in the inventive nutritional composition include catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, epicatechin-3-gal!ate, epigaiiocatechin and gallate. Plants rich in the suitable flavan-3-ols include, but are not limited to, teas, red grapes, cocoa, green tea, apricot and apple.
[0184] Certain polyphenol compounds, in particular flavan-3-ols, may improve learning and memory in a human subject by increasing brain blood flow, which is associated with an increase and sustained brain energy/nutrient delivery as well as formation of new neurons. Polyphenols may also provide neuroprotective actions and may increase both brain synaptogenesis and antioxidant capability, thereby supporting optimal brain development in younger children.
[0185] Preferred sources of flavan-3-ols for the nutritional composition include at least one apple extract, at least one grape seed extract or a mixture thereof. For apple extracts, flavan-3-ols are broken down into monomers occurring in the range 4% to 20% and polymers in the range 80% to 96%. For grape seed extracts flavan- 3-ols are broken down into monomers (about 46%) and polymers (about 54%) of the total favan-3-ols and total polyphenolic content. Preferred degree of polymerization of polymeric flavan-3-ols is in the range of between about 2 and 11. Furthermore, apple and grape seed extracts may contain catechin, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, polymeric proanthocyanidins,
stilbenoids (i.e. resveratrol), flavonols (i.e. quercetin, myricetin), or any mixture thereof. Plant sources rich in flavan-3-ols include, but are not limited to apple, grape seed, grape, grape skin, tea (green or black), pine bark, cinnamon, cocoa, bilberry, cranberry, black currant, chokeberry.
[0186] If the nutritional composition is administered to a pediatric subject, an amount of flavan-3-ols, including monomeric flavan-3-ols, polymeric flavan-3-ols or a combination thereof, ranging from between about 0.01 mg and about 450 mg per day may be administered. In some cases, the amount of flavan-3-ols administered to an infant or child may range from about 0.01 mg to about 170 mg per day, from about 50 to about 450 mg per day, or from about 100 mg to about 300 mg per day.
[0187] In an embodiment of the disclosure, flavan-3-ols are present in the nutritional composition in an amount ranging from about 0.4 to about 3.8 mg/g nutritional composition (about 9 to about 90 mg/100 kcal). In another embodiment, flavan-3-ols are present in an amount ranging from about 0.8 to about 2.5 mg/g nutritional composition (about 20 to about 60 mg/100 kcal).
[0188] In some embodiments, the nutritional composition of the present disclosure comprises flavanones. Non-limiting examples of suitable flavanones include butin, eriodictyol, hesperetin, hesperidin, homeriodictyol, isosakuranetin, naringenin, naringin, pinocembrin, poncirin, sakuranetin, sakuranin, steurbin. Plant sources rich in flavanones include, but are not limited to orange, tangerine, grapefruit, lemon, lime. The nutritional composition may be formulated to deliver between about 0.01 and about 150 mg flavanones per day.
[0189] Moreover, the nutritional composition may also comprise flavonols. Flavonols from plant or algae extracts may be used. Flavonols, such as ishrhametin, kaempferol, myricetin, quercetin, may be included in the nutritional composition in amounts sufficient to deliver between about 0.01 and 150 mg per day to a subject.
[0190] The phytonutrient component of the nutritional composition may also comprise phytonutrients that have been identified in human milk, including but not limited to naringenin, hesperetin, anthocyanins, quercetin, kaempferol, epicatechin, epigallocatechin, epicatechin-gallate, epigallocatechin-gallate or any combination thereof. In certain embodiments, the nutritional composition comprises between about 50 and about 2000 nmol/L epicatechin, between about 40 and about 2000 nmol/L epicatechin gallate, between about 100 and about 4000 nmol/L
epigallocatechin gallate, between about 50 and about 2000 nmol/L naringenin, between about 5 and about 500 nmol/L kaempferol, between about 40 and about 4000 nmol/L hesperetin, between about 25 and about 2000 nmol/L anthocyanins, between about 25 and about 500 nmol/L quercetin, or a mixture thereof.
Furthermore, the nutritional composition may comprise the metabolite(s) of a phytonutrient or of its parent compound, or it may comprise other classes of dietary phytonutrients, such as glucosinolate or sulforaphane.
[0191] In certain embodiments, the nutritional composition comprises carotenoids, such as lutein, zeaxanthin, astaxanthin, lycopene, beta-carotene, alpha- carotene, gamma-carotene, and/or beta-cryptoxanthin. Plant sources rich in carotenoids include, but are not limited to kiwi, grapes, citrus, tomatoes,
watermelons, papayas and other red fruits, or dark greens, such as kale, spinach, turnip greens, coflard greens, romaine lettuce, broccoli, zucchini, garden peas and Brussels sprouts, spinach, carrots.
[0192] Humans cannot synthesize carotenoids, but over 34 carotenoids have been identified in human breast milk, including isomers and metabolites of certain carotenoids. In addition to their presence in breast milk, dietary carotenoids, such as alpha and beta-carotene, lycopene, lutein, zeaxanthin, astaxanthin, and cryptoxanthin are present in serum of lactating women and breastfed infants. Carotenoids in general have been reported to improve cell-to-cell communication, promote immune function, support healthy respiratory health, protect skin from UV light damage, and have been linked to reduced risk of certain types of cancer, and all-cause mortality. Furthermore, dietary sources of carotenoids and/or polyphenols are absorbed by human subjects, accumulated and retained in breast milk, making them available to nursing infants. Thus, addition of phytonutrients to infant formulas or children's products would bring the formulas closer in composition and functionality to human milk.
[0193] Flavonoids, as a whole, may also be included in the nutritional composition, as flavonoids cannot be synthesized by humans. Moreover, flavonoids from plant or algae extracts may be useful in the monomer, dimer and/or polymer forms. In some embodiments, the nutritional composition comprises levels of the monomeric forms of flavonoids similar to those in human milk during the first three months of lactation. Although flavonoid aglycones (monomers) have been identified in human milk samples, the conjugated forms of flavonoids and/or their metabolites may also be useful in the nutritional composition. The flavonoids could be added in the following forms: free, glucuronides, methyl glucuronides, sulphates, and methyl sulphates.
[00194] The nutritional composition may also comprise isoflavonoids and/or isoflavones. Examples include, but are not limited to, genistein (genistin), daidzein (daidzin), glycitein, biochanin A, formononetin, coumestrol, irilone, orobol, pseudobaptigenin, anagyroidisoflavone A and B, calycosin, glycitein, irigenin, 5-0- methylgenistein, pratensein, prunetin, psi-tectorigenin, retusin, tectorigenin, iridin, ononin, puerarin, tectoridin, derrubone, luteone, wighteone, alpinumisoflavone, barbigerone, di-O-methylalpinumisoflavone, and 4'-methyl-alpinumisoflavone. Plant sources rich in isoflavonoids, include, but are not limited to, soybeans, psoralea, kudzu, lupine, fava, chick pea, alfalfa, legumes and peanuts. The nutritional composition may be formulated to deliver between about 0.01 and about 150 mg isoflavones and/or isoflavonoids per day.
[00195] In an embodiment, the nutritional composition(s) of the present disclosure comprises an effective amount of choline. Choline is a nutrient that is essential for normal function of cells. It is a precursor for membrane phospholipids, and it accelerates the synthesis and release of acetylcholine, a neurotransmitter involved in memory storage. Moreover, though not wishing to be bound by this or any other theory, it is believed that dietary choline and docosahexaenoic acid (DHA) act synergistically to promote the biosynthesis of phosphatidylcholine and thus help promote synaptogenesis in human subjects. Additionally, choline and DHA may exhibit the synergistic effect of promoting dendritic spine formation, which is important in the maintenance of established synaptic connections. In some embodiments, the nutritional composition(s) of the present disclosure includes an effective amount of choline, which is about 20 mg choline per 8 fl. oz. (236.6 mL) serving to about 100 mg per 8 fl. oz. (236.6 mL) serving.
[00196] Moreover, in some embodiments, the nutritional composition is nutritionally complete, containing suitable types and amounts of lipids,
carbohydrates, proteins, vitamins and minerals to be a subject's sole source of nutrition. Indeed, the nutritional composition may optionally include any number of proteins, peptides, amino acids, fatty acids, probiotics and/or their metabolic byproducts, prebiotics, carbohydrates and any other nutrient or other compound that may provide many nutritional and physiological benefits to a subject. Further, the nutritional composition of the present disclosure may comprise flavors, flavor enhancers, sweeteners, pigments, vitamins, minerals, therapeutic ingredients, functional food ingredients, food ingredients, processing ingredients or combinations thereof.
[0197] The present disclosure further provides a method for providing nutritional support to a subject. The method includes administering to the subject an effective amount of the nutritional composition of the present disclosure.
[0198] The nutritional composition may be expelled directly into a subject's intestinal tract. In some embodiments, the nutritional composition is expelled directly into the gut. In some embodiments, the composition may be formulated to be consumed or administered enterally under the supervision of a physician and may be intended for the specific dietary management of a disease or condition, such as celiac disease and/or food allergy, for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation.
[0 99] The nutritional composition of the present disclosure is not limited to compositions comprising nutrients specifically listed herein. Any nutrients may be delivered as part of the composition for the purpose of meeting nutritional needs and/or in order to optimize the nutritional status in a subject.
[0200] In some embodiments, the nutritional composition may be delivered to an infant from birth until a time that matches full-term gestation. In some
embodiments, the nutritional composition may be delivered to an infant until at least about three months corrected age. In another embodiment, the nutritional composition may be delivered to a subject as long as is necessary to correct nutritional deficiencies. In yet another embodiment, the nutritional composition may be delivered to an infant from birth until at least about six months corrected age. In yet another embodiment, the nutritional composition may be delivered to an infant from birth until at least about one year corrected age.
[0201] The nutritional composition of the present disclosure may be
standardized to a specific caloric content, it may be provided as a ready-to-use product, or it may be provided in a concentrated form.
[0202] In some embodiments, the nutritional composition of the present disclosure is a growing-up milk. Growing-up milks are fortified milk-based beverages intended for children over 1 year of age (typically from 1-3 years of age, from 4-6 years of age or from 1-6 years of age). They are not medical foods and are not intended as a meal replacement or a supplement to address a particular nutritional deficiency. Instead, growing-up milks are designed with the intent to serve as a complement to a diverse diet to provide additional insurance that a child achieves continual, daily intake of all essential vitamins and minerals, macronutrients plus additional functional dietary components, such as non-essential nutrients that have purported health-promoting properties.
[0203] The exact composition of a nutritional composition according to the present disclosure can vary from market-to-market, depending on local regulations and dietary intake information of the population of interest. In some embodiments, nutritional compositions according to the disclosure consist of a milk protein source, such as whole or skim milk, plus added sugar and sweeteners to achieve desired sensory properties, and added vitamins and minerals. The fat composition is typically derived from the milk raw materials. Total protein can be targeted to match that of human milk, cow milk or a lower value. Total carbohydrate is usually targeted to provide as little added sugar, such as sucrose or fructose, as possible to achieve an acceptable taste. Typically, Vitamin A, calcium and Vitamin D are added at levels to match the nutrient contribution of regional cow milk. Otherwise, in some
embodiments, vitamins and minerals can be added at levels that provide
approximately 20% of the dietary reference intake (DRI) or 20% of the Daily Value (DV) per serving. Moreover, nutrient values can vary between markets depending on the identified nutritional needs of the intended population, raw material
contributions and regional regulations.
[0204] In certain embodiments, the nutritional composition is hypoallergenic. In other embodiments, the nutritional composition is kosher. In still further embodiments, the nutritional composition is a non-genetically modified product. In an embodiment, the nutritional formulation is sucrose-free. The nutritional composition may also be lactose-free. In other embodiments, the nutritional composition does not contain any medium-chain triglyceride oil. In some
embodiments, no carrageenan is present in the composition. In other embodiments, the nutritional composition is free of all gums.
[0205] In some embodiments, the disclosure is directed to a staged nutritional feeding regimen for a pediatric subject, such as an infant or child, which includes a plurality of different nutritional compositions according to the present disclosure. Each nutritional composition comprises a hydrolyzed protein, at least one pre- gelatinized starch, and at least one pectin. In certain embodiments, the nutritional compositions of the feeding regimen may also include a source of long chain polyunsaturated fatty acid, at least one prebiotic, an iron source, a source of β- glucan, vitamins or minerals, lutein, zeaxanthin, or any other ingredient described hereinabove. The nutritional compositions described herein may be administered once per day or via several administrations throughout the course of a day.
[0206] Examples are provided to illustrate some embodiments of the nutritional composition of the present disclosure but should not be interpreted as any limitation thereon. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from the consideration of the specification or practice of the nutritional composition or methods disclosed herein. It is intended that the specification, together with the example, be considered to be exemplary only, with the scope and spirit of the disclosure being indicated by the claims which follow the example.
EXAMPLE 1
[0207] This example illustrates an embodiment of a nutritional composition according to the present disclosure.
Nutrient per 100 kcal
Protein (g) 3
Fat (g) 5.2
Carbohydrates (g) 10.5
Polar lipids (mg) 100
Prebiotic (g) 0.6
Lactoferrin (mg) 90
DHA (mg) 20
ARA (mg) 40
Vitamin A (IU) 1400
Vitamin D (IU) 300
Vitamin E (IU) 8
Vitamin K (meg) 20 Thiamin (meg) 300
Riboflavin (meg) 350
Vitamin B6 (meg) 225
Vitamin B12 (meg) 0.5
Niacin (meg) 5000
Folic acid (meg) 60
Panthothenic acid (meg) 2000
Biotin (meg) 6
Vitamin C (mg) 50
Choline (mg) 22
Calcium (mg) 175
Phosphorus (mg) 100
Sodium (mg) 55
Potassium (mg) 125
Chloride (mg) 100
Iodine (meg) 35
Iron (mg) 2.2
Zinc (mg) 1.7
Manganese (meg) 12
Copper (meg) 150
Selenium (meg) 5
Chromium (meg) 4
Molybdenum (meg) 3
Inositol (mg) 50
Carnitine (mg) 3
Taurine (mg) 10
Adenosine monophosphate (mg) 0.8
Cytidine monophosphate (mg) 4
Guanosine monophosphate (mg) 0.5
Uridine monophosphate (mg) 1 EXAMPLE 2
[0208] This example illustrates another embodiment of a nutritional composition according to the present disclosure.
Nutrient per 100 kcal
Protein (g) 2
Fat (g) 5
Carbohydrates (g) 11
Prebiotic (g) 0.6
Beta glucan (mg) 9
Polar lipids (mg) 100
Lactoferrin (mg) 90
Probiotic(s) (cfu) 1 x 108
DHA (mg) 22
ARA (mg) 40
Vitamin A (IU) 400
Vitamin D (IU) 75
Vitamin E (IU) 2
Vitamin K (meg) 12
Thiamin (meg) 120
Riboflavin (meg) 200
Vitamin B6 (meg) 100
Vitamin B12 (meg) 0.5
Niacin (meg) 1100
Folic acid (meg) 20
Panthothenic acid (meg) 600
Biotin (meg) 4
Vitamin C (mg) 18
Choline (mg) 30
Calcium (mg) 120
Phosphorus (mg) 60
Sodium (mg) 28
Potassium (mg) 140 Chloride (mg) 100
Iodine (meg) 22
Iron (mg) 2
Zinc (mg) 1.2
Manganese (meg) 25
Copper (meg) 100
Selenium (meg) 4
Inositol (mg) 8
Carnitine (mg) 3
Taurine (mg) 8
Adenosine monophosphate (mg) 1
Cytidine monophosphate (mg) 4
Guanosine monophosphate (mg) 0.8
Uridine monophosphate (mg) 1
EXAMPLE 3
[0209] Example 3 illustrates the effect of GOS and PDX on promoting stress resistance in Juvenile Fisher rats, and suggests that the composition of gut bacteria can modulate serotonergic circuits.
[0210] An important neural mechanism for protection against stress-induced anxiety and depression-like behavior is an increase in 5-HTiA receptor expression and function in the dorsal raphe nucleus (especially rostral DRN). The prebiotic diet including GOS and PDX produces stress resistant behavioral effects in mice.
[0211] Example 3 tested whether dietary GOS and PDX initiated during the juvenile period, would produce changes in brain serotonin receptors. Juvenile Fisher rats (PND 24, n=9/grp) were fed a diet containing GOS/PDX (7.0 g/kg each) for either 4 or 9 weeks. Rats were then either exposed to either inescapable tail shock stress (IS; 100 1.5m A tail shocks; a stressor that reliably produces anxiety-like behavior) or remained undisturbed in their home cages.
[0212] GOS/PDX treatment increased mRNA expression of 5-HT1 a receptors in the dorsal raphe nucleus (DRN), a brain region involved in regulating anxiety (See FIG. 1). These results show that GOS/PDX promote stress resistance in Juvenile Fisher rats, and suggest. that gut bacteria can modulate serotonergic circuits regulating mood.
EXAMPLE 4
[0213] Example 4 addressed a problem of constipation which affects up to 30% of children. In this double-blind, randomized, controlled trial, healthy 1-4 year old children were fed 3 servings/day of a cow's milk-based follow-up formula fortified with micronutrients, docosahexaenoic acid, yeast beta-glucan, and the prebiotics polydextrose (PDX) and galactooligosaccharides (GOS) (FF; n=125) or an unfortified cow's milk-based beverage (C; n=131) for 28 weeks. A child was considered constipated if at least 2 of the 3 following symptoms were present for at least 2 uninterrupted weeks: presence of hard stools, difficulty or pain to defecate, and an interval of more than 72 hours without defecation. The children who met these criteria were categorized by age at study entry (12-24 months or 25-48 months) and compared using the Fisher's exact test.
[0214] Twenty four of the 158 children in the 25-48 month age category (15%) met the criteria for constipation. Fewer children in this age category remained constipated at the end of the study in the FF group compared to the control group (See Table 1 ). Although the difference was not statistically significant (p=0.27), it was most likely due to the small number of participants in each formula group who met the criteria for constipation. These results suggest that dietary modification such as addition of the prebiotic fibers PDX and GOS may alleviate functional constipation in children.
Table 1. Constipation status at end of study
Constipation Status at End of Study
Figure imgf000052_0001
EXAMPLE 5
[0215] Example 5 focused on the effect of GOS/PDX and/or lactoferrin (LAC) on the beneficial microbiota, anxiety-like behavior and stress-induced inflammatory response in rats. In summary, diets containing GOS/PDX or combination of GOS/PDX plus LAC significantly increased Lactobacillus spp. and elevated Bifidobacterium spp. in the fecal samples from rats. The same diets also protected against the anxiety -like behavior produced by stress exposure.
[0216] This experiment tested whether GOS and PDX or combination of GOS/PDX plus LAC would attenuate anxiety and depressive-like behavior produced by stressor exposure. Juvenile Fisher rats (PND 24, n=9/grp) were fed a diet containing GOS/PDX (7.0 g/kg each) or GOS/PDX plus LAC (2.6 g/kg). Fecal samples were collected after four weeks on the diet and plated on Lactobacillus spp. and Bifidobacterium spp. specific media. Lactobacillus-specific M-RTLV agar (modified- rhamnose-2,3,5-triphenyltetrazolium chloride-LBS-vancomycin agar) was used as a selective medium to distinguish Lactobacillus spp. species. Bifidobacterium spp. was detected using Bifidobacterium Selective Agar (BIFIDO, Anaerobe Systems) containing Reinforced Clostridial Agar as the basal medium and Polymixin,
Kanamycin, and Nalidixic acid as selective agents. The differential compounds of iodoacetate and 2, 3, 5 - triphenyltetrazolium chloride were also added. Figures 1 and 2 show the significant increase of colony forming units (CFU) of Lactobacillus spp. and Lactobacillus rhamnosus 'm fecal samples collected from rats fed GOS/PDX or combination of GOS/PDX and LAC. (See. Figs. 2 and 3).
[0217] Rats were then either exposed to inescapable tail shock stress (IS; 100 1.5mA tail shocks; a stressor that reliably produces anxiety-like behavior) or remained undisturbed in their home cages (HCC). Subsequently, rats were tested for anxiety/depressive-like behavior using shock-elicited freezing and shuttle-box escape tests 24 h later. Plasma samples were immediately collected from a subset of mice after IS termination. Inflammatory molecule Hsp72 is normally increased by stressor exposure. As seen in FIG. 4, rats fed GOS/PDX showed a tendency to reduce stress- induced Hsp72 responses.
[0218] Anxiety/depressive-like behavior analysis: Briefly, after an 8 minute exploration period, rats were given two foot shocks (0.7 mA). Freezing, a behavioral measure of fear, was scored by a 10 second sampling procedure for 20 minutes. Previous exposure to stress produced exaggerated fear (anxiety) in the control diet rats only (Figure 3). Rats treated with GOS/PDX and/or LAC were protected from the exaggerated fear produced by 100 IS (p = 0.04). After freezing was scored for 20 minutes, rats received 25 foot shocks, escape from which was possible by crossing twice through the shuttle box door (FR-2 contingency). Previous exposure to stress produced a deficit in FR-2 escape latency in the control diet rats only. Rats treated with GOS/PDX and/or LAC were protected from the shuttle box escape deficit produced by 100 IS. (See FIG. 5; p = 0.04; See also FIG. 6).
[0219] Overall, feeding GOS/PDX or GOS/PDX plus lactoferrin increased Lactobacillus spp. within the gut and protected against the behavioral consequences of anxiety-like behavior produced by stress exposure.
[0220] All references cited in this specification, including without limitation, all papers, publications, patents, patent applications, presentations, texts, reports, manuscripts, brochures, books, internet postings, journal articles, periodicals, and the like, are hereby incorporated by reference into this specification in their entireties. The discussion of the references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.
[0221] Although embodiments of the disclosure have been described using specific terms, devices, and methods, such description is for illustrative purposes only. The words used are words of description rather than of limitation. It is to be understood that changes and variations may be made by those of ordinary skill in the art without departing from the spirit or the scope of the present disclosure, which is set forth in the following claims. In addition, it should be understood that aspects of the various embodiments may be interchanged in whole or in part. For example, while methods for the production of a commercially sterile liquid nutritional supplement made according to those methods have been exemplified, other uses are contemplated. Therefore, the spirit and scope of the appended claims should not be limited to the description of the versions contained therein.

Claims

CLAIMS What is claimed is:
1. A method for modulating serotonergic receptor expression in a pediatric subject, the method comprising administering to the pediatric subject a nutritional composition comprising:
a. up to about 7 g/100 kcal of a fat or lipid source, wherein the fat or lipid source comprises milk fat globules derived from an enriched lipid fraction of bovine milk;
b. at least about 15 mg/100 kcal of lactoferrin from a non-human source; and c. about 0.015 g/100 kcal to about 1.5 g/100 kcal of a prebiotic composition, wherein the prebiotic composition comprises poiydextrose, a galacto- oligosaccharide or a combination thereof.
2. The method of claim 1 , wherein the nutritional composition further comprises a source of long chain polyunsaturated fatty acids.
3. The method of claim 2, wherein the source of long chain polyunsaturated fatty acids includes at least one of docosahexaenoic acid, arachidonic acid, and
combinations thereof.
4. The method of claim 2, wherein the source of long chain polyunsaturated fatty acids is present from about 5 mg/100 kcal to about 75 mg/100 kcal.
5. The method of claim 1 , wherein the milk fat globules further comprise gangliosides and phospholipids.
6. The method of claim 1 , wherein lactoferrin is present at a level of about 10 mg/100 kcal to about 200 mg/100 kcal.
7. The method of claim 6, wherein the lactoferrin is bovine lactoferrin.
8. The method of claim 1 , wherein the nutritional composition further comprises a protein source.
9. The method of claim 1 , wherein poiydextrose and galactooligosaccharides comprise at least about 20% of the prebiotic composition.
10. The method of claim 1 , wherein the nutritional composition is an infant formula or a growing up milk.
11. A nutritional composition for reducing constipation in a pediatric subject, comprising: a. up to about 7 g/100 kcal of a fat or lipid source, wherein the fat or lipid source comprises milk fat globules derived from an enriched lipid fraction from bovine milk;
b. up to about 5 g/100 kcal of a protein source;
c. at least about 15 mg/100 kcal of lactoferrin from a non-human source; and d. about 0.015 g/100 kcal to about 1.5 g/100 kcal of a prebiotic composition, wherein the prebiotic composition comprises polydextrose,
galactooligosaccharide or a combination thereof.
12. The composition of claim 11, wherein the nutritional composition further comprises a source of long chain polyunsaturated fatty acids.
3. The composition of claim 12, wherein the source of long chain polyunsaturated fatty acids is present from about 5 mg/100 kcal to about 75 mg/100 kcal.
4. The composition of claim 11, wherein the average diameter of the milk fat globules range is at least about 2 pm.
15. The composition of claim 14, wherein the average diameter of the milk fat globules range is in the range of about 2 μιτι to about 13 μιτι.
16. The composition of claim 11 , wherein the specific surface area of the milk fat globules range is in the range of about 0.9 m2/g to about 3 mVg.
17. The composition of claim 11, wherein lactoferrin is present at a level of about 15 mg/100 kcal to about 300 mg/100 kcal.
18. The composition of claim 1 1 , wherein the lactoferrin is bovine lactoferrin.
19. The composition of claim 11 , wherein polydextrose and
galactooligosaccharides comprise at least about 20% of the prebiotic composition.
20. The composition of claim 1 1 , wherein the nutritional composition is an infant formula or a growing up milk.
PCT/US2015/054628 2014-11-07 2015-10-08 Nutritional compositions containing a prebiotic and lactoferrin and uses thereof WO2016073132A1 (en)

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CN201580060372.6A CN107072282A (en) 2014-11-07 2015-10-08 Alimentation composition and its application comprising prebiotics and lactoferrin
BR112017008747A BR112017008747A2 (en) 2014-11-07 2015-10-08 nutritional compositions containing a prebiotic and lactoferrin and their uses
EP15784863.1A EP3214951A1 (en) 2014-11-07 2015-10-08 Nutritional compositions containing a prebiotic and lactoferrin and uses thereof
SG11201702036QA SG11201702036QA (en) 2014-11-07 2015-10-08 Nutritional compositions containing a prebiotic and lactoferrin and uses thereof
CA2962136A CA2962136A1 (en) 2014-11-07 2015-10-08 Nutritional compositions containing a prebiotic and lactoferrin and uses thereof
AU2015343613A AU2015343613A1 (en) 2014-11-07 2015-10-08 Nutritional compositions containing a prebiotic and lactoferrin and uses thereof
MX2017005783A MX2017005783A (en) 2014-11-07 2015-10-08 Nutritional compositions containing a prebiotic and lactoferrin and uses thereof.
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