WO2024064327A1 - Human milk oligosaccharides for enhancing blood-brain barrier function - Google Patents

Human milk oligosaccharides for enhancing blood-brain barrier function Download PDF

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Publication number
WO2024064327A1
WO2024064327A1 PCT/US2023/033457 US2023033457W WO2024064327A1 WO 2024064327 A1 WO2024064327 A1 WO 2024064327A1 US 2023033457 W US2023033457 W US 2023033457W WO 2024064327 A1 WO2024064327 A1 WO 2024064327A1
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nutritional composition
oligosaccharide
blood
nutritional
amount
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PCT/US2023/033457
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French (fr)
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Rachael Buck
David Hill
Grace MELISI
Anice SABAG-DAIGLE
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Abbott Laboratories
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    • 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
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • 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
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/10—Foods or foodstuffs containing additives; Preparation or treatment thereof containing emulsifiers
    • 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
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
    • A23L29/256—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin from seaweeds, e.g. alginates, agar or carrageenan
    • 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
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • 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
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • 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
    • A23L33/15—Vitamins
    • 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
    • A23L33/15—Vitamins
    • A23L33/155—Vitamins A or D
    • 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
    • A23L33/16—Inorganic salts, minerals or trace elements
    • 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
    • A23L33/175—Amino acids
    • 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
    • A23L33/185—Vegetable 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
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins

Definitions

  • HMOs human milk oligosaccharides
  • the present disclosure relates to nutritional compositions comprising a blend of HMOs that can modulate or improve the health of an individual consumer by treating or preventing one or more symptoms of a disease or condition as described in greater detail herein.
  • BACKGROUND [0003] Breast milk containing HMOs has been associated with enhanced development and balanced growth and maturation of a variety of infant systems including respiratory, gastrointestinal, and immune systems, among others. There are more than 200 known oligosaccharides present in human milk, with various individual classes having been shown to promote health in a variety of ways. It appears that certain infections are prevented by HMOs due to their activity as pathogen receptor analogues (i.e., receptor mimics that prevent pathogenic adhesion).
  • breast milk includes HMOs that not only act as pathogen receptor analogues, but certain structures activate immune factors as well as acting as metabolic fuel in the gut microbiome to preferentially benefit certain beneficial bacteria.
  • HMOs that not only act as pathogen receptor analogues, but certain structures activate immune factors as well as acting as metabolic fuel in the gut microbiome to preferentially benefit certain beneficial bacteria.
  • the general inventive concepts are based, in part, on the surprising discovery that nutritional compositions including at least one fucosylated HMO, at least one sialylated HMO, and at least one N-acetylglucosamine HMO can provide substantial benefits to a consumer including, but not limited to, preventing or reducing damage or degradation to the blood-brain barrier, promoting healing, enhancing nutrient delivery to the brain, enhancing brain development in an infant (including improving cognition), toddler, or child, reducing the effects of neurodegenerative disorders, preventing metastasis of certain cancers, and treating one or more blood-brain barrier (BBB) related conditions or dysfunctions.
  • BBB blood-brain barrier
  • the nutritional compositions described herein include at least one HMO, and in certain instances, a blend of HMOs in combination with one or more nutritionally beneficial ingredients and are useful to treat disorders associated with the blood-brain barrier.
  • HMOs for use in the nutritional compositions, methods, and treatments include, but are not necessarily limited to: sialylated oligosaccharides e.g., 3′-sialyllactose (3′-SL) and 6′-sialyllactose (6′-SL); e.g., N-acetyl-glucosamine oligosaccharides lacto-N-neotetraose (LNnT) and lacto-N-tetraose (LNT); and fucosylated oligosaccharides e.g., 3-fucosyllactose (3-FL) and 2′-fucosyllactose (2FL).
  • HMOs may be present in the nutritional compositions, methods, and treatments in amount of about 0.001 mg/mL to about 20 mg/mL.
  • Aspects of the general inventive concepts are directed to a nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and an N- acetylglucosamine oligosaccharide for use in preventing degradation of barrier function of the blood-brain barrier in an individual in need thereof.
  • aspects of the general inventive concepts are directed to a nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N- acetylglucosamine oligosaccharide for use in treating one or more blood-brain barrier related conditions.
  • a nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N- acetylglucosamine oligosaccharide for use in enhancing cognition in an infant, toddler, or child.
  • aspects of the general inventive concepts are directed to a nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N- acetylglucosamine oligosaccharide for use in enhancing nutrient uptake in an individual in need thereof.
  • aspects of the general inventive concepts are directed to a nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N- acetylglucosamine oligosaccharide for use in enhancing brain chemistry.
  • Fig.1 shows an exemplary image of the component so the blood-brain barrier.
  • Fig.2 shows an exemplary experimental set-up to test BBB permeability.
  • Fig.3 is a graph showing changes in TEER in response to the HMO composition (i.e., 2′- FL, 3-FL, 3′-SL, 6′-SL, and LNT) over time.
  • Fig. 4A is a graph showing changes in 4 kDa FITC-dextran permeability of RBEC monolayers in response to DMNQ in the presence or absence of the HMO composition.
  • Fig.4B is a graph showing changes in 70 kDa TexasRed-dextran permeability of RBEC monolayers in response to DMNQ in the presence or absence of the HMO composition.
  • - 3 - 4859-9621-0305, v.1 [0019] Fig.
  • FIG. 5A is an image of immunofluorescence staining for localization of the claudin-5 protein in RBEC monolayers in control.
  • Fig.5B is an image showing immunofluorescence staining for localization of the claudin- 5 protein in RBEC monolayers with the HMO composition.
  • the nutritional compositions and methods described herein utilize one or more HMOs (e.g., at least one fucosylated HMO, at least one sialylated HMO, and at least one N-acetylated HMO) alone or in combination with long chain polyunsaturated fatty acids, antioxidants, and also including carotenoids, nucleotides, or both, for controlling, preventing, and/or treating a number of diseases and conditions as described in greater detail herein.
  • HMOs e.g., at least one fucosylated HMO, at least one sialylated HMO, and at least one N-acetylated HMO
  • the blood-brain barrier is a highly selective semipermeable membrane or border of cells that regulate the transfer of solutes and chemicals between the circulatory system and the central nervous system.
  • the barrier plays a vital role in early brain development and throughout life.
  • the barrier serves to protect the brain from harmful or unwanted substances in the blood.
  • the BBB is formed by endothelial cells of the capillary wall. The system allows the passage of some small molecules by passive diffusion, as well as the selective and active transport of various nutrients, ions, organic anions, and macromolecules such as glucose and amino acids that are crucial to proper neural development and function.
  • a properly functioning BBB restricts the passage of microorganisms, the diffusion of solutes, and large or hydrophilic molecules present in the blood into the cerebrospinal fluid, while allowing the diffusion of hydrophobic molecules (O2, CO2, hormones) and small non-polar molecules.
  • Cells of the barrier actively transport metabolic products such as glucose across the barrier.
  • the barrier also restricts the passage of peripheral immune factors, like signaling molecules, antibodies, and immune cells, into the CNS, thus insulating the brain from damage due to immune events elsewhere in the body.
  • HMO human milk oligosaccharide
  • exemplary non-limiting HMOs include neutral oligosaccharides (which include fucosylated oligosaccharides and N-acetyl-glucosamine oligosaccharides (e.g., also known as neutral non- fucosylated HMOs or N-acetylated)) and acidic oligosaccharides (which include sialylated oligosaccharides).
  • Specific non-limiting embodiments include: 2′-fucosyllactose (2′-FL), 3- fucosyllactose (3-FL), 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT).
  • HMO blend refers to a combination of fucosylated, sialylated, and N-acetyl-glucosamine HMOs, which may comprise at least two of 2’-FL, 3-FL, 3′-SL, 6′-SL, and LNT, and in certain instances all of 2′-FL, 3-FL, 3′-SL, 6′-SL, and LNT.
  • blood-brain barrier dysfunction refers to a set of conditions and diseases mainly stemming from and/or characterized by disordered or impaired function of the blood-brain barrier (BBB).
  • BBB dysfunction contributes to pathology in a range of neurological conditions including neurodegenerative disease (e.g., Alzheimer’s disease), multiple sclerosis, stroke, epilepsy, and cerebral palsy, among other BBB related conditions.
  • Blood-brain barrier dysfunction is characterized by relative increase in permeability of the BBB (i.e., the junctions between cells are not tight). This dysfunction allows for increased diffusion, including bidirectional diffusion, and less selectivity in passage of blood components, etc., thereby, this negatively impacts nutrient uptake, nutrient retention, and brain chemistry.
  • the term “nutrient uptake” as used herein refers to delivery of nutrients to the brain across the BBB.
  • Enhanced nutrient uptake refers to improved nutrient delivery or treating ineffective nutrient delivery.
  • the term enhanced as used in the present disclosure refers to an improvement in one or more of the associated markers related to the subject condition or disease.
  • Nutrient delivery/uptake can include metabolic substances (e.g., energy), vitamins, and or proper exchange of oxygen and carbon dioxide between the blood and brain tissue.
  • Enhanced nutrient delivery is seen both through a relative increase in desirable nutrient delivery and/or through a reduction in the passage of unwanted or harmful substances across the BBB.
  • Enhanced nutrient uptake or delivery can be shown through in vitro testing showing a reduction in the permeability of the cells making up an exemplary BBB.
  • BBB An important function of the BBB is the selective nature of the barrier, whereby, when functioning properly, essential - 5 - 4859-9621-0305, v.1 or desirable nutrients are transported or otherwise allowed to pass through the barrier and unwanted or harmful materials are excluded.
  • desirable components would include glucose, amino acids, ketone bodies, choline, antioxidant molecules, vitamin B, and purines.
  • undesirable components would be toxins, bacteria, viruses, prions, blood and immune factors such as thrombin, complement, fibrinogen, c-reactive protein (CRP).
  • Enhanced nutrient uptake also includes barrier function that prevents diffusion of desirable nutrients from passing back into the bloodstream after first crossing the BBB.
  • brain chemistry refers to the full assortment of compounds that are circulating in the blood in and around the brain.
  • One function of the BBB is to selectively separate compounds that are circulating in the blood from interacting with neuronal synapses in order to maintain brain chemistry homeostasis.
  • a properly functioning BBB will effectively protect brain chemistry homeostasis and allow for proper functioning of chemical messengers or neurotransmitters that signal between neurons in the brain.
  • blood-barrier integrity refers to the functional permeability of the BBB.
  • a major function of the BBB includes protection of the brain tissue through the functional and structural integrity of the tight junctions between the endothelial cells that make up the vasculature in the brain.
  • a properly functioning BBB includes less permeable junctions between the aforementioned endothelial cells.
  • Improved, enhanced, or treated blood- barrier integrity is generally shown through reduced permeability or a return of permeability from an increased state to a state of lower permeability and may be shown through in vitro testing.
  • blood-brain barrier related condition or “BBB related conditions” as used herein refers to conditions or diseases that are treated or impacted directly by the BBB function, including by either nutrient uptake, brain chemistry, or by the filtration aspect of BBB function (e.g., preventing cancer cells from migrating or preventing microbe infiltration).
  • the conditions are selected from: necrotizing enterocolitis, consequences of preterm birth, blood vessel damage, meningitis, hypoxia, traumatic brain injury, stroke, brain hemorrhage, compromised nervous systems, compromised neuronal blood flow, dysfunctional or impaired BBB, immaturity of the central nervous system, blood-brain barrier dysfunction, - 6 - 4859-9621-0305, v.1 neurodegenerative disease, cancer metastasis, and infectious diseases selected from: Group B Streptococcus, Listeria monocytogenes, Bacillus antracis, Haemophilus influenzae, Neisseria meningitidis, P.
  • the terms “fat” and “oil” as used herein, unless otherwise specified, are used interchangeably to refer to lipid materials derived or processed from plants or animals. These terms also include synthetic lipid materials so long as such synthetic materials are suitable for oral administration to humans.
  • shelf stable refers to a nutritional composition that remains commercially stable after being packaged and then stored at 18-24° C. for at least 3 months, including from about 3 months to about 36 months, including from about 3 months to about 24 months, and also including from about 3 months to about 18 months.
  • the terms “nutritional formulation” or “nutritional composition” as used herein, are used interchangeably and, unless otherwise specified, refer to synthetic formulas including nutritional liquids, nutritional powders, nutritional solids, nutritional semi-solids, nutritional semi-liquids, nutritional supplements, and any other nutritional food product as known in the art.
  • the nutritional powders may be reconstituted to form a nutritional liquid, all of which comprise one or more of fat, protein and carbohydrate and are suitable for oral consumption by a human.
  • the terms “nutritional formulation” or “nutritional composition” do not include human breast milk and do not refer to supplemented milk, whether of human origin or otherwise.
  • infant refers generally to individuals up to age 36 months of age, actual or corrected.
  • all concentrations expressed as “ ⁇ g/liter,” “mg/liter,” “mg/L,” “mcg/L,” “mg/mL” etc. refer to ingredient concentrations within the described nutritional compositions as calculated on an as-fed basis (e.g., reconstituted for consumption in the case of nutritional powders), unless otherwise specified.
  • a suitable amount of liquid typically water
  • a form of nutritional formulation that is not in its ready-to-drink liquid form, such as nutritional powder or a concentrated form of a nutritional liquid, thereby making the nutritional composition ready-to- drink.
  • the individual is at risk due to immaturity of the central nervous system, preterm birth, traumatic brain injury, stroke, infection, etc.
  • the term refers to those having a vulnerability higher than the general population. Vulnerability may not necessarily be due directly to dysfunction of the BBB, but may be due to being at greater risk to serious adverse consequences from dysfunction of the BBB, such as NEC, neurodegenerative disease, infections disease, cancer. - 8 - 4859-9621-0305, v.1 [0042]
  • modulating or “modulation” or “modulate” as used herein, unless otherwise specified, refer to the targeted movement of a selected characteristic or symptom. In certain instances, modulating refers to reducing BBB permeability.
  • an effective amount is intended to qualify the amount of e.g., HMO which will achieve the goal of modulating, preventing or treating a disease or that which will achieve the goal of decreasing the risk that the patient will suffer an adverse health event, including reducing or preventing one or more symptoms, while avoiding adverse side effects such as those typically associated with alternative therapies.
  • the effective amount may be administered in one or more doses.
  • an effective amount is the amount recommended to reduce permeability of the BBB, enhance nutrient uptake, or otherwise treat disfunction of the BBB.
  • the term “food products” as used herein refer to delivery vehicles that contain the HMOs and one or more of fats, amino nitrogen and carbohydrates and provides some or all of the nutritional support for a patient in the recommended daily amounts. Frequently a food product will contain vitamins, minerals, trace minerals and the like to provide balanced nutrition to meal replacements, medical foods, supplements.
  • the food products may be in any typical form such as beverages, powders, bars, juices, carbonated beverages, bottled water, etc.
  • infant formula or “synthetic infant formula” as used herein, unless otherwise specified, are used interchangeably and refer to liquid, solid, semi-solid, and semi-liquid human milk replacements or substitutes that are suitable for consumption by an infant.
  • the synthetic formulas include components that are of semi-purified or purified origin.
  • semi-purified or “purified” refer to a material that has been prepared by purification of a natural material or by synthesis.
  • infant formula or “synthetic infant formula” do not include human breast milk.
  • synthetic pediatric formula refers to liquid, solid, semi-solid, and semi-liquid human milk replacements or substitutes that are suitable for consumption by an infant or toddler up to the age of 36 months (3 years).
  • the synthetic formulas include components that are of semi-purified or purified origin.
  • the terms “semi-purified” or “purified” refer to a material that has been prepared by purification of a natural material or by synthesis.
  • the term “synthetic pediatric nutritional formula” does not include human breast milk.
  • the synthetic formulas include components that are of semi-purified or purified origin.
  • the terms “semi-purified” or “purified” refer to a material that has been prepared by purification of a natural material or by synthesis.
  • the term “synthetic child nutritional formula” does not include human breast milk.
  • human milk fortifier refers to liquid and solid nutritional products suitable for mixing with breast milk or preterm infant formula or infant formula for consumption by a preterm or term infant.
  • All percentages, parts and ratios as used herein, are by weight of the total composition, unless otherwise specified. All such weights, as they pertain to listed ingredients, are based on the active level and, therefore, do not include solvents or by-products that may be included in commercially available materials, unless otherwise specified.
  • Numerical ranges as used herein are intended to include every number and subset of numbers within that range, whether specifically disclosed or not.
  • the general inventive concepts contemplate a nutritional composition for use in preventing, treating, or reducing the effects/symptoms of one or more BBB-related conditions.
  • the HMO-containing nutritional compositions are for use in providing enhancement to BBB function, preventing degradation of the BBB (including reducing current or future degradation of BBB function), reducing permeability of the BBB, or otherwise for treatment of dysfunctional BBB (including repair of damaged BBB) of an individual in need thereof.
  • feeding or otherwise administering to an individual a nutritional composition comprising the HMO compositions described herein improves or enhances BBB function Including a return to proper functioning), or treats, prevents, or repairs at least one aspect of BBB dysfunction.
  • the nutritional composition for use according to the general inventive concepts is a liquid and comprises at least one human milk oligosaccharide selected from 2′-FL, 3-FL, 3′-SL, 6′-SL, LNnT, and LNT in an amount of from about 0.001 mg/mL to about 20 mg/mL, including from about 0.001 mg/mL to about 10 mg/mL, including from about 0.001 mg/mL to about 5 mg/mL, including from about 0.001 mg/mL to less than 2 mg/mL, and including from about 0.01 mg/mL to about 20 mg/mL, including from about 0.01 mg/mL to less than 10 mg/mL.
  • the nutritional composition is a liquid and comprises 2′-fucosyllactose, in an amount of from about 0.001 mg/mL to about 20 mg/mL, including from about 0.001 mg/mL to about 10 mg/mL, including from about 0.001 mg/mL to about 5 mg/mL, including from about 0.001 mg/mL to less than 2 mg/mL, including from about 0.01 mg/mL to about 20 mg/mL, and including from about 0.01 mg/mL to less than 2 mg/mL.
  • the nutritional composition for use comprises at least one and in certain instances each of 2′-FL, 3-FL, 3′-SL, 6′-SL, and LNT.
  • the nutritional composition for use comprises a combination of 2′-FL, 3-FL, 3′-SL, - 11 - 4859-9621-0305, v.1 6′-SL, and LNT in a total amount of HMO of from 1 mg/mL to about 8.2 mg/mL and the nutritional composition prevents, treats, or reduces the symptoms of, at least one of the following blood-brain barrier related conditions: necrotizing enterocolitis, consequences of preterm birth, blood vessel damage, meningitis, hypoxia, traumatic brain injury, stroke, brain hemorrhage, compromised nervous systems, compromised neuronal blood flow, dysfunctional or impaired BBB, immaturity of the central nervous system, blood-brain barrier dysfunction, neurodegenerative disease, cancer metastasis, and infectious diseases selected from: Group B Streptococcus, Listeria monocytogenes, Bacillus antracis, Haemophilus influenzae, Neisseria meningitidis, P.
  • the nutritional compositions for use provide a reduction of permeability of the BBB by from about 10% to about 50%, including 10% to 45%, including 10% to 40%, including 15% to 40%, including 20% to 40%, and including from 25% to about 40%.
  • blood vessel damage may occur through vascular risk factors, such as diabetes, hypertension, and dyslipidemia; environmental risk factors, such as pollution; or genetic risk factors, such as being a carrier of the APOE- ⁇ 4 gene (Alzheimer’s).
  • Blood vessel damage causes BBB dysfunction and decreased brain perfusion. Disruption of the BBB (tight junction integrity) has been associated with the accumulation of blood factors such as complement (an immune factor) in the brain which induces an immunological response.
  • necrotizing enterocolitis is known to increase the risk of brain injury and impaired neurodevelopment. Inflammation originating from the gastrointestinal tract in NEC circulates systemically. Exposure of the neurovascular cells to inflammatory factors - 12 - 4859-9621-0305, v.1 can lead to BBB dysfunction and altered brain chemistry or suboptimal nutrient uptake. This can have direct/immediate effects as well as long term consequences for the developing brain, which may account for the long-term neurocognitive consequences of NEC in some patients.
  • the young/developing brain is believed to be more vulnerable to toxins and disruption. Reducing the permeability (and/or returning the permeability to a normal state) of the BBB in infancy may help in development of the brain.
  • Reducing the permeability (and/or returning the permeability to a normal state) of the BBB in infancy may help in development of the brain.
  • the nutritional compositions according to the general inventive concepts include an HMO component comprising at least one HMO, and in many aspects, a combination of two or more HMOs, including neutral HMOs and acidic HMOs.
  • the composition of human milk oligosaccharides is very complex and more than 200 different oligosaccharide structures are known.
  • the HMOs may be included in the nutritional compositions alone, or in some embodiments, in combination with other functional components (LCPUFAs, antioxidants, nucleotides, etc.) to aid in ameliorating symptoms of the particular condition or disease as described herein.
  • the HMO or HMOs may be isolated or enriched from milk(s) secreted by mammals including, but not limited to: human, bovine, ovine, porcine, or caprine species.
  • the HMOs may also be produced via microbial fermentation, enzymatic processes, chemical synthesis, or combinations thereof.
  • Suitable HMOs for use in the nutritional compositions and HMO components may include neutral oligosaccharides, acidic HMOs, and n-acetyl glucosylated oligosaccharides, and HMO precursors. HMOs may also be characterized as fucosylated, sialylated, and N-acetyl- glucosamine. Specific non-limiting examples of HMOs that may be included individually or in combination in the compositions according to the general inventive concepts include: 2′-FL, 3- FL, 3′-SL, 6′-SL, LNnT, and LNT.
  • HMOs that may be included in certain embodiments include: N-acetylglucosamine (GlcNAc); L-fucose (L-Fuc); D-fucose (D-Fuc); fucosyl oligosaccharides (i.e., Lacto-N- fucopentaose I; Lacto-N-fucopentaose II; Lacto-N-fucopentaose III; Lacto-N-difucohexaose I; and Lactodifucotetraose); non-fucosylated, non-sialylated oligosaccharides (i.e., Lacto-N- neotetraose); sialyl fucosyl oligosaccharides (i.e., 3′-Sialyl-3-fucosyllactose; Disialomonofucosyllacto-N-neohe
  • HMOs lacto-N-fucopentaose V, lacto- - 14 - 4859-9621-0305, v.1 N-hexaose, para-lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-neohexaose, monofucosyllacto-N-hexaose II, isomeric fucosylated lacto-N-hexaose (1), isomeric fucosylated lacto-N-hexaose (3), isomeric fucosylated lacto-N-hexaose (2), difucosyl-para-lacto-N- neohexaose, difucosyl-para-lacto-N-hexaose, difucosyllacto-N-hexaose, lacto
  • the HMOs are present in the nutritional compositions in total amounts of HMO in the composition (mg of HMO per mL of liquid composition) of at least about 0.001 mg/mL to about 20 mg/mL, including about 0.001 mg/mL to about 20 mg/mL, about 0.01 mg/mL to about 20 mg/mL, about 0.1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 10 mg/mL, about 2 mg/mL to about 10 mg/mL, and including from about 2.5 mg/mL to about 8.5 mg/mL.
  • the amount of HMO in the nutritional composition will depend on the specific HMO or HMOs present and the amounts of other components in the nutritional compositions.
  • the nutritional composition includes at least one of 2′-FL, 3-FL, 3′-SL, 6′-SL, and LNT in a total amount of HMO of from about 0.001 mg/mL to about 20 mg/mL, including from about 0.01 mg/mL to about 20 mg/mL, including from about 0.1 mg/mL to about 20 mg/mL, about 0.5 mg/mL to about 20 mg/mL, about 1 mg/mL to about 20 mg/mL, about 2 mg/mL to about 20 mg/mL.
  • the nutritional composition includes at least one of 2′-FL, 3-FL, 3′-SL, 6′-SL, and LNT in a total amount of HMO of from about 0.1 g/L to about 20 mg/mL, including from about 0.1 mg/mL to about 15 mg/mL, including from about 0.1 mg/mL to about 10 mg/mL, including from about 0.1 mg/mL to about 9 mg/mL, including from about 0.1 mg/mL to about 8 mg/mL, including from about 0.1 mg/mL to about 7 mg/mL, including from about 0.1 mg/mL to about 6 - 15 - 4859-9621-0305, v.1 mg/mL, including from about 0.1 mg/mL to about 5 mg/mL, including from about 0.1 mg/mL to about 4 mg/mL, including from about 0.1 mg/mL to
  • the nutritional composition comprises at least one of a fucosylated HMO, a N-acetylated HMO, and/or a sialylated HMO.
  • the nutritional composition comprises a combination of a fucosylated HMO, a sialylated HMO, and an N-acetylated HMO.
  • suitable HMOs include 2′-FL, 3-FL, 3′-SL, 6′-SL, LNnT, and LNT.
  • the nutritional composition comprises a combination of 2′-FL, 3-FL, LNT, 3′-SL, and 6’-SL or the nutritional composition comprises a combination of 2′-FL, 3-FL, LNnT, 3′-SL, and 6′-SL.
  • the nutritional composition comprises 2’-FL in an amount up to 4.15 mg/mL, LNT in an amount up to 2.11 mg/mL, 3-FL in an amount up to 1.17 mg/mL, 3’-SL in an amount up to 0.36 mg/mL, and 6’-SL in an amount up to 0.44 mg/mL.
  • the nutritional composition may include LCPUFAs to provide nutritional support, as well as to enhance growth and functional development of the nervous system.
  • the nutritional composition includes a combination of one or more HMOs and one or more LCPUFAs such that the composition provides a benefit to the end user, such as an improved benefit in modulating or treating one or more of the disease symptoms described herein.
  • Exemplary LCPUFAs for use in the nutritional compositions include, for example, ⁇ -3 LCPUFAs and ⁇ -6 LCPUFAs.
  • LCPUFAs include docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), arachidonic acid (ARA), linoleic acid, linolenic acid (alpha linolenic acid) and gamma-linolenic acid derived from oil sources such as plant oils, marine plankton, fungal oils, and fish oils.
  • the LCPUFAs are derived from fish oils such as menhaden, salmon, anchovy, cod, halibut, tuna, or - 16 - 4859-9621-0305, v.1 herring oil.
  • Particularly preferred LCPUFAs for use in the nutritional compositions with the HMOs include DHA, ARA, EPA, DPA, and combinations thereof.
  • the content of LCPUFAs preferably does not exceed 3% by weight of the total fat content, including below 2% by weight of the total fat content, and including below 1% by weight of the total fat content in the nutritional composition.
  • the LCPUFA may be provided as free fatty acids, in triglyceride form, in diglyceride form, in monoglyceride form, in phospholipid form, in esterified form or as a mixture of one or more of the above, preferably in triglyceride form.
  • the nutritional compositions as described herein will typically comprise total concentrations of ARA, DHA, EPA, and DPA of from about 0.001 g/L to about 1 g/L or more, including from about 0.01 g/L to about 1 g/L, and about 0.1 g/L to about 1 g/L.
  • the nutritional compositions include total long chain ⁇ -6 fatty acids in a concentration of from about 100 to about 425 mg/L or from about 12 to about 53 mg per 100 kcals and/or further include total long chain ⁇ -3 fatty acids in a concentration of from about 40 to about 185 mg/L or from about 5 to about 23 mg per 100 kcals.
  • the ratio of long chain ⁇ -6 fatty acids to long chain ⁇ -3 fatty acids in the nutritional compositions ranges from about 2:1 to about 3:1, preferably about 2.5:1.
  • the nutritional compositions include DHA in a concentration of from about 0.025 mg/mL to about 0.130 mg/mL or from about 3 to about 16 mg per 100 kcals.
  • the nutritional compositions include ARA in a concentration of from about 0.080 mg/mL to about 0.250 mg/mL or from about 10 to about 31 mg per 100 kcals.
  • the nutritional compositions include combinations of DHA and ARA such that the ratio of DHA to ARA ranges from about 1:4 to about 1:2.
  • Antioxidants [0078]
  • the nutritional compositions may comprise one or more antioxidants to provide nutritional support.
  • the nutritional composition includes a combination of HMOs and antioxidants such that the composition provides a benefit to the end user, such as an improved benefit in modulating or treating one or more of the disease symptoms described - 17 - 4859-9621-0305, v.1 herein.
  • the HMO or HMOs is used in combination with carotenoids (and specifically lutein, beta-carotene, zeaxanthin and/or lycopene) to provide the synergistic effect.
  • any antioxidants suitable for oral administration may be included for use in the nutritional compositions of the present disclosure, including, for example, vitamin A, vitamin E, vitamin C, retinol, tocopherol, and carotenoids, including lutein, beta-carotene, zeaxanthin, and lycopene, and combinations thereof, for example.
  • the antioxidants for use in the nutritional compositions may be used with the HMOs, alone, or in combination with HMOs and LCPUFAs and/or nucleotides.
  • the antioxidants for use in the nutritional compositions include carotenoids.
  • the carotenoids are lutein, lycopene, zeaxanthin and/or beta-carotene.
  • the nutritional compositions may comprise at least one of lutein, lycopene, zeaxanthin, and beta-carotene to provide a total amount of carotenoid of from about 0.001 ⁇ g/mL to about 10 ⁇ g/mL.
  • the nutritional compositions may comprise lutein in an amount of from about 0.001 ⁇ g/mL to about 10 ⁇ g/mL, including from about 0.001 ⁇ g/mL to about 5 ⁇ g/mL, including from about 0.001 ⁇ g/mL to about 0.0190 ⁇ g/mL, including from about 0.001 ⁇ g/mL to about 0.0140 ⁇ g/mL, and also including from about 0.044 ⁇ g/mL to about 5 ⁇ g/mL of lutein.
  • the nutritional compositions comprise from about 0.001 ⁇ g/mL to about 10 ⁇ g/mL, including from about 0.001 ⁇ g/mL to about 5 ⁇ g/mL, including from about 0.001 ⁇ g/mL to about 0.0130 ⁇ g/mL, including from about 0.001 ⁇ g/mL to about 0.0075 ⁇ g/mL, and also including from about 0.0185 ⁇ g/mL to about 5 ⁇ g/mL of lycopene.
  • the nutritional compositions comprise from about 1 ⁇ g/mL to about 10 ⁇ g/mL, including from about 1 ⁇ g/mL to about 5 ⁇ g/mL, including from about 0.001 ⁇ g/mL to about 0.025 ⁇ g/mL, including from about 0.001 ⁇ g/mL to about 0.011 ⁇ g/mL, and also including from about 0.034 ⁇ g/mL to about 5 ⁇ g/mL of beta-carotene. Any combination of these amounts of beta-carotene, lutein, zeaxanthin, and lycopene can be included in the nutritional compositions. Other carotenoids may optionally be included in the nutritional compositions.
  • any one or all of the carotenoids included in the nutritional compositions may be from a natural source, or artificially synthesized.
  • RRR-Alpha-Tocopherol - 18 - 4859-9621-0305, v.1 The nutritional compositions described herein may include RRR-alpha-tocopherol in combination with the HMOs to provide nutritional support, as well as to provide an improved benefit to the end user, such as a benefit in modulating or treating one or more of the disease symptoms described herein.
  • the nutritional compositions contain RRR-alpha-tocopherol in concentrations of at least about 3 mg/L, including at least about 5 mg/L, including at least about 7 mg/L, including at least about 8 mg/L, including at least about 9 mg/L, including at least about 10 mg/L, including from at least about 5 mg/L to about 100 mg/L, including from at least about 7 mg/L to about 50 mg/L, including from about 7 mg/L to about 40 mg/L, and including from about 7 mg/L to about 20 mg/L.
  • the total amounts of RRR- alpha-tocopherol include both exogenous and inherent sources of RRR-alpha-tocopherol, as noted below.
  • RRR-alpha-tocopherol refers both to exogenous sources and inherent sources of RRR-alpha-tocopherol and RRR-alpha-tocopherol acetate that are present in a nutritional composition.
  • Inherent sources include RRR-alpha-tocopherol that is inherently present in components that are present in a nutritional composition and may include for example, various oils and fats.
  • Exogenous sources of RRR-alpha-tocopherol include RRR-alpha- tocopherol that is added to the nutritional composition not as part of another component.
  • the nutritional compositions may comprise nucleotides and/or nucleotide precursors selected from nucleosides, purine bases, pyrimidine bases, ribose, and deoxyribose.
  • the nucleotide may be in monophosphate, diphosphate, or triphosphate form.
  • the nucleotide may be a ribonucleotide or a deoxyribonucleotide.
  • the nucleotides may be monomeric, dimeric, or polymeric (including RNA and DNA).
  • the nucleotide may be present in the nutritional composition as a free acid or in the form of a salt, preferably a monosodium salt.
  • the nutritional composition includes a combination of HMOs and nucleotides such that the composition provides a benefit to the end user, such as a benefit in modulating or treating one or more of the disease symptoms described herein.
  • Suitable nucleotides and/or nucleosides for use in the nutritional compositions include one or more of cytidine 5'-monophosphate, uridine 5'-monophosphate, adenosine 5'- monophosphate, guanosine 5'-1-monophosphate, and/or inosine 5'-monophosphate, such as cytidine 5'-monophosphate, uridine 5'-monophosphate, adenosine 5'-monophosphate, guanosine 5'-monophosphate, and inosine 5'-monophosphate.
  • the nucleotides are present in the nutritional compositions in total amounts of nucleotides of at least about 5 mg/L, including at least about 10 mg/L, including from about 10 mg/L to about 300 mg/L, including from about 42 mg/L to about 200 mg/L, including from about 72 mg/L to about 200 mg/L, including from about 72 mg/L to about 120 mg/L and including at least about 72 mg/L of the nutritional product.
  • the nutritional compositions may be formulated to include at least one of protein, fat, and carbohydrate. In many embodiments, the nutritional compositions will contain an HMO or HMOs and comprise at least one of fat, protein, or carbohydrate, including protein, fat, and carbohydrate.
  • total concentrations or amounts of the fat, protein, and carbohydrates may vary depending upon the product type (i.e., human milk fortifier, preterm infant formula, infant formula, etc.), product form (i.e., nutritional solid, powder, ready-to-feed liquid, concentrated liquid, or nutritional bar) and targeted dietary needs of the intended user, such concentrations or amounts most typically fall within one of the following embodied ranges, inclusive of any other essential fat, protein, and/or carbohydrate ingredients as described herein.
  • carbohydrate concentrations most typically range from about 5% to about 40%, including from about 7% to about 30%, including from about 10% to about 25%, by weight; fat concentrations most typically range from about 1% to about 30%, including from about 2% to about 15%, and also including from about 3% to about 10%, by weight; and protein concentrations most typically range from about 0.5% to about 30%, including from about 1% to about 15%, and also including from about 2% to about 10%, by weight.
  • carbohydrate concentrations most typically range from about 10% to about 75%, including from about 10% to about 50%, including from about - 20 - 4859-9621-0305, v.1 20% to about 40%, by weight of the human milk fortifier; fat concentrations most typically range from about 10% to about 40%, including from about 15% to about 37%, and also including from about 18% to about 30%, by weight of the human milk fortifier; and protein concentrations most typically range from about 5% to about 40%, including from about 10% to about 30%, and also including from about 15% to about 25%, by weight of the human milk fortifier.
  • liquid infant formulas both ready-to-feed and concentrated liquids
  • the protein component may comprise from about 7.5% to about 25% of the caloric content of the formula
  • the carbohydrate component may comprise from about 35% to about 50% of the total caloric content of the infant formula
  • the fat component may comprise from about 30% to about 60% of the total caloric content of the infant formula.
  • the protein component is present in an amount of from about 5% to about 35%, including from about 8% to about 12%, and including from about 10% to about 12% by weight of the preterm or term infant formula;
  • the fat component is present in an amount of from about 10% to about 35%, including from about 25% to about 30%, and including from about 26% to about 28% by weight of the preterm or term infant formula;
  • the carbohydrate component is present in an amount of from about 30% to about 85%, including from about 45% to about 60%, including from about 50% to about 55% by weight of the preterm or term infant formula.
  • the nutritional compositions may comprise a source or sources of fat (in addition to the LCPUFAs discussed above).
  • Suitable additional sources of fat for use herein include any fat or fat source that is suitable for use in an oral nutritional product and is compatible with the essential elements and features of such products.
  • the additional fat is derived from long chain polyunsaturated fatty acids and/or short chain fatty acids.
  • suitable fats or sources thereof for use in the nutritional products described herein include coconut oil, fractionated coconut oil, soybean oil, corn oil, olive oil, safflower oil, high oleic safflower oil, oleic acids (EMERSOL 6313 OLEIC ACID, Cognis Oleochemicals, Malaysia), MCT oil (medium chain triglycerides), sunflower oil, high oleic sunflower oil, palm and palm kernel oils, palm olein, canola oil, marine oils, fish oils, fungal oils, algae oils, cottonseed oils, and combinations thereof.
  • the nutritional compositions may comprise a source or sources of protein.
  • suitable proteins or sources thereof for use in the nutritional products include hydrolyzed, partially hydrolyzed or non-hydrolyzed proteins or protein sources, which may be derived from any known or otherwise suitable source such as milk (e.g., casein, whey), animal (e.g., meat, fish), cereal (e.g., rice, corn), vegetable (e.g., soy) or combinations thereof.
  • milk e.g., casein, whey
  • animal e.g., meat, fish
  • cereal e.g., rice, corn
  • vegetable e.g., soy
  • Non-limiting examples of such proteins include milk protein isolates, milk protein concentrates as described herein, casein protein isolates, extensively hydrolyzed casein, whey protein, sodium or calcium caseinates, whole cow milk, partially or completely defatted milk, soy protein isolates, soy protein concentrates, and so forth.
  • the nutritional compositions include a protein source derived from milk proteins of human and/or bovine origin.
  • Carbohydrate [0098] The nutritional composition may comprise, in addition to the HMOs, any carbohydrates that are suitable for use in an oral nutritional product and are compatible with the essential elements and features of such products.
  • Non-limiting examples of suitable carbohydrates or sources thereof for use in the nutritional products described herein may include maltodextrin, hydrolyzed or modified starch or cornstarch, glucose polymers, corn syrup, corn syrup solids, rice-derived carbohydrates, pea- derived carbohydrates, potato-derived carbohydrates, tapioca, sucrose, glucose, fructose, lactose, high fructose corn syrup, honey, sugar alcohols (e.g., maltitol, erythritol, sorbitol), artificial - 22 - 4859-9621-0305, v.1 sweeteners (e.g., sucralose, acesulfame potassium, stevia) and combinations thereof.
  • a particularly desirable carbohydrate is a low dextrose equivalent (DE) maltodextrin.
  • the nutritional compositions may comprise other optional ingredients that may modify the physical, chemical, aesthetic or processing characteristics of the products or serve as pharmaceutical or additional nutritional components when used in the targeted population. Many such optional ingredients are known or otherwise suitable for use in medical food or other nutritional products or pharmaceutical dosage forms and may also be used in the compositions herein, provided that such optional ingredients are safe for oral administration and are compatible with the essential and other ingredients in the selected product form.
  • Non-limiting examples of such optional ingredients include preservatives, emulsifying agents, buffers, fructooligosaccharides, galactooligosaccharides, polydextrose, and other prebiotics (e.g., inulin, oligofructose, polydextrose, pectin hydrolysate, and gums), probiotics (e.g., B. animalis subsp. lactis BB-12, B. lactis HN019, B. lactis Bi07, L. rhamnosus GG, L. rhamnosus HN001, L. acidophilus LA-5, L. acidophilus NCFM, L. fermentum CECT5716, B. longum BB536, B.
  • prebiotics e.g., inulin, oligofructose, polydextrose, pectin hydrolysate, and gums
  • probiotics e.g., B. animalis subsp. lactis BB-12
  • the nutritional compositions may further comprise a sweetening agent, preferably including at least one sugar alcohol such as maltitol, erythritol, sorbitol, xylitol, mannitol, isomalt, and lactitol, and also preferably including at least one artificial or high potency sweetener such as acesulfame K, aspartame, sucralose, saccharin, stevia, and tagatose.
  • a sweetening agent preferably including at least one sugar alcohol such as maltitol, erythritol, sorbitol, xylitol, mannitol, isomalt, and lactitol
  • an artificial or high potency sweetener such as acesulfame K, aspartame, sucralose, saccharin, stevia, and tagatose.
  • sweetener combinations are especially effective in masking undesirable flavors sometimes associated with the addition of vegetable proteins to a liquid beverage.
  • Optional sugar alcohol concentrations in the nutritional product may range from at least 0.01%, including from about 0.1% to about 10%, and also including from about 1% to about 6%, by weight of the nutritional product.
  • Optional artificial sweetener concentrations may range from about 0.01%, including from about 0.05% to about 5%, also including from about 0.1% to about 1.0%, by weight of the nutritional product.
  • a flowing agent or anti-caking agent may be included in the nutritional compositions as described herein to retard clumping or caking of the powder over time and to make a powder embodiment flow easily from its container.
  • Any known flowing or anti-caking agents that are known or otherwise suitable for use in a nutritional powder or product form are suitable for use herein, non-limiting examples of which include tricalcium phosphate, silicates, and combinations thereof.
  • concentration of the flowing agent or anti-caking agent in the nutritional composition varies depending upon the product form, the other selected ingredients, the desired flow properties, and so forth, but most typically range from about 0.1% to about 4%, including from about 0.5% to about 2%, by weight of the nutritional composition.
  • a stabilizer may also be included in the nutritional compositions. Any stabilizer that is known or otherwise suitable for use in a nutritional composition is also suitable for use herein, some non-limiting examples of which include gums such as xanthan gum.
  • the stabilizer may represent from about 0.1% to about 5.0%, including from about 0.5% to about 3%, including from about 0.7% to about 1.5%, by weight of the nutritional composition.
  • the nutritional compositions may further comprise any of a variety of other vitamins or related nutrients, non-limiting examples of which include vitamin A, vitamin D, vitamin K, thiamine, riboflavin, pyridoxine, vitamin B12, carotenoids (e.g., beta-carotene, zeaxanthin, lutein, lycopene), niacin, folic acid, pantothenic acid, biotin, vitamin C, choline, inositol, salts and derivatives thereof, and combinations thereof.
  • vitamins or related nutrients non-limiting examples of which include vitamin A, vitamin D, vitamin K, thiamine, riboflavin, pyridoxine, vitamin B12, carotenoids (e.g., beta-carotene, zeaxanthin, lutein, lycopene),
  • the nutritional compositions may further comprise any of a variety of other additional minerals, non-limiting examples of which include calcium, phosphorus, magnesium, iron, zinc, manganese, copper, sodium, potassium, molybdenum, chromium, chloride, and combinations thereof.
  • Methods of Manufacture [00107] The nutritional composition may be prepared by any known or otherwise effective manufacturing technique for preparing the selected product solid or liquid form. Many such techniques are known for any given product form such as nutritional liquids, powders, and nutritional bars, and can easily be applied by one of ordinary skill in the art to the nutritional compositions described herein.
  • the nutritional composition can be prepared by any of a variety of known or otherwise effective formulation or manufacturing methods.
  • a protein-in-fat (PIF) slurry e.g., canola oil, corn oil, etc.
  • CHO-MIN carbohydrate-mineral
  • PIW protein-in-water
  • the PIF slurry is formed by heating and mixing the oil (e.g., canola oil, corn oil, etc.) and then adding an emulsifier (e.g., lecithin), fat soluble vitamins, and a portion of the total protein (e.g., milk protein concentrate, etc.) with continued heat and agitation.
  • an emulsifier e.g., lecithin
  • a portion of the total protein e.g., milk protein concentrate, etc.
  • the CHO-MIN slurry is formed by adding with heated agitation to water: minerals (e.g., potassium citrate, dipotassium phosphate, sodium citrate, etc.), trace and ultra trace minerals (TM/UTM premix), thickening or suspending agents (e.g. avicel, gellan, carrageenan).
  • minerals e.g., potassium citrate, dipotassium phosphate, sodium citrate, etc.
  • trace and ultra trace minerals e.g. avicel, gellan, carrageenan
  • thickening or suspending agents e.g. avicel, gellan, carrageenan
  • additional minerals e.g., potassium chloride, magnesium carbonate, potassium iodide, etc.
  • carbohydrates e.g., HMOs, fructooligosaccharide, sucrose, corn syrup, etc.
  • the PIW slurry is then formed by mixing with heat and agitation the remaining protein, if any.
  • the resulting slurries are then blended together with heated agitation and the pH adjusted to 6.6-7.0, after which the composition is subjected to high-temperature short-time (HTST) processing during which the composition is heat treated, emulsified and homogenized, and then allowed to cool.
  • HTST high-temperature short-time
  • Water soluble vitamins and ascorbic acid are added, the pH is adjusted to the desired range if necessary, flavors are added, and water is added to achieve the desired total solid level.
  • the composition is then aseptically packaged to form an aseptically packaged - 25 - 4859-9621-0305, v.1 nutritional emulsion.
  • the nutritional solid such as a spray dried nutritional powder or drymixed nutritional powder, may be prepared by any collection of known or otherwise effective technique, suitable for making and formulating a nutritional powder.
  • the spray drying step may likewise include any spray drying technique that is known for or otherwise suitable for use in the production of nutritional powders.
  • One method of preparing the spray dried nutritional powder comprises forming and homogenizing an aqueous slurry or liquid comprising predigested fat, and optionally protein, carbohydrate, and other sources of fat, and then spray drying the slurry or liquid to produce a spray dried nutritional powder.
  • the method may further comprise the step of spray drying, drymixing, or otherwise adding additional nutritional ingredients, including any one or more of the ingredients described herein, to the spray dried nutritional powder.
  • Other suitable methods for making nutritional products are described, for example, in U.S. Pat.
  • the nutritional composition may be formulated and administered in any known or otherwise suitable oral product form. Any solid, liquid, semi-solid, semi-liquid, or powder product form, including combinations or variations thereof, are suitable for use herein, provided that such forms allow for safe and effective oral delivery to the individual of the ingredients as also defined herein.
  • the composition may be formulated as dietary product forms, which are defined herein as those embodiments comprising the ingredients of the present disclosure in a product form that - 26 - 4859-9621-0305, v.1 then contains at least one of fat, protein, and carbohydrate, and preferably also contains vitamins, minerals, or combinations thereof.
  • the nutritional compositions may be formulated with sufficient kinds and amounts of nutrients to provide a sole, primary, or supplemental source of nutrition, or to provide a specialized nutritional product for use in individuals afflicted with specific conditions or with a targeted nutritional benefit as described below.
  • Nutritional liquids include both concentrated and ready-to-feed nutritional liquids. These nutritional liquids are most typically formulated as suspensions or emulsions, although other liquid forms are within the scope of the present disclosure.
  • Nutritional emulsions suitable for use may be aqueous emulsions comprising proteins, fats, and carbohydrates.
  • emulsions are generally flowable or drinkable liquids at from about 1°C to about 25°C and are typically in the form of oil-in-water, water-in-oil, or complex aqueous emulsions, although such emulsions are most typically in the form of oil-in-water emulsions having a continuous aqueous phase and a discontinuous oil phase.
  • the nutritional emulsions may be and typically are shelf stable.
  • the nutritional emulsions typically contain up to 95% by weight of water, including from about 50% to 95%, also including from about 60% to about 90%, and also including from about 70% to about 85%, of water by weight of the nutritional emulsions.
  • the nutritional emulsions may have a variety of product densities, but most typically have a density greater than 1.03 g/mL, including greater than 1.04 g/mL, including greater than 1.055 g/mL, including from about 1.06 g/mL to about 1.12 g/mL, and also including from about 1.085 g/mL to about 1.10 g/mL.
  • the nutritional emulsions may have a caloric density tailored to the nutritional needs of the ultimate user, although in most instances the emulsions comprise generally at least 19 kcal/fl - 27 - 4859-9621-0305, v.1 oz (660 kcal/liter), more typically from about 20 kcal/fl oz (675-680 kcal/liter) to about 25 kcal/fl oz (820 kcal/liter), even more typically from about 20 kcal/fl oz (675-680 kcal/liter) to about 24 kcal/fl oz (800-810 kcal/liter).
  • the 22-24 kcal/fl oz formulas are more commonly used in preterm or low birth weight infants, and the 20-21 kcal/fl oz (675-680 to 700 kcal/liter) formulas are more often used in term infants.
  • the emulsion may have a caloric density of from about 50-100 kcal/liter to about 660 kcal/liter, including from about 150 kcal/liter to about 500 kcal/liter. In some specific embodiments, the emulsion may have a caloric density of 25, or 50, or 75, or 100 kcal/liter.
  • the nutritional emulsion may have a pH ranging from about 3.5 to about 8 but are most advantageously in a range of from about 4.5 to about 7.5, including from about 5.5 to about 7.3, including from about 6.2 to about 7.2.
  • the serving size for the nutritional emulsion can vary depending upon a number of variables, a typical serving size is generally at least 1 mL, or even at least 2 mL, or even at least 5 mL, or even at least 10 mL, or even at least 25 mL, including ranges from 1 mL to about 300 mL, including from about 4 mL to about 250 mL, and including from about 10 mL to about 240 mL.
  • the nutritional solids may be in any solid form but are typically in the form of flowable or substantially flowable particulate compositions, or at least particulate compositions.
  • Particularly suitable nutritional solid product forms include spray dried, agglomerated and/or dryblended powder compositions.
  • the compositions can easily be scooped and measured with a spoon or similar other device and can easily be reconstituted by the intended user with a suitable aqueous liquid, typically water, to form a nutritional composition for immediate oral or enteral use.
  • “immediate” use generally means within about 48 hours, most typically within about 24 hours, preferably right after reconstitution.
  • the nutritional powders may be reconstituted with water prior to use to a caloric density tailored to the nutritional needs of the ultimate user, although in most instances the powders are reconstituted with water to form compositions comprising at least 19 kcal/fl oz (660 kcal/liter), more typically from about 20 kcal/fl oz (675-680 kcal/liter) to about 25 kcal/fl oz (820 kcal/liter), - 28 - 4859-9621-0305, v.1 even more typically from about 20 kcal/fl oz (675-680 kcal/liter) to about 24 kcal/fl oz (800-810 kcal/liter).
  • the 22-24 kcal/fl oz formulas are more commonly used in preterm or low birth weight infants, and the 20-21 kcal/fl oz (675-680 to 700 kcal/liter) formulas are more often used in term infants.
  • the reconstituted powder may have a caloric density of from about 50-100 kcal/liter to about 660 kcal/liter, including from about 150 kcal/liter to about 500 kcal/liter.
  • the emulsion may have a caloric density of 25, or 50, or 75, or 100 kcal/liter.
  • the nutritional compositions of the present disclosure include adult formulas, infant formulas and human milk fortifiers that are orally administered to adults, children, and infants, including preterm or term infants.
  • the nutritional compositions may be administered as a source of nutrition for individuals, to enhance blood flow to the brain (e.g., enhance nutrient uptake or brain chemistry), enhance the BBB function, enhance the BBB integrity (e.g., increase tight junctions or reduce permeability), and/or to treat one or more BBB disorders, conditions, injuries or dysfunctions.
  • One subclass of the general population that can effectively utilize the nutritional compositions described herein include those individuals that are susceptible to, or at a risk of (at an elevated risk as compared to the general infant population) immaturity of the central nervous system and including the brain and the BBB.
  • infants who are susceptible to or at risk of having immaturity of, for example, the brain are herein referred to as “in need of” assistance (or “in need thereof” as referring to the assistance needed) in combating organ development problems such as neural inflammation and BBB disorders or dysfunctions.
  • the methods of the present disclosure are directed to infants during the initial days, weeks or months of life.
  • the nutritional compositions when in the form of an infant formulas described - 29 - 4859-9621-0305, v.1 herein, are administered to the infant for a duration of at least the first week of life, more desirably during at least the first two weeks of life, more desirably during at least the first one or two months of life, more desirably during at least the first four months of life, and more desirably during at least the first six months of life, and including up to the first year of life.
  • the infant may be switched to a conventional infant formula, alone or in combination with human milk.
  • infant formulas described herein can be used alone, or in combination with other infant formulas.
  • the nutritional compositions used in the methods described herein are infant formulas (including human milk fortifiers) and may be in any product form, including ready-to-feed liquids, concentrated liquids, reconstituted powders, and the like as described above.
  • the method may further comprise reconstituting the powder with an aqueous vehicle, most typically water or human milk, to form the desired caloric density, which is then orally or enterally fed to the infant.
  • the present disclosure is directed to a method for the prevention, delay of progression, or the treatment of a circulatory disorder characterized by suboptimal or impaired BBB function.
  • the method comprises administering to an individual in need thereof a nutritional composition comprising a therapeutically effective amount of an HMO or HMO blend.
  • the method comprises identifying an individual in need thereof.
  • administration of the nutritional compositions according to the general inventive concepts leads to improved blood flow, improved or enhanced BBB function or integrity and reduction in one or more symptoms associated with BBB disorders or conditions and may also lead to the prevention, delay of progression, or the treatment of a neurodegenerative disorder or other neuro-associated disorders or injuries.
  • the exemplified compositions are shelf stable nutritional compositions prepared in accordance with the manufacturing methods described herein, such that each exemplified composition, unless otherwise specified, includes an aseptically processed embodiment and a retort packaged embodiment.
  • Examples 1-5 illustrate ready-to-feed nutritional emulsions, the ingredients of which are listed in the table below. All ingredient amounts listed are in kilograms, unless otherwise specified. - 31 - 4859-9621-0305, v.1 Table 1: Examples 1-5 Ingredient Ex.1 Ex.2 Ex. 3 Ex.4 Ex.5 Water Q.S. Q.S. Q.S. Q.S. Q.S. Q.S..
  • the RBEC monolayer assay applies primary rat brain microvascular endothelial cells isolated from three- - 35 - 4859-9621-0305, v.1 week old rats, co-cultured with rat pericytes in the presence of rat astrocyte-secreted factors. Endothelial cells are cultured on the upper side of a semipermeable porous filter, having pericytes on the backside and astrocyte-conditioned media in the bottom of the wells of the culture plate.
  • RBEC monolayers maintain the main characteristics of the cerebral endothelium in vivo.
  • the contribution of single transporters to the active efflux processes can be determined by the application of selective substrates or inhibitors.
  • Cerebral cortices originating from 3-week old rats were cut into small pieces and digested in two steps with collagenase and collagenase/dispase followed by centrifugation on percoll gradient.
  • the cells were cultured on fibronectin/collagen- coated dishes in RBEC-medium: DMEM/F12 (Invitrogen), 10% plasma-derived serum (PDS, First Link, UK) and growth factors. After reaching confluency, endothelial cells were passed onto 24-well Transwell filters (pore size: 0.4 ⁇ m, Costar-Corning) containing pericytes on the backside.
  • the monolayer was supplied with 550 nM hydrocortisone, CPT-cAMP (250 ⁇ M) and 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone (RO) RO-201724 (17.5 ⁇ M; Roche) together with the HMO composition (2′-FL, 3-FL, 3′-SL, 6′-SL, and LNT) for 24 hours.
  • Filter inserts containing RBECs and pericytes were placed into dishes containing glial- conditioned media.
  • TEER was continuously monitored using the cellZscope instrument (24-hour model). In the 12-hour model, TEER was measured with chopstick electrodes at 90 min, 3 hours and 12 hours.
  • Claudin-5 monoclonal antibody (4C3C2) (Thermo Fisher Scientific, cat. no.35-2500) and Zonula occludens protein-1 (ZO-1) polyclonal antibody (Thermo Fisher Scientific, cat. no.61-7300). Nuclei were stained with Hoechst 33342 (Merck-Sigma, cat. no. B2261).
  • Instruments used for detection include a cellZscope automated transepithelial/transendothelial electrical resistance (TEER) measurement system (nanoAnalytics, Weg, Germany) and a FluoStar Optima microplate reader (BMG Labtechnologies, Offenburg, Germany).
  • TEER cellZscope automated transepithelial/transendothelial electrical resistance
  • FIG. 4 shows changes in permeability of RBEC monolayers in response to 2,3- Dimethoxy-1,4-naphthoquinone (DMNQ) in the presence or absence of the HMO composition.
  • Figure 5 shows changes in the tight junction integrity of RBEC monolayers in response to the presence of the HMO composition at 10 mg/mL.
  • Representative images of Immunofluorescence (IF) staining for localization of the claudin-5 protein (green) in RBEC monolayers indicates increased expression of claudin-5 at the tight junctions in the presence of the HMO composition (Fig. 5B) as compared to the control (Fig.5A).
  • IF Immunofluorescence
  • HMOs one or more, including an HMO blend
  • ROS reactive oxygen species

Abstract

A nutritional composition for use in enhancing one or more features or functions of the blood-brain barrier, including promoting healing or improved function of the blood-brain barrier. In certain embodiments, a method comprises administering to an individual in need thereof a nutritional composition comprises at least one human milk oligosaccharide as described herein to treat or prevent one or more blood-brain barrier related conditions.

Description

HUMAN MILK OLIGOSACCHARIDES FOR ENHANCING BLOOD-BRAIN BARRIER FUNCTION CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This non-provisional application claims priority to and the benefit of U.S. Provisional Application No.63/409,010, filed September 22, 2022, the entire content of which is hereby incorporated herein by reference. FIELD [0002] The present disclosure relates to human milk oligosaccharides (HMOs), whether alone or in combination, for nutritionally benefitting an individual consumer, including improving one or more aspects of the individual’s health. More particularly, the present disclosure relates to nutritional compositions comprising a blend of HMOs that can modulate or improve the health of an individual consumer by treating or preventing one or more symptoms of a disease or condition as described in greater detail herein. BACKGROUND [0003] Breast milk containing HMOs has been associated with enhanced development and balanced growth and maturation of a variety of infant systems including respiratory, gastrointestinal, and immune systems, among others. There are more than 200 known oligosaccharides present in human milk, with various individual classes having been shown to promote health in a variety of ways. It appears that certain infections are prevented by HMOs due to their activity as pathogen receptor analogues (i.e., receptor mimics that prevent pathogenic adhesion). Further, breast milk includes HMOs that not only act as pathogen receptor analogues, but certain structures activate immune factors as well as acting as metabolic fuel in the gut microbiome to preferentially benefit certain beneficial bacteria. [0004] Due to the accepted safety of HMOs as a component of an individual’s diet, efforts have been extended to develop the use of HMOs in other therapeutic regimes. However, these efforts have yet to develop fully effective therapies in many areas. Accordingly, there is an unmet need - 1 - 4859-9621-0305, v.1 for nutritional solutions that are effective to treat one or more of the conditions or diseases described herein, including but not, limited to modulating blood-brain barrier function. SUMMARY [0005] The general inventive concepts are based, in part, on the surprising discovery that nutritional compositions including at least one fucosylated HMO, at least one sialylated HMO, and at least one N-acetylglucosamine HMO can provide substantial benefits to a consumer including, but not limited to, preventing or reducing damage or degradation to the blood-brain barrier, promoting healing, enhancing nutrient delivery to the brain, enhancing brain development in an infant (including improving cognition), toddler, or child, reducing the effects of neurodegenerative disorders, preventing metastasis of certain cancers, and treating one or more blood-brain barrier (BBB) related conditions or dysfunctions. [0006] The nutritional compositions described herein include at least one HMO, and in certain instances, a blend of HMOs in combination with one or more nutritionally beneficial ingredients and are useful to treat disorders associated with the blood-brain barrier. Exemplary non-limiting HMOs for use in the nutritional compositions, methods, and treatments include, but are not necessarily limited to: sialylated oligosaccharides e.g., 3′-sialyllactose (3′-SL) and 6′-sialyllactose (6′-SL); e.g., N-acetyl-glucosamine oligosaccharides lacto-N-neotetraose (LNnT) and lacto-N-tetraose (LNT); and fucosylated oligosaccharides e.g., 3-fucosyllactose (3-FL) and 2′-fucosyllactose (2FL). These HMOs may be present in the nutritional compositions, methods, and treatments in amount of about 0.001 mg/mL to about 20 mg/mL. [0007] Aspects of the general inventive concepts are directed to a nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and an N- acetylglucosamine oligosaccharide for use in preventing degradation of barrier function of the blood-brain barrier in an individual in need thereof. [0008] Aspects of the general inventive concepts are directed to a nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N- acetylglucosamine oligosaccharide for use in treating one or more blood-brain barrier related conditions. - 2 - 4859-9621-0305, v.1 [0009] Aspects of the general inventive concepts are directed to a nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N- acetylglucosamine oligosaccharide for use in enhancing cognition in an infant, toddler, or child. [0010] Aspects of the general inventive concepts are directed to a nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N- acetylglucosamine oligosaccharide for use in enhancing nutrient uptake in an individual in need thereof. [0011] Aspects of the general inventive concepts are directed to a nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N- acetylglucosamine oligosaccharide for use in enhancing brain chemistry. [0012] Aspects of the general inventive concepts are directed to a nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N- acetylglucosamine oligosaccharide for use in reducing blood-brain barrier permeability in an individual in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS [0013] These and other features of the present disclosure will become better understood with regard to the following description and accompanying drawings in which: [0014] Fig.1 shows an exemplary image of the component so the blood-brain barrier. [0015] Fig.2 shows an exemplary experimental set-up to test BBB permeability. [0016] Fig.3 is a graph showing changes in TEER in response to the HMO composition (i.e., 2′- FL, 3-FL, 3′-SL, 6′-SL, and LNT) over time. [0017] Fig. 4A is a graph showing changes in 4 kDa FITC-dextran permeability of RBEC monolayers in response to DMNQ in the presence or absence of the HMO composition. [0018] Fig.4B is a graph showing changes in 70 kDa TexasRed-dextran permeability of RBEC monolayers in response to DMNQ in the presence or absence of the HMO composition. - 3 - 4859-9621-0305, v.1 [0019] Fig. 5A is an image of immunofluorescence staining for localization of the claudin-5 protein in RBEC monolayers in control. [0020] Fig.5B is an image showing immunofluorescence staining for localization of the claudin- 5 protein in RBEC monolayers with the HMO composition. DETAILED DESCRIPTION [0021] The nutritional compositions and methods described herein utilize one or more HMOs (e.g., at least one fucosylated HMO, at least one sialylated HMO, and at least one N-acetylated HMO) alone or in combination with long chain polyunsaturated fatty acids, antioxidants, and also including carotenoids, nucleotides, or both, for controlling, preventing, and/or treating a number of diseases and conditions as described in greater detail herein. These and other features of the nutritional compositions and methods, as well as some of the many optional variations and additions, are described in detail hereafter. [0022] The blood-brain barrier is a highly selective semipermeable membrane or border of cells that regulate the transfer of solutes and chemicals between the circulatory system and the central nervous system. The barrier plays a vital role in early brain development and throughout life. The barrier serves to protect the brain from harmful or unwanted substances in the blood. The BBB is formed by endothelial cells of the capillary wall. The system allows the passage of some small molecules by passive diffusion, as well as the selective and active transport of various nutrients, ions, organic anions, and macromolecules such as glucose and amino acids that are crucial to proper neural development and function. [0023] A properly functioning BBB restricts the passage of microorganisms, the diffusion of solutes, and large or hydrophilic molecules present in the blood into the cerebrospinal fluid, while allowing the diffusion of hydrophobic molecules (O2, CO2, hormones) and small non-polar molecules. Cells of the barrier actively transport metabolic products such as glucose across the barrier. The barrier also restricts the passage of peripheral immune factors, like signaling molecules, antibodies, and immune cells, into the CNS, thus insulating the brain from damage due to immune events elsewhere in the body. [0024] The term “human milk oligosaccharide” or “HMO”, as used herein, unless otherwise specified, refers generally to a number of complex carbohydrates found in human breast milk - 4 - 4859-9621-0305, v.1 that can be classified as acidic or neutral, and, in certain instances, to precursors thereof. Exemplary non-limiting HMOs include neutral oligosaccharides (which include fucosylated oligosaccharides and N-acetyl-glucosamine oligosaccharides (e.g., also known as neutral non- fucosylated HMOs or N-acetylated)) and acidic oligosaccharides (which include sialylated oligosaccharides). Specific non-limiting embodiments include: 2′-fucosyllactose (2′-FL), 3- fucosyllactose (3-FL), 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT). The term “HMO blend,” unless specified otherwise, refers to a combination of fucosylated, sialylated, and N-acetyl-glucosamine HMOs, which may comprise at least two of 2’-FL, 3-FL, 3′-SL, 6′-SL, and LNT, and in certain instances all of 2′-FL, 3-FL, 3′-SL, 6′-SL, and LNT. [0025] The term “blood-brain barrier dysfunction,” as used herein refers to a set of conditions and diseases mainly stemming from and/or characterized by disordered or impaired function of the blood-brain barrier (BBB). In general, BBB dysfunction contributes to pathology in a range of neurological conditions including neurodegenerative disease (e.g., Alzheimer’s disease), multiple sclerosis, stroke, epilepsy, and cerebral palsy, among other BBB related conditions. Blood-brain barrier dysfunction is characterized by relative increase in permeability of the BBB (i.e., the junctions between cells are not tight). This dysfunction allows for increased diffusion, including bidirectional diffusion, and less selectivity in passage of blood components, etc., thereby, this negatively impacts nutrient uptake, nutrient retention, and brain chemistry. [0026] The term “nutrient uptake” as used herein refers to delivery of nutrients to the brain across the BBB. “Enhanced nutrient uptake” refers to improved nutrient delivery or treating ineffective nutrient delivery. In general, the term enhanced as used in the present disclosure refers to an improvement in one or more of the associated markers related to the subject condition or disease. Nutrient delivery/uptake can include metabolic substances (e.g., energy), vitamins, and or proper exchange of oxygen and carbon dioxide between the blood and brain tissue. Enhanced nutrient delivery is seen both through a relative increase in desirable nutrient delivery and/or through a reduction in the passage of unwanted or harmful substances across the BBB. Enhanced nutrient uptake or delivery can be shown through in vitro testing showing a reduction in the permeability of the cells making up an exemplary BBB. An important function of the BBB is the selective nature of the barrier, whereby, when functioning properly, essential - 5 - 4859-9621-0305, v.1 or desirable nutrients are transported or otherwise allowed to pass through the barrier and unwanted or harmful materials are excluded. Examples of desirable components would include glucose, amino acids, ketone bodies, choline, antioxidant molecules, vitamin B, and purines. Examples of undesirable components would be toxins, bacteria, viruses, prions, blood and immune factors such as thrombin, complement, fibrinogen, c-reactive protein (CRP). Enhanced nutrient uptake also includes barrier function that prevents diffusion of desirable nutrients from passing back into the bloodstream after first crossing the BBB. Thereby enhanced nutrient uptake via improved BBB function affects/protects brain chemistry in a variety of ways. [0027] The term “brain chemistry” as used herein refers to the full assortment of compounds that are circulating in the blood in and around the brain. One function of the BBB is to selectively separate compounds that are circulating in the blood from interacting with neuronal synapses in order to maintain brain chemistry homeostasis. A properly functioning BBB will effectively protect brain chemistry homeostasis and allow for proper functioning of chemical messengers or neurotransmitters that signal between neurons in the brain. [0028] The term “blood-barrier integrity” as used herein refers to the functional permeability of the BBB. A major function of the BBB includes protection of the brain tissue through the functional and structural integrity of the tight junctions between the endothelial cells that make up the vasculature in the brain. Thus, a properly functioning BBB includes less permeable junctions between the aforementioned endothelial cells. Improved, enhanced, or treated blood- barrier integrity is generally shown through reduced permeability or a return of permeability from an increased state to a state of lower permeability and may be shown through in vitro testing. [0029] The term “blood-brain barrier related condition” or “BBB related conditions” as used herein refers to conditions or diseases that are treated or impacted directly by the BBB function, including by either nutrient uptake, brain chemistry, or by the filtration aspect of BBB function (e.g., preventing cancer cells from migrating or preventing microbe infiltration). In aspects of the present disclosure, the conditions are selected from: necrotizing enterocolitis, consequences of preterm birth, blood vessel damage, meningitis, hypoxia, traumatic brain injury, stroke, brain hemorrhage, compromised nervous systems, compromised neuronal blood flow, dysfunctional or impaired BBB, immaturity of the central nervous system, blood-brain barrier dysfunction, - 6 - 4859-9621-0305, v.1 neurodegenerative disease, cancer metastasis, and infectious diseases selected from: Group B Streptococcus, Listeria monocytogenes, Bacillus antracis, Haemophilus influenzae, Neisseria meningitidis, P. falciparum, Toxoplasma gondii, Acanthamoeba, COVID-19, RSV, Salmonella enterica, and influenza virus. [0030] The terms “fat” and “oil” as used herein, unless otherwise specified, are used interchangeably to refer to lipid materials derived or processed from plants or animals. These terms also include synthetic lipid materials so long as such synthetic materials are suitable for oral administration to humans. [0031] The term “shelf stable” as used herein, unless otherwise specified, refers to a nutritional composition that remains commercially stable after being packaged and then stored at 18-24° C. for at least 3 months, including from about 3 months to about 36 months, including from about 3 months to about 24 months, and also including from about 3 months to about 18 months. [0032] The terms “nutritional formulation” or “nutritional composition” as used herein, are used interchangeably and, unless otherwise specified, refer to synthetic formulas including nutritional liquids, nutritional powders, nutritional solids, nutritional semi-solids, nutritional semi-liquids, nutritional supplements, and any other nutritional food product as known in the art. The nutritional powders may be reconstituted to form a nutritional liquid, all of which comprise one or more of fat, protein and carbohydrate and are suitable for oral consumption by a human. The terms “nutritional formulation” or “nutritional composition” do not include human breast milk and do not refer to supplemented milk, whether of human origin or otherwise. [0033] The term “nutritional liquid” as used herein, unless otherwise specified, refers to nutritional compositions in ready-to-drink liquid form, concentrated form, and nutritional liquids made by reconstituting the nutritional powders described herein prior to use. [0034] The term “nutritional powder” as used herein, unless otherwise specified, refers to nutritional compositions in flowable or scoopable form that can be reconstituted with water or another aqueous liquid prior to consumption and includes both spray-dried and drymixed/dryblended powders. - 7 - 4859-9621-0305, v.1 [0035] The term “nutritional semi-solid,” as used herein, unless otherwise specified, refers to nutritional compositions that are intermediate in properties, such as rigidity, between solids and liquids. Some semi-solids examples include puddings, gelatins, and doughs. [0036] The term “nutritional semi-liquid,” as used herein, unless otherwise specified, refers to nutritional compositions that are intermediate in properties, such as flow properties, between liquids and solids. Some semi-liquids examples include thick shakes and liquid gels. [0037] The term “individual” as used herein, refers generally to a preterm infant, infant, toddler, child, or adult, including elderly adults. [0038] The term “infant” as used herein, refers generally to individuals up to age 36 months of age, actual or corrected. [0039] As used herein, all concentrations expressed as “µg/liter,” “mg/liter,” “mg/L,” “mcg/L,” “mg/mL” etc., refer to ingredient concentrations within the described nutritional compositions as calculated on an as-fed basis (e.g., reconstituted for consumption in the case of nutritional powders), unless otherwise specified. [0040] The term “reconstitute” or various other forms such as “reconstituted” or “reconstituting” all refer to the general act of adding a suitable amount of liquid, typically water, to a form of nutritional formulation that is not in its ready-to-drink liquid form, such as nutritional powder or a concentrated form of a nutritional liquid, thereby making the nutritional composition ready-to- drink. [0041] The terms “susceptible” and “at risk” as used herein, unless otherwise specified, mean having little resistance to a certain condition or disease relative to the general population (e.g., individuals with compromised nervous systems, compromised neuronal blood flow, dysfunctional or impaired BBB) including being genetically predisposed, having a family history of, and/or having symptoms of the condition or disease. In certain instances, the individual is at risk due to immaturity of the central nervous system, preterm birth, traumatic brain injury, stroke, infection, etc. The term refers to those having a vulnerability higher than the general population. Vulnerability may not necessarily be due directly to dysfunction of the BBB, but may be due to being at greater risk to serious adverse consequences from dysfunction of the BBB, such as NEC, neurodegenerative disease, infections disease, cancer. - 8 - 4859-9621-0305, v.1 [0042] The terms “modulating” or “modulation” or “modulate” as used herein, unless otherwise specified, refer to the targeted movement of a selected characteristic or symptom. In certain instances, modulating refers to reducing BBB permeability. [0043] The term “ameliorate” as used herein, unless otherwise specified, means to eliminate, delay, or reduce the prevalence or severity of symptom(s) associated with a condition or disease. [0044] The term “an effective amount” is intended to qualify the amount of e.g., HMO which will achieve the goal of modulating, preventing or treating a disease or that which will achieve the goal of decreasing the risk that the patient will suffer an adverse health event, including reducing or preventing one or more symptoms, while avoiding adverse side effects such as those typically associated with alternative therapies. The effective amount may be administered in one or more doses. In embodiments, an effective amount is the amount recommended to reduce permeability of the BBB, enhance nutrient uptake, or otherwise treat disfunction of the BBB. [0045] The terms “treating” and “treatment” as used herein, unless otherwise specified, includes delaying the onset of a condition, reducing the severity of symptoms of a condition, or eliminating some or all of the symptoms of a condition. [0046] The term “food products” as used herein refer to delivery vehicles that contain the HMOs and one or more of fats, amino nitrogen and carbohydrates and provides some or all of the nutritional support for a patient in the recommended daily amounts. Frequently a food product will contain vitamins, minerals, trace minerals and the like to provide balanced nutrition to meal replacements, medical foods, supplements. The food products may be in any typical form such as beverages, powders, bars, juices, carbonated beverages, bottled water, etc. [0047] The terms “infant formula” or “synthetic infant formula” as used herein, unless otherwise specified, are used interchangeably and refer to liquid, solid, semi-solid, and semi-liquid human milk replacements or substitutes that are suitable for consumption by an infant. The synthetic formulas include components that are of semi-purified or purified origin. As used herein, unless otherwise specified, the terms “semi-purified” or “purified” refer to a material that has been prepared by purification of a natural material or by synthesis. The terms “infant formula” or “synthetic infant formula” do not include human breast milk. - 9 - 4859-9621-0305, v.1 [0048] The term “synthetic pediatric formula” as used herein, unless otherwise specified, refers to liquid, solid, semi-solid, and semi-liquid human milk replacements or substitutes that are suitable for consumption by an infant or toddler up to the age of 36 months (3 years). The synthetic formulas include components that are of semi-purified or purified origin. As used herein, unless otherwise specified, the terms “semi-purified” or “purified” refer to a material that has been prepared by purification of a natural material or by synthesis. The term “synthetic pediatric nutritional formula” does not include human breast milk. [0049] The term “synthetic child formula” as used herein, unless otherwise specified, refers to liquid, solid, semi-solid, and semi-liquid human milk replacements or substitutes that are suitable for consumption by a child up to the age of 12 years. The synthetic formulas include components that are of semi-purified or purified origin. As used herein, unless otherwise specified, the terms “semi-purified” or “purified” refer to a material that has been prepared by purification of a natural material or by synthesis. The term “synthetic child nutritional formula” does not include human breast milk. [0050] The term “preterm infant formula” as used herein, unless otherwise specified, refers to liquid and solid nutritional products suitable for consumption by a preterm infant. [0051] The term “human milk fortifier” as used herein, unless otherwise specified, refers to liquid and solid nutritional products suitable for mixing with breast milk or preterm infant formula or infant formula for consumption by a preterm or term infant. [0052] All percentages, parts and ratios as used herein, are by weight of the total composition, unless otherwise specified. All such weights, as they pertain to listed ingredients, are based on the active level and, therefore, do not include solvents or by-products that may be included in commercially available materials, unless otherwise specified. [0053] Numerical ranges as used herein are intended to include every number and subset of numbers within that range, whether specifically disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, from 5 to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth. - 10 - 4859-9621-0305, v.1 [0054] All references to singular characteristics or limitations of the general inventive concepts 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. [0055] 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. [0056] The general inventive concepts contemplate a nutritional composition for use in preventing, treating, or reducing the effects/symptoms of one or more BBB-related conditions. The HMO-containing nutritional compositions are for use in providing enhancement to BBB function, preventing degradation of the BBB (including reducing current or future degradation of BBB function), reducing permeability of the BBB, or otherwise for treatment of dysfunctional BBB (including repair of damaged BBB) of an individual in need thereof. [0057] Without being bound to a particular theory, it is believed that feeding or otherwise administering to an individual a nutritional composition comprising the HMO compositions described herein improves or enhances BBB function Including a return to proper functioning), or treats, prevents, or repairs at least one aspect of BBB dysfunction. In an aspect, the nutritional composition for use according to the general inventive concepts is a liquid and comprises at least one human milk oligosaccharide selected from 2′-FL, 3-FL, 3′-SL, 6′-SL, LNnT, and LNT in an amount of from about 0.001 mg/mL to about 20 mg/mL, including from about 0.001 mg/mL to about 10 mg/mL, including from about 0.001 mg/mL to about 5 mg/mL, including from about 0.001 mg/mL to less than 2 mg/mL, and including from about 0.01 mg/mL to about 20 mg/mL, including from about 0.01 mg/mL to less than 10 mg/mL. In a specific embodiment, the nutritional composition is a liquid and comprises 2′-fucosyllactose, in an amount of from about 0.001 mg/mL to about 20 mg/mL, including from about 0.001 mg/mL to about 10 mg/mL, including from about 0.001 mg/mL to about 5 mg/mL, including from about 0.001 mg/mL to less than 2 mg/mL, including from about 0.01 mg/mL to about 20 mg/mL, and including from about 0.01 mg/mL to less than 2 mg/mL. In embodiments, the nutritional composition for use comprises at least one and in certain instances each of 2′-FL, 3-FL, 3′-SL, 6′-SL, and LNT. In embodiments, the nutritional composition for use comprises a combination of 2′-FL, 3-FL, 3′-SL, - 11 - 4859-9621-0305, v.1 6′-SL, and LNT in a total amount of HMO of from 1 mg/mL to about 8.2 mg/mL and the nutritional composition prevents, treats, or reduces the symptoms of, at least one of the following blood-brain barrier related conditions: necrotizing enterocolitis, consequences of preterm birth, blood vessel damage, meningitis, hypoxia, traumatic brain injury, stroke, brain hemorrhage, compromised nervous systems, compromised neuronal blood flow, dysfunctional or impaired BBB, immaturity of the central nervous system, blood-brain barrier dysfunction, neurodegenerative disease, cancer metastasis, and infectious diseases selected from: Group B Streptococcus, Listeria monocytogenes, Bacillus antracis, Haemophilus influenzae, Neisseria meningitidis, P. falciparum, Toxoplasma gondii, Acanthamoeba, COVID-19, RSV, and influenza virus. In certain aspects, the nutritional compositions for use provide a reduction of permeability of the BBB by from about 10% to about 50%, including 10% to 45%, including 10% to 40%, including 15% to 40%, including 20% to 40%, and including from 25% to about 40%. [0058] While not wishing to be bound by theory, those of ordinary skill in the art will recognize that certain of the BBB-related conditions described herein will benefit in different manners from enhancing the BBB and/or reducing permeability of the BBB. Certain of the conditions directly result in BBB injury or dysfunction, whereas other conditions described herein will indirectly benefit from enhanced BBB integrity or function due to improved filtration function/reduced permeability or better nutrient selectivity/yield. [0059] For example, blood vessel damage may occur through vascular risk factors, such as diabetes, hypertension, and dyslipidemia; environmental risk factors, such as pollution; or genetic risk factors, such as being a carrier of the APOE-ε4 gene (Alzheimer’s). Blood vessel damage causes BBB dysfunction and decreased brain perfusion. Disruption of the BBB (tight junction integrity) has been associated with the accumulation of blood factors such as complement (an immune factor) in the brain which induces an immunological response. Because of this association, strengthening the integrity of the BBB by reducing the permeability would prevent the continued accumulation of these peripheral proteins in the brain and it is believed inhibit progression of neurodegenerative disease. [0060] As another example, necrotizing enterocolitis (NEC) is known to increase the risk of brain injury and impaired neurodevelopment. Inflammation originating from the gastrointestinal tract in NEC circulates systemically. Exposure of the neurovascular cells to inflammatory factors - 12 - 4859-9621-0305, v.1 can lead to BBB dysfunction and altered brain chemistry or suboptimal nutrient uptake. This can have direct/immediate effects as well as long term consequences for the developing brain, which may account for the long-term neurocognitive consequences of NEC in some patients. Further, the young/developing brain is believed to be more vulnerable to toxins and disruption. Reducing the permeability (and/or returning the permeability to a normal state) of the BBB in infancy may help in development of the brain. [0061] With respect to treating infectious diseases via BBB function, in any bloodstream infection, maintenance of BBB integrity during infection may prevent more serious complications from developing and affecting the CNS. Many types of infections will produce inflammation and the expression of inflammatory factors. Improving BBB integrity and tight junction permeability may reduce inflammation in the brain due to improved separation of blood components from brain tissue. However, these inflammatory factors can result in disruption of BBB integrity, which in turn leads to dysregulated BBB function and the free movement of blood and tissue components (i.e., brain chemistry and nutrient uptake are negatively affected), potentially including infectious bacteria or viruses, between the brain tissue and the blood stream. Certain infections are known to have direct effects on the BBB. For example, COVID-19 infects neurovascular cells causing restructuring of the actin cytoskeleton that leads to breakdown of BBB function. This permits the movement of inflammatory factors into the brain, producing dangerous neuroinflammation. Thus, the repair, improvement, or enhanced BBB function in effect may be viewed as a treatment and/or prevention of infectious disease. [0062] Thus, repairing, improving, or enhancing BBB function can positively affect the health of the consumer in a variety of ways. This can be achieved by administration of the HMO- containing nutritional compositions described herein. Human Milk Oligosaccharides [0063] The nutritional compositions according to the general inventive concepts include an HMO component comprising at least one HMO, and in many aspects, a combination of two or more HMOs, including neutral HMOs and acidic HMOs. The composition of human milk oligosaccharides is very complex and more than 200 different oligosaccharide structures are known. - 13 - 4859-9621-0305, v.1 [0064] The HMOs may be included in the nutritional compositions alone, or in some embodiments, in combination with other functional components (LCPUFAs, antioxidants, nucleotides, etc.) to aid in ameliorating symptoms of the particular condition or disease as described herein. The HMO or HMOs may be isolated or enriched from milk(s) secreted by mammals including, but not limited to: human, bovine, ovine, porcine, or caprine species. The HMOs may also be produced via microbial fermentation, enzymatic processes, chemical synthesis, or combinations thereof. [0065] Suitable HMOs for use in the nutritional compositions and HMO components may include neutral oligosaccharides, acidic HMOs, and n-acetyl glucosylated oligosaccharides, and HMO precursors. HMOs may also be characterized as fucosylated, sialylated, and N-acetyl- glucosamine. Specific non-limiting examples of HMOs that may be included individually or in combination in the compositions according to the general inventive concepts include: 2′-FL, 3- FL, 3′-SL, 6′-SL, LNnT, and LNT. [0066] Other HMOs that may be included in certain embodiments include: N-acetylglucosamine (GlcNAc); L-fucose (L-Fuc); D-fucose (D-Fuc); fucosyl oligosaccharides (i.e., Lacto-N- fucopentaose I; Lacto-N-fucopentaose II; Lacto-N-fucopentaose III; Lacto-N-difucohexaose I; and Lactodifucotetraose); non-fucosylated, non-sialylated oligosaccharides (i.e., Lacto-N- neotetraose); sialyl fucosyl oligosaccharides (i.e., 3′-Sialyl-3-fucosyllactose; Disialomonofucosyllacto-N-neohexaose; Monofucosylmonosialyllacto-N-octaose (sialyl Lea); Sialyllacto-N-fucohexaose II; Disialyllacto-N-fucopentaose II; Monofucosyldisialyllacto-N- tetraose); and sialyl oligosaccharides (i.e., 3′-Sialyllactose; 3-Sialyllactosamine; 6′-Sialyllactose; 6′-Sialyllactosamine; Sialyllacto-N-neotetraose c; Monosialyllacto-N-hexaose; Disialyllacto-N- hexaose I; Monosialyllacto-N-neohexaose I; Monosialyllacto-N-neohexaose II; Disialyllacto-N- neohexaose; Disialyllacto-N-tetraose; Disialyllacto-N-hexaose II; Sialyllacto-N-tetraose a; Disialyllacto-N-hexaose I; and Sialyllacto-N-tetraose b). Also useful are variants in which the glucose (Glc at the reducing end is replaced by N-acetylglucosamine (e.g., 2′-fucosyl-N- acetylglucosamine (2′-FLNac) is such a variant to 2′-fucosyllactose). These HMOs are described more fully in U.S. Patent Application No.2009/0098240, which is herein incorporated by reference in its entirety. Other suitable examples of HMOs that may be included in the compositions according to the general inventive concepts include lacto-N-fucopentaose V, lacto- - 14 - 4859-9621-0305, v.1 N-hexaose, para-lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-neohexaose, monofucosyllacto-N-hexaose II, isomeric fucosylated lacto-N-hexaose (1), isomeric fucosylated lacto-N-hexaose (3), isomeric fucosylated lacto-N-hexaose (2), difucosyl-para-lacto-N- neohexaose, difucosyl-para-lacto-N-hexaose, difucosyllacto-N-hexaose, lacto-N-neoocataose, para-lacto-N-octanose, iso-lacto-N-octaose, lacto-N-octaose, monofucosyllacto-neoocataose, monofucosyllacto-N-ocataose, difucosyllacto-N-octaose I, difucosyllacto-N-octaose II, difucosyllacto-N-neoocataose II, difucosyllacto-N-neoocataose I, lacto-N-decaose, trifucosyllacto-N-neooctaose, trifucosyllacto-N-octaose, trifucosyl-iso-lacto-N-octaose, lacto-N- difuco-hexaose II, sialyl-lacto-N-tetraose a, sialyl-lacto-N-tetraose b, sialyl-lacto-N-tetraose c, sialyl-fucosyl-lacto-N-tetraose I, sialyl-fucosyl-lacto-N-tetraose II, and disialyl-lacto-N-tetraose, and combinations thereof. [0067] The HMOs are present in the nutritional compositions in total amounts of HMO in the composition (mg of HMO per mL of liquid composition) of at least about 0.001 mg/mL to about 20 mg/mL, including about 0.001 mg/mL to about 20 mg/mL, about 0.01 mg/mL to about 20 mg/mL, about 0.1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 10 mg/mL, about 2 mg/mL to about 10 mg/mL, and including from about 2.5 mg/mL to about 8.5 mg/mL. Typically, the amount of HMO in the nutritional composition will depend on the specific HMO or HMOs present and the amounts of other components in the nutritional compositions. [0068] In certain exemplary aspects of the present disclosure, the nutritional composition includes at least one of 2′-FL, 3-FL, 3′-SL, 6′-SL, and LNT in a total amount of HMO of from about 0.001 mg/mL to about 20 mg/mL, including from about 0.01 mg/mL to about 20 mg/mL, including from about 0.1 mg/mL to about 20 mg/mL, about 0.5 mg/mL to about 20 mg/mL, about 1 mg/mL to about 20 mg/mL, about 2 mg/mL to about 20 mg/mL. about 3 mg/mL to about 20 mg/mL, and about 5 mg/mL to about 20 mg/mL. In certain exemplary aspects of the present disclosure, the nutritional composition includes at least one of 2′-FL, 3-FL, 3′-SL, 6′-SL, and LNT in a total amount of HMO of from about 0.1 g/L to about 20 mg/mL, including from about 0.1 mg/mL to about 15 mg/mL, including from about 0.1 mg/mL to about 10 mg/mL, including from about 0.1 mg/mL to about 9 mg/mL, including from about 0.1 mg/mL to about 8 mg/mL, including from about 0.1 mg/mL to about 7 mg/mL, including from about 0.1 mg/mL to about 6 - 15 - 4859-9621-0305, v.1 mg/mL, including from about 0.1 mg/mL to about 5 mg/mL, including from about 0.1 mg/mL to about 4 mg/mL, including from about 0.1 mg/mL to about 3 mg/mL, including from about 0.1 mg/mL to about 2 mg/mL, including from about 0.5 mg/mL to about 4 mg/mL, including from about 0.5 mg/mL to about 3 mg/mL, including from about 1 mg/mL to about 2.5 mg/mL; and including from about 3.1 mg/mL to about 8.2 mg/mL. [0069] In certain exemplary aspects, the nutritional composition comprises at least one of a fucosylated HMO, a N-acetylated HMO, and/or a sialylated HMO. In certain exemplary aspects of the present disclosure, the nutritional composition comprises a combination of a fucosylated HMO, a sialylated HMO, and an N-acetylated HMO. Specific non-limiting examples of suitable HMOs include 2′-FL, 3-FL, 3′-SL, 6′-SL, LNnT, and LNT. [0070] In certain exemplary aspects of the present disclosure, the nutritional composition comprises a combination of 2′-FL, 3-FL, LNT, 3′-SL, and 6’-SL or the nutritional composition comprises a combination of 2′-FL, 3-FL, LNnT, 3′-SL, and 6′-SL. In certain exemplary aspects of the present disclosure, the nutritional composition comprises 2’-FL in an amount up to 4.15 mg/mL, LNT in an amount up to 2.11 mg/mL, 3-FL in an amount up to 1.17 mg/mL, 3’-SL in an amount up to 0.36 mg/mL, and 6’-SL in an amount up to 0.44 mg/mL. Long Chain Polyunsaturated Fatty Acids (LCPUFAs) [0071] The nutritional composition may include LCPUFAs to provide nutritional support, as well as to enhance growth and functional development of the nervous system. In some embodiments, the nutritional composition includes a combination of one or more HMOs and one or more LCPUFAs such that the composition provides a benefit to the end user, such as an improved benefit in modulating or treating one or more of the disease symptoms described herein. [0072] Exemplary LCPUFAs for use in the nutritional compositions include, for example, ω-3 LCPUFAs and ω-6 LCPUFAs. Specific LCPUFAs include docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), arachidonic acid (ARA), linoleic acid, linolenic acid (alpha linolenic acid) and gamma-linolenic acid derived from oil sources such as plant oils, marine plankton, fungal oils, and fish oils. In one particular embodiment, the LCPUFAs are derived from fish oils such as menhaden, salmon, anchovy, cod, halibut, tuna, or - 16 - 4859-9621-0305, v.1 herring oil. Particularly preferred LCPUFAs for use in the nutritional compositions with the HMOs include DHA, ARA, EPA, DPA, and combinations thereof. [0073] In order to reduce potential side effects of high dosages of LCPUFAs in the nutritional compositions, the content of LCPUFAs preferably does not exceed 3% by weight of the total fat content, including below 2% by weight of the total fat content, and including below 1% by weight of the total fat content in the nutritional composition. [0074] The LCPUFA may be provided as free fatty acids, in triglyceride form, in diglyceride form, in monoglyceride form, in phospholipid form, in esterified form or as a mixture of one or more of the above, preferably in triglyceride form. [0075] The nutritional compositions as described herein will typically comprise total concentrations of ARA, DHA, EPA, and DPA of from about 0.001 g/L to about 1 g/L or more, including from about 0.01 g/L to about 1 g/L, and about 0.1 g/L to about 1 g/L. [0076] In one embodiment, the nutritional compositions include total long chain ω-6 fatty acids in a concentration of from about 100 to about 425 mg/L or from about 12 to about 53 mg per 100 kcals and/or further include total long chain ω-3 fatty acids in a concentration of from about 40 to about 185 mg/L or from about 5 to about 23 mg per 100 kcals. In one specific embodiment, the ratio of long chain ω-6 fatty acids to long chain ω-3 fatty acids in the nutritional compositions ranges from about 2:1 to about 3:1, preferably about 2.5:1. [0077] In one specific embodiment, the nutritional compositions include DHA in a concentration of from about 0.025 mg/mL to about 0.130 mg/mL or from about 3 to about 16 mg per 100 kcals. In another embodiment, the nutritional compositions include ARA in a concentration of from about 0.080 mg/mL to about 0.250 mg/mL or from about 10 to about 31 mg per 100 kcals. In yet another embodiment, the nutritional compositions include combinations of DHA and ARA such that the ratio of DHA to ARA ranges from about 1:4 to about 1:2. Antioxidants [0078] The nutritional compositions may comprise one or more antioxidants to provide nutritional support. In some embodiments, the nutritional composition includes a combination of HMOs and antioxidants such that the composition provides a benefit to the end user, such as an improved benefit in modulating or treating one or more of the disease symptoms described - 17 - 4859-9621-0305, v.1 herein. In some embodiments, the HMO or HMOs is used in combination with carotenoids (and specifically lutein, beta-carotene, zeaxanthin and/or lycopene) to provide the synergistic effect. [0079] Any antioxidants suitable for oral administration may be included for use in the nutritional compositions of the present disclosure, including, for example, vitamin A, vitamin E, vitamin C, retinol, tocopherol, and carotenoids, including lutein, beta-carotene, zeaxanthin, and lycopene, and combinations thereof, for example. [0080] The antioxidants for use in the nutritional compositions may be used with the HMOs, alone, or in combination with HMOs and LCPUFAs and/or nucleotides. In one embodiment, the antioxidants for use in the nutritional compositions include carotenoids. In one embodiment the carotenoids are lutein, lycopene, zeaxanthin and/or beta-carotene. [0081] The nutritional compositions may comprise at least one of lutein, lycopene, zeaxanthin, and beta-carotene to provide a total amount of carotenoid of from about 0.001 µg/mL to about 10 µg/mL. In one embodiment, the nutritional compositions may comprise lutein in an amount of from about 0.001 µg/mL to about 10 µg/mL, including from about 0.001 µg/mL to about 5 µg/mL, including from about 0.001 µg/mL to about 0.0190 µg/mL, including from about 0.001 µg/mL to about 0.0140 µg/mL, and also including from about 0.044 µg/mL to about 5 µg/mL of lutein. In another embodiment, the nutritional compositions comprise from about 0.001 µg/mL to about 10 µg/mL, including from about 0.001 µg/mL to about 5 µg/mL, including from about 0.001 µg/mL to about 0.0130 µg/mL, including from about 0.001 µg/mL to about 0.0075 µg/mL, and also including from about 0.0185 µg/mL to about 5 µg/mL of lycopene. In another embodiment, the nutritional compositions comprise from about 1 µg/mL to about 10 µg/mL, including from about 1 µg/mL to about 5 µg/mL, including from about 0.001 µg/mL to about 0.025 µg/mL, including from about 0.001 µg/mL to about 0.011 µg/mL, and also including from about 0.034 µg/mL to about 5 µg/mL of beta-carotene. Any combination of these amounts of beta-carotene, lutein, zeaxanthin, and lycopene can be included in the nutritional compositions. Other carotenoids may optionally be included in the nutritional compositions. Any one or all of the carotenoids included in the nutritional compositions may be from a natural source, or artificially synthesized. RRR-Alpha-Tocopherol - 18 - 4859-9621-0305, v.1 [0082] The nutritional compositions described herein may include RRR-alpha-tocopherol in combination with the HMOs to provide nutritional support, as well as to provide an improved benefit to the end user, such as a benefit in modulating or treating one or more of the disease symptoms described herein. [0083] In certain exemplary aspects of the present disclosure, the nutritional compositions contain RRR-alpha-tocopherol in concentrations of at least about 3 mg/L, including at least about 5 mg/L, including at least about 7 mg/L, including at least about 8 mg/L, including at least about 9 mg/L, including at least about 10 mg/L, including from at least about 5 mg/L to about 100 mg/L, including from at least about 7 mg/L to about 50 mg/L, including from about 7 mg/L to about 40 mg/L, and including from about 7 mg/L to about 20 mg/L. The total amounts of RRR- alpha-tocopherol include both exogenous and inherent sources of RRR-alpha-tocopherol, as noted below. [0084] As used herein, the term “RRR-alpha-tocopherol” refers both to exogenous sources and inherent sources of RRR-alpha-tocopherol and RRR-alpha-tocopherol acetate that are present in a nutritional composition. Inherent sources include RRR-alpha-tocopherol that is inherently present in components that are present in a nutritional composition and may include for example, various oils and fats. Exogenous sources of RRR-alpha-tocopherol include RRR-alpha- tocopherol that is added to the nutritional composition not as part of another component. Nucleotides [0085] The nutritional compositions may comprise nucleotides and/or nucleotide precursors selected from nucleosides, purine bases, pyrimidine bases, ribose, and deoxyribose. The nucleotide may be in monophosphate, diphosphate, or triphosphate form. The nucleotide may be a ribonucleotide or a deoxyribonucleotide. The nucleotides may be monomeric, dimeric, or polymeric (including RNA and DNA). The nucleotide may be present in the nutritional composition as a free acid or in the form of a salt, preferably a monosodium salt. In some embodiments, the nutritional composition includes a combination of HMOs and nucleotides such that the composition provides a benefit to the end user, such as a benefit in modulating or treating one or more of the disease symptoms described herein. - 19 - 4859-9621-0305, v.1 [0086] Suitable nucleotides and/or nucleosides for use in the nutritional compositions include one or more of cytidine 5'-monophosphate, uridine 5'-monophosphate, adenosine 5'- monophosphate, guanosine 5'-1-monophosphate, and/or inosine 5'-monophosphate, such as cytidine 5'-monophosphate, uridine 5'-monophosphate, adenosine 5'-monophosphate, guanosine 5'-monophosphate, and inosine 5'-monophosphate. [0087] The nucleotides are present in the nutritional compositions in total amounts of nucleotides of at least about 5 mg/L, including at least about 10 mg/L, including from about 10 mg/L to about 300 mg/L, including from about 42 mg/L to about 200 mg/L, including from about 72 mg/L to about 200 mg/L, including from about 72 mg/L to about 120 mg/L and including at least about 72 mg/L of the nutritional product. Macronutrients [0088] The nutritional compositions may be formulated to include at least one of protein, fat, and carbohydrate. In many embodiments, the nutritional compositions will contain an HMO or HMOs and comprise at least one of fat, protein, or carbohydrate, including protein, fat, and carbohydrate. [0089] Although total concentrations or amounts of the fat, protein, and carbohydrates may vary depending upon the product type (i.e., human milk fortifier, preterm infant formula, infant formula, etc.), product form (i.e., nutritional solid, powder, ready-to-feed liquid, concentrated liquid, or nutritional bar) and targeted dietary needs of the intended user, such concentrations or amounts most typically fall within one of the following embodied ranges, inclusive of any other essential fat, protein, and/or carbohydrate ingredients as described herein. [0090] For the liquid formulas, carbohydrate concentrations most typically range from about 5% to about 40%, including from about 7% to about 30%, including from about 10% to about 25%, by weight; fat concentrations most typically range from about 1% to about 30%, including from about 2% to about 15%, and also including from about 3% to about 10%, by weight; and protein concentrations most typically range from about 0.5% to about 30%, including from about 1% to about 15%, and also including from about 2% to about 10%, by weight. [0091] For the liquid human milk fortifiers, carbohydrate concentrations most typically range from about 10% to about 75%, including from about 10% to about 50%, including from about - 20 - 4859-9621-0305, v.1 20% to about 40%, by weight of the human milk fortifier; fat concentrations most typically range from about 10% to about 40%, including from about 15% to about 37%, and also including from about 18% to about 30%, by weight of the human milk fortifier; and protein concentrations most typically range from about 5% to about 40%, including from about 10% to about 30%, and also including from about 15% to about 25%, by weight of the human milk fortifier. [0092] In one specific example, liquid infant formulas (both ready-to-feed and concentrated liquids) include those embodiments in which the protein component may comprise from about 7.5% to about 25% of the caloric content of the formula; the carbohydrate component may comprise from about 35% to about 50% of the total caloric content of the infant formula; and the fat component may comprise from about 30% to about 60% of the total caloric content of the infant formula. These ranges are provided as examples only and are not intended to be limiting. Additional suitable ranges are noted in the following table (each numerical value is preceded by the term “about”). [0093] When the nutritional product is a powdered adult, child, toddler, newborn, pediatric, preterm, or term infant formula, the protein component is present in an amount of from about 5% to about 35%, including from about 8% to about 12%, and including from about 10% to about 12% by weight of the preterm or term infant formula; the fat component is present in an amount of from about 10% to about 35%, including from about 25% to about 30%, and including from about 26% to about 28% by weight of the preterm or term infant formula; and the carbohydrate component is present in an amount of from about 30% to about 85%, including from about 45% to about 60%, including from about 50% to about 55% by weight of the preterm or term infant formula. Fat [0094] The nutritional compositions may comprise a source or sources of fat (in addition to the LCPUFAs discussed above). Suitable additional sources of fat for use herein include any fat or fat source that is suitable for use in an oral nutritional product and is compatible with the essential elements and features of such products. For example, in one specific embodiment, the additional fat is derived from long chain polyunsaturated fatty acids and/or short chain fatty acids. - 21 - 4859-9621-0305, v.1 [0095] Additional non-limiting examples of suitable fats or sources thereof for use in the nutritional products described herein include coconut oil, fractionated coconut oil, soybean oil, corn oil, olive oil, safflower oil, high oleic safflower oil, oleic acids (EMERSOL 6313 OLEIC ACID, Cognis Oleochemicals, Malaysia), MCT oil (medium chain triglycerides), sunflower oil, high oleic sunflower oil, palm and palm kernel oils, palm olein, canola oil, marine oils, fish oils, fungal oils, algae oils, cottonseed oils, and combinations thereof. Protein [0096] The nutritional compositions may comprise a source or sources of protein. Any protein source that is suitable for use in oral nutritional compositions and is compatible with the essential elements and features of such products is suitable for use in the nutritional compositions. [0097] Non-limiting examples of suitable proteins or sources thereof for use in the nutritional products include hydrolyzed, partially hydrolyzed or non-hydrolyzed proteins or protein sources, which may be derived from any known or otherwise suitable source such as milk (e.g., casein, whey), animal (e.g., meat, fish), cereal (e.g., rice, corn), vegetable (e.g., soy) or combinations thereof. Non-limiting examples of such proteins include milk protein isolates, milk protein concentrates as described herein, casein protein isolates, extensively hydrolyzed casein, whey protein, sodium or calcium caseinates, whole cow milk, partially or completely defatted milk, soy protein isolates, soy protein concentrates, and so forth. In one specific embodiment, the nutritional compositions include a protein source derived from milk proteins of human and/or bovine origin. Carbohydrate [0098] The nutritional composition may comprise, in addition to the HMOs, any carbohydrates that are suitable for use in an oral nutritional product and are compatible with the essential elements and features of such products. [0099] Non-limiting examples of suitable carbohydrates or sources thereof for use in the nutritional products described herein may include maltodextrin, hydrolyzed or modified starch or cornstarch, glucose polymers, corn syrup, corn syrup solids, rice-derived carbohydrates, pea- derived carbohydrates, potato-derived carbohydrates, tapioca, sucrose, glucose, fructose, lactose, high fructose corn syrup, honey, sugar alcohols (e.g., maltitol, erythritol, sorbitol), artificial - 22 - 4859-9621-0305, v.1 sweeteners (e.g., sucralose, acesulfame potassium, stevia) and combinations thereof. A particularly desirable carbohydrate is a low dextrose equivalent (DE) maltodextrin. Other Optional Ingredients [00100] The nutritional compositions may comprise other optional ingredients that may modify the physical, chemical, aesthetic or processing characteristics of the products or serve as pharmaceutical or additional nutritional components when used in the targeted population. Many such optional ingredients are known or otherwise suitable for use in medical food or other nutritional products or pharmaceutical dosage forms and may also be used in the compositions herein, provided that such optional ingredients are safe for oral administration and are compatible with the essential and other ingredients in the selected product form. [00101] Non-limiting examples of such optional ingredients include preservatives, emulsifying agents, buffers, fructooligosaccharides, galactooligosaccharides, polydextrose, and other prebiotics (e.g., inulin, oligofructose, polydextrose, pectin hydrolysate, and gums), probiotics (e.g., B. animalis subsp. lactis BB-12, B. lactis HN019, B. lactis Bi07, L. rhamnosus GG, L. rhamnosus HN001, L. acidophilus LA-5, L. acidophilus NCFM, L. fermentum CECT5716, B. longum BB536, B. longum AH1205, B. longum AH1206, B. breve M-16V, L. reuteri ATCC 55730, L. reuteri ATCC PTA-6485, L. reuteri DSM 17938), postbiotics (metabolites of probiotics), antioxidant/anti-inflammatory compounds including tocopherols, ascorbate/ vitamin C, ascorbyl palmitate, polyphenols (e.g., curcumin), glutathione, and superoxide dismutase (melon), milk protein of human and/or bovine origin, soy protein, pea protein, other bioactive factors (e.g., growth hormones, cytokines, TFG-β) of human and/or bovine origin, tributyrin or other SCFA-containing mono-, di-, or triglylcerides, human milk- derived lipids, free amino acids or peptides (e.g., HMB, arginine, leucine, and/or glutamine), lactose, other water- and fat-soluble vitamins, minerals, and trace elements, pharmaceutical actives, additional nutrients as described herein, colorants, flavors, thickening agents and stabilizers, emulsifying agents, lubricants, and so forth. [00102] The nutritional compositions may further comprise a sweetening agent, preferably including at least one sugar alcohol such as maltitol, erythritol, sorbitol, xylitol, mannitol, isomalt, and lactitol, and also preferably including at least one artificial or high potency sweetener such as acesulfame K, aspartame, sucralose, saccharin, stevia, and tagatose. These - 23 - 4859-9621-0305, v.1 sweetening agents, especially as a combination of a sugar alcohol and an artificial sweetener, are especially useful in formulating liquid beverage embodiments of the present disclosure having a desirable favor profile. These sweetener combinations are especially effective in masking undesirable flavors sometimes associated with the addition of vegetable proteins to a liquid beverage. Optional sugar alcohol concentrations in the nutritional product may range from at least 0.01%, including from about 0.1% to about 10%, and also including from about 1% to about 6%, by weight of the nutritional product. Optional artificial sweetener concentrations may range from about 0.01%, including from about 0.05% to about 5%, also including from about 0.1% to about 1.0%, by weight of the nutritional product. [00103] A flowing agent or anti-caking agent may be included in the nutritional compositions as described herein to retard clumping or caking of the powder over time and to make a powder embodiment flow easily from its container. Any known flowing or anti-caking agents that are known or otherwise suitable for use in a nutritional powder or product form are suitable for use herein, non-limiting examples of which include tricalcium phosphate, silicates, and combinations thereof. The concentration of the flowing agent or anti-caking agent in the nutritional composition varies depending upon the product form, the other selected ingredients, the desired flow properties, and so forth, but most typically range from about 0.1% to about 4%, including from about 0.5% to about 2%, by weight of the nutritional composition. [00104] A stabilizer may also be included in the nutritional compositions. Any stabilizer that is known or otherwise suitable for use in a nutritional composition is also suitable for use herein, some non-limiting examples of which include gums such as xanthan gum. The stabilizer may represent from about 0.1% to about 5.0%, including from about 0.5% to about 3%, including from about 0.7% to about 1.5%, by weight of the nutritional composition. [00105] The nutritional compositions may further comprise any of a variety of other vitamins or related nutrients, non-limiting examples of which include vitamin A, vitamin D, vitamin K, thiamine, riboflavin, pyridoxine, vitamin B12, carotenoids (e.g., beta-carotene, zeaxanthin, lutein, lycopene), niacin, folic acid, pantothenic acid, biotin, vitamin C, choline, inositol, salts and derivatives thereof, and combinations thereof. - 24 - 4859-9621-0305, v.1 [00106] The nutritional compositions may further comprise any of a variety of other additional minerals, non-limiting examples of which include calcium, phosphorus, magnesium, iron, zinc, manganese, copper, sodium, potassium, molybdenum, chromium, chloride, and combinations thereof. Methods of Manufacture [00107] The nutritional composition may be prepared by any known or otherwise effective manufacturing technique for preparing the selected product solid or liquid form. Many such techniques are known for any given product form such as nutritional liquids, powders, and nutritional bars, and can easily be applied by one of ordinary skill in the art to the nutritional compositions described herein. [00108] The nutritional composition can be prepared by any of a variety of known or otherwise effective formulation or manufacturing methods. In one suitable manufacturing process, for example, at least three separate slurries are prepared, including a protein-in-fat (PIF) slurry, a carbohydrate-mineral (CHO-MIN) slurry, and a protein-in-water (PIW) slurry. The PIF slurry is formed by heating and mixing the oil (e.g., canola oil, corn oil, etc.) and then adding an emulsifier (e.g., lecithin), fat soluble vitamins, and a portion of the total protein (e.g., milk protein concentrate, etc.) with continued heat and agitation. The CHO-MIN slurry is formed by adding with heated agitation to water: minerals (e.g., potassium citrate, dipotassium phosphate, sodium citrate, etc.), trace and ultra trace minerals (TM/UTM premix), thickening or suspending agents (e.g. avicel, gellan, carrageenan). The resulting CHO-MIN slurry is held for 10 minutes with continued heat and agitation before adding additional minerals (e.g., potassium chloride, magnesium carbonate, potassium iodide, etc.), and/or carbohydrates (e.g., HMOs, fructooligosaccharide, sucrose, corn syrup, etc.). The PIW slurry is then formed by mixing with heat and agitation the remaining protein, if any. [00109] The resulting slurries are then blended together with heated agitation and the pH adjusted to 6.6-7.0, after which the composition is subjected to high-temperature short-time (HTST) processing during which the composition is heat treated, emulsified and homogenized, and then allowed to cool. Water soluble vitamins and ascorbic acid are added, the pH is adjusted to the desired range if necessary, flavors are added, and water is added to achieve the desired total solid level. The composition is then aseptically packaged to form an aseptically packaged - 25 - 4859-9621-0305, v.1 nutritional emulsion. This emulsion can then be further diluted, heat-treated, and packaged to form a ready-to-feed or concentrated liquid, or it can be heat-treated and subsequently processed and packaged as a reconstitutable powder, e.g., spray dried, drymixed, agglomerated. [00110] The nutritional solid, such as a spray dried nutritional powder or drymixed nutritional powder, may be prepared by any collection of known or otherwise effective technique, suitable for making and formulating a nutritional powder. [0100] For example, when the nutritional powder is a spray dried nutritional powder, the spray drying step may likewise include any spray drying technique that is known for or otherwise suitable for use in the production of nutritional powders. Many different spray drying methods and techniques are known for use in the nutrition field, all of which are suitable for use in the manufacture of the spray dried nutritional powders herein. [0101] One method of preparing the spray dried nutritional powder comprises forming and homogenizing an aqueous slurry or liquid comprising predigested fat, and optionally protein, carbohydrate, and other sources of fat, and then spray drying the slurry or liquid to produce a spray dried nutritional powder. The method may further comprise the step of spray drying, drymixing, or otherwise adding additional nutritional ingredients, including any one or more of the ingredients described herein, to the spray dried nutritional powder. [0102] Other suitable methods for making nutritional products are described, for example, in U.S. Pat. No.6,365,218 (Borschel, et al.), U.S. Patent 6,589,576 (Borschel, et al.), U.S. Pat. No. 6,306,908 (Carlson, et al.), U.S. Patent Application 20030118703 A1 (Nguyen, et al.), which descriptions are incorporated herein by reference to the extent that they are consistent herewith. Product Form [0103] The nutritional composition may be formulated and administered in any known or otherwise suitable oral product form. Any solid, liquid, semi-solid, semi-liquid, or powder product form, including combinations or variations thereof, are suitable for use herein, provided that such forms allow for safe and effective oral delivery to the individual of the ingredients as also defined herein. [0104] The composition may be formulated as dietary product forms, which are defined herein as those embodiments comprising the ingredients of the present disclosure in a product form that - 26 - 4859-9621-0305, v.1 then contains at least one of fat, protein, and carbohydrate, and preferably also contains vitamins, minerals, or combinations thereof. [0105] The nutritional compositions may be formulated with sufficient kinds and amounts of nutrients to provide a sole, primary, or supplemental source of nutrition, or to provide a specialized nutritional product for use in individuals afflicted with specific conditions or with a targeted nutritional benefit as described below. [0106] Some exemplary, non-limiting, examples of specific products that may be suitable for use in accordance with the present disclosure include preterm infant formulas, term infant formulas, human milk fortifiers, pediatric formulas, adult nutritional formulas, older adult nutritional formulas, medical formulas, geriatric nutritional formulas, diabetic nutritional formulas, nutritional bar, and the like. Nutritional Liquids [0107] Nutritional liquids include both concentrated and ready-to-feed nutritional liquids. These nutritional liquids are most typically formulated as suspensions or emulsions, although other liquid forms are within the scope of the present disclosure. [0108] Nutritional emulsions suitable for use may be aqueous emulsions comprising proteins, fats, and carbohydrates. These emulsions are generally flowable or drinkable liquids at from about 1°C to about 25°C and are typically in the form of oil-in-water, water-in-oil, or complex aqueous emulsions, although such emulsions are most typically in the form of oil-in-water emulsions having a continuous aqueous phase and a discontinuous oil phase. [0109] The nutritional emulsions may be and typically are shelf stable. The nutritional emulsions typically contain up to 95% by weight of water, including from about 50% to 95%, also including from about 60% to about 90%, and also including from about 70% to about 85%, of water by weight of the nutritional emulsions. The nutritional emulsions may have a variety of product densities, but most typically have a density greater than 1.03 g/mL, including greater than 1.04 g/mL, including greater than 1.055 g/mL, including from about 1.06 g/mL to about 1.12 g/mL, and also including from about 1.085 g/mL to about 1.10 g/mL. [0110] The nutritional emulsions may have a caloric density tailored to the nutritional needs of the ultimate user, although in most instances the emulsions comprise generally at least 19 kcal/fl - 27 - 4859-9621-0305, v.1 oz (660 kcal/liter), more typically from about 20 kcal/fl oz (675-680 kcal/liter) to about 25 kcal/fl oz (820 kcal/liter), even more typically from about 20 kcal/fl oz (675-680 kcal/liter) to about 24 kcal/fl oz (800-810 kcal/liter). Generally, the 22-24 kcal/fl oz formulas are more commonly used in preterm or low birth weight infants, and the 20-21 kcal/fl oz (675-680 to 700 kcal/liter) formulas are more often used in term infants. In some embodiments, the emulsion may have a caloric density of from about 50-100 kcal/liter to about 660 kcal/liter, including from about 150 kcal/liter to about 500 kcal/liter. In some specific embodiments, the emulsion may have a caloric density of 25, or 50, or 75, or 100 kcal/liter. [0111] The nutritional emulsion may have a pH ranging from about 3.5 to about 8 but are most advantageously in a range of from about 4.5 to about 7.5, including from about 5.5 to about 7.3, including from about 6.2 to about 7.2. [0112] Although the serving size for the nutritional emulsion can vary depending upon a number of variables, a typical serving size is generally at least 1 mL, or even at least 2 mL, or even at least 5 mL, or even at least 10 mL, or even at least 25 mL, including ranges from 1 mL to about 300 mL, including from about 4 mL to about 250 mL, and including from about 10 mL to about 240 mL. Nutritional Solids [0113] The nutritional solids may be in any solid form but are typically in the form of flowable or substantially flowable particulate compositions, or at least particulate compositions. Particularly suitable nutritional solid product forms include spray dried, agglomerated and/or dryblended powder compositions. The compositions can easily be scooped and measured with a spoon or similar other device and can easily be reconstituted by the intended user with a suitable aqueous liquid, typically water, to form a nutritional composition for immediate oral or enteral use. In this context, “immediate” use generally means within about 48 hours, most typically within about 24 hours, preferably right after reconstitution. [0114] The nutritional powders may be reconstituted with water prior to use to a caloric density tailored to the nutritional needs of the ultimate user, although in most instances the powders are reconstituted with water to form compositions comprising at least 19 kcal/fl oz (660 kcal/liter), more typically from about 20 kcal/fl oz (675-680 kcal/liter) to about 25 kcal/fl oz (820 kcal/liter), - 28 - 4859-9621-0305, v.1 even more typically from about 20 kcal/fl oz (675-680 kcal/liter) to about 24 kcal/fl oz (800-810 kcal/liter). Generally, the 22-24 kcal/fl oz formulas are more commonly used in preterm or low birth weight infants, and the 20-21 kcal/fl oz (675-680 to 700 kcal/liter) formulas are more often used in term infants. In some embodiments, the reconstituted powder may have a caloric density of from about 50-100 kcal/liter to about 660 kcal/liter, including from about 150 kcal/liter to about 500 kcal/liter. In some specific embodiments, the emulsion may have a caloric density of 25, or 50, or 75, or 100 kcal/liter. Methods of Use [0115] The nutritional compositions of the present disclosure include adult formulas, infant formulas and human milk fortifiers that are orally administered to adults, children, and infants, including preterm or term infants. The nutritional compositions may be administered as a source of nutrition for individuals, to enhance blood flow to the brain (e.g., enhance nutrient uptake or brain chemistry), enhance the BBB function, enhance the BBB integrity (e.g., increase tight junctions or reduce permeability), and/or to treat one or more BBB disorders, conditions, injuries or dysfunctions. One subclass of the general population that can effectively utilize the nutritional compositions described herein include those individuals that are susceptible to, or at a risk of (at an elevated risk as compared to the general infant population) immaturity of the central nervous system and including the brain and the BBB. For example, infants who are susceptible to or at risk of having immaturity of, for example, the brain are herein referred to as “in need of” assistance (or “in need thereof” as referring to the assistance needed) in combating organ development problems such as neural inflammation and BBB disorders or dysfunctions. [0116] Based on the forgoing, because some of the method embodiments of the present disclosure are directed to specific subsets or subclasses of individuals (that is, the subset or subclass of infants that are “in need” of assistance in BBB disorders or function) in those embodiments, not all individuals will benefit from all method embodiments described herein as not all individuals will fall within the subset or subclass of individuals (e.g., infants) as described herein. [0117] In certain exemplary embodiments, the methods of the present disclosure are directed to infants during the initial days, weeks or months of life. Desirably, in certain exemplary embodiments, the nutritional compositions, when in the form of an infant formulas described - 29 - 4859-9621-0305, v.1 herein, are administered to the infant for a duration of at least the first week of life, more desirably during at least the first two weeks of life, more desirably during at least the first one or two months of life, more desirably during at least the first four months of life, and more desirably during at least the first six months of life, and including up to the first year of life. Thereafter, the infant may be switched to a conventional infant formula, alone or in combination with human milk. It should be understood by one skilled in the art based on the disclosure herein that the infant formulas described herein can be used alone, or in combination with other infant formulas. [0118] The nutritional compositions used in the methods described herein, unless otherwise specified, are infant formulas (including human milk fortifiers) and may be in any product form, including ready-to-feed liquids, concentrated liquids, reconstituted powders, and the like as described above. In embodiments where the infant formulas are in powder form, the method may further comprise reconstituting the powder with an aqueous vehicle, most typically water or human milk, to form the desired caloric density, which is then orally or enterally fed to the infant. The powdered formulas are reconstituted with a sufficient quantity of water or other suitable fluid to produce the desired caloric density, as well as the desired feeding volume suitable for one infant feeding. The infant formulas may also be sterilized prior to use through retort or aseptic means. [0119] In certain exemplary embodiments, the present disclosure is directed to a method for the prevention, delay of progression, or the treatment of a circulatory disorder characterized by suboptimal or impaired BBB function. The method comprises administering to an individual in need thereof a nutritional composition comprising a therapeutically effective amount of an HMO or HMO blend. In certain exemplary embodiments of the methods described herein, the method comprises identifying an individual in need thereof. [0120] In certain exemplary embodiments, administration of the nutritional compositions according to the general inventive concepts leads to improved blood flow, improved or enhanced BBB function or integrity and reduction in one or more symptoms associated with BBB disorders or conditions and may also lead to the prevention, delay of progression, or the treatment of a neurodegenerative disorder or other neuro-associated disorders or injuries. - 30 - 4859-9621-0305, v.1 EXAMPLES [0121] The exemplified compositions are shelf stable nutritional compositions prepared in accordance with the manufacturing methods described herein, such that each exemplified composition, unless otherwise specified, includes an aseptically processed embodiment and a retort packaged embodiment. Examples 1-5 [0122] Examples 1-5 illustrate ready-to-feed nutritional emulsions, the ingredients of which are listed in the table below. All ingredient amounts listed are in kilograms, unless otherwise specified. - 31 - 4859-9621-0305, v.1 Table 1: Examples 1-5 Ingredient Ex.1 Ex.2 Ex. 3 Ex.4 Ex.5 Water Q.S. Q.S. Q.S. Q.S. Q.S. 86.64 86.64 86.64 86.64
Figure imgf000034_0001
54.80 54.80 54.80 54.80 High oleic safflower oil 14.10 14.10 14.10 14.10 14.10 Soybean oil 10.6 10.6 10.6 10.6 10.6 Coconut oil 10.1 10.1 10.1 10.1 10.1 HMO Blend 0.049 0.097 0.245 0.490 3.92 Galactooligosaccharides (GOS) 3.92 3.92 3.92 3.92 0 Whey protein concentrate 6.40 6.40 6.40 6.40 6.40 Potassium citrate 478.9 g 478.9 g 478.9 g 478.9 g 478.9 g Calcium carbonate 448.28 g 448.28 g 448.28 g 448.28 g 448.28 g Soy lecithin 355.74 g 355.74 g 355.74 g 355.74 g 355.74 g Stabilizer 355.74 g 355.74 g 355.74 g 355.74 g 355.74 g ARA oil 368.01 g 368.01 g 368.01 g 368.01 g 368.01 g Nucleotide/chloride premix 293.26 g 293.26 g 293.26 g 293.26 g 293.26 g Potassium chloride 226.45 g 226.45 g 226.45 g 226.45 g 226.45 g Ascorbic acid 445.94 g 445.94 g 445.94 g 445.94 g 445.94 g Vitamin mineral premix 142.88 g 142.88 g 142.88 g 142.88 g 142.88 g DHA oil 137.8 g 137.8 g 137.8 g 137.8 g 137.8 g Carrageenan 180.0 g 180.0 g 180.0 g 180.0 g 180.0 g Magnesium chloride 55.0 g 55.0 g 55.0 g 55.0 g 55.0 g Ferrous sulfate 58.0 g 58.0 g 58.0 g 58.0 g 58.0 g Choline chloride 53.9 g 53.9 g 53.9 g 53.9 g 53.9 g Vitamin A, D3, E, K1 premix 47.4 g 47.4 g 47.4 g 47.4 g 47.4 g Citric acid 29.77 g 29.77 g 29.77 g 29.77 g 29.77 g Mixed carotenoid premix 26.40 g 26.40 g 26.40 g 26.40 g 26.40 g Sodium chloride AN AN AN AN AN L-carnitine 3.31 g 3.31 g 3.31 g 3.31 g 3.31 g Tricalcium phosphate 15.65 g 15.65 g 15.65 g 15.65 g 15.65 g Potassium phosphate monobasic 13.67 g 13.67 g 13.67 g 13.67 g 13.67 g Riboflavin 2.42 g 2.42 g 2.42 g 2.42 g 2.42 g Potassium hydroxide AN AN AN AN AN AN = as needed Examples 6-10 [0123] Examples 6-10 illustrate ready-to-feed nutritional emulsions of the present disclosure, the ingredients of which are listed in the table below. All ingredient amounts listed are in kilograms, unless otherwise specified. - 32 - 4859-9621-0305, v.1 Table 2: Examples 6-10 Ingredient Ex.6 Ex.7 Ex. 8 Ex.9 Ex.10 Water Q.S. Q.S. Q.S. Q.S. Q.S. Condensed Skim Milk 86.64 86.64 86.64 86.64 86.64 Lactose 54.80 54.80 54.80 54.80 54.80 High oleic safflower oil 14.10 14.10 14.10 14.10 14.10 Soybean oil 10.6 10.6 10.6 10.6 10.6 Coconut oil 10.1 10.1 10.1 10.1 10.1 2’-fucosyllactose (2’FL) 0.049 0.097 0.245 0.490 3.92 Galactooligosaccharides (GOS) 3.92 3.92 3.92 3.92 0 Whey protein concentrate 6.40 6.40 6.40 6.40 6.40 Potassium citrate 478.9 g 478.9 g 478.9 g 478.9 g 478.9 g Calcium carbonate 448.28 g 448.28 g 448.28 g 448.28 g 448.28 g Soy lecithin 355.74 g 355.74 g 355.74 g 355.74 g 355.74 g Stabilizer 355.74 g 355.74 g 355.74 g 355.74 g 355.74 g ARA oil 368.01 g 368.01 g 368.01 g 368.01 g 368.01 g Nucleotide/chloride premix 293.26 g 293.26 g 293.26 g 293.26 g 293.26 g Potassium chloride 226.45 g 226.45 g 226.45 g 226.45 g 226.45 g Ascorbic acid 445.94 g 445.94 g 445.94 g 445.94 g 445.94 g Vitamin mineral premix 142.88 g 142.88 g 142.88 g 142.88 g 142.88 g DHA oil 137.8 g 137.8 g 137.8 g 137.8 g 137.8 g Carrageenan 180.0 g 180.0 g 180.0 g 180.0 g 180.0 g Magnesium chloride 55.0 g 55.0 g 55.0 g 55.0 g 55.0 g Ferrous sulfate 58.0 g 58.0 g 58.0 g 58.0 g 58.0 g Choline chloride 53.9 g 53.9 g 53.9 g 53.9 g 53.9 g Vitamin A, D3, E, K1 premix 47.40 g 47.40 g 47.40 g 47.40 g 47.40 g Citric acid 29.77 g 29.77 g 29.77 g 29.77 g 29.77 g Mixed carotenoid premix 26.40 g 26.40 g 26.40 g 26.40 g 26.40 g Sodium chloride AN AN AN AN AN L-carnitine 3.31 g 3.31 g 3.31 g 3.31 g 3.31 g Tricalcium phosphate 15.65 g 15.65 g 15.65 g 15.65 g 15.65 g Potassium phosphate monobasic 13.67 g 13.67 g 13.67 g 13.67 g 13.67 g Riboflavin 2.42 g 2.42 g 2.42 g 2.42 g 2.42 g Potassium hydroxide AN AN AN AN AN AN = as needed Examples 11-15 [0124] Examples 11-15 illustrate ready-to-feed nutritional emulsions, the ingredients of which are listed in the table below. All ingredient amounts listed are in kilograms, unless otherwise specified. - 33 - 4859-9621-0305, v.1 Table 3: Examples 11-15 Ingredient Ex.11 Ex.12 Ex.13 Ex.14 Ex.15 Water Q.S. Q.S. Q.S. Q.S. Q.S. Condensed Skim Milk 86.64 86.64 86.64 86.64 86.64 Lactose 54.80 54.80 54.80 54.80 54.80 High oleic safflower oil 14.10 14.10 14.10 14.10 14.10 Soybean oil 10.6 10.6 10.6 10.6 10.6 Coconut oil 10.1 10.1 10.1 10.1 10.1 6’ sialyllactose (6’SL) 0.049 0.097 0.245 0.490 0.98 2’fucosyllactose (2’FL) 0.049 0.097 0.245 0.490 0.98 Lacto-N-tetraose (LNT) 0.049 0.097 0.245 0.490 0.98 Galactooligosaccharides (GOS) 3.92 3.92 3.92 1.96 0.98 Whey protein concentrate 6.40 6.40 6.40 6.40 6.40 Potassium citrate 478.9 g 478.9 g 478.9 g 478.9 g 478.9 g Calcium carbonate 448.28 g 448.28 g 448.28 g 448.28 g 448.28 g Soy lecithin 355.74 g 355.74 g 355.74 g 355.74 g 355.74 g Stabilizer 355.74 g 355.74 g 355.74 g 355.74 g 355.74 g ARA oil 368.01 g 368.01 g 368.01 g 368.01 g 368.01 g Nucleotide/chloride premix 293.26 g 293.26 g 293.26 g 293.26 g 293.26 g Potassium chloride 226.45 g 226.45 g 226.45 g 226.45 g 226.45 g Ascorbic acid 445.94 g 445.94 g 445.94 g 445.94 g 445.94 g Vitamin mineral premix 142.88 g 142.88 g 142.88 g 142.88 g 142.88 g DHA oil 137.8 g 137.8 g 137.8 g 137.8 g 137.8 g Carrageenan 180.0 g 180.0 g 180.0 g 180.0 g 180.0 g Magnesium chloride 55.0 g 55.0 g 55.0 g 55.0 g 55.0 g Ferrous sulfate 58.0 g 58.0 g 58.0 g 58.0 g 58.0 g Choline chloride 53.9 g 53.9 g 53.9 g 53.9 g 53.9 g Vitamin A, D3, E, K1 premix 47.40 g 47.40 g 47.40 g 47.40 g 47.40 g Citric acid 29.77 g 29.77 g 29.77 g 29.77 g 29.77 g Mixed carotenoid premix 26.40 g 26.40 g 26.40 g 26.40 g 26.40 g Sodium chloride AN AN AN AN AN L-carnitine 3.31 g 3.31 g 3.31 g 3.31 g 3.31 g Tricalcium phosphate 15.65 g 15.65 g 15.65 g 15.65 g 15.65 g Potassium phosphate monobasic 13.67 g 13.67 g 13.67 g 13.67 g 13.67 g Riboflavin 2.42 g 2.42 g 2.42 g 2.42 g 2.42 g Potassium hydroxide AN AN AN AN AN AN = as needed Examples 16-20 [0125] Examples 16-20 illustrate liquid formulations, the ingredients of which are listed in the table below. All ingredient amounts listed are in kilograms, unless otherwise specified. Table 4: Examples 16-20 Ingredient Ex.16 Ex.17 Ex.18 Ex.19 Ex.20 Water Q.S. Q.S. Q.S. Q.S. Q.S. Sugar 87.7 87.7 87.7 87.7 87.7 Corn maltodextrin 70.7 70.7 70.7 70.7 70.7 Milk protein concentrate 26.2 26.2 26.2 26.2 26.2 - 34 - 4859-9621-0305, v.1 Soy oil 13.4 13.4 13.4 13.4 13.4 Soy protein isolate 10.5 10.5 10.5 10.5 10.5 Pea protein concentrate 6.2 6.2 6.2 6.2 6.2 Canola oil 5.4 5.4 5.4 5.4 5.4 Corn oil 4.1 4.1 4.1 4.1 4.1 Magnesium phosphate 2.9 2.9 2.9 2.9 2.9 Potassium citrate 2.1 2.1 2.1 2.1 2.1 Cellulose gel 2.0 2.0 2.0 2.0 2.0 Natural and artificial flavor 2.0 2.0 2.0 2.0 2.0 Calcium carbonate 1.0 1.0 1.0 1.0 1.0 Potassium chloride 1.0 1.0 1.0 1.0 1.0 Calcium phosphate 960.0 g 960.0 g 960.0 g 960.0 g 960.0 g Sodium citrate 800.0 g 800.0 g 800.0 g 800.0 g 800.0 g Salt 710.0 g 710.0 g 710.0 g 710.0 g 710.0 g Choline chloride 480.0 g 480.0 g 480.0 g 480.0 g 480.0 g Ascorbic acid 468.7 g 468.7 g 468.7 g 468.7 g 468.7 g Cellulose gum 360.0 g 360.0 g 360.0 g 360.0 g 360.0 g Monoglycerides 286.6 g 286.6 g 286.6 g 286.6 g 286.6 g Soy lecithin 286.6 g 286.6 g 286.6 g 286.6 g 286.6 g Carrageenan 240.0 g 240.0 g 240.0 g 240.0 g 240.0 g Potassium hydroxide 145.4 g 145.4 g 145.4 g 145.4 g 145.4 g Ferrous sulfate 59.8 g 59.8 g 59.8 g 59.8 g 59.8 g dl-alpha-tocopheryl acetate 54.8 g 54.8 g 54.8 g 54.8 g 54.8 g Zinc sulfate 45.6 g 45.6 g 45.6 g 45.6 g 45.6 g Niacinamide 25.9 g 25.9 g 25.9 g 25.9 g 25.9 g Manganese sulfate 17.6 g 17.6 g 17.6 g 17.6 g 17.6 g Calcium pantothenate 16.7 g 16.7 g 16.7 g 16.7 g 16.7 g Cupric sulfate 9.2 g 9.2 g 9.2 g 9.2 g 9.2 g Vitamin A palmitate 4.3 g 4.3 g 4.3 g 4.3 g 4.3 g Thiamine chloride hydrochloride 4.3 g 4.3 g 4.3 g 4.3 g 4.3 g Pyridoxine hydrochloride 4.1 g 4.1 g 4.1 g 4.1 g 4.1 g Riboflavin 3.3 g 3.3 g 3.3 g 3.3 g 3.3 g Folic acid 580.0 mg 580.0 mg 580.0 mg 580.0 mg 580.0 mg Chromium chloride 561.0 mg 561.0 mg 561.0 mg 561.0 mg 561.0 mg Biotin 504.0 mg 504.0 mg 504.0 mg 504.0 mg 504.0 mg Sodium molybdate 441.0 mg 441.0 mg 441.0 mg 441.0 mg 441.0 mg Potassium iodide 207.0 mg 207.0 mg 207.0 mg 207.0 mg 207.0 mg Sodium selenate 195.0 mg 195.0 mg 195.0 mg 195.0 mg 195.0 mg Phylloquinone 81.3 mg 81.3 mg 81.3 mg 81.3 mg 81.3 mg Vitamin D3 13.3 mg 13.3 mg 13.3 mg 13.3 mg 13.3 mg Cyanocobalamin 11.4 mg 11.4 mg 11.4 mg 11.4 mg 11.4 mg Blend (2’-FL, 3-FL, DFL, LNnT, LNT, 3’-SL, 6’-SL) 0.392 1.96 3.92 7.84 15.68 Example 21 [0126] Materials and Methods [0127] Fig.2 shows an exemplary experimental set-up to test BBB permeability. The RBEC monolayer assay applies primary rat brain microvascular endothelial cells isolated from three- - 35 - 4859-9621-0305, v.1 week old rats, co-cultured with rat pericytes in the presence of rat astrocyte-secreted factors. Endothelial cells are cultured on the upper side of a semipermeable porous filter, having pericytes on the backside and astrocyte-conditioned media in the bottom of the wells of the culture plate. Brain endothelial cells in contact with pericytes and astrocytic factors form a tight monolayer which separates two solute compartments, above and below the cell layer, that corresponds to the capillary lumen (apical side) and the brain interstitium (basolateral side), respectively. Under appropriate cell culture conditions, RBEC monolayers maintain the main characteristics of the cerebral endothelium in vivo. Moreover, the contribution of single transporters to the active efflux processes can be determined by the application of selective substrates or inhibitors. [0128] Primary cerebral endothelial cells were isolated according to a procedure developed in the Biological Research Centre, Szeged. Cerebral cortices originating from 3-week old rats were cut into small pieces and digested in two steps with collagenase and collagenase/dispase followed by centrifugation on percoll gradient. The cells were cultured on fibronectin/collagen- coated dishes in RBEC-medium: DMEM/F12 (Invitrogen), 10% plasma-derived serum (PDS, First Link, UK) and growth factors. After reaching confluency, endothelial cells were passed onto 24-well Transwell filters (pore size: 0.4 μm, Costar-Corning) containing pericytes on the backside. After reaching confluency, the monolayer was supplied with 550 nM hydrocortisone, CPT-cAMP (250 μM) and 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone (RO) RO-201724 (17.5 μM; Roche) together with the HMO composition (2′-FL, 3-FL, 3′-SL, 6′-SL, and LNT) for 24 hours. Filter inserts containing RBECs and pericytes were placed into dishes containing glial- conditioned media. TEER was continuously monitored using the cellZscope instrument (24-hour model). In the 12-hour model, TEER was measured with chopstick electrodes at 90 min, 3 hours and 12 hours. [0129] Before induction of oxidative stress using 2,3-Dimethoxy-1,4-naphthoquinone (DMNQ), media from both the top and the bottom compartments were collected and stored at -20°C. Selected filters received 25 μM DMNQ in the top compartment. TEER was continuously monitored with the cellZscope for 24 hours. [0130] Filters were removed from the dishes/cellZscope. After washing of the cells, permeability incubations were carried out in phenol red-free DMEM at 37°C with gentle shaking. Media - 36 - 4859-9621-0305, v.1 containing FITC-dextran 4 kDa (200 μg/ml) and TexasRed-dextran 70 kDa (200 μg/ml) were added into the top compartment, the bottom compartment containing phenol red-free DMEM. Samples were collected from the basolateral receptor compartment at the end of the incubation (30 min). The donor compartments were sampled at t0 min and t30 min. Samples were analyzed with a FLUOstar Optima instrument. [0131] Immunofluorescence (IF) assay: Cells were fixed with ethanol/acetic acid. IF was be performed using standard procedures. The following primary antibodies were used: Claudin-5 monoclonal antibody (4C3C2) (Thermo Fisher Scientific, cat. no.35-2500) and Zonula occludens protein-1 (ZO-1) polyclonal antibody (Thermo Fisher Scientific, cat. no.61-7300). Nuclei were stained with Hoechst 33342 (Merck-Sigma, cat. no. B2261). Reagents Chemical Supplier Catalogue No DMNQ Merck Sigma D5439 FITC-dextran 4 kDa Merck Sigma FD4 TexasRed-dextran 70 kDa Thermo Fisher Scientific D1830 [0132] Instruments used for detection include a cellZscope automated transepithelial/transendothelial electrical resistance (TEER) measurement system (nanoAnalytics, Münster, Germany) and a FluoStar Optima microplate reader (BMG Labtechnologies, Offenburg, Germany). [0133] Changes in TEER in response to the HMO composition (i.e., 2’-FL, 3-FL, 3’-SL, 6’-SL, and LNT) over time. TEER was continuously monitored using the cellZscope instrument. Incubation over 12 hour time period with 5HMO increases TEER resistance as compared to the control. See Fig.3. [0134] Figure 4 shows changes in permeability of RBEC monolayers in response to 2,3- Dimethoxy-1,4-naphthoquinone (DMNQ) in the presence or absence of the HMO composition. A) 4 kDa FITC-dextran B) 70 kDa TexasRed-dextran. Samples were collected from the basolateral receptor compartment at the end of the incubation (30 min). The presence of the HMO composition results in less permeability after disruption of the BBB with DMNQ as compared to the DMNQ only control group. - 37 - 4859-9621-0305, v.1 [0135] Figure 5 shows changes in the tight junction integrity of RBEC monolayers in response to the presence of the HMO composition at 10 mg/mL. Representative images of Immunofluorescence (IF) staining for localization of the claudin-5 protein (green) in RBEC monolayers indicates increased expression of claudin-5 at the tight junctions in the presence of the HMO composition (Fig. 5B) as compared to the control (Fig.5A). [0136] These experiments demonstrated that pre-treatment with the HMO compositions reduced the permeability of the blood brain barrier caused by hypoxic injury, simulated by the reactive oxygen species (ROS) produced during DMNQ incubation, and showed increase in tight junction proteins localized at the junction. Further, in certain instances administration of HMOs (one or more, including an HMO blend) results in restoration of blood-brain barrier after hypoxic injury, treating or ameliorating one or more symptoms of blood-brain barrier dysfunction. [0137] While the present disclosure has been illustrated by description of several embodiments and while the illustrative embodiments have been described in considerable detail, it is not the intention of the applicant to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications may readily appear to those skilled in the art. - 38 - 4859-9621-0305, v.1

Claims

What is claimed is: 1. A nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and an N-acetylglucosamine oligosaccharide for use in preventing degradation of barrier function of the blood-brain barrier in an individual in need thereof.
2. A nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N-acetylglucosamine oligosaccharide for use in treating one or more blood-brain barrier related conditions.
3. A nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N-acetylglucosamine oligosaccharide for use in enhancing cognition in an infant, toddler, or child.
4. A nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N-acetylglucosamine oligosaccharide for use in enhancing nutrient uptake in an individual in need thereof.
5. A nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N-acetylglucosamine oligosaccharide for use in enhancing brain chemistry.
6. A nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N-acetylglucosamine oligosaccharide for use in reducing blood-brain barrier permeability in an individual in need thereof.
7. The nutritional composition of any preceding claim, wherein the nutritional composition is one of an infant formula, a pediatric formula, a medical food, and an adult nutritional composition.
8. The nutritional composition of claim 2, wherein the one or more blood-brain barrier related conditions is selected from necrotizing enterocolitis, consequences of preterm birth, blood vessel damage, meningitis, hypoxia, compromised nervous systems, compromised neuronal blood flow, dysfunctional or impaired BBB, immaturity of the central nervous system, - 39 - 4859-9621-0305, v.1 blood-brain barrier dysfunction, traumatic brain injury, stroke, brain hemorrhage, neurodegenerative disease, cancer metastasis, and infectious diseases selected from: Group B Streptococcus, Listeria monocytogenes, Bacillus antracis, Haemophilus influenzae, Neisseria meningitidis, P. falciparum, Toxoplasma gondii, Acanthamoeba, Salmonella enterica, COVID- 19, RSV, and influenza virus.
9. The nutritional composition of any preceding claim, wherein the fucosylated oligosaccharide comprises at least one of 3-fucosyllactose (3-FL) and 2′-fucosyllactose (2′-FL); the sialylated oligosaccharide comprises at least one of 3’-sialyllactose (3′-SL) and 6′-sialyllactose; and the N-acetylglucosamine oligosaccharide comprises at least one of lacto-N- neotetraose (LNnT), lacto-N-tetraose (LNT).
10. The nutritional composition of any preceding claim, wherein the nutritional composition comprises total human milk oligosaccharides in an amount of from about 0.001 mg/mL to about 20 mg/mL.
11. The nutritional composition of any preceding claim, wherein the nutritional composition comprises about 0.001 mg/mL to about 20 mg/mL of a combination of 2′-fucosyllactose, 3- fucosyllactose, 3′-sialyllactose, 6′-sialyllactose, and lacto-N-tetraose.
12. The nutritional composition of claim 11, wherein the nutritional composition comprises 2′-FL, 3-FL, 3′-SL, 6′-SL, and LNT in an amount of from about 1.5 mg/mL to about 8.2 mg/mL.
13. The nutritional composition of claim 11, wherein the nutritional composition comprises 2’-FL in an amount up to 4.15 mg/mL, LNT in an amount up to 2.11 mg/mL, 3-FL in an amount up to 1.17 mg/mL, 3′-SL in an amount up to 0.36 mg/mL, and 6′-SL in an amount up to 0.44 mg/mL.
14. The nutritional composition of any preceding claim, wherein the nutritional composition comprises at least one of protein, carbohydrate, and fat.
15. The nutritional composition of any preceding claim, wherein the nutritional composition comprises protein, carbohydrate, and fat. - 40 - 4859-9621-0305, v.1
16. The nutritional composition of any preceding claim, wherein the nutritional composition comprises ARA, DHA, EPA, and DPA of from about 0.001 g/L to about 1 g/L or more, including from about 0.01 g/L to about 1 g/L, and about 0.1 g/L to about 1 g/L.
17. The nutritional composition of any preceding claim, wherein the nutritional composition comprises vitamin A, vitamin E, vitamin C, retinol, tocopherol, and carotenoids, including lutein, beta-carotene, zeaxanthin, and lycopene, and combinations thereof, for example.
18. The nutritional composition of any preceding claim, wherein the nutritional composition comprises comprise lutein in an amount of from about 0.001 µg/mL to about 10 µg/mL.
19. The nutritional composition of any preceding claim, wherein the nutritional composition comprises nucleotides in an amount of from about 10 mg/L to about 300 mg/L.
20. The nutritional composition of any preceding claim, wherein the nutritional composition is in the form of a liquid or reconstitutable powder comprising carbohydrate in an amount of from about 5% to about 40%; fat in an amount of from about 1% to about 30%; and protein in an amount of about 0.5% to about 30% by weight.
21. The nutritional composition of claim 1, wherein preventing degradation of barrier function of the blood-brain barrier comprises reducing permeability of the blood-brain barrier.
22. The nutritional composition of claim 2, wherein treating one or more blood-brain barrier related conditions comprises reducing permeability of the blood-brain barrier.
23. The nutritional composition of claim 2, wherein cognition is enhanced by at least one of reducing permeability of the Blood-brain barrier and enhancing nutrient uptake.
24. A method of preventing degradation of barrier function of the blood-brain barrier in an individual in need thereof, the method comprising administering a nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and an N- acetylglucosamine oligosaccharide to the individual.
25. A method of treating one or more blood-brain barrier related conditions in an individual in need thereof, the method comprising administering a nutritional composition comprising a - 41 - 4859-9621-0305, v.1 fucosylated oligosaccharide, a sialylated oligosaccharide, and a N-acetylglucosamine oligosaccharide to the individual.
26. A method of enhancing cognition in an infant, toddler, or child, the method comprising administering a nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N-acetylglucosamine oligosaccharide to the infant, toddler, or child.
27. A method of enhancing nutrient uptake in an individual in need thereof, the method comprising administering a nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N-acetylglucosamine oligosaccharide to the individual.
28. A method of enhancing brain chemistry in an individual in need thereof, the method comprising administering a nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N-acetylglucosamine oligosaccharide to the individual.
29. A method of reducing blood-brain barrier permeability in an individual in need thereof, the method comprising administering a nutritional composition comprising a fucosylated oligosaccharide, a sialylated oligosaccharide, and a N-acetylglucosamine oligosaccharide to the individual.
30. The method of any one of claims 24-29, wherein the nutritional composition is one of an infant formula, a pediatric formula, a medical food, and an adult nutritional composition.
31. The method of claim 25, wherein the one or more blood-brain barrier related conditions is selected from necrotizing enterocolitis, consequences of preterm birth, blood vessel damage, meningitis, hypoxia, compromised nervous systems, compromised neuronal blood flow, dysfunctional or impaired BBB, immaturity of the central nervous system, blood-brain barrier dysfunction, traumatic brain injury, stroke, brain hemorrhage, neurodegenerative disease, cancer metastasis, and infectious diseases selected from: Group B Streptococcus, Listeria monocytogenes, Bacillus antracis, Haemophilus influenzae, Neisseria meningitidis, P. falciparum, Toxoplasma gondii, Acanthamoeba, Salmonella enterica, COVID-19, RSV, and influenza virus. - 42 - 4859-9621-0305, v.1
32. The method of any one of claims 24-31, wherein the fucosylated oligosaccharide comprises at least one of 3-fucosyllactose (3-FL) and 2′-fucosyllactose (2′-FL); the sialylated oligosaccharide comprises at least one of 3’-sialyllactose (3′-SL) and 6′-sialyllactose; and the N- acetylglucosamine oligosaccharide comprises at least one of lacto-N-neotetraose (LNnT), lacto- N-tetraose (LNT).
33. The method of any one of claims 24-32, wherein the nutritional composition comprises total human milk oligosaccharides in an amount of from about 0.001 mg/mL to about 20 mg/mL.
34. The method of any one of claims 24-33, wherein the nutritional composition comprises about 0.001 mg/mL to about 20 mg/mL of a combination of 2′-fucosyllactose, 3-fucosyllactose, 3′-sialyllactose, 6′-sialyllactose, and lacto-N-tetraose.
35. The method of claim 34, wherein the nutritional composition comprises 2′-FL, 3-FL, 3′- SL, 6′-SL, and LNT in an amount of from about 1.5 mg/mL to about 8.2 mg/mL. 36. The method of claim 34, wherein the nutritional composition comprises 2’-FL in an amount up to 4.15 mg/mL, LNT in an amount up to 2.11 mg/mL, 3-FL in an amount up to 1.17 mg/mL, 3′-SL in an amount up to 0.
36 mg/mL, and 6′-SL in an amount up to 0.44 mg/mL.
37. The method of any one of claims 24-36, wherein the nutritional composition comprises at least one of protein, carbohydrate, and fat.
38. The method of any one of claims 24-37, wherein the nutritional composition comprises protein, carbohydrate, and fat.
39. The method of any one of claims 24-38, wherein the nutritional composition comprises ARA, DHA, EPA, and DPA of from about 0.001 g/L to about 1 g/L or more, including from about 0.01 g/L to about 1 g/L, and about 0.1 g/L to about 1 g/L.
40. The method of any one of claims 24-39, wherein the nutritional composition comprises vitamin A, vitamin E, vitamin C, retinol, tocopherol, and carotenoids, including lutein, beta- carotene, zeaxanthin, and lycopene, and combinations thereof, for example. - 43 - 4859-9621-0305, v.1
41. The method of any one of claims 24-40, wherein the nutritional composition comprises comprise lutein in an amount of from about 0.001 µg/mL to about 10 µg/mL.
42. The method of any one of claims 24-41, wherein the nutritional composition comprises nucleotides in an amount of from about 10 mg/L to about 300 mg/L.
43. The method of any one of claims 24-42, wherein the nutritional composition is in the form of a liquid or reconstitutable powder comprising carbohydrate in an amount of from about 5% to about 40%; fat in an amount of from about 1% to about 30%; and protein in an amount of about 0.5% to about 30% by weight.
44. The method of claim 24, wherein preventing degradation of barrier function of the blood- brain barrier comprises reducing permeability of the blood-brain barrier.
45. The method of claim 25, wherein treating one or more blood-brain barrier related conditions comprises reducing permeability of the blood-brain barrier.
46. The method of claim 25, wherein cognition is enhanced by at least one of reducing permeability of the Blood-brain barrier and enhancing nutrient uptake. - 44 - 4859-9621-0305, v.1
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6306908B1 (en) 1997-02-21 2001-10-23 Abbott Laboratories Methods for reducing the incidence of necrotizing enterocolitis
US6365218B1 (en) 2000-02-04 2002-04-02 Abbott Laboratories Pediatric formula and methods for providing nutrition and improving tolerance
US20030118703A1 (en) 2001-12-12 2003-06-26 Nguyen Minhthy Le Methods and compositions for brightening the color of thermally processed nutritionals
US20090098240A1 (en) 2007-05-17 2009-04-16 The Regents Of The University Of California Human milk oligosaccharides to promote growth of beneficial gut bacteria
US20150342974A1 (en) * 2012-12-18 2015-12-03 Abbott Laboratories Nutritional compositions comprising neuroprotective dietary oligosaccharides
US20160296540A1 (en) * 2013-11-15 2016-10-13 Nestec S.A. Compositions for use in the prevention or treatment of necrotizing enterocolitis in infants and young children
EP3494805A1 (en) * 2017-12-08 2019-06-12 Jennewein Biotechnologie GmbH Spray-dried tetrasaccharides
US20200260771A1 (en) * 2015-12-15 2020-08-20 Bruce McConnell Mixture of hmos

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6306908B1 (en) 1997-02-21 2001-10-23 Abbott Laboratories Methods for reducing the incidence of necrotizing enterocolitis
US6365218B1 (en) 2000-02-04 2002-04-02 Abbott Laboratories Pediatric formula and methods for providing nutrition and improving tolerance
US6589576B2 (en) 2000-02-04 2003-07-08 Abbott Laboratories Pediatric formula and methods for providing nutrition and improving tolerance
US20030118703A1 (en) 2001-12-12 2003-06-26 Nguyen Minhthy Le Methods and compositions for brightening the color of thermally processed nutritionals
US20090098240A1 (en) 2007-05-17 2009-04-16 The Regents Of The University Of California Human milk oligosaccharides to promote growth of beneficial gut bacteria
US20150342974A1 (en) * 2012-12-18 2015-12-03 Abbott Laboratories Nutritional compositions comprising neuroprotective dietary oligosaccharides
US20160296540A1 (en) * 2013-11-15 2016-10-13 Nestec S.A. Compositions for use in the prevention or treatment of necrotizing enterocolitis in infants and young children
US20200260771A1 (en) * 2015-12-15 2020-08-20 Bruce McConnell Mixture of hmos
EP3494805A1 (en) * 2017-12-08 2019-06-12 Jennewein Biotechnologie GmbH Spray-dried tetrasaccharides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AL-KHAFAJI ALIA H ET AL: "The potential of human milk oligosaccharides to impact the microbiota-gut-brain axis through modulation of the gut microbiota", JOURNAL OF FUNCTIONAL FOODS, ELSEVIER BV, NL, vol. 74, 23 September 2020 (2020-09-23), XP086272909, ISSN: 1756-4646, [retrieved on 20200923], DOI: 10.1016/J.JFF.2020.104176 *
GAO XIAOXIANG ET AL: "Antiviral effects of human milk oligosaccharides: A review", INTERNATIONAL DAIRY JOURNAL, vol. 110, 1 November 2020 (2020-11-01), GB, pages 1 - 10, XP055913316, ISSN: 0958-6946, DOI: 10.1016/j.idairyj.2020.104784 *

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